EAFP 2021

20 - 23 September 2021 ● Virtual

Abstract Book

Page 0
Welcome

Dear Conference delegates,

Welcome to the European Association of Fish Pathologists Conference. Of course this is not the scenario any
of us wanted when we started planning the Conference almost three years ago, we were very much looking
forward to welcoming you to the UK and of course to our fantastic City of Aberdeen and beautiful country
of Scotland.

We thank you for joining us virtually, our exciting programme will present the very latest research and
discoveries and we hope you will find the whole event inspiring, informative and stimulating - we have nearly
400 delegates, 200 speakers and 120 poster presentations. All the recordings will be loaded after they have
been delivered and will be able to be viewed on the portal for an additional 3 months for you to catch up
and enjoy at your leisure.

Thank you to our keynote speakers for sharing their vast knowledge, to all those that submitted an abstract
and are presenting a paper or a poster and also to our supporter Pathovet.

We hope you will embrace and enjoy this year’s virtual meeting and look forward to welcoming you in person
when we host the next EAFP Conference in Aberdeen in 2023.

Best wishes

Prof Pieter van West

Director of the International Centre of Aquaculture Research and Development

University of Aberdeen

Page 1
Keynote Speakers

Listed in presenting order Page 2

Michalis Pavlidis
Michalis, Biologist, is a Professor of “Biology-Physiology of Marine Organisms”
at the Department of Biology, University of Crete, Greece.

His current research is focusing on the molecular, neuro-endocrine and

environmental interactions involved in the regulation of the stress response, in
relation to individual differences in coping strategies, and on the development
of non-invasive reliable welfare indicators in fish species.

He has participated, as coordinator or principal investigator, in more than 30

European, bilateral and National RTD projects and networks and has more than
80 publications in peer-reviewed journals.

Co-editor of the books “The Welfare of Fish” (Springer, 2020) and “Sparidae: Biology and aquaculture of
gilthead sea bream and other species” (Wiley-Blackwell, 2011). Co-author of the HAPO study
“Mediterranean Fish Welfare: Guide to good practices and assessment indicators” (2019). He coordinates
the Erasmus Plus Joint Master Degree in “Aquaculture, Environment and Society”. He also coordinates the
annual FELASA Accredited Course “Care and use of laboratory animals: mice, rats and zebrafish”. National
Representative at the SCAR (Standing Committee on Agricultural Research) Fish Strategic Working Group
(March 2017-today). Since September 2020, vice rector of Research and Development at the University of
Crete.

Welfare of farmed fish: moral considerations, science, and problems of implementation

The fish welfare concept was first raised in 1990s and after the turn of the millennium, it was used by more
and more scientists in the field of aquaculture, animal advocates, politicians, legislators, the fish farming
industry, and consumers. The interaction between empirical scientific data, contemporary animal welfare
concept, and normative ethical theories will be presented. Critical questions and characteristic examples on
the complex interplay among stress, allostatic mechanisms, individual coping strategies, susceptibility to
infectious diseases, health and fish welfare will be given.

Apart from definition problems, theoretical arguments, and societal perspectives on whether fish are
consciousness animals and/or can feel pain (in comparison to terrestrial farmed mammals and poultry birds
for which broad consensus exists), there is an urgent need for a scientific based assessment of welfare
through the development of validated, reliable, and applicable on farm welfare indicators and tools. How do
we define/evaluate fish coping abilities under intensive culture conditions? How do we measure welfare?
Recent advances in the development of direct (fish-based) and indirect (environmental-based), laboratory
and operational fish welfare indicators and technological advancements (e.g. artificial intelligence, machine
learning and digital applications, novel sensors) will be presented.

Overall, the “fish welfare” challenge may essentially be seen under the One Health principle, i.e. “the
collaborative, multisectoral and transdisciplinary approach to achieving beneficial health and well-being
outcomes for producers, people, non-human organisms and their shared environment”.

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Grant Stentiford
Grant is Healthy Seafood Theme lead at Cefas, Head of the OIE Collaborating
Centre for Emerging Aquatic Animal Diseases and, co-Director of the Sustainable
Aquaculture Futures Centre at the University of Exeter, UK. He was Director of
the European Union Reference Laboratory (EURL) for Crustacean Diseases on
behalf of DG SANCO of the European Commission between 2008 and 2018 and
Science lead for Aquatic Animal Health at Cefas from 2016 to 2019.

He has a BSc (first class) in Life Sciences from the University of Nottingham, UK
(1993-1997) and a PhD in invertebrate pathology from the University of Glasgow
(1997-2000). He was elected a Fellow of the Royal Society of Pathology in 2014
and a fellow of the Linnean Society in 2016.

His work focuses on the identification and impact of aquatic animal diseases in farmed and wild
environments. He has led the development of the One Health Aquaculture concept within Cefas, working
with a diverse array of specialists in aquatic animal and human health hazards to build the concept into
current UK and overseas programme work. He tweets on One Health Aquaculture at: @grantstentiford

Sustainable aquaculture through the One Health lens

Aquaculture is one of the fastest growing and highly traded global food sectors and is set to dominate the
supply of aquatic protein by 2050. For aquaculture to deliver significantly enhanced volumes of food in a
sustainable manner, however, appropriate account now needs to be taken of its impacts on the
environment, the welfare of organisms cultured, and human health outcomes of consuming seafood from
this sector.

The One Health Aquaculture concept argues that the sector requires an evidence-policy refresh, bringing in
a wider array of industry, government and societal stakeholders to ensure that key ‘success metrics’ spanning
human, environmental and organismal health are ‘designed-in’ to current and future aquaculture systems -
effectively operationalising a One Health approach to food production from the sector. Sustainable
aquaculture success metrics, applied to a given sub-sector of the industry (e.g., seaweed, molluscs, shrimp,
finfish) and assessed according to the availability of research, evidence, policy and legislation specific to those
metrics can provide a simple framework around which potential negative impacts associated with production
can be defined and reduced or averted. As aquaculture positions to dominate supply of seafood in the next
three decades, the application of One Health principles to the sector provides a means to optimise the
environmental, human, and organismal health benefits of food production from water.

In addition, the increasing recognition of aquaculture as a major global food sector will necessitate a broader
debate on its interaction with land-based food production systems and must underpin appropriate national
and international science and policy strategies to support improved food system design. In particular, the
intricate interaction between aquatic animals and their environment will necessitate closer alignment of
environmental and food policy and, better appreciation of the role of chemical and pathogen hazards in
disrupting aquatic food supplies.

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Hynek Mazanec
Hynek is a Ph.D. student at the Institute of Parasitology, Czech Academy of
Sciences & University of South Bohemia.

He started his Ph.D. in 2018 with a focus on extracellular vesicles (EVs) of

parasitic helminths and the establishment of their life cycles in laboratory
conditions. Together with his colleagues, he is trying to elucidate the role of EVs
during the ontogeny of fish and mammalian helminths and their possible
interplay with the surrounding host environment.

Furthermore, through a combination of ultrastructural and proteomic studies he

is exploring the mechanisms of biogenesis of EVs in helminths and their similarity
to EVs of their hosts.

Diversity and role of extracellular vesicles in the host-parasite crosstalk

Intercellular communication is a fundamental mechanism necessary for all living organisms to synchronize
cellular activities, regulate cell proliferation, and respond to environmental changes. Traditionally,
intercellular signaling was associated solely with soluble excretory-secretory products (ESP) such as proteins,
lipids, glycans and nucleic acids. However, recent studies are elucidating a major role of extracellular vesicles
(EVs) in the cell-cell crosstalk. EVs are lipid-bilayer delimited structures secreted from cells/organisms
through an endosomal pathway (,,exosome”) or a direct budding of the membrane (,,microvesicles”). Despite
the major attention being given to mammalian research, EVs have found their way to the studies of parasitic
diseases in the last two decades.

Based on the viable data we see similarities in terms of the basic biology of EVs across different parasitic
groups and also when compared to mammalian systems. Endocytic pathway, as well as direct shedding of
vesicles, from the surface have been observed among protozoans, nematodes as well as platyhelminths.
Furthermore, common markers of their biogenesis and cargo loading, such as ESCRT complex, syntenin, VPS4
or annexin A1, have been observed. However, the effect on targeted cells differs across various species, life
cycle stages and even specific subpopulations of secreted EVs. It is the specific reactions that they induce
when exposed to the host immune system, that are being exploited in a hope of better understanding the
host-parasite relationship and developing new drugs or vaccines.

In my talk we will cover: (i) the basic biology of extracellular vesicles, common populations observed and
their markers; (ii) challenges in isolation and observation of EVs in parasitic organisms; (iii) recent
developments in the field and possible next steps in the future.

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Ryan Carnegie
Ryan is a marine biologist and a Research Professor at the Virginia Institute of
Marine Science, William & Mary, in Virginia, USA.

He received his PhD from the University of Maine, USA, in 2000, and after
postdoctoral appointments at the Pacific Biological Station in Nanaimo, British
Columbia, Canada, and the Medical University of South Carolina, USA, began
working at VIMS in 2002.

Ryan's research focuses on the diversity and distributions of marine pathogens,

the evolutionary ecology of infectious diseases, and the management of aquatic
animal health in the context of fisheries, aquaculture, and shellfish restoration.

He is widely engaged in aquatic animal health science and management internationally, including in his work
as a reference expert for haplosporidiosis and perkinsosis within the World Organisation for Animal Health
(OIE), and on the ICES Working Group on Pathology and Diseases of Marine Organisms.

Intersections of historical and evolutionary ecology shape the long-term trajectory of an

oyster host-parasite system

Understanding the ecology and impacts of major diseases is essential for effective management of affected
resource species. This is particularly important for marine bivalve molluscs like oysters that are affected by
numerous major pathogens, and that are the focus of restoration efforts financed by substantial public
funding. In the eastern USA, improvement in the status of eastern oyster Crassostrea virginica resources,
both in aquaculture and the wild, is held as a global model both for overcoming disease in aquaculture and
for successful, large-scale shellfish restoration, because of the development of effective approaches to
mitigate disease impacts where natural processes had failed to do so over many decades. New perspective
emerging from long-term study of diseases in the Chesapeake Bay region of the USA is providing a far more
nuanced perspective, however.

The present, improved status of oyster resources may be less a product of human innovation, and more a
function of evolutionary changes both in pathogens and the oyster host that have been set in motion by
historical as well as continuing human impacts on oyster populations. The new insight into these dynamics
highlights the fundamental importance of long-term, ecological and evolutionary perspective for all of our
managed systems. This presentation will describe the trajectory of this system as we can presently perceive
it, and the potential promise of new molecular approaches to provide even more improved resolution.

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Workshop Abstracts

Listed in presenting order Page 7

Diseases of ornamental and laboratory fishes

Ornamental and laboratory fish are extensively traded globally, and can spread pathogens around the world.
Laboratory fish are also widely used as models for biomedical research, which can be impacted by
underestimated health conditions affecting the models. Ornamental fish diseases can spread undetected
between artificially recreated ecosystems, posing threats that are difficult to eradicate when established, or
when contaminating natural water systems. This virtual workshop focuses on the pathobiology and
diagnostics of infectious threats that could be spread through ornamental fishes, and those impacting
research using laboratory fish models. This is an open workshop. A joint article from this workshop will be
published in the EAFP Bulletin.

Chair: Bartolomeo Gorgoglione

[email protected]

Major fish diseases of laboratory fishes: zebrafish and killifish

Olga Haenen, Wageningen Bioveterinary Research, Netherlands

[email protected]

Laboratory fish, like zebrafish (Danio rerio) and African turquoise killifish (Nothobranchius furzeri) are world
widely used as models for veterinary and biomedical research . Zebrafish is used in experimental studies on
genetics, biology, and immunology/diseases of humans and animals. Genetically modified fluorescing
zebrafish, so-called GloFish is also used. The African turquoise killifish is the shortest-living vertebrate bred
in captivity, with a lifespan of three to nine months, ideal for research on aging. Transgenic strains of killifish
are also used.

Although these fish species are ideal model animals for these themes of disease research, they are also
vulnerable to certain fish pathogens. Some of these pathogens may cause chronic, and often neglected, or
only lately clinically manifesting fish diseases, like fish tuberculosis, Pleistophora hyphessobryconis or
Pseudoloma neurophilia in zebrafish, and neoplasia, or Glugea in killifish.

In case experimental laboratory fish suffer even non-clinical fish disease, they should not be used as
experimental model fish, as fish disease will change the physiology and immune biology of the fish. This
artefact will influence the outcome of the experimental model research on for instance human disease. This
may have a big negative impact, as data would be peer reviewed published, but being biased, would be non-
reliable for practice.

Therefore it is of utmost importance, to start with specific pathogen free (SPF) laboratory fish, and have
quarantaine facilities and a preventive monitoring for fish disease in place during laboratory fish rearing and
experiments.

In this lecture, major fish diseases occurring in zebrafish and African turquoise killifish, and how to recognize
and diagnose them, are presented.

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Mycobacteriosis in laboratory fishes: A model system using fluorescent mycobacterium
chelonae and transparent zebrafish

Christopher Whipps, State University of New York, USA

[email protected]

Laboratory zebrafish (Danio rerio) have become an important model organism for a broad array of research
including genetics, development, toxicology, immune system function and infectious disease. Zebrafish can
also be infected with a range of their own pathogens, with impacts ranging from sub-clinical to severe. These
infections can have a confounding impact on research, by directly causing mortality and by contributing to
unanticipated experimental variation when infections are not apparent.

Mycobacteriosis, caused by Mycobacterium species, is a commonly occurring infection in zebrafish. Some

species cause acute and severe disease, which can be devastating for a facility, but several species are
associated with the chronic, sub-clinical infections that persist unnoticed. Good biosecurity to prevent
introductions is of great importance, and should include quarantine and disinfection of eggs. Monitoring
within the facility is also important to catch any infections that may be in the main facility. One
Mycobacterium species that may be encountered even with good biosecurity, is M. chelonae. This species is
associated with chronic infections and is widespread. Because of this, we began evaluating a system using a
GFP expressing M. chelonae, and adult casper zebrafish that lack skin pigment. The advantage of this system
is that infections can be visualized under fluorescence in live fish, avoiding the need to euthanize zebrafish
to determine their infection status. Using this system we have evaluated transmission of mycobacteria to
zebrafish via live feeds, transmission to and from zebrafish and surface biofilms, and treatment of infected
fish with antibiotics. Live feeds have the potential to transmit bacteria, but this risk likely depends more on
the care and maintenance of live feeds.

Some candidate antibiotics are effective at reducing severity of infection and may be appropriate for use in
a targeted manner, like with brood stock of a rare or valuable line. Surface biofilm studies show that
transmission between fish and the environment can occur rapidly, highlighting the importance of removing
sick fish and regular system maintenance. The lessons learned with this model have utility beyond laboratory
zebrafish, including those working on other aquatic models, or in aquaculture.

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From catcher to keeper, the complex trade in ornamental aquatic animals

Nick Stinton, Cefas, UK

[email protected]

Ornamental fish keeping is one of the most popular forms of pet keeping in the world and generally just
behind cats and dogs in their popularity. The trade in ornamental aquatic animals is truly global. Live animals
are shipped across the world, ultimately destined for aquaria and ponds in people’s homes. Owners vary
from average families to the most dedicated of aquatic hobbyists. However, the trade is highly complex, not
only in terms of the range of animals traded, the number of sources, the multiple business models and stages
involved in the supply that enter the trade.

Animals are sourced from both wild capture and aquaculture, which often is dictated by the type or species
of animals traded. For example, most tropical marine species traded are wild caught, whereas most traded
tropical freshwater species are cultured. The debate over the relative ethics of wild capture animals is
continuously debated and there are increasing efforts to ensure a sustainable future for such sources. This
is especially important, as the industry needs a consistent supply and also a very important provider of
income to the poorest of communities.

The culture of ornamental animals can be significantly important to the economies of many countries and
regions across the world and can range from the simplest of ‘back-yard’ or home scale operations, to purpose
built sophisticated re-circulation systems. Hundreds of species, and often in a wide variety of colour-forms
and body shapes are cultured. The routes and stages of supply from these sources to the end-user are equally
complex and diverse, and all have overcome the logistical challenges that allow the shipment of aquatic
animals from one side of the world to another but ensuring the maximum survivability and quality of animals
that reach the hobbyist.

All too often the end point of the animals, the hobbyist, has little or no awareness of when they choose their
animal from their retailer, how complex the trade is and how or where the animals may have come from and
how they made it to them!

Page 10
Transboundary spreading of fish viral disease via ornamental fish

Takafumi Ito, Japan Fisheries Research and Education Agency, Japan

[email protected]

It is known that many aquatic animal pathogens and diseases have spread across the borders by trade in live
aquatic animals. This is not only the case for aquaculture but also for ornamental fish trade. In particular,
pathogens and diseases in ornamental fish are easily neglected, since these fish are reared in closed
environments, such as small tanks, mainly for hobbyists. Freshwater and marine ornamental fish are traded
and transported between more than 125 countries with a retail value of more than US 10 billion $. Thus,
economic damage due to fish disease may be huge.

Previously, we detected cyprinid herpesvirus 2 (CyHV-2) in 4 of 8 batches of imported goldfish imported into
the Netherlands from countries outside the European Union. Our results prove that one of the routes of
spread of various CyHV-2 strains is through the global trade of apparently healthy infected goldfish. The virus
also causes severe mortality to gibel/prussian carp, an ancestral species of goldfish, in natural waters in
Europe and Asia.

Carp edema virus (CEV) detected in koi and common carp in multiple countries is also thought to be spreading
by trade of koi carp. This year, we reported the entire genome of CEV, with more molecular epidemiological
studies to follow on this virus.

Infectious spleen and kidney necrosis virus has been isolated from a few kinds of imported ornamental fish
such as angelfish and dwarf gourami. Infection experiments have been reported that the dwarf gourami
isolate is virulent to murray cod. Movements of non-native species may drive host switching.

Import of infected ornamental fish not only directly affects the fish of hobbyists, but may even cross-infect
aquaculture fish at the culture or transfer facility, and even wild fish in open water, through the water drain
or released diseased fish. Therefore, general hygiene awareness and measures must be in place in the whole
chain of ornamental fish trade, from catch or culture up to hobbyist.

Page 11
Reviewing treatments against common ectoparasites in freshwater ornamental fish

Christina Dover, Michigan State University, USA

[email protected]

The global ornamental fish industry deals with a huge diversity of fish species. High mortality rate often
causes significant losses, linked to polymicrobial infections facilitated by stressful conditions compromising
host health, although accurate data on ornamental fish trade losses remain difficult to retrieve. Ectoparasites
are among the most common causes of disease in freshwater ornamental fish, being easily transmitted
between individuals, particularly when highly stressed, and at high stocking densities. Ectoparasite
treatments for ornamental fish remain limited, often relying on commercial products with undefined
composition, lacking precise dosages and information on target species and efficacy, and posing concerns
about their toxicity, safety, and disposal.

We reviewed treatment methods available against some of the most common ectoparasites affecting a wide
range of species, including selected protists and monogeneans: Ichthyophthirius multifiliis, trichodinids,
Tetrahymena sp., gill (Dactylogyrus sp.) or skin (Gyrodactylus sp.) flukes. We investigated suitable methods
that could be reasonably applied by aquatic veterinarians to ornamental fish in a typical retail setting.
Selected treatments were tested on a commercial stock of fantail goldfish (Carassius auratus) during their
quarantine upon arrival in a pet shop, after 5 days of acclimation.

Naturally occurring infections with Gyrodactylus sp. were morphologically identified with light microscopy,
confirming an even infection before allocating fish to each treatment tank, respectively with FMC (10 ppm),
potassium permanganate (3.6 ppm), Praziquantel (13.5 ppm dissolved in ethanol), Levamisole (10 ppm),
while using ethanol (229 ppm) as a drug vehicle control. 24 h bath treatments were administered at day 0,
3, 7, and 10. Mucus scrapes were sampled prior to each treatment on days 0, 3, 7, and 10, and lastly on day
17.

All treatments were easily applied and well tolerated by goldfish, but none of them resulted in 100% efficacy.
Over our 3-week treatment trial, Levamisole potentially showed the most promising results in reducing the
skin mucus load of Gyrodactylus sp. flukes, while no trichodinids were detected after using potassium
permanganate. Future research should assess specificity, efficacy, and dosages of medications against
ectoparasites in ornamental fishes as well as their toxicity and safety for hosts, operators, and the
environment upon disposal.

Page 12
Parasitic infestation in Corydoras sp. in northern Peruvian Amazon

Jefferson Yunis Aguinaga, IMARPE, Peru

[email protected]

The aim of this study was to determine the seasonal occurrence of the main parasite species in Corydoras
sp. from rivers of the Northern Peruvian Amazon. This study was carried out during 3 years, 38 214 Corydoras
sp. of 22 different species were analyzed, which were obtained from artisanal fishing. After euthanasia with
a benzocaine solution (1: 10,000) diluted in 98 ° ethanol (0.1 g/ml), the specimens were examined at the
macro and microscopic level to detect the presence of ectoparasites. Skin smears were performed using a
sterile scalpel to obtain mucus samples, these samples were placed on slides by adding a drop of 0.85% saline
solution and then compressed with a coverslip for microscopic examination. In addition, the branchial arches
were sectioned and placed in a Petri dish adding 0.85% saline solution to observe them with the stereoscope.

The taxonomic identification of the ectoparasites followed the previously established guidelines. For the
statistical analysis, the percentage of parasitism of each species analyzed was calculated. It was found that
5397 fish were parasitized (14.12%). In both seasons (rainy and dry), similar amounts of parasites were
observed. However, some species showed more parasites in some seasons. C. blochi (40.38%), C. virginiae
(28.99%), C. melanotaenia (18.57%), C. julii (16.00%), and C. arcuatus (10.48%) showed the highest rates of
parasitism, while C. rabauti (5.52%), C. bilineatus (5.26%), C. narcissus (3.93%), C. loretoensis (2.40%), and C.
pygmaeus (1.21%) presented the lowest rates of parasitism.

On the other hand, the Monogenea class was the most abundant group of parasites, occurring in 48.4% of
the parasitized animals. The percentage of these parasites might be related to the time of year, origin,
individual susceptibility and the handling of the animals during transport by fishermen.

Page 13
Antimicrobial susceptibility of bacteria isolates from intestinal flora of dwarf gourami
(Colisa lalia)

Sandrine Baron, French Agency for Food, Environmental and Occupational Health & Safety, France

[email protected]

Bacterial diseases are among the more significant health problems associated with ornamental fish culture
and export worldwide. This can result in frequent and often unregulated use of antibiotics in the absence of
appropriate diagnostic testing. Such routine unregulated application during normal farming has been linked
to the development of antibiotic-resistant strains of bacterial pathogens and bacterial isolates with antibiotic
resistance profiles have become common within the ornamental fish industry. Furthermore, a number of
common bacterial isolates from ornamental fish also possess zoonotic potential and there have been
multiple reports of human pathogens isolated from aquarium fish and their water and human bacterial
infections associated with aquarium keeping.

Methodology
In this study, we characterized the bacteria recovered from the intestinal flora of the dwarf gourami (Colisa
lalia), a popular ornamental species originating from India, Bangladesh, and Pakistan. Fourty-two isolates
were collected from the intestinal track of nine fish and identified by Maldi-Tof. Susceptibility testing was
performed based on agar diffusion using CLSI or EUCAST methods, depending on the genus. The
antimicrobial agents were chosen based on the agents used in aquaculture according to CLSI list and WHO
(critical for human).

Results
The 42 isolates included representants from 12 genera. The four most abundant genera were Citrobacter
(38.0%, n =13), Aeromonas (19.4%, n=8), Bacillus (16.6%, n=7), and Pseudomonas (9.5%, n=4). The eight
other genera were only represented by one isolate.

Among the 13 Citrobacter spp., the most frequent resistances were to acid nalidixic acid (84.7%) cefoxitin,
amoxicillin-acid clavulanic (76.9%), and ampicillin (53.8%). One isolate was resistant to all the betalactamins
tested. Aeromonas isolates were susceptible to most of the antimicrobial agents tested. The most frequent
resistance was to carbepenem (n=3), which in Aeromonas is often linked to the chromosomic gene cphA.

Page 14
Intra vitam detection of cyprinid herpesvirus 3 (CyHV-3) in koi carp for export from Japan

Kei Yuasa, Japan Fisheries Research and Education Agency, Japan

[email protected]

Koi herpesvirus (KHV) disease, which is listed by the OIE as a notifiable disease, is the infection with cyprinid
herpesvirus 3 (CyHV-3) to common carp Cyprinus carpio and its varieties, subspecies and hybrids.

Koi carp Cyprinus carpio koi is one of the most sensitive subspecies to KHV and is being traded at high prices
throughout the world. Therefore, intra vitam assays for detecting KHV in targeted fish with a high detection
efficiency is essential for its trading.

In this study, PCR detection from the gills 1), scales 2) and mucosal epithelium 3) of infected fish, and PCR
detection from the gills 4) of carp cohabited with infected fish were compared. To observe effects on fish by
each biopsy, a control group (non-infection group) was prepared for each assay of 1), 2) and 3). The results
suggest that PCR with 1) the gills or 2) scales was superior to the other two methods in detection efficiency
of the virus. Neither mortality nor any adverse effects including secondary bacterial infection, haemorrhage,
inflammation were observed in the control groups.

According to this result, Ministry of Agriculture, Forestry and Fisheries (MAFF) in Japan mentioned a
necessary implementation of PCR with the gills randomly sampled from all tanks/aquarium for export in each
establishment, according to the OIE code which should be continued twice a year with at least 3 month-
interval as a procedure to demonstrate free from KHV in each establishment of the farm in the guideline for
exporting koi carp.

Page 15
Persistent like carp edema virus - a case study of koi sleepy disease affected fish population

Mikolaj Adamek, University of Veterinary Medicine Hannover, Germany

[email protected]

Poxviruses are known for persistence in the environment and ability to reinfect their hosts at a suitable
timepoint. Carp edema virus (CEV) is a very successful fish poxvirus with worldwide distribution driven by
the international koi and common carp trade. In Germany, the virus was repeatedly found in fish shipments
which were completely asymptomatic even though CEV often causes very severe disease with high mortality.
Furthermore, some fish farmers and ornamental fish keepers experienced repeated outbreaks of the disease
without introduction of new fish. This raised the question if the virus could persist for longer time in the
population of fish after the outbreak.

Methodology
Ornamental fish population consisting of 23 koi was followed up after an outbreak of koi sleepy disease
(KSD). The virus load was measured by qPCR in the gill swabs collected during routine health check-ups. The
fish have been individualised by recognition of their colour pattern.

Results
The fish population initially experienced mild outbreak of KSD in May/June with the fish showing lethargic
behaviour characteristic for this disease. All 23 fish were confirmed to be positive for CEV with the virus load
from 5 to 1600 virus copies. Immediately after diagnosis, a 12-day long salt bath (0.5%) was administered,
which led the clinical signs to subside and only one fish died. However, after this treatment 14 from 22 fish
remained positive for CEV with virus load from 3 to 160 copies of the virus while being clinical signs free.
During routine autumn health check performed in October 3 from 11 fish remained CEV positive, despite no
clinical signs with, and a virus load from 12 to 251 copies of the virus.

Conclusions
Following a small population of fish, it could be shown that CEV seems to be persisting in fish for at least 5
months with some individuals being long haulers of the virus. That long persistence of the virus could explain
the successful spread of the virus and should be an imperative for checking fish before purchase as the virus
seems to be difficult to get cleared from a susceptible population.

Page 16
Aquatic animal health education

Collaborating Organisations: European Association of Fish Pathologists (EAFP), European Association of

Establishments for Veterinary Education (EAEVE), Federation of Veterinarians in Europe (FVE), European
College of Aquatic Animal Health (ECAAH), International Partnership for Aquatic Veterinary Education (i-
PAVE) and World Aquatic Veterinary Medical Association (WAVMA).

Workshop objectives: With animal diseases being one of the greatest obstacles to aquaculture development
and sustainability, a well-trained workforce of aquatic veterinarians and other aquatic animal health (para-
veterinary) professionals is essential. This session will outline several efforts to understand what educational
programs exist, how these provide and recognize competency in diverse knowledge, skills and experience
needed for this workforce, and, with panelists and audience input, develop possible future actions to ensure
aquatic animal industries are well supported.

An overview of efforts and needs for well trained global aquatic animal health workforce
David Scarfe, University of Pretoria, South Africa
[email protected]

Available Training and Education Opportunities for Aquatic Animal Health Professionals in Europe
Francesc Padros, University of Barcelona, Spain
[email protected]

European aquatic veterinary medicine training programs - connecting blue and green
through one health approach
Despoina Iatridou, FVE, Belgium
[email protected]

What is needed for ensuring a well-trained aquatic health workforce? Discussion

EAEVE Accreditation of Veterinary Education Establishments

Ana Bravo, Universidad de Santiago de Compostela, Spain
[email protected]

Credentialing programs recognizing day-1 and advanced competencies in aquatic

veterinary medicine
Dušan Palić, Ludwig Maximilians University, Germany
[email protected]

Knowledge, skills, and experiences in aquatic animal health curricula and continuing
education programmes, discussion
Session summary: the way forward: paths toward ensuring a well-educated aquatic animal
health workforce
Andreas Fabris, API, Italy
[email protected]
Page 17
Myxozoan research forum

Apart from the myxozoan sessions with regular talks we are planning to have a relaxed meeting with
discussions on relevant topics in myxozoan research. There will be short introductions to these topics and
then we will go through any related issues and hope to get views and potential solutions from different
workgroups. This forum further opens the possibility for troubleshooting in different research
methodologies, and it can simply be a point for getting together with researchers working in your field.
Though this is a bit different in an online meeting, after a year of training I am sure we can make this work.

Join for a relaxed but lively discussion on hot topics of research in Myxozoa and anything relevant for the
study of these fish parasites in their hosts.

Topics will include:

• Recent genomic and proteomic advances
• Key molecules of host-parasite interactions
• Species diversity

Stephen Atkinson, Oregon State University, USA

[email protected]

Ivan Fiala, Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Czech Republic
[email protected]

Jason Holland, School of Biological Sciences, University of Aberdeen, UK

[email protected]

Astrid Holzer, Institute of Parasitology, Biology Centre of The Czech Academy Of Sciences, Czech Republic
[email protected]

Pavla Sojkova
Institute of Parasitology, Biology Center of the Czech Academy of Sciences, Czech Republic
[email protected]

Page 18
How outputs from EU projects can upgrade health management in Mediterranean
aquaculture

Snježana Zrnčić, Francesc Padros, Edgar Brun, Croatia

[email protected]

Aquaculture of European sea bass and gilthead sea bream is an essential activity in the Mediterranean basin
and many EU projects are focusing on the improvement of its performances. This workshop aims to display,
share and discuss projects' achievements among interested colleagues engaged in fish diseases. Starting by
alphabetical order, AdriAquaNet, an Italy-Croatia Interreg project focuses on the identification and
characterization of marine natural products (MNPs) expressing immunoregulatory and antimicrobial activity
against bacterial pathogens Vibrio spp. and Photobacterium spp. Project also successfully tested on-farm an
autochthonous Bacillus sp. isolate as supplement in novel feed formulation, evidencing positively altered
host microbiome and expression of genes related to gut epithelial integrity and immunity.

The Horizon 2020 project FutureEUAqua is exploiting the potential of the Internet of Things (IoT) to address
the challenges of a sustainable and resilient aquaculture system that ensures profitability, maintains healthy
aquatic ecosystems and strengthens capacity for adaptation to climate change. H2020 project MedAID is
aiming at establishing a prophylactic strategy for sea bass against viral nervous necrosis (VNN) by testing an
innovative recombinant vaccine in dose-response and time-course vaccination trials under experimental
conditions. The capsid protein auto-assembled into virus-like particles (VLPs) induced a strong antibody
response and long-lasting protection.

Besides, a systematic biosecurity scoring system was developed for sea bass and sea bream farms to provide
a way to evaluate and identify quantifiable biosecurity measures to be applied to improve the farm´s overall
biosecurity. ParaFishControl (H2020), among the rest, developed an extensive guide for sea bass and sea
bream farmers on how to combat parasitic infections. PerformFISH (H2020) evaluated relevant factors and
provided more efficient tools for improvement of productive KPI’s, to enable deeper insight into
identification, tracking, prevention and control of the most relevant diseases for sea bream and sea bass
farming.

It identified and developed reliable diagnostic methods; generated preventive strategies based on
immunoprophylaxis and efficient treatments; outlined methodologies for fish welfare awareness at farm
level and developed health management strategies for the industry to improve the current KPI’s (mortality,
morbidity and economic indexes).

MedAID - A systematic approach for quantifying biosecurity measures

Saraya Tavornapanich, NVI, Norway

[email protected]

MedAID - Vaccination against VNN

Niels Lorenzen, DTU-Aqua, Denmark

[email protected]

Page 19
PerformFish - Current developments in seabream and seabass health management
strategies for the Mediterranean farming: a view from PerformFish
Francesc Padrós, Maria Letizia Fioravanti, Spain

[email protected]

AdriAquaNet – Novel natural compounds as potential therapeutics in aquaculture

Donatella Volpatti, UNIUD, Italy

[email protected]

AdriAquaNet - Novel feed formulation supplemented with autochthonous Bacillus sp.

positively altered host-microbiome
Jerko Hrabar, IZOR, Croatia

[email protected]

FutureEUAqua Project: How the Internet of Things can enhance aquaculture farms
productivity and ensure sustainability?
Amedeo Manfrin, IZSVE, Italy

[email protected]

ParaFishControl – From practical guides to combat parasitic infections of gilthead sea

bream and sea bass to recommendations for policymakers
Ariadna Sitjà-Bobadilla, IATS-CSIC, Spain

[email protected]

Page 20
Oral Abstracts

Listed in presenting order

Page 21
Monday 20th September 2021
1.1 Aquatic animal welfare

MyFishCheck: A model to assess fish welfare in aquaculture

Linda Tschirren1,2, David Bachmann1, Ali Cem Güler1, Oliver Blaser1, Nicola Rhyner3, Andreas Seitz1, Erich
Zbinden4, Thomas Wahli2, Helmut Segner2 and Dominik Refardt1

1Zurich University of Applied Sciences, Institute of Natural Resource Sciences, Research Group for
Aquaculture Systems, Switzerland
2University of Berne, Centre for Fish and Wildlife Health, Switzerland
3Zurich University of Applied Sciences, Institute of Natural Resource Sciences, Research Group for

Environmental Genomics and Systems Biology, Switzerland

4Zurich University of Applied Sciences, Institute of Applied Simulation, Research Group for Knowledge

Engineering, Switzerland

[email protected]

Welfare in animal husbandry includes considerations of biology, ethics, ecology, law and economics. These
diverse aspects must be translated into common quantifiable parameters and applicable methods to
objectively assess welfare in animals. To assist this process in the field of aquaculture, where such methods
are largely missing, we developed a model to assess fish welfare. A network of information was created to
link needs, i.e. fundamental requirements for welfare, with parameters, i.e. quantifiable aspects of welfare.

From this ontology 80 parameters that are relevant for welfare, have practicable assessment methods and
deliver reliable results were selected and incorporated into a model. The model, named MyFishCheck, allows
the evaluation of welfare in five distinct modules: farm management, water quality, fish group behaviour,
fish external and fish internal appearance, thereby yielding five individual grades categorising welfare from
critical, poor, acceptable to good. To facilitate the use of the model, a software application was written.

With its adaptability to different fish species, farming systems, regulations and purposes as well as its user-
friendly digital version, MyFishCheck is a next step towards improved fish welfare assessment and provides
a basis for ongoing positive developments for the industry, the farmers and the fish.

Page 22
1.1 Aquatic animal welfare

Understanding drivers for daily mortality in Norwegian salmon farming

Ingunn Fride Tvete1, Magne Aldrin1, Britt Bang Jenssen2

1The Norwegian Computing Center, Norway

2The Norwegian Veterinary Institute, Norway

[email protected]

Excess mortality in production of farmed salmonids can indicate suboptimal fish welfare, cause excessive
waste of fish feed, and reduce the economic turnover.

We analysed daily production data of full production cycles, from stocking in sea to slaughter in 41 Atlantic
salmon farms located in mid-western coastal Norway.

Methodology
We developed a model for the daily mortality, considering days since stocking, sea temperature, salinity, day
within year, weight at stocking, three types of treatments against sea lice and the possible occurrence of
pancreas disease (PD). The model, a logistic, binomial, additive regression model, allowed for non-linear
descriptions of how factors related to daily mortality.

We categorized treatments against sea lice as medical (bath) or non-medical, where non-medical treatments
were further divided into thermal (heated seawater) and freshwater treatments.

Day within year was treated as a periodic variable, a smooth function over the year. For each sea lice
treatment, we assumed increased mortality on treatment day, followed by a smooth decay. We defined one
variable for PD presence if occurring before October 2017 and another if occurring in October 2017 or later,
as testing regimes for PD then changed. Since PD infection occurs before detection, we allowed for increased
mortality prior to when PD was reported.

Results
We predicted daily mortality without lice treatments (an “ideal situation”) and compared the total mortality
with the corresponding total mortality including lice treatments. The total mortality could be reduced by
around 20% if one could avoid all lice treatments. Similarly, the total mortality could be reduced by
approximately the same if no PD infections had occurred. If avoiding both PD and all lice treatments, the
total mortality could be reduced by around 35%.

Conclusions
Calculations like these are useful for the prioritization of management strategies in fish farming. For example,
it can be included in an assessment whether to vaccinate for PD or not.

Page 23
1.1 Aquatic animal welfare

Fish welfare evaluation index based on the prevalence and severity of external
morphological damage

Lina Weirup1,2, Carsen Schulz1,2, Henrike Seibel1,2

1Gesellschaft für Marine Aquakultur mbH, Germany

2Institute of Animal Breeding and Husbandry, University of Kiel, Germany

[email protected]

Scientific studies regarding fish being sentient raised the need to quantify fish welfare accurately by
implementing standard procedures for welfare assessment in aquaculture. Aim of this study was to define
welfare indicators that can be reliably and practicably assessed directly on farm and to set up a welfare
evaluation index based on the example of rainbow trout (Oncorhynchus mykiss) in flow-through systems in
Germany. Potential on-farm welfare indicators assessed on nine farms included management (tank design,
water supply and -exchange, stocking density, feeding frequency), water quality (temperature, oxygen, pH,
nitrogen compounds), behaviour and health observations (disease- and low welfare indicating behaviours,
social interactions, activity level, external damage), macroscopic organ health (liver colour, gill health),
condition factor, somatic organ indices (SSI, HSI, CSI), and external damage (skin-, fin- and eye injuries,
skeletal deformities, emaciation).

To gain additional in-depth insights into the effects caused by differences in management and water quality,
various health and stress parameters (osmolality, histology, scale cortisol, molecular marker) were assessed
and correlated to on-farm welfare indicators. Principal component analysis followed by multiple linear
regression showed that management regarding water supply, -supply/kg fish and -exchange, ammonia,
nitrite, estimated activity level which was highly correlated to stocking density, and scale cortisol as indicator
for long-term stress had the strongest impact on the occurrence of external damage. Results further showed
positive correlations between external damage and histopathological changes, as indicator for health.
Findings demonstrate external damage to be highly valid and relevant welfare indicators.

Depending on the rearing system, evaluation of crucial welfare indicators such as management, water quality
and behaviour can be problematic within the use of a welfare index regarding reliability or practicability,
while external damage can reliably be assessed using photographic images displaying different severity
grades of impairment. Such method will be easy and fast to learn and can be applied by fish farmers,
monitoring- or certification programs. The welfare evaluation index summarizes the external damage
including severity and prevalence, resulting in categories of good, moderate and poor welfare. Depending
on the results of the welfare evaluation index further measurements and adjustment can be necessary to
improve welfare.

Page 24
1.1 Aquatic animal welfare

Interactions of predominantly plant-based feeding and handling stress on the expression

of selected immune markers in rainbow trout (Oncorhynchus mykiss)

Henrike Seibel1, Ksenia Krassilnikova3, Finn-Thorbjörn Fichtner-Grabowski2, Alexander Rebl4, Carsten

Schulz3, Stéphanie Céline Hornburg2

1Gesellschaft für Marine Aquakultur mbh Büsum, Germany

2Christian-Albrechts-Universität zu Kiel, Institut für Tierernährung und Stoffwechselphysiologie, Germany
3Christian-Albrechts-Universität zu Kiel, Institut für Tierzucht und Tierhaltung, Germany
4Leibniz Institute for Farm Animal Biology, Institute of Genome Biology, Fish Genetics Unit, Germany

[email protected]

Nutrition has a decisive influence on the well-being of organisms. As the exchange of fishmeal in aquafeeds
is still important in the context of sustainability and cost efficiency in aquaculture, the impact of a
predominantly plant-based diet on fish health is yet not fully understood. Therefore, this study examined the
effect of a primarily plant-based diet on the expression of selected immune markers in rainbow trout
(Oncorhynchus mykiss) under the influence of handling stress.

Methodology
In a feeding trial, a predominantly vegetarian diet, where the amount of fish meal was reduced up to 80%
(“veggie”), and a mainly fishmeal-based diet (“fishmeal”) were compared. Half each of the veggie and
fishmehl groups have been stressed, by shooing in the tank twice a day for 50 days. The mRNA expression
from whole blood samples of tumor necrosis factor (TNF) α , interleukin (IL) 1β, immunglobulin (Ig) T and IgD
were evaluated using a standard real time qRT-PCR. Standard performance parameters such as FQ, SWG, K
were evaluated.

Results
Significant differences between the two dietary treatments “fishmeal” and “veggie” were found in
expression of IL1β (p=0.048) and IgD (p=0.0327) for non-stressed fish and in expression of TNFα (p=0.041),
IgT (p=0.0068) and IgD (p=0.0070) for stressed fish, while the stressed “veggie” group significantly
downregulated the genes in each case. No significant differences in gene expression were seen between
non-stressed and stressed fish fed the same experimental diet.

Conclusions
Under repetitive stress mainly fishmeal-based and predominantly plant-based fed trout show contrasting
gene expression patterns whereby the “veggie” group downregulated IgT, IgD and TNFα significantly when
stressed.

The gene expression of investigated immune parameters is significantly impacted by the feed formulation.
The more fishmeal the more pro-inflammatory cytokines, while the performance of fish under stress was
better than with a predominantly vegetarian diet. Without stress the plant-based feed performs better.
Results show, that fish performance under the influence of stress should not be ignored when looking for
alternatives to fishmeal. We need to better understand the impact of plant-based protein sources on, for
example, gut health and microbiome as one of the most important immune modulators in the body of
carnivorous fish like rainbow trout.

Page 25
1.1 Aquatic animal welfare

Effects of acute stress on cyprinids

Constanze Pietsch, Bern Applied University

Bern Applied University

[email protected]

Large numbers of cyprinids are used for aquaculture. Despite the intense growth of the aquaculture sector,
clear guidelines for responsible practices and animal well-being are often lacking. Distress is defined as a
condition that interferes with the well-being of an animal if the adaptation processes of the animal fail to
return the organism to physiological and/or psychological homeostasis.

Fish in aquaculture are often subjected to distress for short periods (acute stress), but can also be exposed
to stressors for longer periods (chronic stress), meaning that the body’s biological functions are sufficiently
altered and its coping mechanisms overwhelmed.

Gene expression and behavioural studies are fundamental tools for an improved understanding of welfare,
but optimal stress markers in aquaculture fish species are poorly described. A comparative study on the
acute stress responses of cyprinids is therefore an important step towards better welfare and welfare
assessment in farmed fish.

Page 26
1.2 Viral diseases I

Assessment of macrophage polarization and activated CD8+ cells in heart and skeletal
muscle inflammation (HSMI) and melanized focal changes in Atlantic salmon (Salmo salar)

Muhammad Salman Malik1, Håvard Bjørgen3, Ingvild Berg Nyman1, Øystein Wessel1, Erling Olaf Koppang3,
Maria Dahle2, Espen Rimstad1

1Section of Virology, Faculty of Veterinary Medicine, Norwegian University of Life Sciences (NMBU), Norway
2Department of Fish Health, Norwegian Veterinary Institute (NVI), Norway
3Section of Anatomy, Faculty of Veterinary Medicine, Norwegian University of Life Sciences (NMBU), Norway

[email protected]

Piscine orthoreovirus (PRV-1) is ubiquitous in farmed Atlantic salmon (Salmo salar) in the marine phase and
is the etiological agent of heart and skeletal muscle inflammation (HSMI). PRV-1 is also associated to the
development of granulomatous changes of melanized spots that can be observed in the white muscle of
Atlantic salmon at slaughter. The melanized focal changes (black spots) develop from red focal changes (red
spots). This study aimed to characterize immune cells involved in development of HSMI and black spots,
focusing on macrophage polarization, i.e. the M1 and M2 phenotypes, and the cellular immune response,
i.e. CD8+ and MHC-1 expressing cells, in order to understand disease pathogenesis and links to PRV-1
infection.

Methodology
Heart and skeletal muscle tissues from experimentally induced HSMI were analyzed at peak PRV-1 load and
when severe histopathological lesions were present. Macroscopic red and black spots samples were
collected from fish in commercial aquaculture. H&E staining of the samples were performed for histological
examination followed by multiplex fluorescent in situ hybridization (FISH) method for the detection of
various cellular targets. Expression analysis was measured by RT-qPCR method.

Results
FISH assays revealed that PRV-1 infected M1 macrophages were a dominating cell type in the early phase of
red spot development, indicating a pro-inflammatory microenvironment. Moreover, PRV-1 infected
melanized M2 macrophages, associated with repair and regeneration, appeared during transformation from
red into black spots. In HSMI, on the other hand, PRV-1 infected M1 macrophages were hardly observed,
while M2 macrophages were a prevalent cell type in the HSMI lesions. This indicated that M1 macrophages
were not associated to the development of HSMI, and the numerous M2 macrophages indicated a good
regeneration potential of the heart. Co-localization of PRV-1 in MHC-1 cells and targeting of activated CD8+
cells is related to a reduction in PRV-1 level both in HSMI and spot formation. Gene expression of polarized
macrophages and cell mediated immunity, as measured by RT-qPCR, mirrored the in situ findings.

Conclusion
Taken together, PRV-1 infection is associated with M1 macrophage polarization in black spot formation, but
less so in HSMI. The M2 macrophage was a prominent phenotype in heart tissue. A cell mediated immune
response was characteristic feature for HSMI.

Page 27
1.2 Viral diseases I

Sequence variation in the full genome and ORF3 of PMCV sampled from field outbreaks of
CMS

Aase B. Mikalsen1, Sunil K. Mor2, Vikash K. Singh2, Øystein Evensen1

1Norwegian University Of Life Sciences, Norway

2University of Minnesota, USA

[email protected]

Cardiomyopathy syndrome (CMS) is a severe heart disease in Atlantic salmon in sea phase of production.
Affected fish might have enlarged ventricle or ruptured atrium with blood filled heart cavity. Histology shows
heart tissue with heavy myocarditis in atrium and spongy parts of ventricle, with full muscle fibers almost
depleted and replaced by inflammatory cells.

CMS is caused by piscine myocarditis virus (PMCV). The virus shares some similarities to the viruses of
Totiviridae, infecting uni-cellular organisms. These viruses are all simple viruses build from several copies of
one capsid protein enclosing genomic RNA, also including a few copies of a polymerase responsible for
copying the genome in an infected cell. In addition, PMCV also include a separate third open reading frame
(ORF) following ORFs for capsid and polymerase. It is expected that the resulting protein encoded could have
a role in infection of or spread from cells due to the more advanced salmon host than the traditional
totiviruses have.

Previous results focusing on smaller parts of the genome/selected proteins, like ORF3, showed that PMCV
varies between individuals in the same disease outbreak, but also that some variants always seem to be
included. This initiated a need to achieve full knowledge on how the virus may vary over the full-length
genome, including both protein encoding and non-coding parts, between individuals in an outbreak and
between separate outbreaks.

In a project collaboration with University of Minnesota we have used next generation sequencing (NGS) to
sequence full-length genomes of PMCV from several individuals of eight different CMS outbreaks. The
samples are collected during 2017 and 2018 and compared to samples from an outbreak in 2011 and the
only full-length genomic sequence published for PMCV – i.e. the PMCV ALV-708 isolate originating from an
outbreak in 2007. Subsequently, ORF3 and encoded protein have been given special focus by phylogenetic
studies involving all available sequences with origin between 2007 and 2019.

The results of the full genome sequences confirm that each CMS outbreak is characterized by several strains
included. The use of sequence information to study phylogenetic relation will be discussed.

Page 28
1.2 Viral diseases I

In vitro cytotoxicity of piscine myocarditis virus ORF3 protein linked to specific amino acid
residues

Racheal Amono, Snøa Fredlund, Aase B. Mikalsen, Øystein Evensen

Norwegian University of Life Sciences

[email protected]

Cardiomyopathy syndrome (CMS) is one of the major health problems affecting salmon fish farms after sea
transfer and is caused by piscine myocarditis virus (PMCV). PMCV is a non-segmented double stranded RNA
virus that is composed of three open reading frames (ORFs), i.e. ORF1 that encodes a putative capsid protein,
ORF2 that encodes the RNA dependent RNA polymerase (RdRp) and ORF3 encoding a protein which lacks
homology to any other known proteins, except for small domain in the N-terminal part that shows some
homology to chemokines. The protein is believed to be a non-structural protein and we have shown by
recombinant expression analyses in cell culture that such expression is toxic in a number of fish cell lines.
The genetic variation in Norwegian PMCV isolates is limited and isolates seem to constitute one genogroup
with the most divergent still sharing up to 98.6% nucleotides. Still, we have found ORF3 field variants that
vary in toxicity to cells, documented through recombinant in vitro expression studies.

ORF3 variants with toxic and non-toxic in vitro profiles were included in these studies and based on sequence
alignments, we identified putative determinants of toxicity. Our interest was to perform gain- and loss-of-
function studies with an aim to identify amino acid residues that could be linked to in vitro toxicity.

These studies show that the toxic characteristics of the ORF3 protein are shared between the majority of
different field variants of ORF3, and similarly, variants were documented to be atoxic to the cells. Through
knock-out and knock-in studies a specific amino acid residue in the ORF3 protein was found to explain toxicity
in vitro. These findings can assist in identifying markers of virulence of different PMCV strains plus provide
guidance as concerns development of recombinant PMCV vaccines in Atlantic salmon.

Page 29
1.2 Viral diseases I

Comparative analysis of the course of infection and the immune response in rainbow trout
(Oncorhynchus mykiss) infected with the 5 genotypes of infectious hematopoietic necrosis
virus

Lénaïg Louboutin, Joëlle Cabon, Estelle Vigouroux, Thierry Morin, Morgane Danion

Anses Unité Pathologies Virales Des Poissons, National Reference Laboratory (NRL) for listed fish diseases,
France

[email protected]

Infectious hematopoietic necrosis virus (IHNV) is a pathogen of importance for salmonid aquaculture. In a
recent study, we aimed to characterize virus behavior and defense mechanisms developed in rainbow trout
(RT, Oncorhynchus mykiss) experimentally infected with isolates belonging to the five described genotypes
of IHNV, i.e. L, U, M, E and J. Mortality was monitored for two months, and blood and target organs were
sampled at different times post-infection to assess viral load and cellular and humoral immune responses.

The five studied strains were assigned as “avirulent”, “moderately virulent”, or “highly virulent”, and
virulence level was found to be highly linked to precocious viral replication but also to the innate and specific
immunity elicited in the host. The standard Sero Neutralization Test (SNT) exhibited reliable results, with
neutralizing action of antibodies “specific” to strains from other genogroups. The tests performed with
homologous viruses were not as conclusive. Clues for genetic divergence between genotype J and the others
were strengthened when the test with the RtNag0a strain was found to be entirely specific to its genogroup.

These data bring new insights about IHNV/RT interaction and reinforce the interest of SNT as preventive and
epidemiological tool.

Page 30
1.2 Viral diseases I

An outbreak of IHN in rainbow trout farmed in seawater in Croatia

Snježana Zrnčić1, Dražen Oraić1, Željko Pavlinec1, Ivana Giovanna Zupičić1, Tine Moesgaard Iburg2, Lone
Madsen2, Argelia Cuenca2, Niccoló Vendramin2

1Croatian Veterinary Institute, Croatia

2DTU Aqua National Institute of Aquatic Resources, Denmark

[email protected]

Cage farming of rainbow trout (Oncorhynchus mykiss) in seawater in Croatia started several years ago. Fry
intended for growing in the sea cages was imported from two different hatcheries and situated in the
rainbow trout farm in the hinterland for several months. The water temperature on the farm was 10 oC the
same as the sea temperature in spring 2020 when batches were transferred into sea cages. Shortly
mortalities have started and swabs were taken from the kidneys of moribund fish, placed into transport
media and delivered for analysis. Upon receipt, swabs were streaked onto bacteriological media and viral
RNA was extracted on KingFisher Duo Prime Purification System using MagMAX CORE Nucleic Acid
Purification Kit following manufacturer’s instructions. RNA was tested for viral hemorrhagic septicaemia virus
(VHSV) and infectious hematopoietic necrosis virus (IHNV) using RT qPCR.

All samples tested negative for VHSV and positive for the presence of IHNV. Bacterial colonies also appeared
on the solid media and were identified as Vibrio spp. using API20E. The affiliation to Vibrio anguillarum was
confirmed by PCR using amiB gene amplification and 16S rRNA protocols and sequencing. After obtaining
preliminary results sampling of fish from the cages was performed. Sampled rainbow trouts weighing 50-300
grams showed loss of scales, dark gills, haemorrhages in the mouth, on the fin basis and skin of opercula and
vent. Necropsy revealed haemorrhages in the fat tissue, pyloric caeca, peritoneum and intestines, swollen
and dark spleen and liver.

Organ pools were tested on the cell cultures that resulted by virus isolation and identification by ELISA
followed by RT qPCR. “Mid-G“ region of the G gene of isolated IHNV was amplified and sequenced. The
obtained sequence showed the affiliation to the IHNV genotype E. Phylogenetic analysis of sequences
available at the NCBI database and sequences from previous IHNV outbreaks in Croatia showed the presence
of ”Croatian clade”. It means that sequences from this outbreak clustered with sequences from other IHNV
outbreaks in Croatia from 2005, 2014 and 2015. It may be concluded that the virus has been circulated in
the country rather than being imported from other countries.

Page 31
1.3.1 Diseases of wild and ornamental fish

Does migration barriers prevent spreading of Proliferative Kidney Disease?

Balance between disease emergence and habitat connectivity

Heike Schmidt-Posthaus1, Ernst Schneider2, Nils Schölzel3, Regula Hirschi1, Moritz Stelzer1, Armin Peter3

1Centre for Fish and Wildlife Health, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty,
University of Bern, Switzerland
2Alte, Switzerland
3 FishConsulting GmbH, Switzerland

[email protected]

Many European streams are obstructed by artificial migration barriers, often preventing the migration of fish
in upstream habitats. However, natural and uninterrupted water courses are important for biodiversity and
fish population stability. Upstream areas often serve as spawning habitats for many fish species. On the other
hand, pathogen transport in former free areas by migrating fishes is still a point of concern. The aim of this
study was to investigate the possible consequences of a migration barrier removal for the upstream
transport of Tetracapsuloides bryosalmonae, the causative agent of Proliferative Kidney Disease (PKD).

Methodology
To test this question, a river system was selected with a migration barrier separating a PKD positive river
section from a PKD negative tributary. After removal of the barrier, PKD prevalence and pathology was
examined during five years.

Results
In the tributary, no PKD was recorded at any time of the survey. By means of unidirectional PIT (passive
integrated transponder)-tagging, we confirmed upstream migration of adult brown trout into the tributary,
mainly during winter months, presumably for spawning. By eDNA, we confirmed presence of T. bryoalmonae
and Fredericella sp., the definitive host, DNA in water from the PKD positive river stretch, but not in the PKD
negative tributary.

Conclusions
Our study illustrates the importance of the connectivity of streams for conservation of spawning habitats.
Although migration of (adult) brown trout from a PKD-positive river into a PKD-negative tributary was
confirmed, upstream spreading of PKD was not observed.

Page 32
1.3.1 Diseases of wild and ornamental fish

Cutaneous angiomatosis in koi carp: clinico-pathological investigations and ongoing

insights

Luciana Mandrioli1, Tim Barbé2, Jos Derks3, Marco Bonazza Foselli4, Ginevra Brocca5, Ranieri Verin5, Rubina
Sirri1, Francesca Errani1, Enrico Volpe1, Francesco Montesi6, Jane Budai6, Sara Ciulli1

1Department of Veterinary Medical Sciences, University of Bologna, Italy

2DAP Vet pract, Belgium
3Vet pract, FishHealth, Nederland
4Vet pract, Italy
5Department of Comparative Biomedicine and Food Science, University of Padova, Italy
6Istituto Zooprofilattico Sperimentale delle Venezie, Padova, Italy

[email protected]

In the period April-September of 2019 and 2020, five koi carp from different places (Belgium, The
Netherlands and Italy) presented with multifocal, irregularly round, few mm to 1 cm, variably raised
cutaneous reddened areas, located on the dorsal and ventral body aspects, without any apparent association
with coloured areas of the skin. The fish displayed good general conditions, normal behaviour and appetite.
After 7-12 months, the skin lesions underwent remission in three out of five carp regardless the treatments.
Carp were reared in ponds supplied with water at temperature not controlled, ranging between 18 °C and
25 °C; carp of two ponds were treated with drugs against gyrodactylosis.

A clinical-pathological investigation was performed to study the skin lesions. Cutaneous swabs were cultured
at 28 °C for 36 hours on blood agar. Skin biopsies were obtained and preserved in buffered formalin and/or
alcohol ≥96%. In addition to routine Hematoxylin & Eosin stain to study the morphology of the skin lesions,
special stainings (Gram, acid-fast, Warthin-Starry) to investigate potential bacterial agents were performed.
Molecular analyses for koi herpesvirus, carp edema virus, Cyprinid herpesvirus 1, Rickettsia-like organism
were performed; the samples were also tested with 16S universal bacterial primers.

Based on the molecular analyses, viral and bacterial fish pathogens were not found. Bacterial cultures from
swabs were negative for primary pathogenic bacteria. Skin parasites or fungi were not detected
histologically. The dermis presented a multifocal inflammatory process focused in the dermis characterised
by well demarcated round to oval clusters measuring 30-400 µm in diameter, composed of densely packed
vascular structures, occasionally forming whorls and inconstantly surrounded by fibrosis. The vascular spaces
were occupied by a florid reduplication of small capillaries with aggregates of plump to elongate cells
resembling endothelium or pericytes without atypia. In hypocellular areas, a continuum of the lesions could
be appreciated, ranging from irregular clusters of plump cells to well-developed micro-vascular structures.
Gross and histological findings, common to all cases, were consistent with a process centred on dermal
vessels and morphologically consistent with angiomatosis reported in other species. Further morphological
investigations are tentatively ongoing in order to elucidate the origin of proliferating vascular/perivascular
structures.

Page 33
1.3.2 Parasitic diseases I

Impact and management of Argulus infections in still water fisheries

Connor Harvey1, Hannah Bradley1, Yasmin Sik-Harris1, Andrew P Shinn2,3, Chris F Williams1

1 Environment Agency, UK
2INVE (Thailand), Thailand
3Faculty of Veterinary Medicine, Chiang Mai University, Thailand

[email protected]

Ectoparasites of the genus Argulus cause disease in a wide range of fish species globally. Infections can be
particularly damaging in sport fisheries, resulting in annual mortalities, loss of reputation, poor angler
satisfaction and economic loss. The management of this parasite has long been problematic, emphasising
the need for simple, cost-effective and practical options to monitor and control the parasite.

Methodology
A survey of 938 still water trout fisheries in England was conducted to assess the impact of Argulus and detail
current management practices. Here we describe the control and monitoring potential of a novel submerged
artificial substrate promoting egg deposition and removal through desiccation. Initial trials were conducted
on 4 study sites with egg counts recorded biweekly over a three-year period between 2017 and 2020. Two
sites where selected for more detailed analysis to assess pipe performance as a means of parasite control.

Results
Responses from 237 fisheries suggest 38% experience disease problems from Argulus and 11% consider the
parasite to be a significant threat to the economic viability of these waters. Application of pipes in a trial site
with a history of persistent disease problems enabled removal of up to 400,000 eggs per pipe in a two-week
period and over 13 million eggs in a single year. Preferable positioning of pipes directly correlates with
preferable host habitat, highlighting gravel, bank slope and water temperature as key indicators of successful
pipe placement. The persistent use of pipes (over multiple years) significantly (F1,1375=26.31, p<0.001)
reduced egg deposition, although the rate of reduction decreased with each year thereafter.

Conclusions
Survey responses highlight the persistent impact of Argulus in still water fisheries and the urgency for
practical, cost-effective management solutions. The described substrate approach offers a simple, non-
intrusive, biological means for monitoring Argulus infections and reducing parasite numbers. This proof of
concept has generated promising data with which to access parasite seasonality, inform management
practices (e.g., timing of stocking events) and control of infections when applied in combination with good
fishery management approaches.

Page 34
1.3.2 Parasitic diseases I

Unveiling the blood-feeding behaviour of the gill parasite Sparicotyle chrysophrii

Enrique Riera-Ferrer, Itziar Estensoro, Carla Piazzon, Raquel Del Pozo, Oswaldo Palenzuela,
Ariadna Sitjà-Bobadilla

Fish Pathology Group, Institute of Aquaculture Torre de la Sal (IATS-CSIC), Consejo Superior de
Investigaciones Científicas, Spain

[email protected]

Sparicotyle chrysophrii is a polyopisthocotylean monogenean (Microcotylidae) parasite of gilthead sea bream

(Sparus aurata) (GSB), attaching to the host with numerous haptor clamps. Sparicotylosis is widespread
across the Mediterranean, and it has been directly linked to severe anaemia, inflicting direct and indirect
economic losses in the GSB farming industry. It is assumed that the anaemia is caused by the hematophagous
behaviour of most polyopisthocotylean monogeneans, but some authors have pointed out that it could also
be due to the haemorrhages inflicted by the clamps. To date, the blood-feeding behaviour of S. chrysophrii
has not been demonstrated. Thus, the aim of the current study was to elucidate this putative
hematophagous behaviour and to develop a method to determine how much blood is indeed taken by the
parasite.

S. chrysophrii specimens were manually detached from dissected gills and observed thoroughly for
internalised blood cells under the stereomicroscope. Later, we proceeded to intravenously inject Sparicotyle-
infected GSB (n=5; mean weight = 148 g) with a solution of 1 μm-ø fluorescent microspheres (FMs) (1.81 x
10^10 FMs/ml-1) at a dose of 2µl/g-1 of body weight. Blood was withdrawn from the fish at 3 and 18 hours
post-injection (hPI). Parasites were collected from injected fish at 18 hPI and individually digested with lysis
buffer overnight at 38 °C. FMs in the host’s blood and in each S. chrysophrii specimen were counted using
fluorescent microscopy. The blood intake by individual parasites was calculated according to the mean FM
concentration present in the peripheral vascular system at 3 and 18 hPI.

The FMs were successfully injected into the caudal vein and recovered from the peripheral vascular system.
S. chrysophrii specimens with internalised blood cells and FMs were observed and photographed. FMs were
recovered from all the lysed parasites and the calculations revealed that the mean blood intake per parasite
was 4.31 μl24/h-1. Therefore, the blood-feeding behaviour of the monogenean as well as its daily feeding
rate have been demonstrated. The calculated average individual blood drawn per parasite explains GSB
anaemia due to sparicotylosis.

Page 35
2.1.1 Case Reports

First report of Vibrio vulnificus infection in a farmed neotropical catfish (Pseudoplatystoma

corruscans) from Brazil

Peter Charrie Janampa-Sarmiento1, Francisco Yan Reis1, Renata Egger1, Fernanda Dorella1, Santiago De
Pádua2, Guilherme Tavares1, Henrique Figueiredo1

1Federal University of Minas Gerais, Belo Horizonte, Brazil

2Biovet Vaxxinova, São Paulo, Brazil

[email protected]

Vibrio vulnificus has been indicated as an opportunistic pathogen for humans and pathogenic for several
aquaculture fish species. In Brazil, there are no reports of V. vulnificus outbreaks in aquacultured fish so far.
On September 2019, spotted sorubim (Pseudoplatystoma corruscans) on grow-out phase at 24 °C freshwater
temperatures exhibited clinical signs of infection (erratic swimming, melanosis and acute anorexia).
Mortality occurred (approximately 5,400 out of 13,500 fish population) during one week after the disease
onset.

This study aimed to identify and characterize the etiologic agent of this outbreak. For that, five moribund
fish were sampled for bacteriologic study and strains were isolated from brain and kidney on Columbia Blood
agar after a 24 hours incubation at 28 °C. Bacterial identification by MALDI-TOF/MS resulted in score values
above 2.00 for V. vulnificus according to MALDI Biotyper database. Then, one representative isolate was used
for biochemical characterization by API20E, identification by 16S rRNA gene sequencing, and antimicrobial
susceptibility testing by disc diffusion method following the CLSI-VET-03 guidelines.

Furthermore, an experimental challenge (Ethics-Committee Protocol Nº315/2019) occurred via

intracoelomic injection (IC, 107 CFU.fish-1, n=6) and 30-minutes immersion bath (IB, 106 CFU/mL-1, n=6) on P.
corruscans juveniles (88 g). Fish on experimental challenge were kept in 45 L glass aquaria (one aquarium by
group) with flow-through freshwater at 28 °C. Positive values for six biochemical tests (ONPG, GEL, GLU,
MAN, AMY, and ARA) were obtained. The 16S rRNA gene sequence was identified as V. vulnificus (99.7% of
similarity) after checked on BLAST webserver. The antimicrobial susceptibility profile showed resistance to
Lincomycin and Neomycin; intermediate resistance to Erythromycin; and susceptibility to Amoxicillin,
Sulfamethoxazole/Trimethoprim, Nitrofurantoin, Norfloxacin, Oxitetracycline and Florfenicol. Mortality rate
of 100% and 0% was obtained on IC (<24 hrs after inoculation) and IB groups (7 day-observation period),
respectively. In addition, V. vulnificus recovering from IC fish organs (brain, liver, spleen and kidney) was
possible after cultivation in Marine agar and incubation by 24 hours at 28 °C. Control (n=4) and IB fish groups
did not develop clinical signs and any bacterial growth at the end of assay. This is a case report of pathogenic
V. vulnificus for farmed P. corruscans.

Funding
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil
(CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico – Brasil (CNPq).

Page 36
2.1.1 Case Reports

Tumors in broodstock fish from an Italian farm

Carmelo Iaria1, Sabrina Natale1, Fabiano Capparucci1, Jessica Maria Abbate2, Rosanna Panebianco3, Giovanni
De Benedetto2, Marco Albano1, Elisabetta Antuofermo4

1Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Italy
2Department of Veterinary Sciences, University of Messina, Italy
3Panittica
Italia, Italy
4Department of Veterinary Medicine, University of Sassari, Italy

[email protected]

The study and research of neoplasms in fish stock have gained over the years considerable importance for
the understanding of possible common mechanisms involved in cancer development at various phylogenetic
levels. Fish from aquaculture farm productions are characterized by a shorter lifespan in comparison with
broodstock fish, which could be more prone to diseases related to senescence. Fish maintenance procedures
and hormone treatments on broodstock through hormonal injection in aquaculture farms, to induce egg
deposition and to improve reproduction performances could be often related to oncogenesis. In the present
work a testicular leiomyoma related to an unusual case of spermatogenic failure syndrome, and a gastric
leiomyoma in broodstock European sea bass (Dicentrarchus labrax) from an Italian aquaculture farm were
reported. The testicular leiomyoma neoplasm was multilobular with a solid-cystic appearance, pale in
colour, slightly vascularized, and with necrotic area in the middle-lower part of the gonad. At the microscopic
examination, the neoplasm was circumscribed and characterized by spindle cells arranged in a whirling
pattern mixed with healthy testicular tissue. There was no evidence of necrotic cells and metastases.
Mallory's trichrome staining marked neoplastic areas in red, as muscle tissue, surrounded by abundant
fibrous tissue. Seminiferous tubules showed severe azoospermia with the absence of sperm cells even closer
to the neoplasia.

The gastric leiomyoma, a non-infiltrative neoplasm of gastric tunica, at microscopical examination was
circumscribed and non-encapsulated composed of spindle cells arranged in parallel interlacing bundles or,
occasionally, in a whirling pattern. Cells had a small quantity of eosinophilic cytoplasm with distinct cell
borders. The tissue didn’t show malignancy index. The mucosal epithelium was intact without infiltration by
the neoplasm. Nuclei contained elongated, cigar-shaped nucleoli with blunt or rounded ends. In addition, no
invasion and metastases of the tumor cells was observed.

In both the neoplasms, the neoplastic cells were immunohistochemically positive for alpha-SMA, desmin,
vimentin, and negative for S-100 confirming the diagnosis of testicular and gastric leiomyomas. These works
could be a starting point to study oncogenesis in broodstock fish, possibly related to fish maintenance and
hormonal treatments that could economically affect aquaculture production.

Funding
Research funds were provided by PO FEAMP 2014-2020 2.56 FISH PATH NET, grant n. G47B18000130009
Project code SIPA 01/MS/18.

Page 37
2.1.2 Environmental and toxicological diseases

Organochlorine pesticides and PCBs in European eel (Anguilla anguilla) from Southeastern
Spanish coastal lagoons

Alonso Pérez-Vegas1, Marcos Pérez-López2, Elena Barcala3, Elena Trofimova1, Diego Romero1, Pilar Muñoz1

1Universidad de Murcia, Spain

2Universidad de Extremadura, Spain
3Centro Nacional Instituto Español de Oceanografía (CSIC), Spain

[email protected]

It is well known that certain pollutants derived from human activities have an impact in wildlife, such is the
case of contaminants which end in the rivers, seas and oceans. These compounds have an outstanding
potential to harm the environment due to the quick spread and the multiple ways of being incorporated by
fish, such as through gills, skin or contaminated food.

Organochlorine compounds are known as important pollutants, which have been proved to produce several
adverse effects on wildlife. In this study, European eels (Anguilla anguilla) from two Southeastern Spanish
Mediterranean coastal lagoons were captured, specifically from Valencia’s coastal lagoon and “El Hondo” in
Alicante (n=30 from each ecosystem). From these eels, muscle tissue was collected to determine the
concentration of twenty organochlorine pesticides (OCPs) and seven polychlorinated biphenyls (PCBs). The
analysis were performed using a Bruker Scion 456 triple quadrupole gas chromatograph mass spectrometer.

Among the twenty analyzed OCPs, eleven of them were detectable in at least one of the samples, being
remarkable that DDT 2,4’ and Aldrin were those with the highest percentage of individuals with
concentrations above the detection limit (43.3% and 21.7 % respectively), DDD 2,4’ and Endrin were detected
in 15% of the samples. The rest of OCPs were detected in less than 5% of the samples, having only DDE 4’4
and dieldrin mean concentrations above 0.5 μg/kg (means of 2 .228 μg/kg and 2.041 μg/kg respectively). In
the case of the PCBs, they were not detected in any sample.

This study provides information about concentrations of organochlorine and PCBs compounds in European
eel from two Spanish aquatic ecosystems, which could be useful to improve management programs of this
endangered species.

Page 38
2.1.2 Environmental and toxicological diseases

Lead, cadmium and mercury in European eels (Anguilla anguilla) from two Mediterranean
ecosystems

Alonso Pérez-Vegas1, Elena Barcala2, Elena Trofimova1, Diego Romero1, Pilar Muñoz1

1Universidad de Murcia, Spain

2Centro Nacional Instituto Español de Oceanografía (CSIC), Spain

[email protected]

Heavy metals are of great importance in all ecosystems and species. Among them, lead (Pb), cadmium (Cd)
and mercury (Hg) are non-essential elements and pollutants which constitute a threat to many marine
organisms and are regarded as priority toxicants by international agencies and by Spanish legislation.

The European eel (Anguilla anguilla) is an endangered species and heavy metal pollution is related to the
decline of its population. In the present study, we analyzed Pb, Cd and Hg concentrations in liver and kidney
of eels from two Spanish Mediterranean coastal lagoons located in the Balearic Islands, the S'Albufereta
Natural Reserve (Mallorca) (N=28) and the Natural Park l’Albufera des Grau (Menorca) (N=30).

In liver, all analyzed specimens (from both ecosystems) had Pb and Hg concentrations above the detection
limit, whereas only Hg was detected in kidney of all specimens. For the three pollutants, the mean
concentrations were low, but some differences between tissues were observed: in liver, the element with
the highest concentration was Pb; in kidney, the specimens from Mallorca had more Cd, while the specimens
from Menorca had higher median concentration of Hg.

However, there were several specimens with some of the highest renal concentration of Pb in the Albufera
des Grau (Menorca). The relation of metals with the size and degree of silvering of eels is discussed.

Page 39
2.1.3 Cleaner fish diseases

Whole cell immuno-proteomic profiling of atypical Aeromonas salmonicida isolates from

cleaner fish in the UK

Elizabeth Buba, Sean Monaghan, Andrew Desbois, Kerry Bartie, Alexandra Adams

Institute of Aquaculture, University of Stirling, UK

[email protected]

Cleaner fish such as ballan wrasse (Labrus bergylta) are a biological approach to controlling sea lice in Atlantic
salmon (Salmo salar). However, cleaner fish are susceptible to bacterial infections, especially by atypical
strains of A. salmonicida (aAs) that causes atypical furunculosis. Little is known of the diversity of aAs strains
causing problems and no commercially licensed vaccine is available. Instead, multivalent autogenous
vaccines are used to control disease incidences, although outbreaks may still occur. Understanding of aAs
antigens and their role in stimulating protective immune response against infection is limited and more
studies are warranted. The aim of this study was to determine the whole cell protein profiles of 77 aAs
isolates from cleaner fish in the UK.

Methodology
Immuno-proteomic profiling of the aAs isolates was performed by One Dimensional Sodium Dodecyl Sulfate-
Polyacrylamide Gel Electrophoresis (1D SDS-PAGE) to determine the protein profiles of each isolate. 2D SDS-
PAGE was performed for 11 aAs and one typical A. salmonicida isolate. Western blotting was conducted for
the 11 aAs isolates following 1D SDS-PAGE protein separation, and one aAs isolate following 2D SDS-PAGE.
Thereafter, several immuno-reactive protein spots were analysed by matrix-assisted laser
desorption/ionization-time of flight (MALDI-TOF) mass spectrometry.

Results
Six protein profiles were identified for the 77 aAs UK isolates by 1D SDS-PAGE. The 2D SDS-PAGE analysis
permitted further discrimination and significant differences in protein expression were detected for 44
protein spots. Twenty immuno-reactive protein spots were identified by 2D SDS-PAGE western blot, with six
spots common amongst the 12 isolates and one spot shared amongst the 11 aAs isolates. Two immuno-
reactive proteins were identified by MALDI-TOF-MS, including the tetragonal virulence array protein (vapA).

Conclusions
The identification of proteomic and immunogenic protein profiles of aAs isolates will be useful in determining
aspects of aAs virulence, immune-related functions and for optimal selection of antigens for inclusion in
vaccines to protect against atypical furunculosis.

Page 40
2.2 Viral diseases II

Epitope mapping of monoclonal antibody MAb IP5B11 used for detection of viral
haemorrhagic septicaemia virus by applying NGS on carpione rhabdovirus

Takafumi Ito1, Tohru Mekata1, Niels Lorenzen2, Niels Jørgen Olesen2

1Pathology Division, Fisheries Technology Institute, Japan Fisheries Research and Education Agency, Japan
2National Institute of Aquatic Resources (DTU Aqua), Technical University of Denmark, Denmark

[email protected]

The monoclonal antibody MAb IP5B11 have since the 1990ties been used world wide for diagnostic detection
of viral haemorrhagic septicaemia virus in immunochemical tests such as ELISA, IFAT. The MAb has been
tested against thousands of VHSV isolates representing all know genotypes and subtypes and against a large
variety of heterologous virus isolates with 100% specificity and no cross-reactions to other known viruses
except for the unique novirhabdovirus, carpione rhabdovirus. The MAb was previously defining VHSV in the
OIE Aquatic Manual. We originally reported that MAb IP5B11 recognizes a linear epitope of the VHSV N-
protein, but the epitope specificity at amino acid level has remained unknown until this study. A unique virus
isolate named carpione rhabdovirus was isolated back in 1988 from carpione (Salmo carpio), a salmonid
species endemic to Lake Garda in Italy. Some minor serological cross reactivity with VHSV was observed, but
cross reaction with MAb IP5B11 was particularly strong. We therefore hypothesised that the epitope of MAb
IP5B11 could be detected by aligning the amino acid (AA) sequences of the N-proteins of VHSV and carpione
rhabdovirus.

Methodology
The full genome of carpione rhabdovirus was obtained by NGS and the encoded AA-sequence of the N
protein was aligned with those of other fish pathogenic rhabdoviruses. Oligopeptides of regions shared by
the N proteins of VHSV and carpione rhabdovirus were then tested in dot blotting against MAb IP5B11.

Results
By dot-blot analysis of purified VHSV, Carpione rhabdovirus, Hirame rhabdovirus and 4 synthetic
oligopeptides the epitope recognized by MAb IP5B11 was determined to be within the region from AA N219
to N233 of the viral N-protein.

Conclusion
The epitope specificity of the most widely used MAb for identification of VHSV, IP5B11, has finally been
determined and among related fish rhabdoviruses the sequence of the N-gene encoding the corresponding
AA region 219-233 was shown to be shared only with carpione rhabdovirus.

Funding
This study was supported by a JSPS KAKENHI Grant (number 20K06218).

Page 41
2.2 Viral diseases II

Transmission of reassortant Nervous necrosis virus (NNV) strains between Senegalese sole
and gilthead seabream

Lucía Vázquez-Salgado, José Olveira, Carlos Dopazo, Isabel Bandín

Instituto de Acuicultura, Departamento de Microbiología y Parasitología, Universidade de Santiago de

Compostela, Santiago de Compostela, Spain

[email protected]

Global aquaculture is currently undergoing a great expansion that have led to the development of optimized
farming strategies and among them, polyculture of different species in the same area is being assayed. The
implementation of this strategy, that optimizes resources to maximize the production, might be
compromised due to pathogen interspecies transmission. Nervous necrosis virus (NNV) is a viral pathogen
that threatens Mediterranean aquaculture.

The virus is classified in four genotypes: barfin flounder-, red-spotted grouper-, striped jack- and tiger puffer
nervous necrosis virus (BFNNV RGNNV, SJNNV and TPNNV, respectively) and in recent years reassortants
between RGNNV and SJNNV have arisen as a main pathogenic problem in gilthead sea bream and Senegalese
sole farming. Cohabiting these NNV susceptible species in the same area could enhance reassortant NNV
spreading and thus trigger a VER outbreak in the facility. Therefore, in this work we have evaluated whether
two reassortant RGNNV/SJNNV strains isolated from diseased Senegalese sole and gilthead seabream could
be cross transmitted between juveniles of both species.

Our results showed moderate cumulative mortality (50 %) in both species in bath-challenges performed with
sole and gilthead seabream-derived isolates and that the two NNV strains replicated in a higher extent in
sole than in gilthead seabream. In addition, we also demonstrated NNV interspecies transmission, in
cohabitation experiments with infected and naïve individuals. In the cohabitation challenges an earlier onset
of mortality and a significantly higher cumulative values than in the bath challenge were recorded in both
species, although the difference was higher in Senegalese sole. Because gilthead seabream showed an
aggressive behavior towards sole cohabitants that resulted in an altered and quick swimming in the flatfish,
the possible effect of the stress in the mortality was analyzed. Results obtained showed that the hsp70 gene,
a stress biomarker, was overexpressed in cohabitant sole, especially on the first day post cohabitation. To
conclude, notwithstanding the multiple benefits of the polyculture, the introduction of this technique in the
farming of NNV-susceptible species might represent a threat for the production due to the virus interspecies
transmission, especially when species showing different behavioral patterns must cohabit.

Funding
This research was funded by the Ministerio de Ciencia, Innovación y Universidades (MCIUI), the Agencia
Estatal de Investigación (AEI) and FEDER under Grant RTI2018-094687-B-C21 and also by the Interreg VA
Spain-Portugal cooperation program (POCTEP) 2014-2020, 0474_BLUEBIOLAB project, co-funded by FEDER
with a research contract granted to L. Vázquez- Salgado.

Page 42
2.2 Viral diseases II

Betanodavirus infection and larval enteropathy in juvenile gilthead sea breams

Serena Savoca1, Simone Palazzolo2, Carmelo Iaria1, Miriam Abbadi3, Cristian Salogni4, Fabiano Capparucci1,
Rosita Quartesan3, Giovanni Loris Alborali4, Anna Toffan3, Fabio Marino1

1Department of Chemical, Biological, Pharmaceutical, and Environmental Sciences, University of Messina, Italy
2Veterinary practitioner, Italy
3OIE Reference Laboratory for Viral Encephalo-Retinopathy, Istituto Zooprofilattico Sperimentale delle Venezie,
Italy
4IZS Lombardia ed Emilia Romagna, Brescia, Italy

[email protected]

The nervous necrosis virus (NNV, Nodaviridae family, Betanodavirus genus), the aetiological agent of viral
encephalo-retinopathy (VER), is one of the most devastating infectious agents in marine aquaculture,
affecting a wide range of wild fish species and responsible for massive mortalities in a large number of
economically relevant farmed fish species. Recently, RGNNV/SJNNV reassortant Betanodavirus has been
reported as a possible cause of disease in Sparus aurata.

Methodology
In this study, mortality events occurred in cultured gilthead sea bream from 19 to 26 days of age, coming
from an Italian aquaculture farm, are described. Samples of diseased larvae, showing lethargy, abnormal
swimming, loss of balance, but also an unusual distended abdomen, were collected and underwent
histopathological, bacteriological and virological analyses. Water quality parameter were fully investigated
as well in order to exclude environmental problems.

Results
Lesions related to VNN and larval enteropathy (LE) were found. Histopathology showed abdominal
distension in more than 60% of the specimens, with severe mucosal degeneration. Neuronal degenerative
vacuolations were observed in the brain and in the eye in 55% of the specimens analysed. Molecular methods
as well as viral isolation clearly confirmed positivity for NNV, and identified the strain as reassortant
RGNNV/SJNNV virus. Phenotypic characterization of the isolated bacterial strains from the intestine showed
the following profiles: API 20E: 7054744, % ID Vibrio alginolyticus 98.1%, API 20NE: 4347124, % ID V.
alginolyticus 75.8% and vibrio static agent O129 (10) R; 0129 (150) S, profile compatible with V. alginolyticus.
No chemical-physical anomalies in the water were found and ion and pollutant concentrations from all water
samples examined were within normal range.

Conclusions
In conclusion, our results showed that VER, caused by reassortant RGNNV/SJNNV betanodavirus can be
found in association with LE syndrome which could, in synergy or simultaneously, elicite severe mortality in
Sparus aurata larvae.

Funding
The viral characterization was funded by the Italian Ministry of Health RC IZSVE 09/18. Research funds were
also provided by PO FEAMP 2014-2020 2.56 FISH PATH NET and Ministry of Education, University and
Research, Italy (grant No. J68G17000050007)

Page 43
2.2 Viral diseases II

2.2 Viral diseases II

Isolation and characterization of hirame aquareovirus (HAqRV): a new Aquareovirus

isolated from diseased hirame Paralichthys olivaceus

Yasuhiko Kawato Y1, Tohru Mekata1, Toyohiro Nishioka1, Ikunari Kiryu1, Takamitsu Sakai1, Tomoki Maeda1,
Satoshi Miwa1, Kanae Koike2, Masahiro Sadakane3, Koh-ichiro Mori1

1FisheriesTechnology Institute, Japan Fisheries Research and Education Agency, Japan

2Technical Center, Hiroshima University, Japan
3Department of Applied Chemistry, Graduate School of Advanced Science and Engineering, Hiroshima

University, Japan

[email protected]

Hirame or Japanese flounder (Paralichthys olivaceus) is an important flatfish for aquaculture in Japan. Mass
mortality of larvae or juveniles suffering from reovirus-like infection have emerged in the flounder hatcheries
since the 2000s. However, the causative virus has not been identified because the virus could not be isolated
with conventional fish cell lines.

In the present study, we successfully isolated a novel Aquareovirus (hirame aquareovirus: HAqRV) from the
diseased flounder using hirame natural embryo (HINAE) cell line. A cytopathic effect characterized by
aggregation of the virus-infected cells was observed 20 days onward after virus inoculation. In electron
microscopy, the spherical virion (75 nm in diameter) was observed with multi-layered capsid structure. The
viral genome consisted of 11 segments and regions encoding 7 virion structural proteins (VP1 - VP7) and 5
non-structural proteins (NS1 - NS5) were predicted. The deduced amino acid sequences of those proteins
were highly similar to those of the viruses of genus Aquareovirus. The terminal sequences (5’-GUUUUA and
UCAUC-3’) of each segment were conserved among the 11 segments and they were identical to those of
Aquareovirus A and G. However, the similarity of complete genome sequence between the HAqRV and other
aquareoviruses was less than 60%. Phylogenetic analyses based on the deduced amino acid sequences of
VP2 (RNA dependent RNA polymerase), VP7 (outer capsid protein), and NS5 (fusion associated
transmembrane protein) suggest that the HAqRV is not classified into the known species of Aquareovirus.
Pathogenicity of HAqRV was clearly demonstrated in accordance with Koch’s postulates by experimental
infection using Japanese flounder. HAqRV had specific tissue tropism to the epithelium of the digestive tract
including hepatocytes and caused syncytia in the infected tissues.

The results suggest that the HAqRV is a new Aquareovirus species which is highly virulent for the Japanese
flounder at early life stages.

Page 44
2.2 Viral diseases II

Feeling dizzy – brain as a secondary site of koi sleepy disease related pathology

Mikolaj Adamek1, Urszula Orzechowska2, Maria Zawisza2, Krzysztof Rakus2, Magdalena Chadzińska2, Verena
Jung-Schroers1, Felix Teitge1, Veronika Piackova3 , David Gela3, Martin Kocour3, Dieter Steinhagen1

[email protected]

1FishDisease Research Unit, University Of Veterinary Medicine, Germany

2Department of Evolutionary Immunology, Institute of Zoology and Biomedical Research, Jagiellonian
University, Poland
3South Bohemian Research Centre of Aquaculture and Biodiversity of Hydrocenoses, Faculty of Fisheries and

Protection of Waters, University of South Bohemia in Ceske Budejovice, Czech Republic

Gill diseases impair respiration, excretion of harmful metabolites and osmo-regulation. This can have
consequences in other tissues including brain. Carp edema virus is causing koi sleepy disease (KSD) by
primarily infecting gills of common carp. The disease however is related to clear neurological clinical signs
like loss of body balance and sleepiness. Therefore, in the brain pathological and immune responses were
studied during KSD to better elucidate the cause of the onset of clinical signs.

Methodology
Brains from koi and common carp infected with CEV genogroup IIa were compared to non-infected fish and
studied for pathological changes, including water content, histopathology, and immune responses with RT-
qPCR. The results were related to differences in sodium and ammonia concentrations in blood.

Results
Fish displaying clinical signs of KSD had unusual blood parameters, which can be attributed to this gill disease:
the sodium concentration was significantly lower (136.6 mmol/L in controls, 88.9 mmol/L in CEV infected
koi) while the ammonia concentration was significantly elevated from 211.8 µmol/L to 416.4 µmol/L.
Although brains of infected koi harbored an over 100-fold lower virus load when compared to gills, it still
showed an increased antiviral response with an upregulation of several antiviral genes (gig1, vig1, mx2). The
infection lowered the expression of cfos, a marker for neuron activity. The inflammation and microglia
activation was increased upon CEV infection. The increase of glutamate dehydrogenase and glutamine
synthetase expression was most likely a hallmark of the exposure to increased ammonia. Furthermore, the
CEV infection caused a significant accumulation of water by 20% from 4.09 mL/g dry weight to 5.09 mL/g dry
weight indicating a brain swelling which was confirmed by histology.

Conclusions
Gill diseases can have severe implication in off-target tissues like brain. Clinical signs in koi affected by KSD,
in particular, the disturbed righting behavior and the lying on one body side most likely can be related to
brain edema resulting from hyponatremia and hyperammonemia caused by an impairment of gill function.

Funding
This research was supported by Deutsche Forschungsgemeinschaft (DFG project number 426513195).

Page 45
2.2 Viral diseases II

Discovering Tilapia parvovirus in Thailand within co-infection patterns dominated by

Tilapia tilapinevirus

Win Surachetpong1, Jidapa Yamkasem1, Puntanat Tattiyapong1, Bartolomeo Gorgoglione2

1Department of Veterinary Microbiology and Immunology, Faculty of Veterinary Medicine,

Kasetsart University, Thailand
2Aquatic Animal Health Laboratory, Dept. of Pathobiology and Diagnostic Investigation, CVM & Dept. of

Fisheries and Wildlife, CANR - Michigan State University, USA

[email protected]

Tilapia parvovirus (TiPV) is the first known parvovirus confirmed to infect fish, recently discovered during
mortality events in farmed tilapia in China. We document for the first time the concomitant occurrence of
TiPV and Tilapia tilapinevirus (TiLV) in farmed red hybrid tilapia, together with the first occurrence of TiPV in
Thailand. The presence of TiLV was widespread across the fish farms investigated, although TiPV was also
detected. Clinical signs of affected fish included abdominal swimming, skin and muscle hemorrhaging, scale
protrusion, exophthalmos, and pale skin. Histologically, infiltration of lymphocytes in anterior kidney and
spleen, erythrocyte depletion in the spleen, and syncytial cells in the liver were observed in moribund fish.

Both viruses were detected in an array of organs by qPCR, suggesting that they can distribute systemically,
thus reaching any fish tissue. The comparison of TiPV sequences isolated from Thailand and China revealed
98.74% sequence identity, with a highly conserved nucleotide and amino acids identity. The 4159 bp
nucleotide partial sequence of the new Thai isolate, “Tilapia parvovirus-strain KU01-TH/2020” (MW685502)
showed a highly conserved and comparable genomic organization, but with a 3 nucleotides deletion in the
5-UTR.

Overall, our results provide the first detection of TiPV outside China and for the first time in Thailand. Further
studies await investigation, including about TiPV pathobiology, genomic analysis, and to define their role in
polymicrobial infection patternswith other tilapia pathogens. Understanding the outcome of TiPV infection
and desing of appropriate control measures could reduce the impact of this emerging virus on global tilapia
aquaculture.

Funding
This project is funded by the National Research Council of Thailand: NRCT5-RSA63002-04. This research was
supported by Kasetsart University Research and Development Institute, under the project number FF(KU)
25.64.

Page 46
2.3 Parasitic diseases II

Unveiling the elaborate process of encystment in Spironucleus vortens: novel techniques,

sequential events, remaining mysteries

Sarah Poynton, Kenneth Witwer

Johns Hopkins University School Of Medicine, USA

[email protected]

Encystment is a fundamental, highly orchestrated, process in the life cycle of many pathogenic flagellates,
providing transition from motile trophozoite to immobile, resistant cyst for transmission. Encystment is
poorly understood in Spironucleus: limited to S. elegans (TEM), S. muris (light microscopy, TEM), and S.
salmonis (light microscopy); contrasting with abundant information about Giardia cysts. However,
understanding encystment in Spironucleus is essential for developing life cycle interrupting strategies. We
sought to develop a novel suite of light microscopy techniques, combine them with ultrastructure, and
document key stages of encystment. This knowledge will be valuable per se, and is essential for monitoring
effects of experimental interventions.

Methodology
We cultured S. vortens (ATCC 50386 from Angelfish, Pterophylum scalare) axenically in modified liver digest,
yeast extract, iron (LYI) medium, at 22oC in the dark. We observed unstained cells, and applied two
fluorescent stains, NucBlue Live Ready Probes Reagent (nucleus), and ViaFluor (cytoskeleton), to live cells.
We also employed scanning electron microscopy.

Results
In early encystment, six anterior flagella moving sinuously upon the cell surface were gradually covered with
distendable cyst wall; their profiles were evident, with those of the compound tufted lateral ridges. During
maturation, the cyst became spherical, the wall no longer distendable; two extended posterior flagella lay
exteriorly. NucBlue showed: (i) young cyst - pair of nuclei, (ii) maturing cyst - longitudinal binary fission,
yielding two adjacent pairs of nuclei, (iii) mature cysts - two distant pairs of nuclei, indicating motile
trophozoites. ViaFluor showed young cysts contained a longitudinally symmetrical cytoskeleton, with three
pairs of microtubular bands, and characteristic S. vortens elements; maturing cysts contained two
cytoskeletons.

Conclusions
We described key sequential events of S. vortens encystment for the first time. Two individual trophozoites
within the mature cyst is consistent with S. salmonis and Giardia spp. We propose: anterior flagella were
covered by developing cyst wall from the trophozoite, as they lay sinuously on the cell surface, facilitated by
their helical beats from their sliding motor; however, posterior flagella were not covered, they lay away from
the encysting trophozoite, being passive and trailing; their fate at completion of encystment is unknown.

Page 47
2.3 Parasitic diseases II

Ichthyophthirius multifiliis proteins associated with the common carp (Cyprinus carpio) skin
mucus

Mona Saleh1, Abdel-Azeem S. Abdel-Baki2,3, Mohamed A. Dkhil2,4, Saleh Al-Quraishy2 Mansour El-Matbouli1

1Clinical
Division of Fish Medicine, University of Veterinary Medicine, Austria
2Zoology Department, College of Science, King Saud University, Saudi Arabia
3Zoology Department, Faculty of Science, Beni-Suef University, Egypt
4Department of Zoology and Entomology, Faculty of Science, Helwan University, Egypt

[email protected]

The fish first line of defence against infections through the skin epidermis is the skin mucus. In a previous
study, we have investigated the skin mucus proteome of common carp (Cyprinus carpio) at 1 day and 9 days
post-exposure with Ichthyophthirius multifiliis. Using nano-LC ESI MS/MS, we have earlier identified that the
abundance of 44 skin mucus proteins has been differentially regulated, including proteins associated with
host immune response and wound healing.

Herein, in skin mucus samples, we identified six proteins of I. multifiliis, associated with the skin mucus in
common carp. Alpha and beta tubulins were detected, in addition to elongation factor alpha, 26S
proteasome regulatory subunit, 26S protease regulatory subunit 6B, and the heat shock protein 90. The
identified proteins are likely involved in motility, virulence and general stress during parasite growth and
development after parasite attachment and invasion. Two KEGG pathways: phagosome and proteasome
were identified among these parasite proteins, mirroring the proteolytic and phagocytic activity of this
parasite during growth and development required representing a plausible host invasion strategy of this
parasite.

The results obtained from this study can support us reveal molecular aspects of the interplay between carp
and I. multifiliis. This may help us understand I. multifiliis invasion strategy at the skin mucus barrier. The
data may support the development of novel drugs, vaccines, and diagnostics suitable for management and
prevention of ichthyophthiriosis in fish.

Page 48
2.3 Parasitic diseases II

Application of Saprolegnia challenge models in eggs, and parr-presmolts

Mohammad Nasif Sarowar, Mark Braceland

The Center for Aquaculture Technologies, Canada

[email protected]

Saprolegniasis, caused by multiple species of the genus Saprolegnia, is a serious problem in Atlantic salmon
(Salmo salar L.) aquaculture operation worldwide, specifically affecting freshwater stages. It is a conserve
estimate that 10% of all salmon in hatcheries will succumb to the disease resulting in losses worth millions
of dollars to the industry every year. Formalin bath remains the principal treatment despite it being a human
carcinogen, hence expected to be banned in near future from aquaculture operation. Alternative
therapeutants for example, bronopol and peracetic acid showed potential but have not displaced formalin.
With further intensification of culture conditions through incorporation of RAS, the problem is only expected
to be worse. Therefore, to test efficacy and toxicity of alternative therapeutants for different life stages of
Atlantic salmon and related species, disease challenge model is of urgent need.

Methodology
At CATC, we have established saprolegniosis challenge models for different life stages of Atlantic salmon,
e.g., eggs and parr. The models were executed by introducing trojans and zoospore/cysts suspensions
generated from pathogenic isolates of Saprolegnia to healthy population of eggs and parr under laboratory
condition, respectively. Standard formalin treatment was used as positive control yielding expected efficacy
in all experiments.

Results and Conclusions

100% infection was observed in fish within 5 – 7 days post challenge while eggs yielding 60 – 75%
entanglement to mycelia extended from trojan. The infection rates in different life stages remained
consistent across various experiments with typical presentation of the disease pathology in the form of
cottony wool-like mycelial growth. Both fish and egg challenges simulated typical saprolegniosis aetiology
observed in aquaculture operation placing the models to test range of different chemicals and reconfirm in
vitro results. The models enable to build a stronger understanding of disease in fish with potential of
treatment administration and the disease pathogenicity which in turn can help develop sustainable
prevention and control measures of industrial implications.

Page 49
2.3 Parasitic diseases II

Histological analysis of the infective process of Anisakis spp. in gilthead seabream (Sparus
aurata L.)

Lopez-Verdejo Alejandro1, Born-Torrijos Ana2, Raga Juan Antonio1, Montero Francisco Esteban1

1University of Valencia, Spain

2Biology Centre of the Czech Academy of Sciences, Ceske Budejovice, Czech Republic

[email protected]

Anisakis spp. (Nematoda, Anisakidae) are parasites known by their economic and health impact as their L3
larval stages infect a vast range of fish species, most of them commercial species, causing sometimes
zoonotic episodes by consumption of raw or undercooked fish. The aim of this study is to determine the
infection process and potential effects of Anisakis spp. on gilthead seabream (Sparus aurata L.), an important
fish species in Mediterranean aquaculture, through periodic histological monitoring of the infective process.

Methodology
Fish were experimentally infected and periodically analysed for L3 larvae for 8 days and up to 6 months.
Samples were obtained at different hours post ingestion (hpi), 3, 6, 12, 18, 24, 36, 48, 72, 96, 120, 144, 168,
192 and 6 months post ingestion. All samples were observed under the stereomicroscope and later fixed for
their histological examination.

Results
Anisakis spp. larvae were found only on the visceral surface and mesenteric tissue, but never free or encysted
in the muscle. Anisakis spp. larvae were observed within the coelomic cavity for the first time 6 hpi, being
found up to 48 hpi. While the earliest evidence of fibrocytes surrounding Anisakis spp. L3 larvae was
observed 18 hpi, the complete spiral cyst formation occurred around 72 hpi. No damage was observed in the
tissue surrounding the cysts.

Conclusions
Despite the infection of the gilthead seabream by Anisakis spp. larvae is feasible, it rather seems unlikely to
happen, especially in aquaculture given the sanitary controlled feeding regimes. In case of infection, the
transmission rate would likely be despicable due to the poor condition in which the specimens of Anisakis
spp. are found. Furthermore, as no larvae were detected in the fish’s muscle, human infections seem unlikely
to occur.

Funding
Study supported by the projects TREPAQUAMED (PID2019-110730RB-I00, Ministry of Science and
Innovation, Spanish Government/UE) and ANITEST PLEAMAR (Biodiversity Foundation, Ministry for the
Ecological Transition and the Demographic Challenge/UE).

Page 50
2.3 Parasitic diseases II

First report of Anguillicoloides crassus in the European eel (Anguilla anguilla) from two
Spanish coastal lagoons in the Western Mediterranean Sea.

Elena Trofimova1, Alonso Perez-Vegas1, Diego Romero1, Elena Barcala2, Pilar Muñoz1

1Universidad de Murcia, Spain

2Centro Nacional Instituto Español de Oceanografía, Spain

[email protected]

Anguillicoloides crassus (Nematoda, Dracunculoidea) is a very efficient invasive parasite species. It´s range
has expanded throughout the world, from its original ecosystems in Asia and it has parasitized five species
of eel within the last three decades. Infection by this nematode is one of the many threats hanging over the
European eel (Anguilla anguilla). In the framework of eel management information regarding parasitological
status of anguilla populations is required. Thus, the aim of the present work was to determine whether the
nematode had already invaded El Hondo-Salinas de Santa Pola Natural Park, wetland in south-eastern Spain,
and the Natural Park l’Albufera des Grau, coastal lagoon on the northeast coast of Menorca (Balearic Islands),
two ecosystems with no information regarding the eel health status.

A total of 30 European eels from each ecosystem were sampled between January and February 2020 using
traditional gear. Swimbladders were dissected and pre-adult and adult A. crassus were removed from the
lumen; swimbladders were then enzymatically digested with hydrochloric acid and pepsin to allow counting
of A. crassus in the wall. Prevalence, mean intensity and mean abundance were calculated. Additionally, we
calculated the swimbladder degenerative index (SDI), (range from 0 without pathological signs of infection,
to 6 when the swimbladder is severely damaged).

The nematode was present in eels in both sampled habitats, with prevalence of 96.67% (29/30) in both
wetlands. In the Hondo-Salinas de Santa Pola wetland environment, we observed a mean intensity of 5.95
worms per infected eel (range 1-16), and a mean abundance of 4.37, the mean SDI was 2.32. In L’Albufera
des Grau coastal lagoon mean intensity was 2.5 worms per infected eel (range 1-9), mean abundance was
1.17, the mean SDI was 1.23.

These results represent the first recorded occurrence of A. crassus in each of these wetland and coastal
lagoon ecosystems. The high prevalence might indicate a recent introduction, as the dynamic pattern
observed elsewhere in Europe shows a rapid spread following the introduction of the parasite, and then
stabilization around ceiling levels.

Funding
This work was supported by The Ministry of Science and Innovation of Spain (Grant RTI2018-097228-B-I00).

Page 51
3.1 Salmonid viral diseases

Infectious salmon anemia virus (ISAV) genotyping to gain new insights on virulence and
virus transmission on Atlantic salmon (Salmo salar) farms in Canada

Delphine Ditlecadet, Kristine Gagnon, Nellie Gagné

Fisheries and Oceans Canada, Canada

[email protected]

Infectious Salmon Anaemia Virus (ISAV) is the causative agent of infectious salmon anemia, that cost millions
in CAD$ in fish loss in the late 1990’s and continues to affect the industry to this day. Changes in management
practices resulted in a significant decrease of the number and severity of outbreaks but ISAV remains under
active surveillance. The virus is still regularly detected in Atlantic Canada, including both HPR0 (not known to
cause disease) and HPRΔ (can cause disease) strains.

Currently, genotyping is done on the hypervariable region (HPR) of segment 6. Genotyping indicates that
new HPRΔ isolates are produced, likely through the mutation of HPR0 strains. Occasionally, identical
genotypes are identified based on the criteria used to classify ISAV strains, but these viruses may have
acquired a similar deletion pattern by chance, and may not be directly related. The level of virulence within
the HPRΔ viruses cannot be predicted solely based on segment 6 sequences. A second marker of virulence
located on segment 5, encoding the fusion protein, has been identified but is rarely sequenced in routine
detection. There are indications that some variability exist, and that sequencing of segment 5 could bring
additional information.

We will present a revised phylogeny of ISAV based on sequences of both segment 5 and 6 obtained from
multiple archived diagnostic cases and will discuss its relevance and new insights on virus movements,
relationship between isolates, virulence level and mechanisms leading to the transition from HPR0 to HPRΔ.

Page 52
3.1 Salmonid viral diseases

The physiological cost of a sub-lethal infection with Infectious salmon anemia virus (ISAV)
in wild Atlantic salmon (Salmo salar)

Nellie Gagné1, Travis Melanson2, Delphine Ditlecadet1, Steve Leadbeater2, Tillmann Benfey3

1Fisheriesand Oceans Canada, Moncton, Canada

2Fisheriesand Oceans Canada, St-Andrews, Canada
3University of New Brunswick, Canada

[email protected]

Infectious Salmon Anemia Virus (ISAV) is an important pathogen affecting farmed Atlantic salmon and
infectious to wild salmon. Wild Atlantic salmon in the inner Bay of Fundy of Canada’s East Coast are an
important component of biodiversity in aquatic ecosystems and are listed as endangered. The physiological
cost of acquiring an infection such as ISAV needs to be established within these populations as scientific
literature is lacking. Here, the effects of sub-lethal ISAV infection are quantified in wild-type Atlantic salmon
(Tobique River strain) using an ISAV strain first isolated in Nova Scotia, Canada, in 2012.

All fish were intraperitoneally injected with the virus or sham-injected with physiological saline, and anemia
caused by ISAV was assessed at intervals using red blood cell count, hematocrit, and hemoglobin
concentration. The effects of ISAV on metabolic rates at peak infection (16 days post-infection (DPI)) and
post-peak infection (30 DPI) were measured using whole-tank intermittent stop-flow respirometry to obtain
post-stress aerobic metabolic scope (PSAMS) and excess post-stress oxygen consumption rates (EXPOC)
using a 2 minute net chase as the stressor.

In a separate ISAV challenge using fish from the same strain, rates of protein synthesis were determined in
kidney tissue samples at 7, 17 and 78 DPI to assess the effects of sub-lethal ISAV infection on the protein
synthesis/degradation cycle. Sub-lethal infection was achieved and had mild to no effect on the parameters
outlined above. Sub-lethal infection with ISAV therefore may not affect wild salmon going through short-
term physical stress.

Page 53
3.1 Salmonid viral diseases

Susceptibility of brown trout (salmo trutta) to piscine orthoreovirus genotype 3 (PRV-3)

Argelia Cuenca1, Anne Berit Olsen2, Arvind Sundaram3, Maria Dahle3, Niels Jørgen Olesen1, Niccolò
Vendramin1

1National Institute For Aquatic Resources, DTU Aqua, Technical University of Denmark, Denmark
2Norwegian Veterinary Institute, Denmark
3University of Oslo, Norway

[email protected]

Piscine orthoreovirus (PRV) is a relevant pathogen for salmonid aquaculture worldwide. Currently, three
distinct PRV genotypes are described with relatively discrete host niche. PRV-1 infects predominantly Atlantic
salmon (Salmo salar) and some variants cause Heart and Skeletal Muscle Inflammation (HSMI). PRV-2 is
reported to cause erythrocytic inclusion body syndrome EIBS in Coho salmon (Onchorhynchus kisutchi) in
Japan. PRV-3, discovered in Norway in 2015 and detected for the first time in Denmark in 2017 in association
with complex disease cases in rainbow trout (O. mykiss) in recirculating aquaculture systems (RAS). PRV-3
has been associated with proliferative darkening syndrome (PDS), a condition affecting wild brown trout in
the pre-Alpine rivers of Austria, Germany, and Switzerland with nearly 100% mortality. The causative
relationship between PRV-3 and PDS remains, however, unclear. In this study, we assess the susceptibility of
brown trout to infection with PRV-3.

Methodology
Three experimental groups of specific pathogen free brown trout were included in our challenge trial: 1) a
negative control, 2) a group challenged with infected PRV-3 blood from rainbow trout, and 3) a group
challenged with gradient purified PRV-3 particles. The challenge model to study infection and pathogenesis
was cohabitation. Shedders were intra-peritoneal injected, tagged and maintained in tanks with non-tagged
cohabitant fish. The experiment run for 10 weeks, with sampling every two weeks to assess viral load (RT-
qPCR), histopathological changes, and host immune response by RNAseq.

Results
No reduced survival was observed along the experiment, but PRV-3 successfully replicated in shedders and
transferred horizontally to cohabitants. After peak of viremia heart lesions consistent with those observed
in HSMI in Atlantic salmon were present in both, shedders and cohabitant brown trout. Prevalence of heart
lesions was higher in fish challenged with infected blood possibly due to exposure to a higher virus load. No
lesions consistent with PDS were observed. Changes in gene expression were quite different between
challenged and control groups at 8 weeks post challenge.

Conclusions
This study documents the susceptibility of brown trout to PRV-3 and its pathogenesis. This allow to a better
understanding of the role of brown trout in the epidemiology of PRV-3.

Page 54
3.1 Salmonid viral diseases

Effect of water temperature on infection kinetics with Piscine orthoreovirus genotype 3 in

rainbow trout

Juliane Sørensen1, Niccolò Vendramin1, Argelia Cuenca1, Kerstin Skovgaard2, Niels Jørgen Olesen1

1National Institute for Aquatic Resources DTU Aqua, Technical University of Denmark
2DTU Bioengineering, Technical University of Denmark

[email protected]

Piscine orthoreovirus genotype 3 (PRV-3) was reported in 2017 in Denmark in association with disease
outbreaks in rainbow trout (Oncorhynchus mykiss). A surveillance program revealed that the virus is
widespread within the country. Notably, disease outbreaks associated with detection of PRV-3 occurred only
in recirculating aquaculture systems. Disease associated with PRV-3 is predominantly observed during winter
and characterized by severely increased mortality.

Methodology
In order to understand the effect of water temperature, an in vivo cohabitation trial was conducted with
rainbow trout. The experimental setup included three different temperatures: 5 °C, 12 °C, and 18 °C each
with both a control tank (mock-injected) and a tank with fish exposed to PRV-3 (shedder:cohabitant ratio
was 50:50). Samples were collected from all the experimental groups every second week post challenge
(WPC) until trial termination at week 12.

Results
Preliminary data shows that, in heart tissue, PRV-3 peaked at 6 WPC in cohabitants maintained at 12 °C and
18 °C, while it peaked at 12 WPC in cohabitants maintained at 5 °C. Interestingly, the highest viral load
reached in the 5 °C group was significantly higher than that at 12 °C and 18 °C. Additionally, area under the
curve analysis shows that there is a significant difference between the viral load of the three temperatures
across the trial, with the highest viral load at 5 °C.

Shedders at 12 °C and 18 °C cleared the infection considerably faster than the group at 5°C. While shedders
have cleared most of the virus at 4 WPC and 6 WPC for the groups kept at 18 °C and 12 °C respectively, high
viral load persisted in the shedders kept at 5°C at 12 WPC.

Additionally, a significant reduction in haematocrit value was observed in the group exposed to PRV-3
maintained at 12 °C in correlation with the peak viraemia at 6 WPC. No changes were observed in the
haematocrit at 18 °C.

Conclusions
These data show that low water temperature allow significantly higher PRV-3 replication in rainbow trout.
Although no mortality was observed in the experimental trial, these data comply with field observations of
clinical outbreaks during cold months. Future studies will investigate host immune response involved in
clinical disease development.

Page 55
3.1 Salmonid viral diseases

Experimental infection with PMCV in Atlantic salmon – insights into pathogenesis

Niccoló Vendramin1, Andrew Tighe2, Tine Moesgaard Iburg1, Charlotte Axén3, Neil Ruane2, Argelia Cuenca1

1Unitfor Fish and Shellfish diseases, DTU Aqua, Kgs Lyngby Denmark
2Marine Institute, Oranmore, Ireland
3SVA, Sweden

[email protected]

Piscine myocarditis virus (PMCV) is recognised as the aetiological agent of cardiomyopathy syndrome (CMS)
in Atlantic salmon (Salmo salar). This disease has been described since the mid 80’ies in Norway and later on
in several countries farming Atlantic salmon including Ireland, Faroe Islands and Scotland.

Research on PMCV and CMS is currently hampered by the lack of in vitro culture systems.

In order to gain knowledge on the pathogenesis of CMS in relation to PMCV infection we conducted an
experimental infection trial in Atlantic salmon.

The experiment was conducted using a cohabitation challenge model. Two experimental groups of Atlantic
salmon juveniles (landlocked from Sweden) were included, one exposed to high dose and one to low dose.
Shedders were tagged by clipping the adipose fin and IP injected with supernatant of heart tissue obtained
from CMS outbreak. After screening the inoculum tissue for salmon pathogens, two dilutions were prepared,
with a difference of 1:1000 in PMCV concentration. A negative control group was also included, using heart
supernatant from negative fish. After injection shedders were cohabited with naïve fish, which were not
tagged. The experiment was run for 12 weeks.

At selected time points, 6 shedders and 6 cohabitants from each of the infected groups, and 2 shedders and
2 cohabitants from the negative control were sampled, heart was dissected and tested for qPCR for assessing
PMCV load and for histopathological lesions.

An overview of viral load fluctuations over time in the different groups will be presented, it was possible to
observe transfer of viral RNA from shedders to cohabitants both in high and low dose, although a large
variation was observed within the same time point was observed.

Heart pathology consistent with CMS was observed in shedders and cohabitants of both groups, in the late
phase of the experiment; the majority of affected fish was observed in the high dose group.
At the end of the trial a panel of heart RNA was selected to conduct further genomic characterization of
PMCV RNA recovered from fish with high viral load without CMS lesions and fish with high viral load and CMS
lesions.

Page 56
3.1 Salmonid viral diseases

The effects and genetic diversity of salmon gill poxvirus disease in freshwater Atlantic
salmon in Scotland

Roas Allshire1, Manfred Weidmann2, Jessica Benkaroun1, Ola Wands3, Rhona Robertson3, Johanna Baily1

1Institute
of Aquaculture, UK
2Brandenburg Medical School Theodor Fontane, Germany
3Cooke Aquaculture Scotland, UK

[email protected]

Salmon gill poxvirus is an emerging viral disease causing significant losses in the Scottish Atlantic salmon
industry. Reported in all stages of production, clinical signs consist primarily of gill pathology, however little
is known about the effects of the virus in other organs or about the variation in clinical presentation of
disease. The presence of SGPV was monitored in two cohorts of fry from stocking into two RAS systems until
seawater transfer, through a naturally occurring SGPV infection and subsequent SGPVD outbreak. Although
both groups tested positive for the virus, the presentation varied widely from high mortality and severe
pathology in one cohort, to no clinical signs of disease at all in the other. The effects of infection in these fish
will be presented through histological and molecular results.

The variation of presentation and mortality levels across outbreaks, as well as a molecular population study
published on Norwegian viral samples using genotyping, suggests that diversity in SGPV in different Scottish
sites is likely. Therefore, a collection of samples from geographically distinct SGPVD outbreaks across
Scotland in the last three years were gathered for whole genome sequencing and phylogenetic analysis.
Results of these analyses will be presented and discussed within the wider geographical context.

Funding
Cooke Aquaculture and the University of Stirling.

Page 57
3.1 Salmonid viral diseases

Investigations of compound influences on mortality induced during challenge of Atlantic

salmon (Salmo salar L.) with infectious pancreatic necrosis virus (IPNV)

M Braceland1, M Booman1, J Poley1, E Munro2

1The Center for Aquaculture Technologies, Canada

2Marine Scotland, Scotland

[email protected]

Infectious pancreatic necrosis virus (IPNV) is again emerging as an economic concern in Atlantic salmon
(Salmo salar L.) aquaculture. While the industry has benefitted greatly from the availability of IPN resistant
genotypes, in recent years outbreaks have increased in occurrence throughout the industry. This has led to
investigations here on the effects of host and virus genotypes, and environment on infection, pathogenesis,
and elicited mortality.

Methodology
In brief, the effects of resistant and susceptible genotypes of fish belonging to multiple strains and
geographical origins were challenged with IPNV via IP and cohabitation routes. In addition, environmental
and IPNV virulence markers were identified effects on resulting mortalities.

Results and Conclusions

This study demonstrates the compound effects of host and viral genotypes, environment, and challenge
route on mortality in populations. The predictive nature of resistant fish genotype on survival post challenge
was found to be variable dependent on fish strain, thus potentially highlighting key differences in marker
associated disease outcomes. The development of this highly reproducible model allows for the testing of
multiple technologies to mitigate the impacts of IPNV on the industry. In addition, gives further insight on
the risk factors associated with outbreaks.

Page 58
Tuesday 21st September 2021
4.1 Aquatic animal epidemiology I

Advances in data-driven fish health surveillance

Britt Bang Jensen

Section for Epidemiology, Norwegian Veterinary Institute, Norway

[email protected]

In aquaculture, the foundations of using data for fish health surveillance is presently being explored. For
example, increases in mortality detected in data collected on production in official registries can possibly be
used as an indicator of an adverse health problem that should be investigated. Sensor data from water
quality sensors collected in online systems might be used to detect issues with oxygenation or temperature
increases in RAS-farms.

During the last decade, systems for early detection of syndroms has begun to be used in the surveillance of
diseases in production animals and in human health. Such systems are set up to give out a warning when an
unusual pattern or occurrence is identified in a non-specific health indicator. Non-specific health indicators
could be production loss (milk reduction, reduced growth and mortality), increased use of antibiotics or
increased reportings of patients with ie respiratory syndroms. Data for syndromic surveillance can come from
sources like reporting’s from veterinary clinics or physicians, drug registers and abattoirs, or from production
registers. The advantage of syndromic surveillance systems is thus that they rely on data that are already
being collected, but for other purposes.

There are some caveats for implementation of such systems. The biggest issues to be addressed is data
accessibility and data rights. Aquaculture production is diverse and the technology levels range from very
low to highly advanced, when it comes to data collection, reporting and analyses.

Within the latest year, there has been a surge in development of software technology for presenting trends
and predictions of infection and disease due the ongoing Covid-19 pandemic. The general public has become
used to read and interpret interactive graphs and dashboards and farmers are asking for better information
based on the data they can provide. We can use this momentum to create solutions that will be of benefit
to the industry, authorities and fish welfare.

In this presentation, some of the recent advances on the use of data for fish health surveillance will be
discussed, and perspectives for the future presented.

Page 59
4.1 Aquatic animal epidemiology I

Investigating risk factors for fish mortality in Norwegian salmon farms

Victor HS Oliveira1, Katharine R Dean1, Lars Qviller1, Carsten Kirkeby2, Britt Bang Jensen1

1Section
for Epidemiology, Norwegian Veterinary Institute, Norway
2Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of
Copenhagen, Denmark

[email protected]

High mortality of Atlantic salmon (Salmo salar L.) causes major economic losses to the aquaculture industry
and is an indicator of poor fish welfare. Over the last five years, salmon mortality has increased in Norway.
Producers reported more than 52 million dead salmon in 2020. Our objective was to evaluate potential farm-
level risk factors associated with salmon mortality, using monthly reported production, environmental, and
fish health data.

The data were from salmon in the marine phase, produced between January 2014 and December 2019. The
study population comprised of 642 commercial farms located across all of the 13 production zones in
Norwegian coastal waters. We built a mortality model with the monthly death counts as the response
variable. We found a median mortality rate of 3 deaths per 1,000 fish-months at risk (interquartile range:
1.6−6.5). The explanatory variables of our model significantly associated with salmon mortality (P ≤ 0.05)
were month at first stocking, fish weight, production zone, sea lice treatments, and sea surface temperature
and salinity. The transfer of fish with higher weight to sea was also associated with higher mortality,
especially during certain times of the year.

In general, more frequent sea lice treatments, particularly non-medicinal treatments, led to increased
mortality of the salmon. The mortality situation was worse in productions zones that experienced more
outbreaks of important viral diseases, including pancreatic disease. We found a non-linear relationship
between temperature and mortality, in which temperatures below 5 ˚C and above 10 ˚C were suboptimal in
that they increased mortality. Additionally, higher salinity was more detrimental to salmon survival as
compared to lower salinity. Our results can be used to predict mortality using routinely collected data and
to identify risk factors to target for interventions in order to reduce salmon mortality in the future.

Page 60
4.1 Aquatic animal epidemiology I

Mortality patterns in salmon farming

David Persson1, Arnfinn Aunsmo1,2, Ane Nødtvedt1, Marit Stormoen1

1Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Norway

2Laksar Fiskeldi, Iceland

[email protected]

The aims of this study were to describe patterns of mortality in commercial salmon farming and to investigate
sources of variation in mortality during the first 180 days of the marine phase.

Daily mortality records, including cause-specific registrations, were retrieved from two salmon farming
companies in Norway. Data included 21 mill salmon from ten hatcheries, stocked in 136 cages at 21 farms
over the years 2017/2018, and followed until harvested (“full dataset”). Overall total mortality from sea-
transfer until harvest was 8.7% (equal to 1.8 mill dead fish). A subset of the “full dataset” was created to
investigate sources of variation in mortality (“early mortality”, n=121). Fish-groups (fish transferred to the
same cage from the same hatchery) traceable until 180 days in production were used to build a cross-
classified multilevel linear regression model, with “fish-group” nested within “farm” and cross-classified with
“hatchery” (using MLwiN v. 3.05, University of Bristol).

The mortality classification in the “full dataset” showed a pattern where causes related to smolt quality (10%
of dead fish) dominated the start of the production, and mortality due to handling (29% of dead fish) was
evident from mid-production until harvest. Infectious disease mortality (17% of dead fish) was mainly
identified in the start and towards the end of production. Fish-groups in “early mortality” had a mean
mortality of 2.7% (range: 0.3-21.2%) and mortality caused by smolt quality, infectious diseases and handling
identified 32%, 17% and 1.5% of the dead fish respectively. “Fish-group” explained most of the variation in
mortality in the final model (70.2%) followed by hatchery (23.4%) and farm (6.4%). Together, our findings
indicate that smolt related mortality is one of the major causes of death in the first part of the production,
and in specific fish-groups also the overall main cause of death. However, handling was by far the most
important cause of mortality across all fish-groups.

The study indicates that mortality varies by fish-group to a large extent. This means that targeted preventive
strategies against mortality at the fish-group might be more effective than overall measures at farm or
hatchery level.

Page 61
4.1 Aquatic animal epidemiology I

Mortality patterns during the freshwater production phase of salmonids in Norway

Siri Kristine Gåsnes1, Victor H.S. Oliveira1, Kristine Gismervik1, Ashley Ahimbisibwe1,2, Brit Tørud1, Britt Bang
Jensen1

1Norwegian Veterinary Institute, Norway

2Institute of Basic Medical Sciences, Oslo Centre for Biostatistics and Epidemiology, Department of
Biostatistics, University of Oslo, Norway

[email protected]

Mortality of salmonids in Norwegian aquaculture have major influence on fish welfare and
represents economic losses for producers. Norwegian fish farmers are obliged to submit monthly reports
with the number of fish, average weight and mortality for the freshwater farms since 2010. The reported
numbers are on a production unit level (tank) from the start-feeder stage until they transfer fish to sea.

Methodology
In this study, we have estimated the monthly mortality for freshwater farms with Atlantic salmon (AS) and
rainbow trout (RT) between 2011 and 2019. We calculated farms’ monthly mortalities as the proportion of
dead fish out of the standing stock per weight groups (i.e. 2-12.2 g, 12.3-34.2 g, 34.3-89.1 g, and >89.1 g). We
built a regression model for mortality with the variables year, weight group, season, region, farm size and
farm.

Additionally, we distributed questionnaires to farmers to gather further information regarding potential

causes of mortalities. The final data set comprised reports from more than 5,000 tanks with AS and more
than 600 tanks with RT.

Results
The following results refer to the median mortalities for AS. The analysis of production data showed that the
mortality increased from 0.15% in 2011 to 0.25% in 2019. There were significant regional
differences, particularly comparing the North region with highest mortality (0.27%) and
the Southwest region with the lowest mortality (0.16%). The mortality was lowest in winter (0.12%) and
highest in summer (0.24%). The mortality was highest in smaller fish (3-12.2 g) and lowest in fish from 34.3-
89.1 g. Results from the questionnaire showed that infectious or non-infectious diseases were the most
commonly reported cause of mortality in this study.

Conclusions
Analysis of the data presented shows that mortality varies with fish weight, region, season and year. The
mortality patterns described in this study identifies several important risk factors for mortality, which is an
important step to reducing mortality in the freshwater production phase of salmonids in Norway.

Funding
This study was funded by the Norwegian Animal Protection Alliance's Research Fund (project “SETFISKVEL”)
and by the Norwegian Research Council (project number 294647 “Understanding and monitoring mortality
in farmed fish towards sustainable growth in aquaculture”). We thank the farmers who participated in this
study for answering the questionnaires.

Page 62
4.1 Aquatic animal epidemiology I

Modelling scenarios for control and mitigation of Infectious salmon anaemia (ISA)

Mona Dverdal Jansen1, Magne Aldrin2, Ragnar Bang Huseby2, Britt Bang Jensen1

1Norwegian Veterinary Institute, Norway

2Norwegian Computing Center, Norway

[email protected]

With the implementation of the new EU animal health law, different ways to mitigate and control listed
diseases are being explored. Control of Infectious salmon anaemia (ISA) in Norway is currently by use of a
stamping-out strategy: if a farm experiences a clinical ISA outbreak, the farmer is usually required to
slaughter all fish at the farm within a few weeks.

We have developed a model for the spread of infectious diseases between and within marine aquaculture
farms in Norway. The estimated model was then used for scenario simulation, or what-if analysis, to
investigate the effects of potential strategies to combat ISA, including screening, vaccination and culling.

From the model, we found the estimated relative importance for each of the various sources of infection.
Spread of ISA-virus (ISAV) from infected neighbouring farms accounts for around 50% of the infections.
Infection from ”non-specified sources” accounts for around 40% of the infections. For ISAV we believe that
the most important of these are viruses mutating from the non-virulent virus variant (ISAV HPR0) to the
virulent variant (ISAV HPRdel). Infected cohorts moved from another farm accounts for 8%, and most of
these are presumably originally either infected by other farms or by ”non-specified sources” before they
were moved. Infection as a result of stocking smolts infected by virulent ISAV HPRdel accounts for around
1% of all infections in the model.

From the scenario simulations, we found that omitting the mandatory culling without any other preventive
measure will increase the number of outbreaks dramatically, and is probably a non-viable option.
Introducing mandatory vaccination may be equally effective as today's culling strategy alone depending on
the obtained field-efficacy of a vaccine. Introducing mandatory screening combined with culling will bring
the number of outbreaks to a low number because infected cohorts are slaughtered before an outbreak
occurs.

However, the selection of a new strategy also involves both economic and political considerations. Thus,
using scenario simulations of different strategies provides one valuable input to support decision making.

Page 63
4.2 Bacterial diseases I

Whole genome comparison of Vibrio harveyi strains from the Mediterranean Sea

Željko Pavlinec1, Robert Vaser2,3, Ivana Giovanna Zupičić1, Snježana Zrnčić1, Dražen Oraić1, Mile Šikić2,3

1Croatian
Veterinary Institute, Croatia
2Faculty
of Electrical Engineering and Computing, University of Zagreb, Croatia
3Genome Institute of Singapore, A*STAR, Singapore

[email protected]

In recent years, outbreaks of vibriosis are getting more common in the Mediterranean aquaculture, causing
severe losses and threatening the long-term sustainability. In fact, in a three year period, from 2015 to 2017,
the main reported disease in sea bass aquaculture in Mediterranean was vibriosis. This disease is found in all
parts of the Mediterranean Sea, and it is the most reported disease in the western, southern and eastern
Mediterranean. Today Vibrio harveyi is recognized as one of the major causes of vibriosis, but there is a great
heterogeneity in the virulence of different strains of V. harveyi. Unfortunately, little is known about the
genomic differences in the strains of V. harveyi originating from different parts of the Mediterranean.

We used eleven strains of V. harveyi isolated from fish originating from different parts of the Mediterranean
Sea: four strains from Croatia, two from France, two from Italy, and one strain from Spain, Tunisia and Turkey.
To obtain high quality complete genome assemblies we performed high throughput sequencing using MiSeq
(Illumina) and MinION (Oxford Nanopore Technologies) sequencers. For MiSeq sequencing genomic DNA
was extracted from bacterial cultures using NucleoSpin Microbial DNA Mini kit, and the sequencing libraries
were prepared using NexteraXT Library Prep Kit. For MinION sequencing DNA was extracted using Nanobind
CBB Big DNA Kit and the libraries were prepared using Rapid Barcoding Kit. Whole genomes were assembled
using Flye and Medaka tools with nanopore reads, and corrected using Racon polishing tool with Illumina
reads. Larger deletions, inversions and translocations were detected using MAUVE multiple genome
alignment. Virulence factors were identified in all of the genomes using VFDB pathogenomic platform, and
the resistance genes were identified using CARD RGI tool.

The genomes of all eleven strains were successfully assembled, with BUSCO scores ranging from 97.58% to
100%. Comparison of the strains using the genomic determinants stated above will be presented in detail.
Exploring the differences between strains of V. harveyi originating from different parts of the Mediterranean,
and increasing the number explored strains, we will obtain knowledge helpful in determination of both local
and regional management of vibriosis.

Page 64
4.2 Bacterial diseases I

ToxRS and AraC1 are involved in the temperature dependent expression of the piscibactin
siderophore system in Vibrio anguillarum

Marta Afonso Lages, Manuel L Lemos, Miguel Balado

University of Santiago de Compostela, Spain

[email protected]

The High-Pathogenicity Island irp-HPI is widespread among Vibrionaceae and encodes the piscibactin
siderophore system, a major virulence factor of relevant fish pathogens such as V. anguillarum and
Photobacterium damselae subsp. piscicida. Recent works showed that the expression of piscibactin genes
(irp genes) is favoured by low temperatures. Nonetheless, not much is known about the regulatory
mechanism behind irp-HPI gene expression.

Methodology
Since toxRS is a global regulator system that regulates the expression of some virulence factors in response
to external stimuli, a V. anguillarum toxRS defective mutant was constructed to analyse the expression
pattern of piscibactin siderophore system. In addition, irp-HPI includes two putative AraC-like transcriptional
regulators dubbed AraC1 and AraC2. Therefore, V. anguillarum araC1 and araC2 single and double mutant
strains were constructed and the role of each gene in irp-HPI expression was analysed.

Results
The results showed that ToxRS exerts a temperature dependent repression on the irp genes. Additionally,
while araC1 is required for the expression of irp-HPI genes, araC2 is not involved in this process. Notably,
araC1 expression is strongly up-regulated at low temperatures and it is the transcriptional activator required
for the expression of irp-HPI by inducing the transcriptional activity of frpA promoter (PfrpA), the gene
encoding the piscibactin outer membrane receptor.

Conclusions
All results showed that, although AraC1 is a key activator required for the expression of irp-HPI genes, ToxRS
does participate directly or indirectly in the temperature dependent expression of the piscibactin
siderophore system. Thus, the irp-HPI expression pattern is the result of a complex regulatory circuit
emerging from unidentified element(s) likely encoded outside the High-Pathogenicity Island.

Funding
This work was supported by grants PID2019-103891RJ-100 and RTI2018-093634-B-C21/C22 (AEI/FEDER, EU),
co-funded by the FEDER Programme from the European Union and the State Agency for Research (AEI) of
Spain. The work was also supported by grant GRC2018/018 from Xunta de Galicia (Spain).

Page 65
4.2 Bacterial diseases I

Structural and functional characterization of NiTox: a new AB-toxin from Vibrio

Nigripulchritudo lethal to shrimp

Johnny Lisboa1,2, Inês Lua Freitas1,2, Ana do Vale1,2, Nuno MS dos Santos1,2

1Fish Immunology and Vaccinology Group, i3S - Instituto de Investigação e Inovação em Saúde, Universidade
do Porto, Portugal
2Fish Immunology and Vaccinology Group, IBMC – Instituto de Biologia Molecular e Celular, Universidade do

Porto, Portugal

[email protected]

Shrimp aquaculture has been increasing over the last decades, and currently provides half of the world
shrimp supplies. White-leg shrimp (Penaeus vannamei) has become the main cultured shrimp species and
accounts for 53% of the total crustacean aquaculture production. However, the intensification of shrimp
aquaculture gave rise to severe disease outbreaks that threaten production and cause huge economic losses.

Recently, we identified NiTox, a new AB-toxin of 96 kDa secreted by Vibrio nigripuchritudo, a Gram-negative
bacterium that causes shrimp vibriosis, leading to massive mortalities. In vivo experiments showed that the
injection of the NiTox in the Penaeus vannamei hemothorax results in 80% mortality. By solving NiTox tri-
dimensional structure, we found that similarly to most AB-toxins, NiTox has three distinct domains: a
catalytic domain (C, NiToxM621-P887), a putative translocation domain (T, NiToxQ38-P519) and a receptor-binding
domain (R, NiToxV520-G620). The C domain is structurally similar to the catalytic domain of AIP56, an AB-toxin
secreted by Photobacterium damselae subsp. piscicida that cleaves NF-kB p65, with a conserved zinc-
metalloprotease signature 781HEIMH785. As expected, considering the similarities to AIP56, NiTox is able to
cleave NF-kB p65 and Dorsal, the p65 homologue of drosophila and shrimp.

Interestingly, NiTox presents an additional zinc-metalloprotease signature 678HEALH682 in its C domain,

absent in AIP56. Mutations into the first or second metalloprotease signatures of NiTox does not affect the
lethality of the toxin for Penaeus vannamei. However, when both catalytic signatures are simultaneously
inactivated, lethality is abolished. These results show the importance of both zinc-metalloprotease
signatures for the NiTox toxicity. To further understand the molecular details beneath the lethal activity of
this toxin, we are currently investigating the NiTox’s second zinc-metalloprotease signature target and
mechanism of action.

Page 66
4.2 Bacterial diseases I

Antimicrobial susceptibility of Vibrio spp. Isolates collected from Brazilian shrimp fishfarms

Culot Antoine1,3, Grippon Pauline2, Quentin Bruey1, Le Devendec Laëtitia2, Clarisse Techer1, Larvor Emeline2,
Stephane Frouel1, Jouy Eric2, Kempf Isabelle2, Gautier Michel3, Sandrine Baron2

[email protected]

1Mixscience SAS, 2 Avenue de Ker Lann, 35170 Bruz, France

2The French Agency for Food, Environmental and Occupational Health & Safety (ANSES), Ploufragan France
3Laboratoire de Microbiologie d’Agrocampus Ouest, INRAE, Institut Agro, STLO, Rennes, France

Vibrio spp. is a genus of ubiquitous bacteria found in a wide variety of aquatic and marine habitats. Some
species are pathogenic for humans and/or aquatic animals like shrimps. Susceptibility testing of non-cholera
Vibrio spp. isolated from aquatic animals or the aquatic environment may be undertaken for two main
reasons: either to inform about the selection of agents to be used in the therapy of aquatic animal diseases,
or as part of a food safety monitoring of the potential impact of aquatic animal products for human health.

Methodology
Seventy-four Vibrio isolates were collected in 2018 from 50 Brazilian shrimp ponds and identified at genus
level by Maldi-Tof Bruker. Antimicrobial susceptibility testing was performed by agar diffusion according to
CLSI methods. Antimicrobial agents tested were those, which are critical for human health (list WHO - panel
1) and those used in aquaculture according to the list defined by CLSI (panel 2). Panel 1 (human) included
seven antibiotics: ampicillin, amoxicillin-clavulanic acid, cefotaxime, ceftazidime, imipenem and meropenem
tested after incubation at 35°C during 16/18h (CLSI, M45). Panel 2 (aquaculture) included ten agents:
ampicillin, chloramphenicol, florfenicol, enrofloxacin, erythromycin, fosfomycin, gentamicin, oxolinic acid,
oxytetracycline and trimethoprim-sulfamethoxazole. For this second panel, incubation was performed at
28 °C during 24/28h as defined in previous study. For both antibiotics panels, quality control strains were
included.

Results
Only 67 isolates grew at 35°C (panel 1). Based on the clinical breakpoints defined by CLSI, 62.7% (n=42) were
resistant to ampicillin and all the isolates were susceptible to the five left agents tested. All strains were
susceptible to first-line treatment options used in case of human vibriosis.

At 28 °C, all the isolates grew, which confirms the interest of this incubation temperature to test the
susceptibility of Vibrio isolates from aquaculture environment. Moreover, for incubation at this temperature,
CLSI has defined quality control values for the reference strains for eight of the ten antimicrobials tested,
which is an essential condition to determine epidemiological cutoffs values. As no breakpoint values are
available, MIC50 was calculated for each antimicrobial agent. Apart of ampicillin, MIC50 range from 25 to 34
mm.

Conclusions
The data generated in this study (panel 2) can now be used to determine provisional epidemiological value
at genus level (1st step). Further works are in progress concerning identification at species level and detection
of antibiotic associated resistance genes.

Page 67
4.2 Bacterial diseases I

Structural and functional characterization of a PirAB-like binary toxin from Photobacterium

damselae subsp. piscicida

Ana do Vale1,2, Inês Loureiro1,2, Johnny Lisboa1,2, Alba V Barca3, Alexandra Teixeira1,2, Carlos R. Osório3, Nuno
M. S dos Santos1,2

1i3S- Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal

2FishImmunology and Vaccinology Group, IBMC – Instituto de Biologia Molecular e Celular, Universidade do
Porto, Portugal
3Departamento de Microbioloxía e Parasitoloxía, Instituto de Acuicultura, Universidade de Santiago de

Compostela, Spain

[email protected]

Photobacterium damselae subsp. piscicida (Phdp) causes a bacterial septicemia that affects a wide variety of
marine fish, representing a serious threat to aquaculture. The extracellular products (ECP) secreted by Phdp
are known to be crucial for the bacterium virulence, but apart from the apoptogenic toxin AIP56 and the
PnpA peptidoglycan hydrolase, no other relevant ECP components were identified.

Recently, we performed a proteomic analysis of Phdp ECPs and identified a 61 kDa putative pore-forming
toxin with similarity to delta-endotoxin and an 11 kDa protein with unknown function. We found that the
secretion of both proteins was impaired in a epsL deletion mutant, indicating that they are secreted by the
Phdp type II secretion system (T2SS). In silico analysis suggested that the genes encoding the proteins
constituted an operon and predicted that the proteins associate to form a binary toxin structurally similar to
the Vibrio parahaemolyticus PirAB toxin (VpPirAB). Following these findings, we developed several in vitro
and in vivo studies to characterize the biological function of this Phdp putative toxin (hereafter named
PhdpPirAB) and crystalized and solved its 3D structure.

PhdpPirA (11 kDa) and PhdpPirB (61 kDa) 3D structures are very similar to the structures of VpPirA and VpPirB,
respectively, despite the low sequence identity (15% for PhdpPirA/VpPirA and 24% for PhdpPirB/VpPirB). In vivo
assays showed that the co-injection of recombinant PhdpPirB and PhdpPirA proteins in sea bass and sea bream
resulted in 100% mortality and abundant cell death in several organs, while injection of each protein
individually did not induce mortality or significant histopathological alterations. To investigate if PhdpPirAB
was involved in Phdp virulence, we generated a Phdp pirAB knock-out strain and tested it in vivo. Deletion of
PhdppirAB attenuated lethality, confirming that PhdpPirAB is a virulence factor of Phdp. In vitro/ex-vivo
cytotoxicity tests showed that PhdpPirAB is highly toxic to sea bass macrophages, but does not displays toxicity
to epithelial (EPC) and fibroblast (SAF-1) cell lines. PhdpPirAB is also toxic to mouse bone-marrow derived
macrophages, suggesting that the molecular targets of the toxin are evolutionarily conserved.

Page 68
4.3 Gill diseases

Longitudinal analysis of the gill microbiome of farmed Atlantic salmon in Scotland to

monitor changes occurring in relation to Complex Gill Disease

Kelly Stewart1, Cindy Smith1, Umer Ijaz1, Annette Boerlage2, George Gunn2, Aaron Reeves2, Angela Ashby3

1School
of Engineering, University of Glasgow, UK
2EpidemiologyResearch Unit, SRUC, UK
3PHARMAQ Analytiq UK, UK

[email protected]

Scotland is home to a significant Atlantic salmon aquaculture industry, creating approximately 10,000 jobs
and valued at £601 million in 2017. Gill disease is now a prominent issue, leading to major mortalities.
Recently, complex gill disease (CGD), thought to be multifactorial in nature with a wide range of clinical
presentations, has emerged, with little know about the cause or development. We aim to characterize and
investigate the role of the gill microbiome in gill health in relation to CGD in farmed salmon production cycles
from hatchery to harvest.

Methodology
To achieve this, we tracked the gill microbiome of farmed salmon from two Autumn’18 inputs at two
locations along the Scottish coast, from their freshwater hatcheries to their seawater farms for 16 and 17
months. One farm was sampled monthly, and the other intensively every two weeks, both through swabbing
the gills, resulting in 378 fish for the study. Characterisation of the gill microbiome was performed by 16S
rRNA gene amplicon sequencing targeting the V1-V2 region. The primer choice was optimised to reduce the
volume of salmon host DNA contamination.

Results
The results from these longitudinal studies aid in characterising the gill microbiome of farmed salmon before,
during and after cases of CGD. As a result, changes in the microbiome composition can be tracked to aid in
determining its role in CGD. This was carried out through techniques such as the analysis of the ‘core’
microbiome of the gills, the comparison of diseased fish microbiomes to non-diseased counterparts, and
analysis using the vast volume of health and environmental meta-data that was gathered throughout the
project.

Conclusions
In this study, we were able to successfully map the microbiome changes undergoing in the gills of farmed
Atlantic salmon before, during and after CGD cases. Furthermore, the microbiome of the farmed salmon
gills changes in composition over the course of their terms at sea, as well as upon their transfer from
freshwater hatchery to their seawater cages.

Page 69
4.3 Gill diseases

Investigating the aetiology of nodular gill disease (NGD) in rainbow trout (Oncorhynchus
mykiss) farmed in Switzerland

Chloe English1, Stefania Vannetti2,4, Irene Adrian-Kalchhauser2, Christine Huynh3, Ralph Knüsel4, James
Wynne5, Heike Schmidt2

1The University of Queensland, Australia

2University of Bern, Switzerland
3Nautilus Collaboration, Australia
4fishdoc GmbH, Switzerland
5Commonwealth Scientific and Industrial Research Organisation, Australia

[email protected]

Nodular gill disease (NGD) is a form of branchitis associated with amoebae which impacts primarily
freshwater salmonids globally. NGD has been ascribed to four species of amoebae based on one-off case
studies in New Zealand, Germany, Czech Republic, Canada and the USA. Despite these reports, it is unknown
whether NGD can be caused by multiple amoeba species within the one region or whether particular causal
agents are regionally specific.

Moreover, the taxonomic identity of the agents cited in most NGD publication are uncertain because the
assessment was based on morphology alone and this is unreliable because different species often look
extremely similar. In recent years, several outbreaks of severe gill disease have impacted rainbow trout
farmed in Switzerland in early spring, causing up to 50% mortality in affected fish. Histology revealed
proliferation of the gill epithelium, often in a nodular manner at the filament tips, and associated amoebae.
Prior to this project, attempts to isolate and culture the amoebae were unsuccessful, hence no morphological
or genetic information was available to accurately identify the amoeba species.

This collaborative project aimed to investigate the aetiology of NGD-affecting farmed rainbow trout in
Switzerland using a combination of morphological and sequence-based taxonomic methods. Culturing and
morphological assessment have identified a number of amoeboid morphotypes on affected gills. Next
generation sequencing targeting the 18s rRNA gene was used to profile the amoeba community of NGD-
affected and asymptomatic rainbow trout from four farms sampled in Switzerland. This data helps link
amoeba identity, prevalence and abundance with gill pathology to provide supporting evidence of possible
causal agents.

Funding
This project was supported by the EAFP Small Grant Scheme.

Page 70
4.3 Gill diseases

Scoring the scores: diagnostic sensitivity and specificity of gross gill scores for AGD and CGD
in Scotland’s salmon aquaculture

Annette Boerlage, Jude Eze, Aaron Reeves

Scotland's Rural College, UK

[email protected]

Amoebic gill disease (AGD) and complex gill disease (CGD) have established themselves among the most
significant diseases in Scotland’s marine farmed salmon industry. Most salmon producers use repeated gross
gill observations throughout a production cycle to monitor gill disease status of fish, but there is uncertainty
about the ability of gross gill scores to designate gill disease correctly. In the autumn of 2018, most of
Scotland’s farmed salmon producers embarked on a research project focusing on the epidemiology of
complex gill disease. As part of this project, eight salmon producing sites from across Scotland were sampled
bi-weekly by a variety of tests, among which gross gill scores.

To obtain a better understanding of the ability of gross gill scores to designate gill disease correctly, we
compared for AGD: gross AGD scores (0-5), PCR test, and histopathology. For CGD we compared: gross
proliferative gill disease (PGD) scores (0-5), gross total gill scores (0-5), and histopathology. We used BaYesian
latent class models so we could estimate diagnostic sensitivity (DSe) and specificity (DSp) in the absence of
a gold standard tests. Reason was that histopathology, which has been used as gold standard in the past,
was taken from the second gill arch on the right and not the most severe gill arch.

We showed that DSp was close to 1 for all gill scores for a medium or severe stringent case definition (score
≥2 or ≥3), implying that almost all negative fish were correctly designated negative and there were almost
no false positives. DSe was highest for gross total gill score to detect CGD (DSe ≥ 0.8) implying most of the
positive fish were designated positive gill score, but about 20% as false negatives. DSe for gross PGD scores
to detect CGD and gross AGD scores to detect AGD scored lower, but above 0.5. Results help to interpret and
understand gross gill scores as test method. This is the first attempt to estimate of gross PGD scores and
gross total gill scores for diagnosis of CGD.

Page 71
5.1 Aquatic animal epidemiology II

Chasing ISAV: the challenge of detecting ISAV in infected populations

Mona Dverdal Jansen1, Torfinn Moldal1, Maria Aamelfot2, Marta Alarcon3, Siw Larsen1, Lisa Furnesvik1,
Knut Falk1

1NorwegianVeterinary Institute, Norway

2NorwegianInstitute of Public Health, Norway
3Pharmaq Analytic, Norway

[email protected]

Infectious salmon anaemia (ISA) is a viral disease of Atlantic salmon caused by infectious salmon anaemia
virus (ISAV). It is listed by the European Union and the World Organization for Animal Health (OIE) and
therefore has both welfare and trade implications. Standard sampling procedures for ISAV-screening consists
of tissue samples from heart and mid-kidney. While a positive result provides evidence of clinical infection
with ISAV, the lethal nature of this sampling may limit screening frequency and sample size, particularly in
large, high value fish. While the objective of this study was to investigate the use of skin- and gill swabs in
relation to population-level screening for ISAV, the results provided important knowledge related to the
challenge of detecting ISAV in infected populations.

Five Atlantic salmon seawater sites with ongoing ISA-outbreaks were sampled. At each site three fish groups
from two different cages were sampled: a) fish with clinical signs of ISA from a cage with a confirmed ISA-
diagnosis, b) apparently healthy fish from the same ISA-affected cage and c) apparently healthy fish from a
cage without suspicion of ISA. Skin – and gill swabs on RLT buffer, as well as kidney tissue on RNAlater and
formalin, were collected. Swabs and kidney tissue were tested by PCR while formalin-fixed tissues were
tested by immunohistochemistry.

Swabs were found to perform well in relation to determining the ISAV-status at population level. More
importantly, the results illustrated the challenge of detecting ISAV in several of these populations,
particularly in fish without clinical signs of ISA. This supports findings from the field, where challenges with
confirming some suspected ISA outbreaks subsequently limits the management options available. A more
thorough understanding of the distribution of ISAV within infected populations may potentially aid the
refinement of sampling schemes to increase the likelihood of correctly determining ISAV-status at population
level.

Page 72
5.1 Aquatic animal epidemiology II

Bioeconomic evaluation of different treatment strategies against salmon lice in Norway

Cecilie Walde 1, Marit Stormoen 2, Jostein Mulder Pettersen 3, Britt Bang Jensen 1

1Norwegian Veterinary Institute, Norway

2Department of Production Animal Clinical Sciences, Faculty of Veterinary Medicine, Norwegian University
of Life Sciences, Norway
3Patogen AS, Norway

[email protected]

The extensive use of treatments against salmon lice in Norway has a severe financial impact. The total cost
of controlling salmon lice in Norway was estimated to NOK 5 billion per year in 2017. However, the current
cost calculations of delousing are uncertain due to lack of detailed information on costs of the effect of
delousing on death, reduced growth and slaughter quality of the salmon.

Methology
In a recent study, we have therefore estimated cage-level mortality distributions after six immediate
delousing methods: thermal, mechanical, hydrogen peroxide, medicinal, freshwater and combination of
medicinal treatments. Three Norwegian aquaculture companies supplied daily production data. After data
management the dataset includes 4,644 immediate delousing events in 1,837 cohorts in 159 unique marine
salmon producing sites within the period 2014 to 2019. Key variables in the dataset are region, site, cage and
fishgroup identification number, delousing method, mortality, number of fish, feed amount, temperature,
and date. Mortality is expressed as a mortality rate, taking into account the number of dead fish and the
standing stock in a cage. Mortality rate within 7 days before delousing served as baseline mortality, and we
estimated distributions of the difference in mortality 1, 7 and 14 days after each of the delousing events
categorised into the six different delousing method.

Results
The results show that we can expect mortality after all of the delousing methods, where the median
difference in mortality rates after thermal and mechanical delousing are 5.4 and 6.3 times higher than
medicinal treatment, respectively for the 2017 year-class. We also find wide variability in the change in
mortality within the different delousing methods. The knowledge of variability makes it possible to account
for uncertainty in an economic cost estimation. In an ongoing study, we are exploring the effect these six
delousing methods has on growth. The result from the recent study on mortality and the ongoing study on
growth will provide a fundament for an up-to date cost estimation.

Page 73
5.1 Aquatic animal epidemiology II

Farmed salmon redistribution may reduce salmon lice infestations and treatment
frequency, a simulation study

Lars Qviller, Katharine R Dean, Britt Bang Jensen

Section for Epidemiology, Norwegian Veterinary Institute, Norway

[email protected]

Host density is a key driver in parasite population dynamics, and the number of parasites increase rapidly
when host density increases. This mechanism leads to a disparity between the desire to increase cultured
salmon production, and the negative effects from parasite infestations. This mechanism can lead to an
increase in salmon lice, the predominant ectoparasite in salmonids in the northern hemisphere. Increases in
salmon lice density can cause spillover to wild salmon and negatively impact the animal welfare of farmed
salmonids due from salmon lice treatments.

Here, we examine how a redistribution of the salmonid farms may hamper exchanges of infective salmon
lice larvae between farms, and consequently reduce the salmon lice burdens and treatment frequencies.
More specifically, we use a simulation model to examine how the lice abundances and treatments changed
when the biomass in the system was redistributed to fewer, but larger farms.

We simulated several scenarios, where an increasing number of farms were closed, and their biomass were
redistributed to other farms with matching cohorts. The results indicate that fewer and larger farms are
beneficial, reducing both lice abundance and treatment frequency.

The results further indicate that a strategic removal of farms based on an assessment of their importance for
connectivity in an oceanographic lice dispersal network, improve this effect. The results highlight some core
mechanisms that should be considered in regional production planning, and in the allocation of production
concessions in salmonid farming.

Page 74
5.1 Aquatic animal epidemiology II

Genetic diversity of the carp edema virus in France

Marine Baud1, Laurane Pallandre1, Fabrice Almeras1, Loeiz Maillet1, David Stone2, Laurent Bigarré1

1ANSES, France
2CEFAS, UK

[email protected]

The carp edema virus (CEV) has emerged worldwide, causing massive mortalities in two varieties of Cyprinus
carpio, common carps and koi. The sleepy disease of carp was observed on farms and in ponds in France
since the 2010s, and CEV was first confirmed in 2013. We studied the genetic diversity of the viral samples
found in France from 2013 to 2020.

Methodology
Samples of CEV collected in France over a period of eight years were characterized at the molecular level by
Sanger-sequencing of a 890-long fragment of the p4a gene.

Results
All the sequences, except one, fell into two well-defined genogroups. Sequences obtained from CEV detected
in common carps generally clustered in genogroup I, and sequences from CEV detected in the koi were
assigned to genogroup II. A particular sample was different to all the others and represented a putative new
genogroup. Detected in diseased common carps, it possibly arose from a recombination event between a
genogroup II sequence and a sequence from an unknown genogroup. Compared with sequences from CEV
of other countries, most of the French sequences exhibited high degree of DNA identities with those
published previously, suggesting identical sources of viruses and the sequence diversity suggests multiple
introductions of the viruses in France rather than a spread of infection. Among the French sequences, two
genogroup-specific molecular markers were identified. One was an insertion/deletion identified within a
microsatellite. Another was a group of single nucleotide polymorphisms.

Conclusions
Two common genogroups of CEV each represented by a number of variants were found in France. The
existence of a third new genogroup was suspected. Genogroup-specific molecular markers were identified
for the first time. Like other viruses from the Poxvirinae family, CEV seems to generate genetic diversity via
diverse mechanisms: substitutions, indels and recombination events. This evolutionary potential likely
facilitates adaption to different hosts and environments.

Page 75
5.1 Aquatic animal epidemiology II

Molecular epidemiology of megalocytivirus in Malaysian freshwater angelfish

(Pterophyllum scalare)

Che Azarulzaman Che Johan1, Sandra Catherine Zainathan1,2

1Faculty of Fisheries and Food Science, University Malaysia Terengganu, Malaysia

2Institute of Marine Biotechnology, University Malaysia Terengganu, Malaysia

[email protected]

Malaysia has more than 630 culturists that are involved in the ornamental fish industry for culturing 250
species including local and exotic species. However, megalocytiviruses have been associated globally with
severe systemic disease and economic loss in ornamental fish. The intensity of Megalocytivirus infection in
Pterophyllum scalare is unknown in Malaysia.

Thus, the objectives of this study include the detection of the presence or absence of Megalocytivirus,
determination of genotypes and the major risk factors that contribute to precipitation of disease outbreaks
in P. scalare and lastly, the evaluation of non-invasive sampling detection (swabbing method) in P. scalare
broodstock. A total of six pairs of freshwater angelfish broodstock were used. The water samples such as
inlet river, inlet well, broodstock ponds, breeding pails, growth out ponds, outlet water, and other samples
such as mucus swabs, gill swabs, freshwater angelfish eggs, fries, juvenile, snail, snail eggs, live feed (tubifex
worms and moina), sediment samples (from river, broodstock ponds, growth out ponds, outlet) and wild fish
were collected from the farm from day 0 until day 60. These samples were extracted and proceeded using
nested polymerase chain reaction (PCR) analysis.

A total of 159 samples included both inlet river and well, broodstock ponds, breeding pails, growth out ponds,
outlet water sample, mucus and gill swabs, the eggs fries and juvenile of P. scalare, snail and snail eggs,
tubifex worm, moina and all of sediment samples showed positive results for megalocytivirus. In conclusion,
the results showed the existence of different possible routes of megalocytivirus distribution in the farm.

Page 76
5.2 Bacterial diseases II

Isolation and Characterization of Outer Membrane Vesicles from Photobacterium

damselae subsp. piscicida

Alexandra Teixeira1,2, Ines Loureiro1,2, Nuno dos Santos1,2, Ana do Vale1,2

1i3S– Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal

2FishImmunology and Vaccinology Group, IBMC – Instituto de Biologia Molecular e Celular, Universidade do
Porto, Portugal

[email protected]

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative bacterium that infects a wide variety
of warm water fish species worldwide, causing a huge negative impact in the aquaculture industry. It is
known for long that Phdp extracellular products (ECP) have an essential role for Phdp virulence and contain
antigens required for vaccine efficacy, but most of their components remain undisclosed. We recently
performed a detailed analysis of Phdp ECP and found that they contain high amounts of outer membrane
vesicles (OMVs). These nanosized spherical bilayered particles are receiving a huge attention in the field of
vaccinology, since they present antigens in native conformations and are strongly immunogenic, without
requiring adjuvants. This prompted us to isolate and characterize the OMVs secreted by Phdp.

We started by developing an optimized protocol for obtaining high amounts of pure Phdp OMVs. First, the
bacteria-free supernatant from a Phdp late-exponential culture was harvested, concentrated and dialyzed,
to eliminate soluble proteins and then was ultracentrifuged, to collect the OMVs. The obtained OMVs
preparations were analyzed by Dynamic Light Scattering (DLS), indicating that the main population of vesicles
has sizes around 30-50 nm, although residual populations with different sizes are also present. Transmission
electron microscopy showed that the vesicles have a single bilayered lipid membrane and confirmed the size
distribution obtained by DLS. Proteomic analysis of the OMVs revealed the presence of the apoptogenic toxin
AIP56 and the peptidoglycan hydrolase PnpA, as well as several uncharacterized proteins, including a
putative pore-forming toxin, a putative adhesin/invasin and several Outer Membrane Proteins (OMPs) with
molecular weights ranging from 18 to 64 kDa. Amongst these are several OMPs with an OmpA-like structure
that may act as adhesins, and a 64 kDa OMP predicted to be structurally similar to the BtuB vitamin B12
outer membrane receptor present in several bacterial species. Preliminary in vivo studies suggest that some
of these OMVs-associated proteins may be involved in Phdp virulence.

Page 77
5.2 Bacterial diseases II

A highly unstable plasmid encoding the type III secretion system contributes to virulence
of Photobacterium damselae subsp. piscicida for fish

Carlos R Osorio, Saqr Abushattal, Ana Vences

University of Santiago de Compostela, Institute of Aquaculture, Spain

[email protected]

The marine pathogenic bacterium Photobacterium damselae subsp. piscicida (Pdp), the causative agent of
photobacteriosis in fish, causes important financial loses in marine aquaculture. Recently, it was shown that
genes of a type III secretion system (T3SS) are highly prevalent in Pdp strains. Here, we demonstrate that the
T3SS is encoded within a novel plasmid of 133 Kb (pPDPT3) in Pdp strain PP3. The most prominent feature
of pPDPT3 is the presence of two paralogous copies of twenty-four T3SS-related genes (vscNOPQRSTU, vscXY,
yopN, tyeA, scyN, vcrDRGVH, vopBD, yopH, tir, exsA and vscW), and twelve genes (vscABCDEFGHIJKL) in single
copy.

The plasmid also carries two paralogous genes encoding a YopH-like tyrosine phosphatase effector and a
single copy gene of a YadA-like adhesin. pPDPT3 showed to be highly unstable, its loss being detected after
a single subculture step on agar plates. Plasmidless strains resulting from spontaneous curing of pPDPT3
under laboratory conditions, exhibited reduced virulence levels for European sea bass (Dicentrarchus labrax)
compared to the parental strain, suggesting for the first time a role of T3SS in Pdp virulence for fish. When
we attempted to construct T3SS mutants by insertion of antibiotic resistance cassettes into each of the two
paralogous vcrD genes, we observed that the pressure for plasmid maintenance forced a series of genome
reorganizations. These included in some cases, the integration of pPDPT3 in the chromosomes, and such
mutants maintained the plasmid genes stable for generations. In other cases, pPDPT3 was found to undergo
events of gene gain and loss. Invariably, the vcrD mutants were non virulent for fish even at very high doses,
being more impaired in virulence than the plasmid-cured strains.

This observation might be explained by disruption of virulence gene functions caused by plasmid integration,
and/or by virulence gene loss upon genome rearrangements. We also demonstrated that a collection of Pdp
strains recently isolated from diseased fish, all tested positive for T3SS genes by PCR, but such genes were
no longer detected by PCR in a fraction of the isolated colonies formed after a single subculture step on agar
plates. This clearly suggests the existence of mechanisms that promote plasmid maintenance in vivo but not
in vitro. Collectively, our results provide evidence that the T3SS constitutes a novel, hitherto unreported
virulence factor of Pdp that is encoded within a highly unstable plasmid. Most notably, our findings warn
that the picture of Pdp virulence gene content has been historically biased and underestimated, as the loss
of plasmid-encoded T3SS genes during bacterial isolation and culturing may have occurred systematically in
laboratories for decades.

Page 78
5.2 Bacterial diseases II

Pasteurellosis in norwegian atlantic salmon

Hanne Nilsen1, Snorre Gulla2, Sverri Strøm3, Caroline Piercey Åkesson4, Farah Manji5, Anne Berit Olsen1,
Duncan Colquhoun2

1Norwegian Veterinary Institute, Norway

2Norwegian Veterinary Institute, Norway
3FOMAS A/S, Norway
4PHARMAQ Analytiq A/S, Norway
5Mowi ASA, Norway

[email protected]

Bacteria belonging to the genus Pasteurella are known to cause disease in fish. Pasteurella skyensis causes
intermittent outbreaks with significant mortalities in farmed Atlantic salmon in Scotland, while in both
Norway and Scotland infection with a yet un-named Pasteurella sp. causes serious disease in lumpsucker
used as cleaner fish. In farmed Norwegian Atlantic salmon, pasteurellosis was first diagnosed in 1989 and
has since occurred irregularly with many years between outbreaks. Since 2018, however, the disease has
increased annually in impact with 57 sites known to be affected in 2020. All post-2018 outbreaks have been
located on the south-western coast of Norway.

Macroscopic and histopathological findings were consistent with previous pasteurellosis cases in salmon.
Bacterial culture from affected fish revealed rich growth of small, grey, slightly alpha-haemolytic colonies.
To further characterise the bacteria involved and answer important questions concerning routes of infection
and host specificity, we whole-genome sequenced 88 isolates from Norwegian and Scottish salmon and
lumpsucker.

The results (to be presented in detail) reveal that, apart from two cases involving Pasteurella skyensis, the
ongoing epizootic in Norwegian salmon is caused by an apparently salmon specific variant of the same
Pasteurella species causing disease in Norwegian and Scottish lumpsucker.

Page 79
5.2 Bacterial diseases II

Pasteurella spp. infections in Atlantic salmon and lumpsucker

Nina Sandlund1, Anita Rønneseth2, Rebecca Marie Ellul2, Stian Nylund3, Liv Sandlund3

1Institute
of Marine Research, Norway
2Departmentof Biological Sciences, Norway
3PHARMAQ Analytiq, Norway

[email protected]

In Norwegian aquaculture, the use of cleaner fish as a delousing method has increased over the last few
years which has led to the emergence of a new large industry of farming lumpsuckers (Cyclopterus lumpus).
However, bacterial diseases that causes high mortalities is a challenge and pasteurellosis has become one of
the major emerging diseases. During the past few years, outbreaks of pasteurellosis in farmed Atlantic
salmon (Salmo salar) have become more frequent adding to the concern of pasteurellosis as an emerging
disease. The purpose of this study was to investigate the susceptibility of Atlantic salmon to Pasteurella spp.
infection and the possibility of lumpsuckers transmitting pasteurellosis to Atlantic salmon.

Methodology
Atlantic salmon were experimentally challenged, either by bath or by cohabitation with challenged
lumpsuckers, using two different strains of Pasteurella spp. originating from lumpsucker and Atlantic salmon,
respectively. The bath-challenge dose used were 1.0 x 106 bacteria/ml for one hour. Unchallenged cohabiting
Atlantic salmon were transferred to tanks containing challenged lumpsuckers one day post challenge.

Results
No clinical signs of pasteurellosis were observed for any of the Atlantic salmon. The lumpsuckers were,
however, equally susceptible to both isolates. In addition, clear differences in histopathological changes were
observed between individuals challenged with the two isolates.

Conclusions
Our findings indicate that lumpsuckers are equally susceptible to Pasteurella-strains isolated from Atlantic
salmon and lumpsucker, however, the isolate originating from Atlantic salmon seems more virulent. The
absence of clinical signs related to pasteurellosis in the Atlantic salmon under the given experimental
conditions, could indicate that outbreaks of pasteurellosis in salmon farms is the result of an opportunistic
infection rather than disease caused by Pasteurella spp. as a primary pathogen.

Page 80
5.2 Bacterial diseases II

Tenacibaculosis in Atlantic salmon (Salmo salar L.) it’s rotten

Mark Braceland, Marije Booman, Lyndsay Ferguson

The Center for Aquaculture Technologies, Canada

[email protected]

The aetiological agent of tenacibaculosis (or yellow mouth, or mouth rot) Tenacibaculum spp. infect multiple
fin fish species globally and is a major pathogen of sea bass (Dicentrarchus labrax), sea bream (Sparus
aurata), turbot (Psetta maxima), and Atlantic salmon (Salmo salar L.) aquaculture. In Atlantic salmon, the
disease and associated mortalities occur both in smolts soon after transfer to sea, and in larger adults. While
the disease exhibits variable pathological manifestations, yellow mouth lesions, fin erosion, and skin lesions
are characteristic of the disease. In Canada, the disease causes significant losses to the industry and is a
priority issue with annual cost of outbreaks for one Canadian company cited as $1.6 million. Moreover,
instances of outbreaks in other geographical regions, e.g. Chile, Norway, and Scotland have increased in
recent years. At current, there is no commercially available vaccines for use in salmonids and a reliance on
therapeutic use of antibiotics has led to a strong need for the industry to establish efficacious alternatives.

Methodology
Multiple isolates of T. maritimum, T. finnmarkense, and T. dicentrarchi, and one potentially novel species of
Tenacibaculum originating from Western Canada (BC) were utilised in these studies. Investigations focussed
on understanding the virulence, and compound factors that influence virulence, of the different isolates and
species. Furthermore, the clinical presentation of the isolates, and understanding how challenge methods
can be manipulated for the testing of different technologies and treatments are explored.

Results and Conclusions

Isolates, within and between, species of Tenacibaculum have significantly different virulence and clinical
presentations. Furthermore, it has been identified that while most environmental, and other compound
factors, influence infection and disease outcomes some are unique. The development of these disease
models is an important step in the development of technologies to mitigate its impacts on the culture of
Atlantic salmon. However, tenacibaculosis is a complex disease, and often is not exclusive to the presence/
infection of fish by a single species. Therefore, current developments are focussed on multiple species
infection models for use in the industry.

Page 81
5.2 Bacterial diseases II

Tenacibaculosis in recently sea-transferred Norwegian Atlantic salmon (Salmo salar) is

primarily associated with Tenacibaculum finnmarkense genomovar finnmarkense

Anne Berit Olsen, Bjørn Spilsberg, Hanne Nilsen, Snorre Gulla, Karin Lagesen, Mona Dverdal Jansen, Saraya
Tavornpanich, Hilde Sindre, Duncan Colquhoun

Norwegian Veterinary Institute, Norway

[email protected]

Infectious skin conditions including those caused by various Tenacibaculum spp., constitute a significant
threat to the health and welfare of sea-farmed Atlantic salmon (Salmo salar) in Norway, in addition to
significant economic losses. Here, the Tenacibaculum species involved in 15 outbreaks of presumptive
tenacibaculosis, distributed along the Norwegian coast during the late winter and spring of 2018, were
characterised to better understand the underlying aetiology.

The affected fish mainly displayed lesions of the head/jaw and histopathological examination confirmed the
presence of filamentous bacterial cells consistent with Tenacibaculum spp. in skin lesions. Bacteriological
cultures from lesions were dominated by yellow-pigmented colonies. Seventy-six bacterial isolates,
representing all cases, were analysed by whole-genome sequencing and MALDI-TOF. Speciation by average
nucleotide identity (ANI) and MALDI-TOF gave identical results, and identified 97% (N=74) of the studied
isolates as T. finnmarkense and 3% (N=2) as T. dicentrarchi. Core genome multilocus sequence typing
(cgMLST) identified the presence of both recognised genomovars of T. finnmarkense, with T. finnmarkense
genomovar finnmarkense dominating in most farms. While considerable dissimilarity was identified amongst
individual T. finnmarkense genomovar ulcerans isolates, T. finnmarkense genomovar finnmarkense
separated largely into a number of relatively genetically distinct but internally closely related clusters.

While the genetic diversity amongst Tenacibaculum spp. present in most outbreaks was considerable,
members of a highly related, possibly clonal strain of T. finnmarkense gv. finnmarkense were present in 13
of the 15 outbreaks investigated across the whole geographic range studied. To study the population
structure of T. finnmakense gv. finnmarkense more closely a SNP based tree was generated. This confirmed
the topology of the cgMLST tree and the within-cluster variation of the putatively clonal group of isolates
averaged 34.0 SNPs. Thus, while fish suffering tenacibaculosis appear to be susceptible to colonisation by a
number of Tenacibaculum species and strains, the data suggests that a particularly homogenous strain of
T. finnmarkense gv. finnmarkense may be linked to serious outbreaks of tenacibaculosis in newly sea-
transferred smolts in Norway.

Page 82
5.3.1 Genomic approach related to fish pathology

Predicting Atlantic salmon (Salmo salar) smoltification readiness using gill gene expression
profiles

Jordan Poley, Holly MacDonald, Lyndsay Ferguson, Mark Braceland

Center for Aquaculture Technologies, Canada

[email protected]

Smoltification of anadromous salmonids is a complex physiological process required for migration from
freshwater to seawater environments. This process includes changes in fish condition factor, body color,
behavior, and molecular profiles of muscle, brain, gill, and plasma, among other tissues. For wild fish, the
transition to higher salinity environments is often gradual, with various amounts of time spent in estuaries
prior to full seawater migration. However, smolts produced for aquaculture in land-based, freshwater
hatcheries are transferred directly to seawater without brackish acclimation. This transfer also includes
additional stressors such as crowding, transport, and handling. Thus, seawater transfer of salmonids in
aquaculture is a key event that, if done sub-optimally, can lead to compromised immunity, lower
osmoregulatory capacity, stunted growth, and increased rates of mortality. Several biochemical tests based
on Na+/K+ ATPase (NKA) activity and mRNA abundance of NKA isoforms and related cotransporters are used
by the industry to predict the readiness of smolts for seawater introduction.

Methodology
In the present study, a novel mRNA transcript target predictive of Pacific salmon smoltification was used to
determine an Atlantic salmon ortholog and smolt readiness tests were benchmarked against industry
standard markers using RT-qPCR.

Results
Multiple studies examining the expression of NKA isoforms and the novel gene of interest from Pacific
salmonids from non-lethally sampled gill lamellae show that the predictive power in determining the life
stage, smolt readiness, and response to environmental factors influencing smoltification (e.g.
photomanipulation, functional feeds, etc.) are improved using the novel smolt gene. Furthermore,
introduction of smolts to different salinities resulted in dose-response expression curves of the novel gene,
with 100- and 300-fold increases in expression three days and three weeks post-seawater (25 ppt) transfer.

Conclusions
This work demonstrates improvements to traditional smolt test markers and suggests that the use of
additional genes to NKA isoforms may offer improved insights on Atlantic salmon responses to seawater
transfer.

Page 83
5.3.1 Genomic approach related to fish pathology

Typing of Streptococcus agalactiae isolated from fish by protein mass spectrometry

Henrique Lopes Costa¹, Fernanda Alves Dorella¹, Felipe Luiz Pereira², Guilherme Campos Tavares¹, Henrique
César Pereira Figueiredo¹

¹Federal University of Minas Gerais (UFMG), Brazil

²Federal Institute of Santa Catarina (IFC), Brazil

[email protected]

Streptococcus agalactiae is an important pathogen that can affect several hosts including fishes, causing
outbreaks that lead to mortalities. Multilocus Sequence Typing (MLST) is a typing tool used for molecular
epidemiology studies and it is considered the gold standard for S. agalactiae typing, however, it is a laborious
and time-consuming method. An alternative method would be protein typing using MALDI-ToF MS that is
faster and less labor intensive. Our objective was to validate MALDI-ToF MS as an alternative protein typing
method for S. agalactiae strains using the Main Spectra Profile (MSP) and to compare with different methods
such as Serotyping and MLST.

Methodology
Therefore, 43 strains, previously serotyped, of S. agalactiae from 11 Brazilian states were selected.
Experiments were conducted using different numbers of MSPs strains to evaluate their technical (n=6) and
biological (n=10) reproducibility. For each category analyzed, three replicates of each strain were used.
Subsequently, the data were evaluated for Dice similarity using the MSP-Share software. For 23 strains that
did not have sequence typing (ST), DNA sequencing of seven MLST genes was performed and evaluated by
the PubMLST platform. Then, eBURST was performed using all fish STs reported in the literature to define
their clonal complexes (CC). Finally, 69 MSPs were generated from the 43 selected strains and the MSPs were
grouped in a dendrogram using the MSP-Share (DD) and the Maldi Biotyper 3 (BD) software. The
discriminatory power of these typing techniques was estimated by Simpson's Diversity Index (SDI) and the
agreement rates were determined by the Wallace coefficient (WC) (ρ <0.05).

Results
The average similarities for technical and biological replicates were 0.989±0.005 and 0.86±0.046,
respectively. The MLST and eBURST analyzes identified a new ST, a new Non-typeable group and the clonal
complex CC1525. The SDIs ranged from 0.503 to 0.833 and MLST was the most effective discriminatory
method. The Wallace coefficient indicated that the concordances between BD and the other typing methods
were almost zero, while high agreement between DD×Serotype (WC=0.930), and low agreement between
DD×ST (WC=0.138) and DD×CC (WC=0.229) was demonstrated.

Conclusions
It is concluded that MALDI-ToF MS is suitable for S. agalactiae typing, however, it has little correlation with
molecular typing methods.

Funding
This study was funded by Coordination for the Improvement of Higher Education Personnel – CAPES,
National Council for Scientific and Technological Development – CNPq.

Page 84
5.3.2 Innovation in disease control

A system for classifying and real-time monitoring causes of mortality and losses in salmon
farming

Arnfinn Aunsmo1, Marit Stormoen1, David Persson1, Sturia Romstad2, Paul Midtlyng1

1Norwegian University of Life Sciences, Faculty of Veterinary Medicine, Oslo, Norway

2Aqua Kompetanse AS, Norway

[email protected]

Among the novel aspects of the system are:

• It is designed a support tool for the animal owners, who are ultimately responsible for health and welfare
of their fish, and the owner of generated data.
• It is operational in real-time, and designed for use at all levels (site, company, corporation) when data
are needed.
• The mortality classification is based on CAUSALITY (the underlying cause of death) instead of on
symptoms, pathology, risk factors or associations.
• Mortalities and losses due to similar causes (e.g: bacterial diseases, viral diseases, parasites) can be
aggregated up to five main categories (e.g infectious diseases, trauma etc.)
• Fish lost due to euthanasia or culling can be specified vs. fish found dead.

According to the Norwegian aquaculture legislation, reports on mortality and losses, and fish inventory must
be submitted to the authorities at the end of each month. Therefore, all computerized farm management
systems have developed functionality to support this periodic reporting to the public sector. Assigning daily
mortalities to various causes of death is being practiced for internal use, where a mini-survey among 9
salmon farming companies revealed that anything between 20 and 50 categories were used, in total
summing up to more than 100.

With support from the Seafood Innovation Cluster in Bergen, we are proposing a novel, uniform system for
assignment of daily mortalities, downgrading at harvest, and other biological losses into a code of underlying
causes in a hierarchical system. Output can be aggregated both spatially and temporally to yield quantitative,
empirical data for prioritizing attention and resources for health management, disease control, and
maintenance of animal welfare.

The International Classification of Disease (ICD) for humans (WHO) provided the main scientific and
structural inspiration for the system we propose. ICD based on centuries of history and as a central part since
the founding of WHO in 1948, provides empirical, quantitative data for increasing the effect of health policies
and of spendings on healthcare systems. Selected details of the proposed system, and a discussion of its
benefits vs. current practices will be presented.

Page 85
5.3.2 Innovation in disease control

The effect of black soldier fly Hermetia illuscens crude protein and Bacillus spp.
supplementation on gilthead sea bream (Sparus aurata) intestinal health status

Jerko Hrabar1, Tina Pavelin1, Tanja Šegvić Bubić1, Slaven Jozić1, Josep Calduch Giner 2, Paul G. Holhorea2,
Jaume Pérez-Sánchez 2, Ivona Mladineo3

1Institute of Oceanography and Fisheries, Croatia

2Institute of Aquaculture Torre de la Sal - CSIC, Spain
3Institute of Parasitology, BC CAS, Czech Republic

[email protected]

In an era of rising antimicrobial resistance and overfished stocks, aquaculture strives to find cost-effective
and environmentally-friendly alternatives for fish meal and chemotherapeutics, while meeting the market
demand for high-quality fish products. Use of alternative protein sources and health-promoting additives,
such as insect-derived proteins and probiotics, has beneficial effects on fish health at both local and systemic
level, although the involved mechanisms are largely unknown.

Methodology
We performed a six-month farm feeding trial with three groups of gilthead sea bream (Sparus aurata) in
duplicate cages using i) feed supplemented with insect protein; ii) feed supplemented with insect protein
plus autochthonous intestinal Bacillus spp.; and iii) standard feed (control). For the second treatment group,
feeding regime during the whole six-month trial included 2-weeks of Bacillus-supplemented diet
administration followed by 2-weeks of feed administration where insect meal was included, i.e. the same
diet used for the first treatment group. Twelve fish per treatment group were sampled at 0.5, 1 and 6 months
after the start of the trial. Samples were collected for whole-intestine microbiota analysis (MiSeq 16s rRNA),
histological analysis (TEM) and gene expression analysis using a customized PCR-array of 44 selected markers
of intestinal function and integrity, nutrient transport, and immune response.

Results
Intestinal epithelium of all treatment groups showed no signs of degenerative or inflammatory changes.
Main changes in microbial community composition occurred already within the first month of feeding trial.
PLS discriminant analysis of gene expression in respect to the dietary treatment allowed the selection of 17
genes, mostly related to epithelia integrity and immune response that showed high contribution to
differences among diets. Heatmap clustering of the selected genes grouped together the experimental diets
apart from the control diet. Many of the epithelia integrity markers were up-regulated with insect diet, and
probiotic supplementation additionally amplified this up-regulation. Probiotic also reversed the up-
regulation of inflammatory/immune-regulatory genes that occured with insect diet.

Conclusions
In conclusion, probiotic supplementation emerges as beneficial for the preservation of gut homeostasis and
innate immunity in fish fed alternative protein sources.

Funding
This research was funded by Interreg Italy-Croatia project AdriAquaNet – Enhancing Innovation and
Sustainability in Adriatic Aquaculture.

Page 86
5.3.2 Innovation in disease control

Discovering a major QTL affecting resistance to Vibrio anguillarum in Rainbow Trout

Asma M Karami1, Jørgen Ødegård2, Moonika H. Marana1, Shaozhi Zuo1, Rzgar Jaafar1, Heidi Mathiessen1,
Louise von Gersdorff Jørgensen1, Per W. Kania1, Inger Dalsgaard3, Torben Nielsen4 and Kurt Buchmann1

1Laboratory of Aquatic Pathobiology, Department of Veterinary and Animal Sciences, Faculty of Health and
Medical Sciences, University of Copenhagen, Denmark
2AquaGen, Norway
3Institute of Aquatic Resources, Technical University of Denmark, Denmark
4Aquasearch Ova ApS, Jelling, Denmark

[email protected]

Vibrio anguillarum is the causative agent of classical vibriosis, the earliest recognized bacterial fish disease
targeting a large number of wild and aquacultured fish species. Genetic selection of disease resistant fish is
a major strategy to improve health, welfare and sustainability in aquaculture.

The aim of this study was to estimate the genetic variation in host resistance and explore the genetic
architecture of resistance to vibriosis by defining relevant quantitative trait loci (QTL). Rainbow trout
fingerlings were exposed to the pathogen V. anguillarum serotype O1 in a solution of 1.5 x 107 cfu/ml and
observed for 14 days. The mortality rate reached 55% within 11 days. DNA was sampled from all fish –
including survivors – and analyzed on a 57 k Affymetrix SNP platform whereby it was shown that disease
resistance was associated with a major QTL on chromosome 21 (Omy 21).

Estimated fractions for genetic and phenotypic variance explained by each top SNP indicated that more than
50% of the resistance trait is controlled by the QTL. Analyses were conducted to disclose immune genes
associated with resistance. Data of the work was published in Frontiers in Genetics, December 2020.

Page 87
6.1 Bacterial Diseases III

The ancestral history of yersiniosis in Norwegian salmon farming

Snorre Gulla1, Eve Marie Louise Zeyl Fiskebeck1, Andreas Riborg1,2, Saima Nasrin Mohammad1, Duncan
Colquhoun1,3

1Norwegian Veterinary Institute, Norway

2Vaxxinova Norway AS, Norway
3University of Bergen, Norway

[email protected]

Yersiniosis in salmonids, also known as enteric redmouth disease (ERM), is caused by the Gram-negative
bacterium Yersinia ruckeri and constitutes a significant fish-health challenge in most countries where
industrialised farming of salmonids is practiced. A recent study carried out at the Norwegian Veterinary
Institute (NVI) verified that a single, clonal Y. ruckeri lineage (termed ‘clonal complex 2’/’CC2’) has spread
nigh globally through international rainbow trout farming, likely via anthropogenic activities (transport of
live fish etc.). The situation in farmed Atlantic salmon seems, in contrast, to be characterised by several
locally endemic strains, confined at the national level.

A single clonal lineage hitherto specific to Norway (termed ’CC1’) has almost exclusively dominated amongst
clinical yersiniosis outbreaks in farmed Norwegian salmon over the last 20-30 years. This is despite the
evident concurrent presence of a wide array of distantly related Y. ruckeri strains found in association with
various salmon farming activities (fish-tank biofilms, screening programs, ovarian fluid etc.), albeit not from
diseased fish. Through an ongoing study, led by NVI and financed by the Norwegian Seafood Research Fund
(FHF), we hope to attain a better understanding of the epizootiological development surrounding yersiniosis
in Norwegian salmon farming over the last decades.

Using a hybrid approach of both short- (Illumina) and long-read (Oxford Nanopore) deep sequencing,
covering approximately 300 Y. ruckeri isolates from the virulent CC1, we are in the process of establishing
the phylogenetic ancestry of this lineage in Norway, over time. Central findings from this ongoing work will
be presented.

Page 88
6.1 Bacterial Diseases III

Toward a selection to improve turbot (Scophthalmus maximus) resistance to

edwardsiellosis

Yoannah François1, Marie Villa3, Bruno Peyrou3, Pierrick Haffray1, Thierry Morin2, Anastasia Bestin1

1SYSAAF (French Poultry and Aquaculture Breeders Technical Centre), INRAE-LPGP, France
2French Agency for Food, Environmental and Occupational Health & Safety (ANSES), Ploufragan-Plouzané-
Niort Laboratory, Unit Virology, immunology and ecotoxicology of fish (VIMEP), France
3France Turbot Ichtus, France

[email protected]

Edwardsiellosis disease caused by the enterobacteria Edwardsiella tarda affects a wide range of animals,
from fish to mammals. This gram negative bacteria has been responsible of severe economic losses in farmed
fish these past years and has become a serious concern. Genetic selection is an interesting strategy among
the different solutions existing against edwardsiellosis, which has already been used on other species and
pathogens. In this study, we developed a protocol to phenotype turbot resistance to edwarsdsiellosis in order
to estimate the possibility of improving this trait by genetic selection.

Methodology
In vivo assays used turbots of around 50g provided by the breeding company France Turbot Ichtus, respecting
a density of 6 to 8 kg/m² per tank. During the protocol development, we compared two contamination routes
(bath and Intra-peritoneal (IP) injections), two water temperatures (15 and 18 °C) and different inoculation
doses. For the phenotyping challenge, 1220 fish were contaminated by IP injection and distributed into seven
tanks. Mortality was monitored daily and survivors at the end of the challenge were euthanized. Caudal fin
of dead and survivor fish were sampled for DNA extraction to establish their respective pedigree and
estimate genetic parameters.

Results
Turbots were not sensitive to the bacteria at 15 °C, neither by bath nor by IP injection. Fish contaminated by
bath started showing clinical signs and mortality 3 weeks after contamination. Among fish contaminated by
injection, we observed a dose-effect in the appearance of mortality since the lower the dose was, the later
the mortality occurred, results that were confident with the literature. Mortality rates by injection were
much higher than by bath, but bath process needs to be improved. During the phenotyping challenge, we
observed the same mortality kinetic than during the development tests and a mortality rate in the range of
90%. Genetic parameters estimation on resistance to edwardsiellosis are in progress.

Conclusions
This study was the first trial of phenotyping turbot’s edwardsiellosis resistance for genetic purpose. It
highlighted parameters that need to be optimized. Indeed, strengthening protocols and improving challenge
efficiency would provide more robust challenge results and permit the more accurate genetic parameters
estimations as possible.

Funding
Project TURBOOST: EMFF 2018.

Page 89
6.1 Bacterial Diseases III

A comparison of the genomes of Icelandic Renibacterium salmoninarum strains to genomes

of North American and European strains

Birkir Thor Bragason, Sigridur Hjartardottir, Snorri Mar Stefansson, Arni Kristmundsson

Keldur Institute for Experimental Pathology, Iceland

[email protected]

Studies have shown that the causative agent of bacterial kidney disease (BKD), the Gram-positive bacteria
Renibacterium salmoninarum (Rs), is endemic in wild salmonids that inhabit Icelandic freshwater systems,
i.e., Arctic char (Salvelinus alpinus), brown trout (Salmo trutta) and Atlantic salmon (Salmo salar). Genetic
studies on Icelandic populations of Arctic char and Atlantic salmon suggest that the species colonized Iceland
during deglaciation in the late Pleistocene. Despite the prevalence of Rs in the wild, macroscopic pathological
signs of BKD have not been observed in wild salmonids since regular monitoring started in 1986 and it has
been suggested that Rs has been endemic in the Icelandic populations for a long time.

The first case of BKD in Iceland was reported in 1968 in association with fish farming and the disease has
since then occurred on a regular basis on fish farms in Iceland. This has been associated with the introduction
of Rs into the farms via intake of freshwater from local water systems or contact with wild fish. Therefore,
the Rs strains that cause disease in the aquaculture environment should represent the wild, endemic, strains.
Culturing of Rs from wild salmonids has not been successful whereas culturing of Rs from aquaculture
outbreaks is successful. The aim of this study was to gain insight into the genome of Icelandic Rs strains and
their relationship to Rs genomes of strains that have been isolated in North America and Europe. Whole
genome sequencing was performed on DNA isolated from 8 Rs strains isolated from BKD affected rainbow
trout and Atlantic salmon from aquaculture farms and stored in the sample bank of Keldur.

Raw whole genome sequencing data for 68 Rs strains from Europe and North America was obtained from
the European Nucleotide Archive. The sequencing data were analysed by alignment to the reference strain,
ATCC33209, as well as by de novo genome assembly. Tree construction based on data obtained from both
analysis methods suggest that the Icelandic Rs strains form a lineage that is distinct compared to the two Rs
lineages previously described.

Page 90
6.1 Bacterial Diseases III

Lactococcus garvieae infection in tropical fish species: experimental infection and ECOFF
for some selected antimicrobials

Renata Egger, Carlos Henrique Assis, Naísa Cristine Passos, Sarah Carneiro, Junia Teixeira, Carlos Augusto
Leal, Henrique César Figueiredo, Guilherme Tavares

Federal University of Minas Gerais, Belo Horizonte, Brazil

[email protected]

In the last years Lactococcus garvieae has been associated with disease outbreaks in Arapaima gigas,
Colossoma macropomum, Lophiosilurus alexandri, Oreochromis niloticus, and Pseudoplatystoma corruscans
in Brazil. This study assessed the antimicrobial susceptibility of 13 L. garvieae isolates recovered from
outbreaks in these fish species in the country (2013–2020). Additionally, Koch's Postulate was used to
evaluate the pathogenicity of one isolate from Nile tilapia (O. niloticus), which is the most produced fish in
the country.

This study was approved by CEUA-UFMG. Antibiograms and provisional epidemiological cut-off values
(ECOFF) were analyzed for Amoxicillin, Erythromycin, Florfenicol, Neomycin, Sulfamethoxazole, and
Tetracycline according to the CLSI guideline VET03-A. ECOFFs were calculated by the NRI (Normalized
Resistance Interpretation) method using automatic and freely available Excel spreadsheet calculators
(www.bioscand.se/nri/). For the disk susceptibility test, each isolate was suspended in saline solution, and
absorbance adjusted to 0.08–0.13 (OD₆₂₅), corresponding to 10⁸ CFU/mL. Nile tilapia (126.61±35.73 g) were
challenged by intraperitoneal injection with 0.2 mL of L. garvieae at 9.6 × 10⁷ CFU/fish (n=6), and control fish
received 0.2 mL of sterile saline solution (n=6). Fish were monitored during eight days for clinical signs and
mortality. All dead or surviving fish were subjected to bacterial isolation followed by identification by MALDI-
TOF MS. The ECOFFs were 17 mm for Amoxicillin, 24 mm for Erythromycin, 21 mm for Florfenicol, 20 mm for
Neomycin, 8 mm for Sulfamethoxazole, and 19 mm for Tetracycline. All isolates were susceptible to
Amoxicillin, and only one was resistant to Florfenicol, while some isolates were resistant to Erythromycin
(15.4%), Neomycin (23.1%), Tetracycline (46.2%), and Sulfamethoxazole (76.9%). Furthermore, three multi-
resistant isolates were identified (resistance to three or more classes of antimicrobials). In experimentally
infected Nile tilapia, 83.3% of mortality occurred 16h after challenge. Dead fish presented extensive internal
haemorrhage. One challenged fish survived and presented lethargy, anorexia, and unilateral exophthalmia
with corneal opacity. All challenged fish were positive to L. garvieae.

Conversely, animals from control did not present mortality, behavioural alterations, nor bacterial infection.
In conclusion, most of L. garvieae isolates tested were susceptible to Amoxicillin and Florfenicol, and this
bacterium was capable of inducing high and acute mortality in experimentally infected Nile tilapia.

Funding
This study was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES, and
Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq.

Page 91
6.1 Bacterial Diseases III

Iron availability and feeding status alter pathogen virulence and host susceptibility in
Aeromonas hydrophila infections of channel catfish (Ictalurus punctatus)

Haitham Mohammed1,2, Eric Peatman3, Benjamin Beck4

1The Center For Aquaculture Technologies, Canada

2Facultyof Veterinary Medicine, Assiut University, Egypt
3School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, USA
4United States Department of Agriculture, Agricultural Research Service, Aquatic Animal Health Research

Unit, USA

[email protected]

Outbreaks of motile Aeromonas septicemia (MAS) in warm-water fishes have triggered significant economic
losses in aquaculture industries worldwide. On catfish farms of the Southeastern United States, startling
outbreaks of the disease have been reported since 2009 with an estimated 2,000 tons of dead market-sized
catfish every year. A virulent Aeromonas hydrophila (vAh) pathotype has been responsible for those
widespread farm losses in the US catfish industry over the last decade. While the genetic and biochemical
attributes of vAh have been thoroughly studied and characterized, our ability to reliably induce the disease
under laboratory conditions has remained limited. A successful and reproducible disease challenge model in
the lab is crucial for the development of new approaches to prevent, treat, and control this disease.

Methodology
Taking cues from observed pond/farm conditions associated with vAh outbreaks, we disturbed iron
scavenging dynamics of the bacteria during the culture growth phase and altered the feeding status of
channel catfish. A waterborne immersion challenge was used to expose catfish to vAh in a low flow-through
tank system.

Results
First, iron limitation by addition of a strong iron chelator, a xenosiderophore called deferoxamine mesylate
(DM), to vAh cultures prior to experimental infection significantly increased vAh induced mortality in several
vAh isolates but not in a non-epidemic strain. DM addition did not affect vAh growth traits or perturb iron-
sensitive gene pathways but did significantly enhance hemolysis of catfish blood. Second, the postprandial
status (hours between the last feeding and immersion challenge) was a key determinant of catfish
susceptibility. Fasted catfish showed significantly higher survival than those with a full gastrointestinal tract.

Conclusions
DM-enhances vAh virulence and feeding status impacts catfish susceptibility to vAh. Taken together, the
results provide a reproducible waterborne challenge model and unveil insights into host-pathogen-
environment interactions underlying vAh disease pathogenesis. More results in this vein will be presented
as well.

Page 92
6.1 Bacterial Diseases III

Antibiotic-resistance patterns of Pseudomonas population isolated from farmed rainbow

trout (Oncorhynchus mykiss)

Kenny Oberlé1, Agnes Bouju-Albert2, Nicolas Helsens1,2, Hervé Prevost2, Catherine Magras2, Ségolène Calvez1

1INRAE, Oniris, BIOEPAR, France

2INRAE, Oniris, SECALIM, France

[email protected]

Pseudomonas genus is ubiquitous in many environments such as soils, river water or associated to aquatic
animals. Fish farms are interfaces connecting aquatic environments and food-producing animals as rainbow
trout (Oncorhynchus mykiss). Associated bacterial ecosystems are exposed to effluents and to antibiotics
used for treatment from human activities and animal production (fish and terrestrial). These ones could
participate to select in the environment antibiotic-resistant bacteria especially within ubiquitous populations
as Pseudomonas. The aims of our study were (i) to characterize antibiotic resistance profiles within
Pseudomonas populations and (ii) to compare the difference of profiles between two fish farms along the
same river.

Methodology
Pseudomonas strains were isolated from 56 rainbow trout with skin sampled according to the ISO 3720
standard. Fifty-one isolates confirmed as belonging to the Pseudomonas genus by specific PCR, were
identified using a MALDI-TOF approach.

Results and Conclusions

The results showed that most of isolates (94.1%) belonged to Pseudomonas fluorescens group. Minimum
Inhibitory Concentration (MIC) to ten antibiotics have been assessed for these 51 Pseudomonas isolates. The
results showed that 31.4% of strains were sensitive to all antibiotics tested based on breakpoint values and
calculated ECOFF (Epidemiological Cut-Off). However, we highlighted that 43.1%, 23,5% and 2.0% of
Pseudomonas strains were resistant to one, two and three antibiotic classes respectively. Seven out of 51
strains were highly resistant to several antibiotics with MIC values ranged from 64 µg/mL-1 to 256µg/mL-1 for
oxytetracycline, from 512 µg/mL-1 to upper than 1024 µg/mL-1 for florfenicol and colistin, from 128/2432
µg/mL-1 to upper than 512/9728 µg/mL-1 for trimethoprim/sulfamethoxazole association. A Multiple
Correspondence Analysis showed that resistance profiles observed were not significantly different between
both farms. At last, we identified seven strains at high MIC values for antibiotics used in aquaculture
(oxytetracycline, florfenicol, sulphonamides) and for human concern (colistin) suggesting that environmental
Pseudomonas populations can be a reservoir of resistant strains. Deeper research would be necessary to
well understand genetic elements responsible of high resistance profiles and their involvement in spreading
to fish pathogenic bacteria.

Funding
This work was financed by the European Union and the “Région Pays de la Loire” with RFI “Food for
Tomorrow” call for proposals 2016 – Contract No 34000656 - Ph.D. grant to Nicolas Helsens.

Page 93
6.1 Bacterial Diseases III

Identification, pathogenicity and antimicrobial susceptibility of Edwardsiella tarda strains

isolated from tambaqui (Colossoma macropomum)

Francisco Yan Reis1, Peter Janampa-Sarmiento1, Henrique Costa1, Naísa Passos1, Carlos Henrique de Assis1,
Brendhal Silva1, Fernanda Dorella1, Márcia Leibowitz1, Ronald Luz1, Felipe Pierezan1, Silvia Gallani2,
Guilherme Tavares1 and Henrique César Figueiredo1

1Federal University of Minas Gerais (UFMG), Brazil

2Nilton Lins University (UNL), Brazil

[email protected]

Tambaqui (Colossoma macropomum) is the second most produced fish in Brazil. Edwardsiella tarda is a fish
pathogenic bacterium, which has a broad host range and causes significant economic losses to the
aquaculture industry worldwide. The aim of this study was to evaluate the susceptibility of tambaqui to E.
tarda and the antimicrobial susceptibility of some selected bacterial isolates. A total of 14 E. tarda strains
isolated from brain, liver, kidney, spleen and intestine of tambaqui cultivated at different states of Brazil
(Amazonas, Rondônia and Minas Gerais) were studied.

Strains were primarily identified by MALDI-TOF and subsequently submitted to identification by sequencing
of dnaJ gene. For the antimicrobial susceptibility test, a disk diffusion assay, performed as recommended by
the guideline VET03-A of the Clinical and Laboratory Standard Institute (CLSI), was applied for all E. tarda
strains. The pathogenicity analysis occurred through bacteriological and histological investigation of
experimentally infected tambaquis. A strain of E. tarda (ED38-17), at 2.4 x 107 CFU/fish, was injected
intraperitoneally in six healthy animals. MALDI-TOF and dnaJ gene sequencing (identities = 96.07% - 100%;
query cover = 99% - 100%) identified all strains as E. tarda.

The ED38-17 strain induced the development of splenomegaly and whitish areas on spleen and kidney, but
neither clinical signs nor death were registered for the experimentally infected fish. The histopathology,
however, revealed granulomatous splenitis, nephritis and hepatitis. Meanwhile, ED38-17 was recovered
from five out of six animals through bacteriology.

The antibiogram revealed that all E. tarda strains are sensitive to florfenicol, neomycin, erythromycin,
oxytetracycline and rifampicin, but some are resistant to metronidazole (78.6%),
sulfamethoxazole/trimethoprim (50%), norfloxacin (28.6%), amoxicillin (28.6%), nitrofurantoin (7.1%) and
cephalothin (7.1%). Multi-resistant strains (strains resistant to two or more antimicrobials) were also
identified, mainly, showing resistance to norfloxacin, sulfamethoxazole/trimethoprim and metronidazole.
Therefore, the antibiogram revealed multiple profiles of antimicrobial susceptibility for the E. tarda strains,
and some are even multi-resistant, including ED38-17. The same strain presented itself as a pathogen to
experimentally infected C. macropomum. These findings raise awareness about potential new sanitary
challenges for the tambaqui aquaculture.

Page 94
6.1 Bacterial Diseases III

Susceptibility of tambaqui (Colossoma macropomum) to Nile tilapia-derived Streptococcus

agalactiae and Francisella orientalis strains

Francisco Yan Reis, Peter Janampa-Sarmiento, Márcia Leibowitz, Ronald Luz, Felipe Pierezan, Guilherme
Tavares and Henrique César Figueiredo

Federal University of Minas Gerais (UFMG), Brazil

[email protected]

Tambaqui (Colossoma macropomum) is the most produced amazon fish in Brazil. Currently, Aeromonas
hydrophila is the only scientifically acknowledged pathogenic bacterium to this fish species. Streptococcus
agalactiae and Francisella orientalis largely affect the production of Nile tilapia (Oreochromis niloticus) in
Brazil, thus their pathogenicity against C. macropomum was evaluated. This project was approved by the
Ethics Committee on the Use of Animals of the Federal University of Minas Gerais (378/2019).

Initially, negative proof for the presence of any bacterial infection was obtained via bacteriology for the
tambaqui specimens. The S. agalactiae (SA95) and F. orientalis (FNO12) isolates were, originally, recovered
from outbreaks in different Brazilian tilapia farms. SA95 and FNO12 were injected intraperitoneally in healthy
juvenile tambaquis at 1.0 x 107 CFU/fish (n=6) and 3.4 x 107 CFU/fish (n=6), respectively, after sedation in a
benzocaine bath (100 mg/L). Control animals were injected with sterile bacterial broth. Fish were also
maintained at water temperatures of 28 oC for the SA95 group and 21 oC for the FNO12 group. The
occurrence of clinical signs and deaths were recorded during a period of 15 days post-inoculation. Dead
animals along with surviving animals, at the end of the experimental period, were necropsied and analyzed
for bacteriology (identification of recovered bacteria was conducted by MALDI-TOF MS) and histology. S.
agalactiae-infected fish developed clinical signs with progression to death in 83.3% of the animals.

By contrast, F. orientalis-infected tambaquis developed no clinical signs or deaths. Control fish did not
develop clinical signs or deaths as well. From SA95 and FNO12 groups, bacterial recovery occurred in five
(83%) and six fish (100%), respectively; and these values were identical for histopathological findings.
Nevertheless, fish from the SA95 group developed neutrophilic and fibrinonecrotic inflammation, whilst
animals from the FNO12 group developed granulomatous nephritis, splenitis and hepatitis.

Accordingly, S. agalactiae and F. orientalis strains, originally isolated from tilapia, were able to infect
tambaqui fish, as well as to develop pathogenic effects. These findings warn for a potential natural
transmission of S. agalactiae and F. orientalis infections between Nile tilapia and tambaqui, this economically
important amazon fish.

Page 95
Wednesday 22 September 2021
7.1 Host-pathogen interactions I

Analysis of the internal distribution of Anisakis simplex in wild Atlantic salmon (Salmo salar
L.) and the relationship with Red Vent Syndrome

Conor McCormick1, Amy Reading1, Niall Cook2, Ian Davidson3, Randolph Richards4 and Chris Williams1

1Environment Agency, England

2Environment Agency, England
3Natural Resources Wales, Wales
4Fishpharm Supplies Limited, Scotland

[email protected]

Red Vent Syndrome (RVS) is a disease condition of unknown aetiology which is expressed through swelling
and haemorrhaging of the vent tissue in adult wild Atlantic salmon (Salmo salar L.) returning to freshwater.
The condition has been associated with aggregations of the marine nematode Anisakis simplex in the vent,
although many aspects of this relationship remain poorly understood.

Methodology
The relationship between A. simplex and RVS was investigated through parasitological, clinical and
pathological examinations of 124 wild S. salar, sampled from the rivers Dee, Wales and Tyne, England
between 2009 and 2013.

Results
All of the salmon examined were infected with A. simplex, with up to 375 nematodes per fish. Salmon
exhibiting vent lesions had significantly higher worm burdens overall, and within the vent, than unaffected
individuals. Of all organs examined, the vent was most heavily infected, irrespective of vent condition, and
harboured up to 95.5% of the total worm burden despite representing <1% of total body weight. The outer
5mm of vent tissue harboured significantly higher worm burdens than any other site, with up to 83.6% of
total worm burdens found within this location.

There was no evidence that nematode aggregations within the vent were a consequence of overspill from
visceral infections, later penetration of the intestine wall, or displacement due to pathological damage
elsewhere, suggesting that the vent is a discrete and potentially favoured site of A. simplex. Pathological
responses to infection varied, with clinical RVS observed in a small number of fish harbouring very low
nematode burdens and conversely, large aggregations of A. simplex within the vent of clinically normal
salmon. In these individuals, inflammation and marked fibrogranulomatous changes were observed in the
absence of haemorrhage, suggesting the potential for under-reporting of clinical disease.

Conclusions
This study provides new insights into the relationship between A. simplex and RVS, and the internal
distribution of A. simplex within wild Atlantic salmon. Atlantic salmon are in a state of decline throughout
much of their biogeographical range, and an understanding of all pressures faced by this species, including
emergence of disease, is important in our attempts to mitigate these declines.

Page 96
7.1 Host-pathogen interactions I

Anisakis pegreffii infection of poikilotherm and homeotherm host - keeping cool makes it
better

Željka Trumbić 2, Ivona Mladineo1, Jerko Hrabar3

1Institute of Parasitology, BC CAS, Czech Republic

2University of Split, Croatia
3Institute of Oceanography and Fisheries, Croatia

[email protected]

Anisakiasis is considered one of the most important foodborne parasitic zoonoses, although the nematode
cannot complete its life cycle in humans. Generation of -omics data has enabled more versatile approache
to study host-parasite interactions. To scrutinise transcriptomic signature of Anisakis pegreffii infective
larvae (L3) during migration through a poikilotherm (fish, paratenic host) and homeotherm accidental host
(rat, model for human infection), we RNA-sequenced (Illumina) isolated L3, performed a differential analysis
of gene expression (DEGs) and calculated the enrichment of GO terms and KEGG pathways within DEGs sets.

We also differentiated between transcripts of successfully migrating L3 vs those that were expelled through
faeces from the host (non-migrating). The final transcriptome, filtered for transcripts with a detectible coding
sequence, resulted in 36,201 transcripts with predicted ORFs and 4,016 proteins represented by nearly full-
length transcripts and >80% alignment coverage. Of these, 1,937 were found differentially expressed (FDR <
0.05) in migrating vs. non-migrating L3 in rat, Rattus norvegicus (1,096 up and 841 down), 484 in sea bass,
Dicentrarchus labrax (328 up and 156 down) and 509 were differentially regulated in the two hosts. The
largest difference in log2FC between migrating L3 in rat and in sea bass was observed for putative cuticle
collagen (CO155) and glucose-6-phosphate exchanger (G6PT1), which were both upregulated in rat and
downregulated in sea bass, while the opposite was observed for NADH-dependent flavin oxidoreductase
(NADA).

In the homeothermic accidental host (rat), Anisakis L3 upregulated ribosome-related genes, cell division,
cuticle constituents, oxidative phosphorylation, probably in an unsuccessful effort to moult to the next
developmental stage. Contrary, in the paratenic poikilotherm host (fish), L3 moderately upregulated or
silenced the main metabolic pathways, preparing for dormancy by triggering autophagy and longevity
pathways. We hypothesise that the failure of L3 to survive in the accidental host is a result of metabolic/
energy exhaustion that occurs from undetermined migration towards a suitable niche that is never to be
achieved, additionally stimulated by host homeothermy. In the poikilotherm paratenic host, moderately
upregulated or silenced metabolism serves to enable larvae to prepare for paratenesis, warranting their
survival.

Page 97
7.1 Host-pathogen interactions I

Immune modulation in rainbow trout during proliferative kidney disease: Utility of RNA-
Seq to characterize early responses in gill tissue following exposure to parasite spores

Jason Holland1,4, Davina Derous1, Konstantinos Apostolakis2, Nicole Strepparava3, Helmut Segner3,
Christopher Secombes1

1School of Biological Sciences, University of Aberdeen, UK

2Aberdeen Oomycete Laboratory, Institute of Medical Sciences, University of Aberdeen, UK
3Center for Fish and Wildlife Health, Vetsuisse Faculty, University of Bern, Switzerland
4Moredun Research Institute, Pentlands Science Park, UK

[email protected]

Proliferative kidney disease (PKD) is a serious climate change-driven disease of farmed and wild salmonid
fish in Europe and USA. A great deal of progress has been made towards understanding the immune
mechanisms underpinning host responses to the causative agent, the myxozoan parasite Tetracapsuloides
bryosalmonae. Following exposure, the parasite targets the kidney that becomes the focus of disease
pathology. Studies, to date, have focused on infected kidney tissues despite the gills being the preferred
portal of parasite entry. To develop a more holistic overview of parasite-mediated immune modulation, we
have undertaken an RNA-Seq study to characterize the early gill responses following parasite exposure.

Methodology
Fish were exposed to T. bryosalmonae mature spores and sub-sampled over four days with mock infected
control fish sampled in parallel. The infection status of gill arches from individual fish was confirmed by
RTqPCR based on the presence of parasite small subunit rDNA (SSU rDNA) in extracted gill genomic DNA.
Day-4 barcoded Illumina cDNA libraries were constructed from purified poly A+ RNA, sequenced on an
Illumina HiSeq 2500 sequencer and single reads aligned to the rainbow trout genome. Differential gene
expression was performed using DESeq2 with gene set enrichment analysis and QuickGO used to determine
transcript and GO term representation linked to specific pathways. RNA-Seq data was further validated by
RTqPCR on a subset of differentially expressed immune transcripts.

Results
Analysis revealed 260 differentially expressed genes in pathways involved in tissue repair and immunity, with
the latter including several members of the suppressors of cytokine signalling (SOCS) family. RTqPCR analysis
of SOCS family genes revealed prolonged up-regulated expression profiles of SOCS3A and CISHa2 over the 4
days of parasite exposure.

Conclusions
Our findings represent the first insights into early immune modulation within the gill mucosa following
exposure to and tissue invasion of T. bryosalmonae infective spores. In common with kidney transcriptional
studies, members of the SOCS family were over-expressed in the gills of exposed fish. This reinforces the
premise that parasite-mediated SOCS modulation may represent an important component of host immune
evasion. Such data may be highly informative in developing future disease mitigation strategies.

Page 98
7.1 Host-pathogen interactions I

Expression profile of B cell associated genes in brown trout, Salmo trutta during
proliferative kidney disease (PKD)

Saloni Shivam, Mansour El-Matbouli, Gokhlesh Kumar

University Of Veterinary Medicine Vienna, Austria

[email protected]

The myxozoan endoparasite Tetracapsuloides bryosalmonae causes a chronic kidney infection in the brown
trout, Salmo trutta. Adaptive immune responses to parasites affect the disease outcome in fish hosts. B cells
are important immune cells with diverse roles such as antigen presentation, chemokine and cytokines
secretion, in addition to antibody production. However, knowledge about the B cell mediated immune
response in brown trout during proliferative kidney disease (PKD) is scarce.

Therefore, the present study investigated the expression of B cell associated genes like CD22, CD79A, CD34,
CD74, BLNK, BCL7 and C4A in the kidney and spleen of infected brown trout using real time quantitative PCR
at different time-points. Brown trout were exposed to T. bryosalmonae spores collected from the infected
bryozoan host Fredericella sultana. Samples were collected at different time points and processed for
histopathology and qRT-PCR. Gene expression analysis showed significant upregulation of CD22, CD79, CD74,
BLNK and C4A in the infected kidney at different time points in contrast to CD34, which was downregulated
at all time-points. Similarly, significant upregulation of CD22, CD34, CD79, BCL7 and BLNK occurred mainly at
8 weeks post exposure, however, C4A was significantly downregulated in the spleen of infected brown trout.

The results from this study provide valuable insights into the processes leading to changes in B cell
development, inflammation and antibody production during the course of PKD in brown trout.

Page 99
7.1 Host-pathogen interactions I

Identification of vivo-induced antigens recognised by sera of infected brown trout with

Tetracapsuloides bryosalmonae

Gokhlesh Kumar, Saloni Shivam, Mansour El-Matbouli

University Of Veterinary Medicine Vienna, Austria

[email protected]

Tetracapsuloides bryosalmonae is a myxozoan parasite and the causative agent of proliferative kidney
disease (PKD) in wild and farmed salmonids in Europe and North America. The life cycle of T. bryosalmonae
completes between bryozoan and mainly brown trout. Brown trout is a native fish species in Europe, and
after recovering from T. bryosalmonae infection, acts an as asymptomatic carrier. Virulence factors of T.
bryosalmonae are induced in the fish during infection or interactions with host cells. T. bryosalmonae genes
expressed in vivo are likely to be important in fish pathogenesis.

Therefore, the identification of in vivo induced genes of T. bryosalmonae related to the disease development
could improve our understanding of pathogenesis and promote the discovery of novel therapeutic targets.
Here, we identify in vivo induced antigens of T. bryosalmonae during infection in brown trout using in vivo-
induced antigen technology. Brown trout (12 cm in size) were exposed to the spores of T. bryosalmonae
overnight. After exposure, fish were maintained at 15 ± 1 °C with running water and fed daily. Samples were
collected from exposed and unexposed control brown trout at 2, 4, 6, 8, 10, 12 and 17 weeks post exposure.
The pooled sera of brown trout were first pre-adsorbed with antigens of T. bryosalmonae and E. coli XL1.
Afterwards, adsorbed sera were used to screen T. bryosalmonae cDNA phage expression library. One
hundred forty five antigens were identified including calmodulin, cdc42 homolog, heat shock protein 70 and
serine/threonine-protein kinase. The identified antigens of T. bryosalmonae were involved in cell invasion,
metabolism, ion-protein binding and hypothetical proteins, of so far unknown functions.

The results may help us to increase our understanding of the parasite pathogenesis during infection. Some
of the antigens found may have significant implications for the discovery of candidate molecules for the
development of potential therapies and preventive measures against T. bryosalmonae in salmonids. The data
we obtained will provide new insights into the pathogenic features of T. bryosalmonae.

Funding
Austrian Science Fund (FWF) Project No. P 30981-B32

Page 100
7.2 Myxozoa I

The genome of Sphaerospora molnari (Myxozoa, Cnidaria). How do the long read sequence data
contribute to assembling of highly derived parasite genomes?

Anush Kosakyan1, Monika Wiśniewska1, Dr Ron Dirks2, Dr Astrid S. Holzer1

1Institute of Parasitology, Biology Center of the Czech Academy of Sciences, The Czech Republic
2Future Genomics Technologies B.V, Netherlands

[email protected]

Myxozoans are metazoan parasites that diverged from their free-living cnidarian ancestors to become
obligatory microscopic endoparasites. Several species cause serious diseases in farmed fish and wild fish
populations. Sphaerospora molnari is a myxozoan parasite infecting common carp and causing up to 100%
mortalities of fingerlings in Central European pond cultures.

We have established Sphaerspora molnari as a lab model to study functional gene groups, parasite strategies
and biology by sequencing and analyzing the genomic data as a fundamental first step. An accurate genome
assembly is a prerequisite for multiple research questions for any group of organism. This is especially critical
for parasites such as myxozoans, which have highly derived genomes and samples are prone to host
contamination, due to the host’s polyploidy and the parasite’s close interaction with host cells and tissues.
In myxozoans, host-parasite separation is generally being performed “bioinformatically” but complete host
sequence removal has not previously been achieved.

Here we used physical separation as the first step and 99.8% purified parasite material served as a template
for DNA isolation and genome sequencing. We combined two different technologies: PromethION-Oxford
Nanopore and Illumina to be able decipher the highly derived myxozoan genome and its gene order. We
used combination of commonly used algorithms, custom-made scripts and pipelines for assembling,
structurally and functionally annotating the genome, as well as for comparing it with other available
cnidarian datasets, to describe the gene repertoires fitted to the myxozoan parasitic life style. We further
analyzed functional gene groups that duplicated in S. molnari and that are known to be involved in parasitic
lifestyles in other protistan and metazoan parasite pathogens.

Funding
Czech Science Foundation (#20-30321Y, #19-28399X), European Commission H2020 (#634429;
ParaFishControl).

Page 101
7.2 Myxozoa I

Comparative genomics of species in an early diverging myxozoan clade - the Malacosporea

Hanna Hartikainen1, Liam Doonan2, Ashlie Hartigan3, Alex Gruhl4, Jason Holland5, Paul Long2, Beth Okamura3

1University Of Nottingham
2Facultyof Life Sciences & Medicine, King’s College London, University of London, UK
3Department of Life Sciences, Natural History Museum, UK
4Max-Planck-Institut für Marine Mikrobiologie, Germany
5University of Aberdeen, UK

[email protected]

Myxozoans are endoparasitic cnidarians that comprise the subclasses Myxosporea and Malacosporea. The
morphologically simplified myxosporeans are characterised by some of the smallest metazoan genomes,
have many fewer genes than their free-living cnidarian relatives and develop exclusively as plasmodia and
pseudoplasmodia. Malacosporeans, however, have retained tissues (epithelia and muscles) and develop
into non-motile “spore sacs” and active “worms”. Despite their significance as early-diverging myxozoans
and as major agents of fish disease (e.g. Proliferative Kidney Disease of salmonids), no malacosporean
genomes have been assembled to date.

We examined the genomes of two malacosporeans to compare genetic complexity and gene reduction
relative to myxosporeans and free-living cnidarians. Tetracapsuloides bryosalmonae (non-motile sacs) and
Buddenbrockia plumatellae (motile worms) were collected from their freshwater bryozoan hosts. DNA was
extracted and variously sequenced (Illumina HiSeq short reads, PacBio long reads and artificial mate pairs).
Assembly was achieved using ABYss and Platanus. Gene models were constructed using the
Augustus/MAKER pipeline. Shared genes were examined using OrthoGroups and BUSCO analyses.
Homeodomains and motility genes were identified by homologous BLAST searches and then assigned by
phylogeny.

Our assemblies reveal the two malacosporean genomes are smaller than those characterised for
myxosporeans (the T. bryosalmonae genome estimated at 12.9Mb and B. plumatellae at 27.7Mb) and exhibit
the highest gene density of all published cnidarian genomes. Few conserved core metazoan genes were
found in BUSCO searches (38.2% and 37.6%, for T. bryosalmonae and B. plumatellae, respectively). Four
shared homeodomains families and a large number of orthogroups exclusive to malacosporeans were
detected. T. bryosalmonae appears to have retained many of the genes associated with muscle and tissue
differentiation, which are also present in B. plumatellae. The retention of genes involved with cell signalling
and patterning in both malacosporeans as well as motility genes in a non-motile species is notable.

Page 102
7.2 Myxozoa I

Genome skimming identifies phylogenetically and functionally informative genes in

vermiform myxozoans (Cnidaria: Myxozoa: Ceratomyxa)

Suellen Aparecida Zatti1,2, Antônio Augusto Mendes Maia1, Edson Aparecido Adriano1, Paul Long2,
Beth Okamura2

1FZEA/Universidade de São Paulo (USP), Brazil

2Natural History Musem, London

[email protected]

Myxozoa are diverse and widely distributed endoparasitic cnidarians with complex life cycles with stages
typically alternating between fish and invertebrate hosts. Some species cause disease outbreaks with serious
economic impacts. Recent genome-level investigations have provided valuable insights into myxozoan
biology. The aim of this study is to use a genome skimming approach to identify genes involved in the
development, evolution, and motility of Ceratomyxa species infecting the gallbladder of South American
freshwater fish. A unique feature of these Ceratomyxa species is the plasmodium which exhibits a vermiform
morphology and associated motility reminiscent of multicellular worm-like metazoans. Genomic libraries
were constructed from five Ceratomyxa species caught from the Amazon and Paraná River basins, Brazil.

Paired-end sequencing was conducted on an Illumina NovaSeq6000. Poor quality data were trimmed using
Trimmomatic. Contigs were assembled using a de novo approach (SPAdes). A database was created for genes
of interest by conducting BLAST searches for homologs from published databases of myxozoan and free-
living cnidarian species. This dataset was used as queries in similarity searches to mine for the target genes.
We also undertook genome sequencing of the fish hosts in order to subtract contaminating fish genes.

Preliminary genomic analyses demonstrated the genomic skimming approach successfully recovered nuclear
ribosomal (ssrRNA, lrRNA, ITS1, and ITS2) and mitochondrial genes that may enable further resolution of
diversity and phylogenetic relationships within this freshwater-specific clade. In addition, we identified genes
that may be involved in motility (F-Actin, G-Actin, Myosin, cytoskeleton-related genes) and that may provide
insights into potential roles for key proteins in the context of motility patterns displayed in vermiform
ceratomyxids.Using a genome skimming approach we were able to retrieve a diversity of nuclear and
mitochondrial genes for vermiform ceratomyxids. We uncovered a large and varied array of motility-
associated genes that have evolved in a clade uniquely characterised by worm-like patterns of movement
but generated in a single cell (the plasmodium).

Page 103
7.2 Myxozoa I

Is anybody out there? Attempt to peek behind the door of Notch signalling in myxozoans
(Cnidaria).

Alena Lövy1, Jiří Kyslík1,2, Lenka Gahurová2, Alena Krejčí2, Astrid S Holzer1

1Institute of Parasitology, Biology Centre, Czech Academy of Sciences, Czech Republic

2Faculty of Science, University of South Bohemia, Czech Republic

[email protected]

Notch signalling is among ancient functional pathways that emerged in Metazoa. This pathway plays a
fundamental role in cell fate determination, cell growth, and division. In a context-dependent manner, there
are canonical and non-canonical pathway mechanisms. In Cnidaria, sister group of the bilaterians, the
function of Notch seems to be conducted via the noncanonical pathway (hairy and enhancer of split (Hes)
and suppressor of hairless (Su(H))- independent pathway). Despite the two key elements of canonical Notch
signalling, Su(H) and mastermind (MAML), being present in Cnidaria, this hypothesis still awaits confirmation.

To extend the knowledge on Notch signalling, we study Myxozoa, Cnidarians, which underwent an extreme
evolutionary transition from free-living organisms to microscopic endoparasites. Two myxozoans species
(Sphaerospora molnari, Myxidium lieberkuehni) with different developmental strategies were selected as the
target species of study. We identified the core Notch pathway components with exploratory high-throughput
screening data searches of both, Myxozoa and Cnidaria. Unexpectedly, myxozoans lost some key
components e.g. transcription coactivator (MAML) and corepressor (SMRT). To determine the role of the
Notch signalling and its primary transcriptional targets, inhibition (DAPT, DFK) / activation (EDTA) assays were
performed. Inhibitors of the γ-secretase complex block the cleavage of Notch intracellular domain (NICD) as
opposed to activators that stimulate the shedding of NICD. With myxozoan being fast-evolving organisms,
the homology searches were challenging and two putative primary targets (Hes) in S. molnari were identified.
Expression patterns of these Hes-related candidates in S. molnari and the nematocyst-related developmental
gene, minicollagen 1 (Ncol-1) in M. lieberkuehni were analyzed. Both Hes-related candidates and Ncol-1 are
upregulated by activation with EDTA (up to 2 fold change). One Hes-related candidate is significantly
downregulated by DAPT (8h) and DFK (24h).

Further, we aim to localize some key components (e.g. NICD) of Notch signalling by immunochemistry and
to elucidate their functions during polar capsule (nematocyst) development. The study will provide another
interesting challenge to the field. Moreover, this knowledge should shed light on the evolution of Notch
signalling at the base of Metazoa.

Funding
This research was funded by the Czech Science Foundation (19-25536Y).

Page 104
7.2 Myxozoa I

Biochemical characterization and localization of a cysteine protease inhibitor from the

myxozoan Sphaerospora molnari

Pavla Bartošová-Sojková1, Daniel Sojka1, Anush Kosakyan1, Jan Kotál1, Larissa Almeida Martins1, Veronika
Urbanová1, Marie Vancová1, Justin Tze Ho Chan1, Tomáš Korytář 1,2, Astrid Sibylle Holzer1

[email protected]

1Instituteof Parasitology, Biology Center of the Czech Academy of Sciences, Czech Republic
2Instituteof Aquaculture and Protection of Waters, Faculty of Fisheries and Protection of Waters, University
of South Bohemia, Czech Republic

Sphaerospora molnari is a myxozoan pathogen of common carp Cyprinus carpio that primarily affects the
fish skin and gills. Heavily infected fish suffer from respiratory and osmoregulatory failure and anemia. The
parasite is also an important cofactor for swim bladder inflammation, the disease responsible for up to 100%
of mortalities in stocks of carp fingerlings in Central Europe. Besides typical myxozoan developmental stages
– spores and plasmodia – the parasite also forms motile extrasporogonic stages in host bloodstream.

Parasite-driven cysteine protease inhibitors, besides their inner housekeeping roles in protein degradation
regulation, are recognized as essential molecules involved in host-parasite interactions. These pathogenicity
and immunogenicity-determining factors have been used as effective diagnostic markers and therapeutic
targets against various parasitic infections. Thus, they would be of interest in aquaculture because we lack
efficient and environmentally sound strategies of myxozoan parasite control in fish destined for human
consumption. S. molnari possesses two cysteine protease inhibitors of the stefin type, one of which has been
studied. This stefin is a single-domain protein with a predicted molecular mass of 13.5 kDa that contains a
cystatin-like domain and, unusually for stefins, carries a signal peptide. We have produced it as an active
recombinant against which we raised the polyclonal antibodies.

Afterward, we have characterized its biochemical properties and localized it using immunoconfocal
fluorescent and immunogold microscopy in the parasite stages. The inhibitor effectively inhibited the
enzymatic activities of cysteine cathepsins H, K, L and S while no inhibition was observed for cathepsin C.
Cathepsin B was inhibited only at a very high inhibitor concentration and no inhibitory activity was
encountered against legumain and serine proteases. The protein localized within the endoplasmic reticulum,
membrane-bound organelles – larger globular granules and small transport vesicles located in the vicinity of
the blood stage primary cell plasma membrane. Future research is aimed at characterizing the immunogenic
and immunoprotective properties of S. molnari stefin to evaluate it as a potential vaccine target. This
research was funded by the Ministry of Education, Youth, and Sports of the Czech Republic (LTAUSA17201)
and the Czech Science Foundation (19-28399X).

Page 105
7.3 Vaccines I

Effect of vaccines against pancreas disease on spinal cross-stitch vertebrae development,

growth and economic impact of cage reared Atlantic salmon

Ragnar Thorarinsson1, Grete Baeverfjord2, Arnfinn Aunsmo3,4, Eystein Skjerve4, Paul Negaard1, Patricio Peña1

1Elanco Animal Health, Norway

2Nofima, Sunndalsøra, Norway
3Laxar Fiskeldi ehf, Iceland
4Faculty of Veterinary Medicine, University of Life Sciences, Norway

[email protected]

A controlled field trial was carried out to assess safety and efficacy of two vaccines against pancreas disease
(PD) using 2018 S0 smolts at two sea cage sites in southern Norway. The fish were immunized using either a
DNA PD vaccine and a 6-component oil-based vaccine (Group A), or a 7-component oil-adjuvanted PD vaccine
(Group B). Group A were adipose fin-clipped during the vaccination process for identification.

The experimental design was functionally blinded and primarily followed two cohabitated cages (15% Group
A, 85% Group B) at each of the two sea sites.

Combining the results from both sites shows that group B developed significantly greater prevalence of a
newly described spinal deformity named cross-stitch vertebrae of 17% compared to 4% in group A (p<0.001).
The prevalence of the most severe manifestations of this condition (scores 3 and 4) averaged 13,5% in group
B compared to 1% in group A.

Significant negative correlation was found between pathology scores ≥2 (p=0.09) and loss of growth with an
average negative impact of 1.75 kg of the fish with scores 4 (p<0.001). This anomaly raises major fish welfare
concerns while simultaneously causing substantial negative economic impact for the salmon producer as the
data to be presented will show.

Page 106
7.3 Vaccines I

Duration of immunity in Atlantic salmon parr vaccinated with CLYNAV using a saltwater
Salmonid alphavirus subtype-3 cohabitation challenge model

Ana Silva3, Lisa Phillips1, Ginny Jones1, Jennifer Harkness1, Paul Tonita1, Jeffrey C. Wolf2

1Elanco Canada Ltd

2ExperimentalPathology Laboratories Inc., Virginia, USA
3 Elanco Denmark ApS, Norway

[email protected]

Elanco Animal Health has developed CLYNAV, a DNA vaccine for Pancreas Disease (PD) in Atlantic salmon,
approved for use in Europe. In order to demonstrate clinically relevant duration of immunity (DOI), the SAV3
cohabitation challenge was optimized and used to challenge Atlantic salmon in saltwater at 6, 9.5 and 12
months post-vaccination. The results, including gross lesions, impact on average weight gain and mortality
for all time points, and cardiac, pancreatic and skeletal muscle histopathologic findings at 12 months will be
presented for the CLYNAV vaccinates, saline control fish and Not Vaccinated Not Challenged (NVNC) controls.

The main findings were that CLYNAV’s duration of immunity was demonstrated at 6, 9.5 and 12 months post-
vaccination. Significantly lower microscopic pathology index scores for pancreas, heart, red skeletal muscle
in the CLYNAV group compared to the saline control group at time points 21 (± 2), 54 (± 2) and 84 (± 5) days
post challenge, white skeletal muscle 54 (± 2) and 84 (± 5) days post challenge for 12-month challenge. No
difference in microscopic pathology index score for pancreas, heart, red and white skeletal muscle in CLYNAV
group compared to NVNC group for all time points for the 12-month challenge.

The relevance to the European salmon farming industry is that the direct prophylactic benefit of CLYNAV is
further strengthened, on the basis that the DOI for the claims for a reduction of impaired daily weight gain
and for a reduction of cardiac, pancreatic and skeletal muscle lesions are demonstrated at 12 months post-
vaccination.

Page 107
7.3 Vaccines I

Safety and efficacy of a DNA and oil-based PD vaccines in Atlantic salmon infected with
SAV3 using a cohabitation challenge model

Ragnar Thorarinsson1, Makoto Inami2, Jeffrey C. Wolf3, Hilde Sindre4, Eystein Skjerve5, Lisa Phillips1, Alicia
Macdonald1, Jose F. Rodriquez1

1Elanco Animal Health, Norway

2VESO Vikan, Namsos, Norway
3Experimental Pathology Laboratories Inc., Virginia, USA
4Norwegian Veterinary Institute, Norway
5Faculty of Veterinary Medisine, Norwegian University of Life Sciences, Norway

[email protected]

Healthy and tightly graded salmon parr were PIT-tagged and two weeks later immunized at random with one
of three different pancreas disease (PD) vaccine combinations currently used in the Norwegian aquaculture
industry; a DNA vaccine (DNAV) administered intramuscularly (i.m.) in addition to an oil adjuvanted (OA)
vaccine without a PD component administered intraperitoneally (i.p.) (group A), and two different OA PD
vaccines (groups B and C). Fish injected i.p. with saline served as negative controls. Temperature of ~12 °C
was maintained throughout the study. Data collected in freshwater at the smolting stage ~ 1020 degree days
(dd) post-vaccination includes weight gain, side effects and in vitro SAV3 neutralization capacity of the
immune plasmas. After 9 days in seawater, ~1040 dd post vaccination, the fish were infected with salmonid
alphavirus, subtype 3, using a cohabitation challenge model. The fish groups were sampled at 19, 54 and 83
days post challenge (dpc).

No differences in growth and side effect development in the peritoneum were measured between the fish
groups after ~1020 dd post vaccination. Only the fish in group A demonstrated significantly higher SAV3
neutralizing antibody titers after the immunization period pre-challenge and significantly lower SAV3 viremia
levels at 19 dpc compared to the controls. The fish in group A had significantly greater weight gain than the
other groups during the challenge period. Prevalence and severity of heart necrosis (at 19 dpc), and loss of
exocrine pancreas tissue (at 54 and 83 dpc) were significantly lower in groups A and B compared to group C
and controls. The cumulative mortality in the negative control group during the challenge period was low
(10.5%). The fish in group A experienced the lowest mortality (6.4%) of the immunized groups but none of
these were statistically significant compared to the controls. The data suggest that use of DNAV may further
reduce the economic impact of PD by protecting against SAV3-induced damage and destruction of the
pancreas tissue and subsequent growth impairment which is the most common and costly clinical outcome
of this disease.

Page 108
7.3 Vaccines I

Effect of vaccines against pancreas disease in commercially reared Atlantic Salmon

Magnus Vikan Røsæg1, Arnfinn Aunsmo3,4, Ragnar Thorarinsson2

1Salmar, Norway
2Elanco Animal Health, Norway
3Barkbekken Drift AS, Norway
4Faculty of Veterinary Medicine University of Life Sciences, Norway

[email protected]

Pancreas disease (PD) caused by salmonid alphavirus (SAV) continues to negatively impact European salmon
farming. Clinical findings of PD include increased mortality and reduced growth. The objectives of this study
were to assess and compare the effect of vaccines against PD, measured in growth and mortality.
Groups of Atlantic salmon 2018 spring smolts (S1’s) immunized with different commercially available
pancreas disease (PD) vaccines were compared throughout normal seawater production cycle and at time of
slaughter. The field study entailed three separate experimental designs, as follows:

1. Within one sea cage, adipose fin-clipped and PIT-tagged fish were immunized using six different
vaccine regimes and compared
2. Inside two separate sea cages, physically marked fish (maxillary clipped) with two vaccine groups
were reared together and compared
3. Cage-to-cage comparison using unmarked fish with three vaccine groups in six sea cages (two cages
per group)

The fish at the sea cage study experienced a clinical PD outbreak caused by salmonid alphavirus, subtypes 2
(SAV2) and 3 (SAV3). Vaccine efficacy criteria including mortality and protection against PD induced growth
impairment will be presented and discussed in addition to vaccine safety data also collected.

Page 109
7.3 Vaccines I

Evaluation of autologous vaccine against Vibrio harveyi in European sea bass (Dicentrarchus
labrax)

Ivana Zupičić, Željko Pavlinec, Dražen Oraić, Snježana Zrnčić

Croatian Veterinary Institute, Croatia

[email protected]

Adriatic marine aquaculture is facing to the similar health challenges as of sea bass farming in other
Mediterranean countries. In last decade, among bacterial diseases the most prevalent are outbreaks caused
by Vibrio harveyi causing serious losses during the summer months. Unfortunatelly, there is no commercial
vaccine against V. harveyi and to prevent the losses in aquaculture of sea bass, we produced and tested
autologous vaccine against this bacteria.

V. harveyi was isolated from eyes, anterior kidneys, hearts and spleens of diseased sea bass from farm on
the Eastern Adriatic on marine agar (MA) and tryptic soy agar (TSA) supplemented with 1.5% sodium
chloride, incubated during 48h at 25 °C. Typical bacterial colonies were purified and affiliation confirmed by
polymerase chain reaction (PCR) using toxR primers and also using 16S rRNA sequencing. Serotyping was
performed, and isolate used for vaccine belongs to the serovar A, known to cause the severe clinical
symptoms. The bacterin was prepared by growing V. harveyi in Tryptic Soy Broth (TSB) with 1.5% NaCl using
a 2 L fermenters for 48h at 24 °C. The culture was inactivated with 0.4% formaldehyde during 48h. Safety
tests of the vaccines and protocols were performed using 20 fish per test group (unvaccinated and
vaccinated) prior the vaccination and no mortalities was recorded in period of 40 days. The vaccination was
performed in laboratory conditions by immersion and intraperitoneal injection (IP). Vaccinated fish were
challenged 36 days after vaccination both by immersion and intraperitoneal injection. Control groups were
mock vaccinated with sterile saline applying both procedures.

Challenged fish in unvaccinated and vaccinated groups showed typical pathological signs of V. harveyi
infection - haemorrhages at the fin bases. Immersion vaccination against V. harveyi of juvenile sea bass
showed satisfactory RPS=63, thirty-six days post-vaccination after IP challenge and survival percentage of
99% (SP=99) after a challenge by immersion. IP vaccination gave RPS=100 with same challenge dose for
testing group immersion vaccination. Autologous vaccine against vibriosis caused by V. harveyi showed to
be effective protection for juvenile sea bass. The results were supported by higher gene expression of
interleukins in spleen, kidney and intestines.

Page 110
8.1.1 Host-pathogen interactions II

Immunohistochemical detection of IgT-positive cells and Flavobacterium psychrophilum in

immersion challenged Atlantic salmon (Salmo salar)

Valeria Macchia1, Fabian Grammes3, Andrew Davie1, Lauren McMillan5, Andrew Reeve4, Thomas Moen3,
Andrew Desbois1, Kim Thompson2, Rowena Hoare1

1Institute
of Aquaculture, University of Stirling, Scotland, UK
2Moredun Research Institute, Pentland Science Park, UK
3AquaGen, Norway
4AquaGen Scotland Ltd, Scotland, UK
5DawnFresh Farming Ltd, Scotland, UK

[email protected]

Flavobacterium psychrophilum is the causative agent of rainbow trout fry syndrome (RTFS), which has caused
substantial losses in the rainbow trout (Oncorhynchus mykiss) industry globally, including the UK, for
decades. The disease is widespread and can cause high mortality in fry in freshwater hatcheries. Antibiotics
are often used to treat affected stock and currently no vaccines are available in the UK. Recently, F.
psychrophilum has also been isolated from Atlantic salmon fry (Salmo salar) in Scotland causing concern for
the industry. Using a newly developed immersion challenge model, we conducted a trial to search for
markers of resistance to F. psychrophilum in a 2018 cohort of Atlantic salmon fry (n=3060). A subsequent
GWAS (genome-wide association study) of the challenged salmon fry revealed a strong quantitative trait
locus (QTL) located on chromosome 9.

This present study aimed to optimise the detection of IgT-positive cells and F. psychrophilum in Atlantic
salmon mucosal tissues by immunohistochemistry (IHC) to test whether the presence of bacteria and IgT-
positive cells correlated to the alleles at the QTL. Whole salmon fry (n=10) were sampled at days 3, 6 and 10
post-challenge, fixed in 10% formalin, embedded in paraffin and sectioned. F. psychrophilum and IgT-positive
cells in mucosal tissues were detected by optimised IHC protocols. DNA was extracted from formalin-fixed
fish tissue to determine the genotype of each individual fish, using a high-density SNP chip (70,000 markers).
Novel monoclonal antibodies (mAbs) for F. psychrophilum were characterised by IHC, ELISA and western
blotting. The mAbs recognised a high molecular weight protein of F. psychrophilum by western blotting. The
mAbs also detected F. psychrophilum by immune-fluorescent antibody technique (IFAT) and ELISA.

The results of this study will aid in diagnostics for RTFS and elucidating the mechanisms of resistance to F.
psychrophilum in Atlantic salmon fry.

Page 111
8.1.1 Host-pathogen interactions II

Apoptosis decrease of peritoneal salmonid macrophages in vitro exposed to Piscirickettsia

salmonis extracellular products

Pedro Smith1, X Farías1, Fernando Duarte1, Carlos Maturana2, Jaime Palomino1, Ana Maria Ramírez1, Pablo
Salgado1

1Faculty of Veterinary Sciences, University of Chile, Santiago, Chile

2Virbac-Centrovet, Chile

[email protected]

Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a systemic disease affecting several teleost
fish species that is particularly important in salmonid fish reared in sea and estuarine waters in Chile.
Although in vivo this bacterium replicates in the cytoplasm of several types of fish cells, macrophages are
probably the main target cells for its multiplication, at least in salmonid fish. To gain a better understanding
of piscirickettsiosis pathogenesis, the in vitro apoptosis modulation of peritoneal macrophages of rainbow
trout (Oncorhynchus mykiss) by the extracellular products (ECPs) of P. salmonis cultured in CHSE-214 cells
was assessed in this work.

Methodology
Leukocyte pools obtained from peritoneal washings from 20 juvenile fish reared in fresh water were used.
Cell suspension aliquots were seeded in ultra-low attachment microplate wells and exposed to P. salmonis
ECPs at different concentrations in two sets of experiments. After exposure times of 20, 24 and 48 h at 10°C,
the suspensions of each well were tested by flow cytometry using the JC-1 cationic dye to detect apoptotic
cells.

Results
It was found that P. salmonis ECPs reduced the apoptosis proportion in macrophage enriched populations
(Kruskall-Wallis test, p ≤ 0.05) and that this reaction would occur in a dose-response manner. Such inhibitory
effect was observed in all the exposure times that were tested.

Conclusions
These findings are consistent with previous results obtained in vivo from salmonid fish and suggest that P.
salmonis ECPs modulate the apoptosis of immunocompetent cells. It is possible that during the course of a
natural infection P. salmonis interferes with the normal apoptotic rate of macrophages thus facilitating its
intracellular multiplication in these phagocytic cells, and therefore it would contribute towards the process
of infectivity in salmonid fish hosts.

Funding
Partially supported by grant EQM120156 (Fondequip) of the National Commission for Science and
Technology (CONICYT-Chile).

Page 112
8.1.2 Immunostimulants, prebiotics & probiotics

The health-promoting potential of fulvic acid in water for yolk-sac larvae

Thora Lieke1,2, Thomas Meinelt1, Klaus Knopf1,2, Werner Kloas1,2

1Leibniz Institute of Freshwater Ecology and Inland Fisheries, Germany

2Humboldt University of Berlin, Germany

[email protected]

Supplementing feed with immunostimulants to improve overall health has become an increasing field of
research. One significant problem is the high mortality of pre-feeding yolk-sac larvae, which cannot be
protected with this approach. At this early life stage, the use of antibiotics against bacterial infections is the
remedy of choice against bacterial problems. We evaluated the potential of an immunostimulatory fulvic
acid (FA) to be used as a bath treatment.

Methodology
Zebrafish larvae were exposed until 144 hpf (hours post fertilization) to 1 to 500 mg C/L of a phenol-rich
fulvic acid (FA) following the Fish Embryo Acute Toxicity Test guidelines. Time of hatching, formation of
edema and hematomas, and mortality were observed every 24 h for six days. Furthermore, the concentration
of reactive oxygen species inside the larvae after 96 h of exposure to 5 mg C/L, 50 mg C/L, 300 mg C/L (start
of detrimental effects in the toxicity test), and 500 mg C/L was measured by the conversion of a fluorescence
dye. Finally, the expression of genes involved in growth (gh, igf-1, he1-α), innate immunity (lyz, mpx), and
anti-oxidative protection (keap1, nrf2, cat, sod-1, sod-2) was analyzed by qRT-PR.

Results
Concentrations between 20 mg C/L and 200 mg C/L of FA significantly increased the hatching rate after 72 h
(> 60% compared to > 35% in the control), while the overall hatching rate at 96 h was not affected by any
concentration. 5 mg C/L and 50 mg C/L increased the expression of gh, and he1-α. While FA did not affect
the respiratory burst activity at any concentration. Concentrations up to 50 mg C/L increased the expression
of lyz and mpx. FA concentrations of 50 mg C/L and higher exerted oxidative stress, as shown by increased
expression of keap1-nrf2 and downstream anti-oxidative enzymes (cat, sod-1, and sod-2). However, while
this activation was enough to protect the larvae at 50 mg C/L, the systems were depleted at higher
concentrations, resulting in edema, hematomas, and mortality.

Conclusions
Phenol-rich fulvic acid can be used at low to medium concentrations to accelerate the hatching, stimulate
immunity, and activate antioxidative protection. However, it becomes detrimental at high concentrations.

Page 113
8.1.2 Immunostimulants, prebiotics & probiotics

Mucosal and systemic immune effects of Bacillus subtilis in rainbow trout (Oncorhynchus
mykiss) point to its potential as an oral adjuvant

Félix Docando1, Cláudia R. Serra2, Paula Arense1, Noelia Nuñez-Ortiz1, Paula Enes2, Aires Oliva-Teles2, Carolina
Tafalla1, Patircia Díaz-Rosales1

1Animal Health Research Center (CISA-INIA-CSIC), Spain

2CIIMAR, University of Porto, Porto, Portugal

[email protected]

Oral vaccines are highly demanded by the aquaculture sector, which requests alternatives to labor-intensive
injectable vaccines that involve individual handling of fish, provoking stress-related immunosuppression and
handling mortalities. Despite this, most attempts to obtain effective oral vaccines have failed in fish and
mammals. In this context, the search for mucosal adjuvants that when orally delivered along with antigens
could help circumvent intestinal tolerance, and thus induce an adequate immune response to the antigen, is
an important step for the development of effective oral vaccines.

Bacillus spp. are well known for their probiotic properties. However, in fish, their potential as mucosal
adjuvants has not yet been studied in depth. In the current work, we performed a series of experiments with
rainbow trout (Oncorhynchus mykiss) aiming at establishing the adjuvant potential of two Bacillus subtilis
spore-forming strains isolated from farmed fish, designated as FI99 and FI162. As an initial step, we evaluated
the transcriptional effects of the two strains on rainbow trout intestinal epithelial cell line RTgutGC, and in
gut tissue sections incubated ex vivo with FI99 and FI162. The capacity of both strains to adhere to RTgutGC
cells was evaluated by flow cytometry. Although both strains had the capacity to modulate the transcription
of several genes related to innate and adaptive immune responses, it was the FI99 strain that provoked more
immune effects, also exerting a higher binding capacity to intestinal epithelial cells. Consequently, we
selected this strain to establish the transcriptional effects in the spleen, kidney, and gut of rainbow trout at
1, 3, and 7 days after a single oral administration of the bacteria. We confirmed that genes involved in
inflammation, antimicrobial genes, and genes involved in T cell responses were induced by the FI99 strain.

Our results provide valuable information regarding how B. subtilis modulates the immune response of
rainbow trout both within the gut and systemically, after a single oral administration, pointing to this
probiotic strain as a good mucosal adjuvant candidate that would help to increase the immune response
mounted against orally-delivered antigens.

Funding
This work was supported by the Spanish Ministry of Science, Innovation and Universities (project AGL2017-
85494-C2-1-R), by the Comunidad de Madrid (grant 2016-T1/BIO-1672).

Page 114
8.1.2 Immunostimulants, prebiotics & probiotics

Study of innate immune response of the European seabass (Dicentrarchus labrax), by

dietary supplementation with Bacillus velezensis

Luis Monzón-Atienza1, Jimena Bravo1, Daniel Montero1, José Ramos-Vivas1, Jorge Galindo-Villegas2,
Félix Acosta1

1 Grupo de Investigación en Acuicultura (GIA), Instituto Ecoaqua, Universidad de Las Palmas de Gran Canaria,
Spain
2 Faculty of Biosciences and Aquaculture, Nord University, Norway

[email protected]

The aquaculture intensification cause stressful conditions in fish, increasing the risk of diseases such as
vibriosis by V. anguillarum. The indiscriminate use of antibiotics generate pathogen resistance as well as food
security and environmental problems. One recently eco-friendly solution are probiotics, such as Bacillus spp.,
trough the capacity to modulate the immune system of fishes against pathogens. Previous studies
demonstrated that B. velezensis improves the resistance of seabass against V. anguillarum but information
about the effects of B. velezensis on immune status in seabass remains limited. The aim of this study was to
evaluated the effects of dietary supplementation with B. velezensis on the immune parameters of European
seabass.

Methodology
The immune parameters evaluated were serum bactericidal, serum lysozyme, serum nitric oxide (NO),
phagocytic activities and the gene expression of interleukin-1 (IL-1), tumor necrosis factor alpha (TNFα),
dicentracin (DIC) and Cyclooxygenase 2 (COX2) in head kidney tissue by quantitative RT-PCR.

Results
The seabass treated with the probiotic increased serum killing, lysozyme activities, nitric oxide and
phagocytic activity. The expressions of the IL-1, TNF and Dic, COX2 genes exhibited higher levels than
control groups. IL-1 showed statistically significant levels only after 48 hours, in the case of TNF, Dic and
COX2 only after 72 hours.

Conclusions
These results clearly indicated that B. velezensis has the potential to be developed as a probiotic agent for
seabass in aquaculture industry due to capacity of immunomodulate.

Page 115
8.1.2 Immunostimulants, prebiotics & probiotics

Probiotics could strength tilapia resistance against Tilapia Lake Virus (TiLV)

Win Surachetpong1, Pitchaporn Waiyamitra1, Mehmet Arif Zoral2, Amorn Luengnaruemitchai3, Olivier
Decamp4, Bartolomeo Gorgoglione2

1Department of Veterinary Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart

University, Thailand
2Aquatic Animal Health Laboratory, Dept. of Pathobiology and Diagnostic Investigation, CVM & Dept. of

Fisheries and Wildlife, CANR - Michigan State University, USA

3Manit Genetic Co Ltd, Thailand
4Inve Technologies, Belgium

[email protected]

Mortality outbreaks caused by Tilapia tilapinevirus or Tilapia lake virus (TiLV), have drawn attention in tilapia
aquaculture worldwide, due to their rapid spreading between farms and high mortality rate. Currently, there
are no control measures or vaccination strategies available against TiLV. The adoption of alternative
strategies should be explored to prevent TiLV infections, including with the use of probiotics or
immunostimulants that could strength the host immunity. Working in this direction, we evaluate the effects
of Bacillus spp. probiotics supplementation on mortality, viral load and expression of immune-related gene
in red hybrid tilapia (Oreochromis spp.) during TiLV infections in Thailand.

Our results revealed that dietary supplementation with 0.5% and 1% probiotic Bacillus spp. for 21 days
elicited less mortality (25% and 24% respectively) than in the control group (32%). Moreover, fish fed with
1% probiotic diet had a significantly lower viral load than those fed with 0.5% probiotic and control diet. The
expression patterns of immune-related genes, including IL-8 (aka CXCL8), IFN-γ, IRF-3, MX, RSAD-2 (aka
VIPERIN) showed significant improvement upon probiotic treatment during the peak of TiLV pathogenesis in
fish fed with the 1% probiotics-supplemented diet. Overall, our study indicated that preventive dietary
supplementation using Bacillus spp. probiotics may have beneficial effects in strengthening tilapia immunity
and resistance against TiLV infections. Therefore, probiotic treatments may be preventively administered as
a strategy aimed at reducing losses to tilapia farms when this emerging viral infection might be predictable.

Funding
This research was funded by the Research and Researchers for Industries (RRI) (grant: MSD62I0028), and
Financial Support from the Faculty of Veterinary Medicine, Kasetsart University. We would like to
acknowledge the financial support received from the National Research Council of Thailand (NRCT) under
the NRCT Mid-Career Research Grant (NRCT5-RSA63002-04).
Conflict: authors declare to have no conflict of interest.

Page 116
8.2 Myxozoa II

Myxozoa build functional worms at the cellular level

Edson Adriano1, Suellen Zatti 1, Beth Okamura2

1Federal University of São Paulo, UNIFESP, Brazil

2University of São Paulo FZEA/USP, Brazil
3Natural History Museum, UK

[email protected]

Recent studies have revealed a lineage of myxozoans belonging to the genus Ceratomyxa that have radiated
as endoparasites in the gall bladder of South American freshwater fishes. A remarkable feature of these
ceratomyxids is their worm-like (vermiform) plasmodia that demonstrate motility similar to that achieved by
multicellular worms that use hydrostatic skeletons involving tissue layers and fluid-filled cavities (e.g.
nematodes). In this study we present representative morphological, behavioural, and molecular data to infer
how these vermiform ceratomyxids have undergone unprecedented variation in form and function to
develop as cellular hydrostats.

Methodology
We use light, confocal, electron microscopy, and videos to characterize anatomy and infer how movement
is achieved. In addition, SSU rDNA sequences were obtained in order to undertake phylogenetic analysis to
characterise the inter-relationships of this ceratomyxid radiation.

Results and Conclusions

Vermiform ceratomyxid plasmodia are characterized by cytoskeletal elements that include net forming
microfilaments and microtubules, long tubular mitochondria, a large internal vacuole, and a relatively rigid
outer glycocalyx. Thus, they share functional elements with multicellular worms but these have enabled
convergence at the cellular level. Such convergence involves repurposing the fundamental building block of
cnidarians: the epitheliomuscular cell. The apparent restriction of these vermiform ceratomyxids to South
American freshwaters suggests an origination via Cretaceous or Miocene marine transgressions and
subsequent radiation.

Funding
FAPESP - São Paulo Research Foundation (Proc. n°s. 2019/17427-3 and 2019/26082-0).

Page 117
8.2 Myxozoa II

Environmental DNA metabarcoding assay for detection of freshwater myxozoan

communities

Ivan Fiala1,2, Martina Lisnerová1,2, Astrid Holzer1

1Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Czech Republic
2Faculty of Science, University of South Bohemia, Czech Republic

[email protected]

Monitoring of parasitic diseases of fish is very important for fish aquaculture. Accurate assessment of fish
parasite distribution and diversity is fundamental for controlling fish diseases and for describing parasite
communities in the geographic areas concerned. The environmental DNA (eDNA) metabarcoding approach
has become a useful tool for the detection of a diversity of many species including parasitic ones. Myxozoa
represents a unique group of morphologically simplified endoparasites infecting mainly fish, with more than
2600 nominal species. However, their diversity is still largely unexplored and undervalued. Parasitological
examination of host tissues is an invasive technique that requires often fish killing or obtaining a fish host
with difficult access. eDNA metabarcoding of infectious myxozoan spore stages occurring in aquatic
environments is a non-invasive method of assessing myxozoan biodiversity on a given locality, yet very little
explored.

We propose a novel eDNA-based methodological approach for studying freshwater myxozoan diversity.
eDNA was isolated from organic materials elutriated from aquatic sediments. Our metabarcoding approach
was based on next-generation sequencing (NGS) of pooled PCR amplicons generated using selected sets of
seven newly designed primer pairs that specifically amplify the V4 region of SSU rDNA of representatives of
all phylogenetic clades of freshwater myxozoans.

Primer pair candidates were thoroughly tested first by in-silico PCR, followed by PCR testing on DNA samples
of fish tissues infected by a wide range of myxozoans, and finally on a range of environmental samples
covering various freshwater habitats. Amplicons were tagged with specific barcodes that enabled us to
analyze a wide spectrum of different samples from different localities at once. Our target DNA marker region
provides sufficient genetic information for determining even very closely related species. Our eDNA analysis
successfully detected large numbers of myxozoan OTUs from all freshwater clades. Myxozoan NGS reads
represented 93.8% of all reads obtained from studied localities.

We believe that this approach enables us to monitor myxozoan distribution in natural or artificial aquaculture
freshwater environments as well as to reveal a yet unexplored myxozoan diversity.

Funding
Czech Science Foundation (#29-28399X).

Page 118
8.2 Myxozoa II

Some like it hot but don’t mind cold: the distribution of myxozoan parasite
Tetracapsuloides bryosalmonae in northernmost Europe

Magnus Lauringson1, Mikhail Ozerov2, María-Eugenia Lopez3, Eero Niemela4, Anti Vasemägi1,3

1Estonian University of Life Sciences, Estonia

2Universityof Turku, Finland
3Swedish University of Agricultural Sciences, Sweden
4Natural Resources Institute Finland (Luke), Finland

[email protected]

Global climate change is altering the abundance and spread of many aquatic parasites and diseases.
Proliferative kidney disease (PKD) of salmonid fishes caused by myxozoan T. bryosalmonae is no exception
to this tendency, as the increase of water temperature is expected to escalate the impact of PKD in northern
latitudes.

Yet, currently we know very little about the distribution and prevalence of T. bryosalmonae in Northern
Europe. Here, we studied 27 rivers flowing to the Barents Sea in northernmost Norway and Finland to
describe T. bryosalmonae infection frequency and patterns in juvenile Atlantic salmon (n=1099), brown trout
(Salmo trutta, n=240) and Arctic char (Salvelinus alpinus, n=50). Molecular genetic analysis was used to
determine the presence of the parasite DNA from kidney tissue samples collected during 2019-2020.

The causative agent of PKD was discovered in 11 rivers out of 27 (40%). The prevalence of T. bryosalmonae
ranged from 4.2 to 55.5% in Atlantic salmon and 5.88 to 75% in brown trout among infected rivers, while all
studied Arctic charr samples were free of myxozoan parasite. In ten studied rivers where brown trout and
Atlantic salmon occurred in sympatry, brown trout was more frequently infected with T. bryosalmonae than
salmon. Analysis of age-specific parasite prevalence patterns revealed that the infection of juvenile fish in
the northernmost Europe predominantly occurs during the second summer. Comparison of the temporal
patterns of T. bryosalmonae over 10 year period did not reveal any signs of contemporary spread of parasite
to previously uninfected rivers.

However, the occurrence of the causative agent of temperature-driven disease in rivers flowing to Barents
Sea suggests that extended heatwaves may cause new PKD outbreaks in northern regions.

Page 119
8.2 Myxozoa II

IgM+ B Cells and the antibody response protect the common carp Cyprinus carpio against
infection by the myxozoan Sphaerospora molnari

Justin Tze Ho Chan, Tomáš Korytář, AS Holzer

Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Czech Republic

Whether through immunization or natural infection, a consequence of immune challenge is the expansion
of the lymphocyte compartment and production of antigen-specific antibodies. We previously demonstrated
these phenomena in a laboratory model of infection of the common carp by the myxozoan Sphaerospora
molnari, the etiological agent of gill and skin sphaerosporosis. What is more difficult to demonstrate is any
protection conferred because immunity to S. molnari is non-sterile and infection is chronic and latent.
Antibodies and activated lymphocytes are rather the most basic prerequisites, and are only correlates of
disease until we can demonstrate activities such as parasite lysis, and prophylaxis.

Methodology
Immunosuppressed or immunocompetent laboratory-reared fish were infected with different doses of
purified blood-stage S. molnari. We verified expansion of the IgM+ B cell compartment by flow cytometry
and designed a direct enzyme-linked immunosorbent assay to monitor anti-S. molnari antibody titers.
Quantitative polymerase chain reaction was used to quantify S. molnari 18S ribosomal DNA and parasitemia.
These techniques were also applied towards testing lytic activity of immune plasma, and towards monitoring
parasitemia after passive immunization.

Results
We measured an increase in anti-S. molnari antibody titers within four weeks of infection. Following
elimination of the B cell compartment, no such titers were detected and parasitemia was exacerbated in
onset and magnitude. When different dosages of the initial parasite inoculum were tested on
immunosuppressed fish, we observed dose-dependent parasitemia. Surprisingly, in immunocompetent
animals, both antigen-specific antibody titers and parasitemia were dose-dependent. Mechanistically, fish
immune plasma demonstrated heat-labile lysis of cultured parasite in vitro. Additionally, immune plasma
conferred passive immunity to animals upon challenge with S. molnari.

Conclusions
The antibody response is protective short-term upon primary infections, and also protective long-term,
keeping the parasite latent. Differentiated (memory) effector subsets of B cells, the B cell precursors
responsible for protection, the antibodies raised, the antigens targeted or not targeted, mechanisms of
antibody-mediated protection – all need to be further studied as our data provide evidence that targeting
the humoral response is a viable strategy for both anti-parasitic therapy and long-term (interseasonal)
protection against the myxozoan.

Funding Source
Czech Science Foundation (GAČR) - 19-25589Y

Page 120
8.2 Myxozoa II

Tetracapsuloides bryosalmonae calreticulin selectively attenuates pro-inflammatory

cytokine activity in rainbow trout, Oncorhynchus mykiss

Jason Holland1, Bei Wang2, Tiehui Wang1, Marc Faber1,4, Beatriz Abos3, Carolina Tafalla3, Christopher
Secombes1

1School of Biological Sciences, University of Aberdeen, UK

2Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animal,

Key Laboratory of Control for Disease of Aquatic Animals of Guangdong Higher Education Institutes, College
of Fishery, Guangdong Ocean University, China
3Centro de Investigación en Sanidad Animal (CISA-INIA), Spain
4Moredun Research Institute, Pentlands Science Park, Scotland, UK

[email protected]

The malacosporean parasite, Tetracapsuloides bryosalmonae, is the causative agent of proliferative kidney
disease (PKD) of farmed and wild fish throughout Europe and the USA. In the absence of sustainable control
strategies, uncovering parasite virulence is an important requisite in developing immunotherapeutic
approaches. As well as being a protein chaperone, the lectin-like protein, calreticulin (CRT), is an important
factor in host immune evasion in some parasites, including fish trypanosomes. The “KDEL” endoplasmic
reticulum retention signal is often modified and rendered non-functional, permitting surface expression.
Here, for the first time in myxozoans, we report the functional characterisation of a T. bryosalmonae
calreticulin (TbCRT) homologue.

Methodology
A full-length CRT transcript was cloned and characterized from a spore sac cDNA library and compared with
other parasite CRTs, including myxozoan homologues. TbCRT transcription was examined in infected
bryozoans and rainbow trout hosts by qRT-PCR and recombinant (r)TbCRT generated in E. coli for functional
analysis. Immune modulatory activity of TbCRT was investigated, in vitro, using the monocyte / macrophage
cell line, RTS-11. Cells were pre-incubated with rTbCRT and stimulated with the immunodulatory trout
cytokines: IL-1β, TNFα, IL-6, and IFNƴ. Western blotting was performed to determine whether fish with
clinical PKD possess endogenous antibodies recognizing TbCRT.

Results
As in other parasites, most myxozoan CRT homologues were found to possess “scrabbled” KDEL retention
signals, implying that they could be classically secreted. Indeed, we were able to demonstrate an endogenous
antibody response to TbCRT in infected fish. TbCRT transcripts were found to be predominantly expressed
in fish hosts relative to bryozoans with expression closely correlating with parasite load at different stages of
PKD. rTbCRT was found to selectively attenuate the activity of rIL-1β and rTNFα in RTS-11 cells, but not rIL-6
and rIFNƴ.

Conclusions
Our findings represent the first insights into the role of myxozoan CRTs in fish immune evasion, which may
represent a mechanism to overcome early innate and pro-inflammatory immune responses important to
parasite survival following host invasion and during clinical disease. The presence of anti-TbCRT antibodies
in infected fish indicates that TbCRT is a useful serological biomarker for PKD exposure.

Page 121
8.2 Myxozoa II

From infected fish blood to samples of host-free parasite: Isolation of myxozoan

proliferative stages

Ana Born-Torrijos1, Anush Kosakyan1, Sneha Patra1,2, Joana Pimentel-Santos1, Brian Panicucci1, Justin Tze Ho
Chan1, Tomáš Korytář1,3, Astrid S. Holzer1

[email protected]

1Instituteof Parasitology, Biology Centre of the Czech Academy of Sciences, Czech Republic
2Global Change Research Institute of the Czech Academy of Sciences, Czech Republic
3Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and

Biodiversity of Hydrocenoses, University of South Bohemia, Czech Republic

Myxozoan (Cnidaria) are an extremely diverse group of endoparasites, some being causative agents of
serious fish diseases. Our laboratory model is Sphaerospora molnari, a myxozoan parasite of common carp
(Cyprinus carpio) that undergoes presporogonic development in the blood, forming blood stages (BS). To
perform myxozoan ‘-omics’ that can aid in the development of antiparasitic strategies, it is crucial to obtain
pure parasite samples. Furthermore, obtaining host-free samples will help culture myxozoans, which is
nowadays not possible.

We used anion exchange chromatography to biochemically isolate myxozoan presporogonic proliferative

stages from infected host blood. A diethylaminoethyl cellulose (DEAE-C) column retained host cells and
eluted the less negatively charged parasites. To maximize the yield and purity of the isolated parasites,
several conditions were optimized: column length, ionic strength and elution buffer composition. The
integrity of the isolated parasites was assessed by analysing their cell number as well as their viability and
infectivity, injecting the obtained BS to uninfected fish. As an alternative to microscopy for quantifying
parasites, a rapid flow cytometry method was developed based on staining of host cells with a polyclonal
pan-carp blood cell-specific antibody.

Our optimized DEAE-C protocol yielded 46% of input impure parasites (maximum 68%), allowing us to isolate
up to 28 million BS/mL of blood at >99% purity and low BS mortality (0 ̶ 1.7%). The variability of parasite
cellularity ranged from 2 to 20 cells and mirrors the natural cellular parasite composition in blood pre-
isolation. Highly multicellular BS are more strongly retained, likely due to their larger surfaces. Furthermore,
isolated BS can successfully infect new fish hosts. Flow cytometry distinguished parasite from host cells in
whole blood such that the measured yields matched those of conventional microscopy.

To our knowledge, this is the first method for isolating the myxozoan proliferative stages with almost a 100%
purity. Further development and application of parasite-specific antibodies will complement efforts to
phenotype parasite stages. This method opens new research avenues based on in vivo and in vitro assays to
study myxozoan-fish host interactions, as well as for the generation of host-free genomic and transcriptomic
data.

Funding
Czech Science Foundation (20-30321Y, EXPRO 19-28399X) and Horizon 2020 Framework Programme
(ParaFishControl, 634429).

Page 122
8.3.1 Vaccines II

Do not let it get under your skin! – Vaccination protects the skin barrier from disruption
caused by cyprinid herpesvirus 3

Mikolaj Adamek1, Marek Matras2, Alexander Rebl3, Magdalena Stachnik2, Lars Schröder4, Walter Fuchs4,
Michal Reichert2, Dieter Steinhagen1

[email protected]

1Fish Disease Research Unit, Institute for Parasitology, University Of Veterinary Medicine Hannover,
Germany
2Laboratory of Fish Diseases, National Veterinary Research Institute, Poland
3Fish Genetics Unit, Leibniz Institute for Farm Animal Biology (FBN), Institute of Genome Biology, Germany
4Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Germany

Vaccination is the best form of protecting fish from viral diseases when the pathogen cannot be contained
by biosecurity. Live attenuated virus based vaccines seem to be most effective for vaccination against
challenging pathogens like cyprinid herpesvirus 3. However, there are still knowledge gaps how these
vaccines effectively protect fish from the deadly disease caused by the epitheliotropic CyHV-3, and which
aspects of non-direct protection are important in the water environment.

Methodology
To elucidate some elements of protection, common carp were vaccinated against CyHV-3 using a double
deletion vaccine virus KHV-TΔDUT/TK in absence or presence of a mix of common carp beta-defensins 1, 2
and 3. The fish were challenged 2.5 months post vaccination with a hyper-virulent Polish isolate of CyHV-3.
Blood, skin, gill, and kidney were collected 2, 7, 14, 28 days post vaccination and post challenge for
monitoring immune responses by SNT, RT-qPCR using Fluodigm, and pathology related to the disease.

Results
Vaccination induced marginal clinical signs, low virus load and minor upregulation of cd8 and igm gene
expression in vaccinated fish while neutralisation activity of blood was rising from 14 days post vaccination.
A challenge infection with CyHV-3 induced a severe disease with 80-100% mortality in non-vaccinated fish
while in vaccinated fish no mortality was recorded and the virus load was >1000-fold lower. Beta-defensin
adjuvation gave no better outcome. Histological analysis showed strongest pathological changes in skin. In
the skin of non-vaccinated fish, T and B cells responses were severely downregulated, inflammation and
stress responses were increased upon challenge, while vaccinated fish had boosted neutrophil, T and B cell
responses. A disruption of skin barrier elements (tight, and adherence junction, desmosomes, mucins) led to
an uncontrolled increase of skin bacteria load which most likely exacerbated the pathology.

Conclusions
Using a live attenuated virus vaccine, we show that increased neutrophil, T and B cell responses provide
protection from CyHV-3 infection and lead to preservation of skin integrity, which supports a successful
protection from additional pathogens in the aquatic environment.

Page 123
8.3.1 Vaccines II

A live attenuated vaccine to combat against KHVD

Sandro Klafack1, Lars Schröder1, Yeonhwa Jin1, Arndt-Christian Hofmann1, Walter Fuchs1, Jean-Christophe
Avarre2, Sven M. Bergmann1

1Friedrich-Loeffler-Institut, Germany
2Institute of Evolutionary Sciences, France

[email protected]

Since the late 1990th, KHV is a major threat for carp and koi industry worldwide. Because of its high fatalities
and live-long persistence, eradication of the agent is hard but necessary. Many attempts were undertaken
to develop a protective vaccine to combat KHVD. Unfortunately, none is approved until now in the EU or
elsewhere. Therefore, a safe and reliable vaccine is needed.

In this talk we will present data on a newly established live vaccine, based on KHV from Taiwan (KHV-T). A
live attenuated KHV was generated by serial passages onto CCB cells at 20 °C. This vaccine virus is applicable
either orally, by immersion or in a combination of both and leads to a 100% survival rate after wild-type virus
challenge. It did not induce any clinical signs. Serological data revealed a successful, humoral immune
response after immunization but also after challenge. Whole genome sequencing revealed some major
differences to the wild type virus, and a differentiation of infected from vaccinated animals (DIVA) is possible
by PCR.

Moreover, a deletion in ORF150 was discovered, probably associated to the attenuation. Tested in carp
model, ORF150 was demonstrated to cause attenuation if deleted. Additionally, a deletion of ORF150
converts KHV-T to an attenuated virus. The naturally occurred mutation in the live attenuated virus seems
to be stable.

Page 124
8.3.2 Microbiomes in relation to fish pathology/health

Skin mucus microbiota modulation in Sparus aurata L. during an episode of chronic stress

María Cámara-Ruiz1, Isabel Cerezo Ortega2, Francisco Antonio Guardiola1, José María García-Beltrán1, María
del Carmen Balebona2, Miguel Ángel Moriñigo2, María Ángeles Esteban1

1Grupo de Inmunobiología para la acuicultura, Universidad de Murcia, Spain

2Grupo de Profilaxis y Biocontrol de Enfermedades de Peces, Universidad de Málaga, Spain

[email protected]

Fish stress and welfare are important subjects for producers due to the fact that growth and disease
susceptibility are directly related to stress conditions. It has been reported that aquaculture practices might
become potential chronic stressors severely affecting fish health status. Recent studies have shifted their
attention towards measuring stress parameters at the mucosal sites, since the collection method is
considered non-invasive. Under stressful situations, the cross talk between de teleost immune system and
the microbiota present at the mucosal sites is thought to shift significantly rendering commensals into
opportunists or pathogens.

Thus, the aim of this study is to characterize skin mucus microbiota using 16S rRNA next generation
sequencing (NGS) of gilthead seabream (Sparus aurata) during an episode of chronic stress. A total of 36 fish
were divided into two groups: control group (C) and chronically stressed group (S). Control fish remained
undisturbed while stressed fish were subjected to 1 min air exposure twice a week for 4 weeks. Skin mucus
samples were collected for microbiota analysis in the beginning of the experiment (t0), after 14 days (t14)
and after 28 days (t28).

The results showed that total expected richness (chao1) was significantly higher in S28 group with respect to
C28. Regarding total expected diversity, S14 and S28 (shannon) were significantly higher than C14 and C28,
respectively. Beta diversity analysis using NMDS showed that there were significant differences (anosim)
across microbial communities between C14-S14 and C28-S28 experimental groups. Taxonomic analysis
showed significant difference at 14 days, NS3a_marine was more abundant in the C14 group and
Pseudoalteromonas was more abundant in the S14 group. These differences persisted after 28 days of
experiencing chronic stress. To conclude, this particular type of stress affected skin microbiota composition.

Page 125
8.3.2 Microbiomes in relation to fish pathology/health

Influencing aquatic bacteria in recirculating aquaculture systems

Verena Jung-Schroers1, Felix Teitge1, Julia Bauer1, Mikolaj Adamek1, Angela Boley2, Christina Peppler3, Dieter
Steinhagen1

1Fish Disease Research Unit, Institute for Parasitology, University of Veterinary Medicine Hannover, Germany
2Institute for Sanitary Engineering, University of Stuttgart, Germany
3Polyplan-Kreikenbaum GmbH, Germany

[email protected]

In aquaculture recirculating systems for aquatic animals, autotrophic bacteria, that are important for the
maintenance of an optimal chemical water quality, and heterotrophic bacteria, that are reducing organic
matter but can also act as facultative pathogens for aquatic animals, can be found. One important goal in
aquaculture is to avoid outbreaks of clinical bacterial infections. By specific management measures the total
bacterial amounts in the systems and especially the amounts of heterotrophic bacteria should be reduced.

Methodology
In this study the influence of different measures on the bacterial populations in aquaculture systems were
examined. In detail, ultrafiltration of recirculating water by a membrane-denitrification-reactor, the use of
UV-light and the application of ozone and peracetic acid in different concentration to the water were
investigated.

Results
Methods that were not selecting more resistant bacteria against the specific measure should be preferred
and the focus should be on the maintenance of a stable and diverse microbiome in the system. Ultrafiltration
by the membrane-denitrification reactor and the application of ozone to the water to separated tanks proved
as the most suitable methods compared to the other tested methods. The results showed that every method
can only be successful in stable systems without additional external influences. Even promising approaches
failed as soon as the amount of organic material in the system increased significantly.

Conclusions
Till now the focus in diagnosing diseases of aquatic animals is often on the aspect that the disease was caused
by one specific pathogen, however disease outbreaks in aquaculture normally occur according to the
composition of bacterial communities. For future studies it is especially important to improve the knowledge
on the relationships between the bacterial species in aquaculture systems. The pathogen related approach
needs to be changed to a more pathobiome related approach. Supporting the naturally occurring
microbiomes in aquaculture systems by stabilizing them and by supporting bacterial diversity should become
the main goal.

Page 126
8.3.2 Microbiomes in relation to fish pathology/health

The effects of florfenicol and phage therapies on the gut microbiota of healthy and
Flavobacterium psychrophilum-infected rainbow trout fry

Valentina L. Donati1, Lone Madsen1, Mathias Middelboe2, Mikael Lenz Strube1, Inger Dalsgaard1

1Technical University of Denmark, Denmark

2University of Copenhagen, Denmark

[email protected]

The constant growth of the aquaculture sector has led to an increased interest in the development of
environmentally friendly therapies to treat and prevent bacterial infections. The assessment of the effects
of various antimicrobial therapies (e.g. antibiotics, bacteriophages) on the fish gut microbiota as a measure
for improving the fish health status had also had increased interest.

Methodology
In this study, we investigated the effects of the antibiotic florfenicol and a two- component mix of phages
targeting Flavobacterium psychrophilum (FpV4 and FPSV-D22) on the gut microbiota of healthy and F.
psychrophilum-infected rainbow trout fry (1-2 g). A fish experimental trial was set up, where fish were fed
continuously with either phage-treated feed or commercial feed. In an additional group, florfenicol was
administered for 10 days starting two days after the infection procedure. The fish distal intestine was
collected over time and the microbial composition assessed by 16S rRNA gene (V3-V4 region) analysis.

Results
Results showed how the infection and the antibiotic therapy altered the microbial composition: The β-
diversity (diversity between groups) analysis highlighted shifts in the overall composition, and the taxonomic
mapping revealed the specific populations affected by the treatments. Measures of α-diversity (diversity
within a group) were only altered in infected fish. When fish recovered from the infection and the antibiotic
treatment was terminated (33 dpi), these changes disappeared. Interestingly, phage addition altered the
microbiota of the fish independently of the presence of their target bacterium. The overall gut bacterial
community in fish fed phage-treated feed was different from the controls (β-diversity analysis). However, it
was not possible to identify specific bacterial populations responsible of these changes.

Conclusions
Overall, the results indicate that the administered phages might affect the complex network of phage-
bacteria interactions in the fish gut. Even though we did not observe negative effects on fish health or
growth, further studies should be directed in understanding if the changes in the gut microbiota are
beneficial or not for the fish health.

Funding
BONUS FLAVOPHAGE project.

Page 127
8.3.2 Microbiomes in relation to fish pathology/health

Antibiotic resistance genes and bacterial communities of farmed rainbow trout fillets
(Oncorhynchus mykiss)

Nicolas Helsens1,2, Ségolène Calvez2, Hervé Prévost1, Agnès Bouju-Albert1, Aurélien Maillet1, Albert Rossero1,
Dominique Hurtaud-Pessel3, Monique Zagorec1, Catherine Magras1

1INRAE,Oniris, SECALIM, France

2INRAE,Oniris, BIOEPAR, France
3ANSES Laboratoire de Fougères, Unité Analyse des Résidus et Contaminants, France

[email protected]

Farmed fish are suspected to be a reservoir of antibiotic resistance genes and resistant bacterial hazards.
With aquaculture products being the food of animal origin most consumed worldwide, the rise of antibiotic
resistance is a challenge for human and animal health treatments. However, few studies aim at investigating
the presence of antibiotic resistance in the bacterial ecosystems of fresh fish fillets (farm environment
including rivers and practices, and factory environment).

The present study aimed at characterizing the antibiotic resistance profiles of the rainbow-trout fillets
bacterial communities. High-throughput methodologies were used, and the data included chemical
quantification of the antibiotic residues in the fillets. A total of 56 fillets (composed of muscle and skin tissue)
from fish raised on two farms on the same river were collected and processed under either factory or
laboratory sterile filleting conditions. We observed a core-bacterial community profile on the fresh rainbow
trout fillets, but the processing conditions of the fillets has a great influence on their mean bacterial load
(3.38 ± 1.01 log CFU/g vs 2.29 ± 0.72 log CFU/g) and on the inter-individual diversity of the bacterial
community. The bacterial communities were dominated by Gamma- and Alpha-proteobacteria,
Bacteroidetes, Firmicutes, and Actinobacteria.

The most prevalent genera were Pseudomonas, Escherichia-Shigella, Chryseobacterium, and

Carnobacterium. Of the 73 antibiotic residues searched, only oxytetracycline residues were detected in
13/56 fillets, all below the European Union maximum residue limit (6.40–40.20 mg/kg). Of the 248 antibiotic
resistance genes searched, 11 were found to be present in at least 20% of the fish population (tetracycline
resistance genes tetM and tetV, β-lactam resistance genes blaDHA and blaACC, macrolide resistance gene
mphA, vancomycin resistance genes vanTG and vanWG and multidrug-resistance genes mdtE, mexF, vgaB
and msrA) at relatively low abundances calculated proportionally to the 16S rRNA gene.

Page 128
9.1 Myxozoa III

Ab-normal erythrocytes in proliferative kidney disease (PKD) – rainbow trout

(Oncorhynchus mykiss) infected by Tetracapsuloides bryosalmonae harbor IgM+ red blood
cells

Justin Tze Ho Chan1, Amparo Picard-Sanchez1, Tomáš Korytář1, Alexander Rebl2, Dirk Koczan3, Astrid Holzer1

[email protected]

1Instituteof Parasitology, Biology Centre of the Czech Academy of Sciences, Czech Republic
2Instituteof Genome Biology, Leibniz Institute for Farm Animal Biology, Germany
3Core Facility for Microarray Analysis, Institute for Immunology, Rostock University Medical Centre, Germany

Tetracapsuloides bryosalmonae is the etiological agent of proliferative kidney disease (PKD) in salmonid
fishes, notably the commercially farmed rainbow trout Oncorhynchus mykiss. Among other signs, infected
fish can be identified by swollen kidneys and abdominal distension. Some of these signs of PKD can be
explained by changes at the cellular level such as B lymphocyte proliferation in the kidneys, but the B cell
response alone does not account for all signs and pathologies associated with PKD such as anemia and pale
gills. Here, we describe an unexpected cellular abnormality in the form of IgM + red blood cells (RBCs) in the
blood of infected commercially farmed rainbow trout in the midst of a seasonal PKD outbreak.

Methodology
We verified the presence of surface IgM via flow cytometry and fluorescence microscopy using anti-trout
IgM clone 1.14, and via mass spectrometry analysis of the surface fraction of the RBC proteome. We
compared the transcriptomes of healthy and PKD fish by microarray analysis.

Results
We repeatedly observed this abnormal population of IgM+ erythrocytes in 19 fish with PKD whereas no IgM
was detected on RBCs from specific pathogen-free fish. The frequency of IgM+ RBCs (up to almost 100% of
RBCs) have not been described in homeostasis nor in other diseases of teleost fishes. The absence of self-
reactive Igs in the plasma of PKD fish, the lack of hemolytic activity in this plasma, and the stability of the
phenotype are evidence that the IgM is not bound to an antigen on the erythrocyte surface, but is instead
captured by an unidentified Ig receptor. Compared to healthy cells, RBCs originating from PKD fish have
altered metabolism, iron homeostasis, adhesion, and signaling.

Conclusions
We have identified an aberrant population of RBCs bearing a potential novel marker of PKD. This marker
needs to be further studied and contextualized with what we know about the humoral response in order to
determine if the IgM is as much a driver of pathology (via interaction with antigen and/or a receptor) as it is
an indicator of disease.

Funding
Ministry of Education, Youth and Sports-Inter-action, USA (MŠMT- LTAUSA) - LTAUSA19108.

Page 129
9.1 Myxozoa III

Protective α-Gal immunity in fish: Immunotherapeutic applications

Astrid Holzer1, Joana Pimentel-Santos1, Manuel Stammler2, Lourdes Mateos-Hernández3, Tomáš Korytář1,
Alejandro Cabezas-Cruz3

1Instituteof Parasitology, Biology Centre Of The Czech Academy Of Sciences, Czech Republic
2Keppler University, Linz, Austria
3Joint Research Unit for Molecular Biology and Parasitic Immunology, INRAe, France

[email protected]

The α-Gal syndrome is a human disease associated with tick salivary glycoproteins containing Galα1-3Galβ1-
(3)4GlcNAc-R (α-Gal), which may result in the production of anti-α-Gal IgE antibodies after tick bites. These
IgEs can cause allergies to red meat, cetuximab, and gelatin. In contrast, anti-α-Gal responses based on IgM
were shown to be protective against vector-borne malaria, Leishmania and Trypanosoma, in humans. In
zebrafish, first trials showed that vaccination with α-Gal protects against mycobacterial infection. In the
present study we set out to determine the protective role of bacterial α-Gal in parasitic infections in fish,
with special focus on myxozoans.

We used different α-Gal-containing bacterial strains and applications (IP injection, gavage, bath). We
challenged fish with blood stages of the myxozoan parasite Sphaerospora molnari, by IP injection, and we
tested which bacterial treatments positively affected the immune response of fish (cytometry, concentration
of sera antibodies) and the number of parasites proliferating in the blood and forming spores in the gills
(estimated by qPCR). To better understand the mechanism of protection, we applied different immune
assays (ELISA, western blot, killing assays).

We demonstrate the immunoprotective effect of bacterial strains containing high concentrations of α-Gal
on their surface, in fish infected with the myxozoan S. molnari. Treated fish showed an improved growth
rate, decreased inflammatory response and lower parasitemia levels, when compared to the untreated
group. We demonstrate the efficacy of gavage and bath applications, suitable for different fish life cycle
stages. We further discuss preliminary data indicating the mechanism of protection.

A considerable body of research demonstrates the efficacy of immunization of humans with the α-Gal
glycotope against several α-Gal-containing pathogens, transmitted by arthropod vectors. We were able to
prove a similar protective effect in fish challenged with a myxozoan infection and we predict a large potential
and wide applicability of treatments based on protective α-Gal immunity for fish diseases in aquaculture.

Funding
Technology Agency of the Czech Republic (GAMA II program).

Page 130
Thursday 24th September 2021
10.1 Bivalve and crustacean diseases I

Preliminary results from the second Irish National Crayfish Plague Surveillance Programme:
lessons in sampling schedules, detection limits, and the use of environmental DNA

Bogna Griffin, Samantha White, Fiona Swords

Marine Institute, Ireland

[email protected]

Austropotamobius pallipes (White-clawed crayfish; WCC) is the only crayfish species indigenous to Ireland.
While the Irish population of WCC is still considered the healthiest in Europe, the latest species assessment
under Article 17 of the EU Directive on the Conservation of Habitats, Flora and Fauna (92/43/EEC), reports
their future prospects and overall status as bad. The main threats to this endangered and protected species
are habitat destruction, the introduction of non-native species and the risk posed by Aphanomyces astaci
(A.astaci), the causative agent of crayfish plague.

Since 2015, several outbreaks of crayfish plague have decimated crayfish populations in some Irish river
systems. In response to the spreading plague, an environmental DNA (eDNA) based National Crayfish Plague
Surveillance Programme was established in 2018. Following the successful first 2-year cycle in 2018-2019, a
new collaboration between the National Parks and Wildlife Service and the Marine Institute was established
with a second 2-year surveillance programme beginning in July 2020. Throughout 2018-2019 608 water
samples from 28 catchments were tested for the presence of A. astaci, A. pallipes and 8 non-native crayfish
species using real-time PCR. Seven new catchments tested positive for A. astaci at at least one site, bringing
the number of crayfish plague affected catchments in Ireland to 12. These results suggested a rapid spread
of the crayfish plague both within and between catchments. Genotyping of crayfish plague-positive samples
from mortality events suggests multiple possible introduction pathways are possible. The distribution of WCC
populations from the 2018-2019 survey confirms the decline described in the Article 17 assessment.

The preliminary results from the first year of the 2020-2021 survey will be presented here. The programme’s
scope has been widened, and the methodology further developed, with efforts to assign a genotype identity
to eDNA samples ongoing as we attempt to pinpoint the origin of the infection. This study aims to further
understand and monitor the outbreaks of crayfish plague in Ireland, its vectors, and its impact on the native
WCC and to aid the relevant bodies in developing disease control measures to protect the endangered WCC
in Ireland.

Page 131
10.1 Bivalve and crustacean diseases I

The first reported detection of infectious hypodermal haematopoietic necrosis virus

(IHHNV) infection in the European Union

Silvia MC Soares, Eann Munro, Jonathan Oladjins, Joseph Triscott, Rebecca McIntosh

Marine Scotland Science, Marine Laboratory, UK

[email protected]

A population of 350,000 post-hatch larval whiteleg shrimps (Litopenaeus vannamei) were imported from a
supplier in Texas, USA on 18 April 2019. A mortality of approximately 50% occurred during transport of the
animals to Scotland. The animals were imported by a small, inland aquaculture site and placed in closed
containment tanks, with artificial heated seawater and recirculation units. Site effluent as well as final
discharge were collected and treated and no live animals were moved off the site. The aquaculture site was
classified as an experimental pilot facility, distantly located from coastal areas and the waters surrounding
the UK do not contain IHHNV susceptible species.

After arrival on site, the whiteleg shrimps demonstrated poor and extremely variable growth. Samples were
sent for diagnostic investigation to a private laboratory who reported the detection of infectious hypodermal
haematopoietic necrosis virus (IHHNV) by conventional PCR on 15 July 2019.

Results
A Marine Scotland Science, Fish Health Inspector, attended the site on 16 July 2019 to initiate a disease
investigation. The remaining stock of whiteleg shrimp were culled later that day. Samples were taken from
30 animals for histopathology examination and molecular screening. No clinical signs of disease or significant
pathology were observed, however, 24/30 individuals tested IHHNV PCR positive. DNA sequence analysis
performed on two samples confirmed the detection of decapod Penstyldensovirus 1 (IHHNV).

A population of 300 animals for broodstock that were also imported from the same facility in Texas but at a
different time point to the infected population were housed in a separate contained area of the facility and
showed no clinical signs of disease or significant mortality. The broodstock population was also culled as a
precautionary biosecurity measure by the company.

Immediately after culling, the site was fully disinfected by high test hypochlorite to achieve 500 ppm free
chlorine in the storage reservoir and pipework.

Infection of animals with IHHNV was subsequently confirmed at the export hatchery in Texas, USA after the
transport of shrimp to Scotland.

Page 132
10.1 Bivalve and crustacean diseases I

White spot syndrome virus and Vibrio parahaemolyticus challenge model development in
whiteleg shrimp (Litopenaeus vannamei)

Marije Booman, Nasif Sarowar, Lyndsay Ferguson, Jordan Poley, Mark Braceland

The Center for Aquaculture Technologies, Canada

[email protected]

Shrimp aquaculture is a 38 billion dollar (USD) global industry, with whiteleg shrimp (Litopenaeus vannamei)
accounting for approximately 80% of production. Due to the intense nature of shrimp aquaculture, disease
outbreaks can have major impact. Pathogens of concern include white spot syndrome virus (WSSV), and
Vibrio parahaemolyticus, the aetiological agent of acute hepatopancreatic necrosis disease (AHPND), both
of which have been reported to cause mortality up to 80-100%. No current treatments are available for
WSSV, and reduced efficacy of antibiotic treatments for V. parahaemolyticus due to development of
resistance, together with a negative market opinion on their use, has led the industry to seek novel treatment
strategies. To facilitate research into the efficacy of novel disease treatments in whiteleg shrimp, challenge
models for WSSV and V. parahaemolyticus were developed.

Methodology
In these challenge model development experiments, methods were established to prepare the WSSV
inoculum, and bacterial culture growth conditions were optimized for V. parahaemolyticus. In addition, the
dose-response relationship and the effect of shrimp size on mortality were characterized.

Results and Conclusions

Per os and intraperitoneal injection challenge models for WSSV, and a per os challenge model for V.
parahaemolyticus were established. Although the challenge models were initially developed for challenge of
individual shrimp housed in separate tanks, experiments showed that these models translate well to
population-based challenges. These allow a larger number of shrimp to be challenged, increasing the
statistical robustness of the study. When testing the efficacy of treatments expected to have a moderate
protective effect (e.g. a relative percent survival of 20-40% as observed in some functional feed studies), use
of a population-based challenge increases the likelihood to detect a significant effect of the treatment on
survival.

Page 133
10.1 Bivalve and crustacean diseases I

Composition, identification methods and occurrence of pathogenicity factors of Vibrio spp.

isolated from recirculating aquaculture systems (RAS) with different salinity levels (15‰
and 30‰) for Pacific white shrimp (Litopenaeus vannamei)

Julia Bauer1, Felix Teitge1, Lisa Neffe1, Mikolaj Adamek1, Arne Jung2, Christina Peppler3, Dieter Steinhagen1,
Verena Jung-Schroers1

[email protected]

1University of Veterinary Medicine Hannover, Institute for Parasitology, Fish Disease Research Unit, Germany
2University of Veterinary Medicine Hannover, Clinic for Poultry, Germany
3Polyplan Kreikenbaum Gruppe, Germany

Vibrio spp. are ubiquitous bacteria in marine and brackish aquatic environments and part of the normal flora
of shrimps´ digestive tract and skin. Some species play an important role as pathogens in aquaculture of
shrimp while others are the cause of wound or diarrhoeal infections in humans. Recirculating aquaculture
systems (RAS) for land-based production of shrimp are often operated at low salinities in order to reduce
costs. Examinations on water samples, swabs from the biofilm of RAS and shrimp surfaces from six RAS at
salinity levels of 15‰ or 30‰ were analysed for Vibrio composition, reliable method of identification and
for the occurrence of pathogenicity factors.

Different methods of identification were compared regarding their suitability for standard diagnostics in
laboratories (16S rRNA and pyrH sequencing, biochemical identification and MALDI-TOF). The presence of
different pathogenicity factors was analysed that are known to induce an elevated pathogenicity in certain
Vibrio spp., for example the toxins PirA and PirB, the aetiological cause for AHPND in shrimp and factors that
elevate the pathogenicity of V. cholerae (VPI, ToxR, ToxS), as well as different haemolysins. The motility of
the isolates and the presence of different flagelline gene loci was investigated. The Vibrio compositions from
all RAS were compared in water samples.

The investigations on the Vibrio isolates from RAS and type strains using the different identification methods
often show divergent results. The 16S rRNA sequencing of the variable regions V1-V8 proved to be the most
reliable method for general diagnostics. Furthermore, using MALDI-TOF mass spectrometry, fast and reliable
results could be obtained, but merely for pathogenic species. PirA and PirB could not be detected, but many
other pathogenicity factors were found in the isolated Vibrio species. Furthermore, in RAS with low salinity
of 15‰ a significant higher number of pathogenic, possibly AHPND-causing Vibrio species were detected.
In conclusion, for the identification of Vibrio spp. using standard diagnostic methods, sequencing of the 16S
rRNA (V1-V8) is recommended. The investigations on isolates from different systems show that potentially
pathogenic Vibrio spp. can be found in RAS, especially in low salinities of 15‰.

Page 134
10.1 Bivalve and crustacean diseases I

Detection and prevalence of a novel viral infection (Carcinus maenas nimavirus (CmNiV)) in
European shore crabs (Carcinus maenas) from the UK

Wafa S Al Arimi1,2, Rose Kerr1, David Bass1,3, Grant D Stentiford1, Len Wilfert4, Ben Longdon2, Barbara
Tschirren2, Ronny Van Aerle1, Kelly S Bateman1

1Centre for Environment, Fisheries and Aquaculture Science (Cefas), UK

2University of Exeter, Penryn campus, UK
3Natural History Museum, UK
4University of Ulm, Germany

[email protected]

The family Nimaviridae consists of a single member, White Spot Syndrome Virus (WSSV); initially discovered
in penaeid shrimp in 1990s, the virus is associated with mass mortalities in shrimp aquaculture and has been
shown to infect a wide range of decapod crustaceans. Nima-like viruses such as Rod-Shaped Virus of Carcinus
maenas (RVCM) and B virus were previously reported in European shore crab, C. maenas, but the prevalence
of these infections is unknown. The current study has examined the prevalence of the novel Carcinus maenas
nimavirus (CmNiV) among shore crab species populations in the UK using a newly developed molecular
diagnostic method and histopathological investigations.

Methodology
A total of 469 shoreline crabs, mainly C. maenas (n=350), were sampled from eight geographic locations in
England and West Wales. All samples were screened for the presence of CmNiV using a newly developed
diagnostic PCR assay. Tissue samples were processed for light-microscopy analysis and investigated for the
presence of WSSV-like and other common infections.

Results
CmNiV was detected only in C. maenas crabs from all locations with a mean prevalence of 60%,
approximately 65 % of CmNiV positive animals by PCR were co-infected with other pathogens when
examined histologically. We found no difference in prevalence across sampling locations and between size
ranges, males and individuals infected with Hematodinium sp. were found to be less likely to be infected with
CmNiV.

Conclusions
Our study demonstrated that CmNiV is prevalent in shore crab populations in the UK. Further research is
ongoing to investigate the pathological impact of the novel virus on European shore crab populations and to
explore its phylogeny.

Funding
We acknowledge the Omani government represented by the Cultural attaché of the Embassy of Oman in
London and the Ministry of the Higher Education, Research, and Innovation, who funded this PhD project.

Page 135
10.2 Fish and shellfish immunology I

The importance of the Atlantic salmon peritoneal cavity B cell response: local IgM secreting
cells are predominant upon Piscirickettsia salmonis infection

Yorick A. van der Wal1,2, Shiferaw Jenberie2, Henriette Nordli2, Linn Greiner-Tollersrud2, Jaap Kool1, Ingvill
Jensen2, Jorunn B. Jørgensen2

1Vaxxinova Research & Development GmbH, Germany

2Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries & Economics, UiT The Arctic
University of Norway, Norway

[email protected]

The intraperitoneal route is favored for administration of inactivated and attenuated vaccines in Atlantic
salmon. Nevertheless, the immune responses in the teleost peritoneal cavity (PerC) are still incompletely
defined. In this study, we investigated the B cell responses after intraperitoneal Piscirickettsia salmonis (P.
salmonis) challenge of Atlantic salmon, focusing on the local PerC response versus responses in the lymphatic
organs: spleen and head kidney.

To this end, we used ELISpot assays to enumerate total IgM, anti-P. salmonis, and anti-Y. ruckeri antibody
secreting cells and ELISA assays to determine anti-P. salmonis, anti-Y. ruckeri, and anti-TNP-KHL IgM in serum.
We observed an increase of leukocytes and antibody secreting cells in the PerC at 3- and 6-weeks post
infection. The serum antibody response included both P. salmonis-specific antibodies and non-specific
antibodies. Finally, we present qPCR-based evidence that supports a putative role for the adipose tissue in
the PerC immune response.

Page 136
10.2 Fish and shellfish immunology I

Antigen-specific antibody secreting cells reside in the peritoneal cavity and systemic
immune sites of Atlantic salmon challenged intraperitoneally with salmonid alphavirus

Shiferaw Jenberie, Shiferaw Jenberie, Henriette Nordli, Guro Strandskog, Linn Greiner-Tollersrud,
Michelle D. Peñaranda, Jorunn B. Jørgensen, Professor Ingvill Jensen

UiT - University of Tromsø, Norway

[email protected]

How the peritoneal cavity (PerC) influences the outcome of immune responses to intraperitoneally (IP)
injected antigens is poorly understood in teleosts. We have recently shown prolonged presence of IgM
antibody secreting cells (ASCs) in the PerC of Atlantic salmon (Salmo salar) in response to IP infection with
salmonid alphavirus subtype 3 (WtSAV3). Here we extended this study to include both WtSAV3 and
inactivated SAV (InSAV) and a boost group (injected twice with InSAV) to study recall B cell responses.

Presence of total and SAV E2-specific ASCs were assessed using ELISpot assays. The results demonstrated an
increase of total and SAV E2-specific ASCs after injection of live WtSAV3. Both responses were most
pronounced and prolonged in PerC compared to systemic immune sites (head kidney/spleen). In the InSAV
group, ASC responses were in general lower and of shorter duration. A E2-specific serum antibody response
was only detected in the WtSAV3 group. The booster injection of InSAV did not increase neither the total nor
the E2-specific ASCs response in PerC or systemic sites or the E2-specific serum antibody, thus indicating no
recall response. PerC adipose tissue (AT) of WtSAV3 infected fish displayed upregulation of a wide array of
immune related genes including antiviral, antigen presenting, B cell, T cell, co-stimulatory and myeloid cell
markers supporting the notion that PerC AT has an active immunological role in fish.

However, whether the PerC micromilieu supports formation of ASCs or PerC ASCs are trafficked from other
immune sites warrants further investigation.

Page 137
10.2 Fish and shellfish immunology I

Pro-inflammatory and B cell regulating capacities of TWEAK in rainbow trout

(Oncorhynchus mykiss)

Carolina Tafalla, Beatriz Abós, Elena Pérez

Animal Health Research Center (CISA-INIA), Valdeolmos 28130, Spain

[email protected]

TNF-like weak inducer of apoptosis, or TWEAK is a member of the TNF superfamily involved in the regulation
of many biological processes. In mammals, TWEAK has been shown to play a role in some autoimmune or
inflammatory conditions such as rheumatoid arthritis as it is known to regulate inflammation. In teleost fish,
although a few studies have identified homologues to mammalian TWEAK, how these cytokines are
modulated during the immune response or their biological effects have never been investigated.

In the current study, we have studied the transcriptional regulation of two TWEAK orthologs (TWEAK 1 and
2) identified in rainbow trout (Oncorhynchus mykiss) throughout different tissues, in response to parasitic or
viral infections, or in head kidney (HK) leukocytes stimulated with different stimuli. Although the
transcription of both isoforms was modulated when HK leukocytes were exposed to several immune stimuli,
only TWEAK 1 was significantly modulated upon pathogenic exposure.

Thus, we performed a characterization of the functions exerted by this cytokine in HK leukocytes.

Recombinant TWEAK 1 strongly up-regulated the transcription of pro-inflammatory genes and antimicrobial
peptides in HK leukocytes, with differential transcriptional effects in IgM + B cells, IgM- lymphocytes and
myeloid cells. TWEAK 1 also increased B cell survival and increased the number of IgM-secreting cells in HK
leukocyte cultures, provoking a significant up-regulation in the transcription of some isoforms of Blimp1 in
IgM+ sorted B cells. Our results demonstrate that in teleost fish, TWEAK 1 is involved in the response to
different types of pathogens, through the modulation of antimicrobial and pro-inflammatory genes in
different leukocytes subsets. Furthermore, a role for TWEAK as a B cell differentiation factor, not previously
reported in mammals, has been demonstrated in rainbow trout.

Page 138
10.2 Fish and shellfish immunology I

IgM+ and IgT+ B cell traffic to the heart during SAV infection in Atlantic salmon

Anne Bakke1, Håvard Bjørgen1, Erling Olaf Koppang1, Petter Frost2, Sergey Afanasyev3, Preben
Boysen1, Aleksei Krasnov4, Hege Lund1

1Faculty of Veterinary Medicine, Norwegian University of Life Sciences (NMBU), Norway

2MSD Animal Health Innovation AD, Norway
3I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Russia
4Nofima AS, Norway

[email protected]

B cells of teleost fish differentiate in the head kidney and spleen, and either remain in the lymphatic organs
or move to the blood and peripheral tissues. There is limited knowledge about piscine B cell traffic to sites
of vaccination and infection and their functional roles at these sites.

In this work, we examined the traffic of B cells in Atlantic salmon challenged with salmonid alphavirus (SAV).

In situ hybridization (RNAScope) showed increased numbers of immunoglobin (Ig)M+ and IgT+ B cells in the
heart in response to SAV challenge, with IgM+ B cells being most abundant. An increase in IgT+ B cells was
also evident, indicating a role of IgT+ B cells in nonmucosal tissues and systemic viral infections. After
infection, B cells were mainly found in the stratum spongiosum of the cardiac ventricle, colocalizing with
virus-infected myocardial-like cells. From sequencing the variable region of IgM in the main target organ
(heart) and comparing it with a major lymphatic organ (the spleen), co-occurrence in antibody repertoires
indicated a transfer of B cells from the spleen to the heart, as well as earlier recruitment of B cells to the
heart in vaccinated fish compared to unvaccinated. Transcriptome analyses performed at 21 days post-
challenge suggested higher expression of multiple mediators of inflammation and lymphocyte-specific genes
in unvaccinated compared to vaccinated fish, in parallel with a massive suppression of genes involved in
heart contraction, metabolism, and development of tissue. The adaptive responses to SAV in vaccinated
salmon appeared to alleviate the disease.

Altogether, these results suggest that migration of B cells from lymphatic organs to sites of infection is an
important part of the adaptive immune response of Atlantic salmon to SAV.

We thank VESO Vikan for conducting the challenge experiments and Tina Søfteland at MSD Animal Health
for organizing and providing samples for the animal study. We also thank Fish Vet group Norway for providing
fixation of the tissue samples and Marianne Hansen (Nofima AS) and Anne Clementine Linde (Faculty of
Veterinary Medicine, NMBU) for their laboratory assistance. We also thank PatoGen for providing RNAlater
for tissue sampling.

Page 139
10.2 Fish and shellfish immunology I

Atlantic salmon pre-smolt survivors of Renibacterium salmoninarum infection show

inhibited cell-mediated adaptive immune response and a higher risk of death during the
late stage of infection at lower water temperatures

Andrea Peña1, Marco Rozas-Serri1,2, Rodolfo Correa1, Ricardo Ildefonso1, Jorge Vásquez1, Victoria Jaramillo1,
Romina Walker1, Francisco Schwerter3

1LaboratorioPathovet, Switzerland
2Newenko Group SpA, Chile
3Hendrix Genetics Aquaculture S.A, Chile

[email protected]

Bacterial kidney disease (BKD) caused by the facultative intracellular bacteria Renibacterium salmoninarum
is worldwide spread causing substantial economic losses for the salmon aquaculture industry. The search of
BKD resistant fish phenotypes is of great interest for salmon breeders. The objective of this study was to
investigate the pathophysiological response and gene expression profiles related to the immune response at
different water temperatures and to identify the best immunopathological biomarkers to define a phenotype
of resistance to BKD.

Methodology
An intraperitoneal challenge was conducted in two identical independent systems at 11 °C and 15 °C,
respectively. Each system considered three replicate tanks with fish inoculated with R. salmoninarum and a
control tank with fish inoculated with sterile saline (0.9%). Abundance of the major soluble antigen (msa,
p57) transcripts of the bacteria was determined in head kidney to evaluate its proliferation. Hemathological
and biochemical blood analysis as well as histopathological examination were performed. Gene expression
profiles related to immune response was determined in head-kidney samples of survivors of each group at
the endtime sampling point.

Results
The abundance of msa transcripts of R. salmoninarum in the head kidney was significantly higher in infected
fish at 11 °C. R. salmoninarum induced significantly more severe kidney lesions, anemia and impaired renal
function at 11 °C. In addition, the expression pattern of the genes related to humoral and cell-mediated
immune responses in infected fish at 11 ° and 15 °C was very similar, although R. salmoninarum induced a
significantly greater downregulation of the adaptive immune response genes at the lower water
temperature. These results could be due to a suppressed host response directly related to the lowest water
temperature and/or associated with a delayed host response related to the lowest water temperature.

Conclusions
Although no significant differences in survival rate were observed, fish infected at the lowest temperature
showed a higher probability of death and delayed the mortality curve during the late stage of infection (35
days after infection). Thirty-three immunopathological biomarkers were identified for potential use in the
search for a resistance phenotype for BKD, and eight were genes related specifically to the adaptive cell-
mediated immune response.

Page 140
10.3 Diagnostics I

Microanalysis of bile pigments in fish blood

Paola Sist1, Antonella Bandiera1, Paola Pelizzo1, Luisa Donini1, Nevenka Medic1, Federica Tramer1, Marco
Stebel1, Chiara Bulfon2, Donatella Volpatti2, Jerko Hrabar3, Ivona Mladineo3, Emilio Tibaldi2,
Sabina Passamonti1

1 Dept. of Life Sciences, University of Trieste, Italy

2 Dept. of Agricultural, Food, Environmental and Animal Sciences, University of Udine, Italy
3 Institute of Oceanography and Fisheries, Croatia

[email protected]

In vertebrates, heme released from the heme proteins is oxidized by heme oxygenase to release biliverdin
(BV), which is reduced by biliverdin reductase to bilirubin (BR). In the liver, BR is conjugated to bilirubin
glucuronide (BRG) and excreted into the bile. BV can be excreted through the bile or, in fish, through the gills
and skin. In some fish species, BV causes the blue-green color of the blood and color changes associated with
sexual dimorphism.

The bile pigments BV and BR act as a redox couple that can scavenge oxygen free radicals. In farmed fish,
infection or contamination can trigger an oxidative stress response leading to hemolysis and liver failure,
with abnormal BV, BR and BRG levels in the blood. In addition, starvation increases the BR/BR ratio in some
fish species. Therefore, analysis of bile pigment levels in the blood of farmed fish can integrate
multiparameter monitoring of fish wellness.

Routine analysis of BR and BRG is performed using automated methods, which, however, cannot detect BV.
We addressed this unmet diagnostic need by developing a high-throughput fluorometric method based on
high-affinity BR binding by the bifunctional recombinant protein HUG. This fusion protein, which can be
readily purified by exploiting the thermoreactive properties of the elastin-like scaffold, binds bilirubin via the
UnaG domain. The stable and very specific complex formation results in a strong fluorescent signal. The assay
of BR in whole blood, serum or plasma can be performed in microtiter plates using a multiplate fluorescence
reader. Both BV and BRG can be enzymatically converted to BR, allowing their simultaneous detection. This
analysis requires <0.1 mL of blood collected from the tail vein of the fish without sacrificing the donor.

We applied this method to sea breams reared at Friskina Farm (Croatia) and to sea breams and sea bass
reared at the experimental farm of the University of Udine (Italy). In all samples studied, BV was significantly
higher than BR or BRG, and differences were observed according to fish species, feed treatment and rearing
location.

This approach can help filling knowledge gaps in the pathophysiology of biliary pigments in fish species.

Funding
INTERREG V-A Italy-Croatia CBC 2014-2020 (AdriAquaNet project, ID ID10045161). We dedicate this work to
the premature loss of Andrea Novelli, manager and administrator of Azienda Agricola Ittica Caldoli srl (Italy),
partner 9 of AdriAquaNet project. This work has been reported in Luisa Donini’s Master thesis (University of
Trieste, 2021).

Page 141
10.3 Diagnostics I

Biolayer interferometry is a fast, highly reproducible method for quantifying specific IgM
response in fish serum and measuring its avidity for antigen in a single-step

Angus Li, Richard Harris, Bryan Fry, Andrew Barnes

School of Biological Sciences, The University Of Queensland, Australia

[email protected]

Quantification of specific antibody responses is critical in determining activation of MHCII-dependent

immune memory and is generally performed by enzyme-linked immunosorbent assay (ELISA). Antibody
avidity to particular antigen may also provide valuable information about the quality of the adaptive immune
response following vaccination.

Avidity can be determined by chaotropic elution ELISA, pre-absorption ELISA, or surface plasmon resonance
(SPR), although polymeric antibodies such as IgM are problematic for SPR. ELISA-based assays are very time
consuming, require secondary antibody reagents, and are poorly repeatable. Here we demonstrate that
biolayer interferometry (BLI) using an Octet HTX instrument can robustly and reproducibly quantify and
determine avidity of IgM for an antigen directly from fish serum in a single step.

We collected sera from giant grouper (Epinephelus lanceolatus) that had been vaccinated with the hapten
2,4-dinitrophenyl conjugated with keyhole limpet hemocyanin (DNP-KLH) and from control fish injected with
phosphate buffered saline. The specific IgM in the serum and its avidity for DNP were quantified via ELISA
and BLI. The quantity and avidity data generated by BLI was sensitive and precise when comparing to those
generated by ELISA. The wet-lab preparation and machine running time for BLI was 3-5 times faster than
ELISA to generate the same amount of data. The ELISA inter-plate variation significantly affected
reproducibility while BLI was consistent and repeatable between samples and plates.

Indeed, the BLI data consistency indicated that technical triplicates were redundant for BLI. Biological
replication alone was efficient to elucidate the effect of treatments. However, BLI required a lower serum
dilution than ELISA, and thus more serum was required to produce high resolution data. BLI is an extremely
high-throughput assay, providing teleost serum IgM quantification and avidity data as a single-step agile
alternative to ELISA.

Page 142
10.3 Diagnostics I

Set up of a rapid identification method for nervous necrosis virus (NNV) variants circulating
in Southern Europe

Sara Ciulli1, Francesca Errani1, Enrico Volpe1, Enrique Riera Ferrer2, Monica Caffara1, Francesc Padros2,
Andrea Gustinelli1, Marialetizia Fioravanti1,

1Department of Veterinary Medical Sciences, University of Bologna, Italy

2Facultat de Veterinària, Universitat Autònoma de Barcelona, Spain

[email protected]

Nervous Necrosis Virus (NNV) represents one of the most threatening pathogens for Mediterranean
aquaculture due to its economic impact in the finfish industry. Currently, the RGNNV genotype and the
RGNNV/SJNNV reassortant are the most widespread strains in Southern Europe with a more limited diffusion
of the SJNNV genotype and the SJNNV/RGNNV reassortant. So far variants identification is based on genome
amplification and sequencing, however this approach is quite time consuming. In recent years, multiplex PCR
assays have gained popularity due to its convenience in terms of cost and time.

The aim of this study was to develop and validate a one-step multiplex RT-PCR (mRT-PCR) as a rapid
diagnostic technique to detect NNV and to discriminate between RGNNV and SJNNV genotypes from their
reassortant strains RGNNV/SJNNV and SJNNV/RGNNV.

Methodology
In order to design optimal primer pairs, NNV genome alignments were generated and primer candidates
were selected according to their position within each genotype’s RNA sequence, melting temperature (Tm)
and generated amplicon size. New primers were combined together and with other seven primers already
described in literature. The primers were tested and the mRT-PCR assay optimization was conducted with
reference strains previously propagated on SSN-1 cell lines. Analytical sensitivity and specificity, and
repeatability were also evaluated. Finally, the method was applied to European seabass (Dicentrarchus
labrax) and gilthead sea bream (Sparus aurata) brain samples collected during disease outbreaks.

Results and Conclusions

The mRT-PCR method efficiently identified the different NNV variants represented by the reference strains
for RGNNV and SJNNV genotypes, and RGNNV/SJNNV and SJNNV/RGNNV reassortant strains producing one
or two specific bands for each of them. The method showed a limit of detection ranging from 10 2.3 to 103.4
TCID50 ml-1 with the highest sensitivity for the RGNNV/SJNNV reassortant strain. Analytical specificity was
tested towards several sea bass and bream pathogens (Lymphocystivirus, Vibrio anguillarum,
Photobacterium damselae subsp. piscicida, Aeromonas veronii) and showed no cross reactivity. The method
resulted to be precise and robust providing overlapping results in intra- and interassay repeatability tests.
In conclusion, the developed mRT-PCR assay has revealed to be an accurate and rapid method for early
identification of NNV variants circulating in Southern Europe.

Funding
PerformFISH EU H2020 project (727610).

Page 143
10.3 Diagnostics I

Development of siderophore-based fluorescent probes for the study and detection of

Aeromonas salmonicida

Diego Rey-Varela1, Javier Cisneros-Sureda2, Jaime Rodríguez2, Miguel Balado1, Carlos Jiménez2, Manuel L
Lemos1

1University of Santiago de Compostela, Departamento de Microbioloxía y Parasitoloxía, Instituto de

Acuicultura, Spain
2University of A Coruña, Departamento de Química Fundamental, Centro de Investigacións Científicas

Avanzadas (CICA), Spain

[email protected]

Aeromonas salmonicida subsp. salmonicida is the causal agent of furunculosis in salmonid and non-salmonid
fish. It produces two catechol siderophores, amonabactin and acinetobactin, which allow the multiplication
of the bacterium within the host. We have previously elucidated the synthesis and transport mechanisms of
both siderophores. In recent years, it was demonstrated that the attachment of different cargos to
siderophore analogues allow the delivery of these compounds to the target bacteria through the siderophore
receptors. We have demonstrated that the amonabactin transporter FstC could efficiently internalize
amonabactin analogues that could be used to deliver attached compounds into A. salmonicida cells.

Methodology
Three different fluorescent conjugates were synthesized based on biscatechol amonabactin analogues. The
conjugates differed in the analogue linker length, in the position where the fluorophore was attached, and
in the fluorophore bound.

Results
The biological activity of the synthetic conjugates indicated that to be transported, the bond between the
analog and the cargo had to mimic the bond of Phe or Trp present in the natural siderophore. Thus, the first
conjugate, which contained a fluorophore NBD (4-nitro-benzo[1,2,5]oxadiazole) linked to the free amino
group of the biscatechol amonabactin analogue, did not display siderophore activity. Although a second
conjugate, with the fluorophore NBD linked to the carboxylic acid functionality showed siderophore activity,
did not show any fluorescence because the iron complexed by the siderophore quenched its fluorescence.
Finally, the third conjugate, containing the fluorophore sulforhodamine B, showed promising results as a
probe since it could be efficiently and specifically transported into A. salmonicida cells through the
amonabactin transporter FstC. The red fluorescence was easily observed by fluorescence microscopy. We
also demonstrated that the probe is specific for species of the genus Aeromonas, since other bacterial fish
pathogens did not transport the probe.

Conclusions
This probe could be useful in future works for a detailed study of siderophore biochemistry, to follow
Aeromonas infections in vivo, as well as for the detection of Aeromonas spp. in fish farms samples to
anticipate disease outbreaks. The results also confirmed the utility of siderophore analogues to develop
novel compounds with biotechnological applications.

Page 144
10.3 Diagnostics I

Development of a real-time PCR assay for the identification of the fish pathogen Pasteurella
skyensis

Catherine Whitby, Rebecca McIntosh, Alison Garden, Eann Munro

Marine Scotland Science, Scotland, UK

[email protected]

Members of the genus Pasteurella are gram-negative, facultative anaerobic bacteria causing the disease
Pasteurellosis in a wide variety of species. In 2002, a new species was formally identified in farmed Scottish
Atlantic salmon (Salmo salar L.) and termed Pasteurella skyensis following a series of disease outbreaks
around the Isle of Skye.

A new quantitative real-time polymerase chain reaction (qPCR) TaqMan probe assay has been developed to
rapidly and specifically detect and quantify P. skyensis from cultured bacteria and salmonid tissue. The qPCR
is able to detect multiple strains of P. skyensis and does not produce a signal when tested against 23 common
fish bacteria. The assay has a high average efficiency of 97.81% and the limit of detection was estimated to
0.656pg uL-1 total DNA and 0.407fg uL-1 bacteria DNA. It was shown to be more sensitive than traditional
bacteria culturing techniques.

Sequencing and phylogenetic analysis of the 16S rRNA and rpoB genes of P. skyensis isolates within the
Marine Scotland culture collection demonstrates the presence of multiple strains of P. skyensis within the
Scottish A. salmon aquaculture industry.

In conclusion, we have developed a rapid, highly specific and sensitive qPCR assay which can be used to
monitor and confirm P. skyensis infections from farmed populations.

Page 145
11.1 Bivalve and crustacean diseases II

Increased resistance against marteiliosis in the cockle Cerastoderma edule population of

the inner area of the Ría de Arousa (Galicia NW Spain) through natural selection.

Antonio Villalba1, David Iglesias1, Damián Costas2, J. Carlos Mariño3, Marta Domínguez4, Eva Cacabelos4,
Emilio Abella5, Marina Pampín6, Paulino Martínez6, Mónica Incera7, Rosa Fernández7

1Centro de Investigacións Mariñas (CIMA), Consellería do Mar da Xunta de Galicia, Spain

2Estación de Ciencias Mariñas de Toralla (ECIMAT), Centro de Investigación Mariña, Universidade de Vigo,
Spain
3Confraría de Pescadores “San Antonio” de Cambados, Spain
4Hydrosphere S.L., Spain
5Confraría de Pescadores “A Pastoriza” de Vilanova de Arousa, Spain
6ACUIGEN, Departamento de Zoología, Genética y Antropología Física, Universidad de Santiago de

Compostela, Spain
7Centro Tecnológico del Mar – Fundación CETMAR, Spain

[email protected]

Infection of cockles Cerastoderma edule with the protistan Marteilia cochillia was first detected in Galicia in
2012; it caused the cockle fishery collapse in the Rías de Arousa, Pontevedra and Vigo. Surveillance of
marteiliosis in the inner area of the Ría de Arousa showed that its prevalence and the cockle mortality
trended to decrease since 2017. Additionally, cockles collected from Noia (Ría de Muros-Noia), where
marteiliosis outbreaks have never been detected, and transplanted into the shellfish bed of Lombos do Ulla
(inner area of Ría de Arousa) in 2017 and 2018, showed higher marteiliosis prevalence and mortality than
the cockles naturally recruited in Lombos do Ulla. These results suggested the hypothesis that marteiliosis-
resistance was being enhanced in the inner area of the Ría de Arousa through natural selection.

A study was launched to (1) test the above mentioned hypothesis, (2) assay every stage in the process of
restoration in marteiliosis-affected cockle beds, involving two shell-fisher associations, and (3) validate fifty
candidate genetic markers of marteiliosis-resistance identified through population genomic and
transcriptomic approaches, which can be used in marker-assisted selection programmes. Adult cockles were
collected from Lombos do Ulla (hypothetically, resistant stock) and from Noia (susceptible stock) in May 2020
and transported to the hatchery facilities of ECIMAT, to be used as broodstock. Various batches of cockle
seed obtained from each stock were transferred to a raft in the Ría de Arousa for outdoor pre-growing in
August 2020. Two months later, batches of cockle seed from each stock were deployed in two shellfish beds
of the Ría de Arousa, namely Ariño and O Sarrido, where mortality and prevalence and severity of marteiliosis
are being surveyed.

Cockles of the susceptible stock have been heavily affected by marteiliosis (prevalence >90%), reaching very
high cumulative mortality (>90%) in each bed, in March and April 2021, respectively, whereas cockles of the
hypothetically resistant stock have been very lightly affected by marteiliosis (prevalence <5%) and show
significantly higher survival in both beds. These results confirm the marteiliosis-resistance of the stock from
the inner area of Ría de Arousa and its potential for restoring marteiliosis-affected beds.

Page 146
11.1 Bivalve and crustacean diseases II

The haemocytes of the cockle Cerastoderma edule: types, immune abilities and influence
of environment and pathological conditions on the haemocyte counts

Antonio Villalba, Silvia Lorenzo Abalde, Asunción Cao, David Iglesias, María J. Carballal

Centro de Investigacións Mariñas (CIMA), Consellería do Mar da Xunta de Galicia, Spain

[email protected]

A study was performed to get knowledge on the cockle Cerastoderma edule immune system, with emphasis
on the main effector cells, the haemocytes. The shellfish bed of Noia (ria of Muros-Noia, Galicia, NW Spain)
was sampled monthly for two years; the haemolymph of the cockles was analysed to estimate the total
haemocyte count (THC) and the differential haemocyte count (DHC). Furthermore, health condition of those
cockles was analysed with histology and the influence of the most serious pathological conditions on THC
and DHC was assessed. The protistan Marteilia cochillia is highly pathogenic for cockles; given that
marteiliosis outbreaks have never been detected in the ria of Muros-Noia, cockles from Noia were
transplanted in Lombos do Ulla (ria of Arousa), a bed heavily affected by marteiliosis, and similar sampling
and analyses were performed until all translocated cockles had died, after 8 months.

The THC ranged from 1.9 x 10⁵ to 9.7 x 10⁶ cells/ml, with the 95% CI from 1.77 to 1.95 x 10⁶ cells/ml. It was
correlated with seawater temperature, thus it showed a seasonal pattern of variation, with minima in
winter–early spring and maxima in summer-early autumn. Six haemocyte types were identified: acidophilic,
basophilic and mixed granulocytes, large hyalinocytes, haemocytes with a large vacuole (HLV), and
haemoblasts. Their 95% CI of relative abundance was 40.7 – 44.2%, 13.0 – 14.6%, 2.0 – 3.0%, 27.6 – 29.7%,
9.5 – 10.5% and 2.0 – 2.4%, respectively. The dominant type, acidophilic granulocyte, showed relative
abundance maxima in autumn and minima in summer. Advanced infection with M. cochillia was associated
with decrease of the THC, marked decrease of the relative abundance of acidophilic and basophilic
granulocytes and marked increase of haemoblasts. Infestation with trematode sporocysts and inflammatory
reactions were also associated with changes in DHC.

Functional differences among haemocyte types were assessed using flow cytometry. Granulocytes showed
the highest ability to phagocytose bacteria, while the hyalinocytes and the haemoblast showed very low
ability and the HLV showed intermediate ability. Every type produced reactive oxygen species, without
significant differences among types. Lysosomal and non-specific esterase contents were higher in
granulocytes and lower in haemoblasts.

Page 147
11.1 Bivalve and crustacean diseases II

A survey of parasites infesting two populations of the freshwater mussel unio crassus

Eva Lewisch1, Farah Arnold1, Hans-Peter Fuehrer2, Josef Harl3, Ursula Reichart4, Stephan Handschuh4, Frankie
Thielen5, Mansour El-Matbouli1

1ClinicalDivision of Fish Medicine, University of Veterinary Medicine, Austria

2Institute of Parasitology, University of Veterinary Medicine, Austria
3Institute of Pathology, University of Veterinary Medicine, Austria
4VetCORE Facility for Research, University of Veterinary Medicine, Austria
5natur & ëmwelt/ Fondation Hëllef fir d’Natur, Luxembourg

[email protected]

The thick shelled river mussel Unio crassus (Philipsson, 1788) is native to many European habitats with
declining populations (Zettler & Jueg 2007). The knowledge of parasitic infestations in these mussels is
scarce. This study aims to identify parasites of two populations of U. crassus.

Methodology
Thirty specimens of Unio crassus were collected from the River Sauer and the River Our (Luxembourg) in May
2019. The mussels were opened with sterile scalpel blades by severing the adductor muscles and
macroscopically visible parasites were recorded. Small tissue pieces (0.5 cm x 0.5 cm) of gills, foot, adductor
muscles, mantle, kidney, digestive gland, gastro intestinal tract and the gonads were transferred separately
to petri dishes. The tissues were examined by stereo- and a light- microscopy and all findings were photo-
documented. Parasite identification was based on morphological criteria, and parasites were counted per
field of view, as required. For molecular genetic identification, DNA was extracted and sections of the nuclear
18S ribosomal RNA gene (Ahuir Baraja et al. 2015) and the mitochondrial cytochrome C oxidase subunit I
gene (COI) (Folmer et al. 1994) were amplified by PCR and sequenced.

Results
In both populations of U. crassus, only few parasites of the genera Trichodina and Conchophthirus were found
on gills, foot and mantle. In the river Our, 46.7 % of the mussels were infested with freshwater mites of the
genus Unionicola, whereas in the river Sauer the prevalence was 93.3%. Infestation sites were the mantel and
the gills. The mites were described morphologically and by DNA barcoding and proposed as a new species U.
sauerensis sp. nov. (Lewisch et al. 2021). In U. crassus from the river Sauer, the digenean Rhipidocotyle sp. was
identified in the ovary of three specimens, whereas this parasite was not detected in mussels of the river Our.

Conclusions
Our results give insights in the infestation of two populations of U. crassus with parasites. In particular, a
different prevalence of water mites of the genus Unionicola and of the bucephalid digenean Rhipidocotyle
sp., which might have detrimental effects on the host and the population justify further investigations.

Funding
The authors would like to thank Michel Frisch, for collecting the mussels from the Rivers Sauer and Our. Parts
of the study were funded by the project “Restoration of Unio crassus rivers in the Luxemburgish Ardennes
LIFE11 NAT/LU/857” and by the Austrian Federal Ministry of Education, Science and Research via an ABOL
(Austrian barcode of Life; http://www.abol.ac.at) associated project within the framework of the
“Hochschulraum-Strukturmittel” Funds. This research was supported using resources of the VetCore Facility
(Imaging) of the University of Veterinary Medicine Vienna.

Page 148
11.1 Bivalve and crustacean diseases II

Multi-pathogen infection and multi-factorial pathogenesis in Pinna nobilis driving to mass

mortality events: A consensus report
Francesca Carella1, Dusan Palic2, Tomislav Saric3, Ivan Zupan3, Bartolomeo Gorgoglione4, Patricia Prado5, Karl
Andree5, Ioannis Giantsis6, Basile Michaelidis7, Athanasios Lattos7, John Theodorou6, Juan Barja Perez8,
Sergio Rodriguez8, Fabio Scarpa9, Marco Casu9, Elisabetta Antuonfermo9, Daria Sanna10, Domenico Otranto11,
Rossella Panarese11, Carmelo Iaria12, Fabio Marino12, Gionata De Vico1
[email protected]

1Department of Biology, University of Naples Federico II, Italy

2Department of Fish Diseases and Fisheries Biology, Faculty of Veterinary Medicine, Ludwig-Maximilians-
University Munich, Germany
3Department of Ecology, Agronomy and Aquaculture, University of Zadar, Croatia
4Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State

University, USA
5IRTA-Sant Carles de la Rapita, Spain
6Department of Animal Science, Faculty of Agricultural Sciences, University of Western Macedonia, Greece
7Department of Zoology, School of Biology, Faculty of Science, Aristotle University of Thessaloniki, Greece
8Department of Microbiology and Parasitology, University of Santiago de Compostela, Spain
9Department of Veterinary Medicine, University of Sassari, Italy
10Department of Biomedical Sciences, University of Sassari, Italy
11Department of Veterinary Medicine, University of Bari Valenzano, Italy
12Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Italy

The noble pen shell Pinna nobilis,one of the largest native bivalves in the Mediterranean Sea, is currently facing
extinction due to ongoing Mass Mortality Events (MMEs) that started in 2016. Initial diagnostic investigations
focused on a novel protozoan parasite, Haplosporidium pinnae. Subsequent studies described polymicrobial
infections involved in large MMEs, with or without H. pinnae.

Methodology
Moribund specimens collected during MMEs in the Tyrrhenian Sea (Campania, Italy) showed a systemic
syndrome associated to the presence of the Mycobacterium simiae complex. Further reports published by
research groups from other Mediterranean coastal areas confirmed the presence of both H. pinnae and
mycobacterial pathogens in multiple MMEs, also adding the concomitant occurrence of other pathogens like
Vibrio mediterranei and other Vibrio species. It was recently observed that H. pinnae is not an exclusive pathogen
of P. nobilis, as it can infect other marine species. Therefore, the initial hypothesis of a single protozoan pathogen
causing the extinction of P. nobilis populations in the Mediterranean Sea needs to be reconsidered in the light of
recent evidence and new hypothesis, supported by standardized investigation protocols.

Results and Conclusions

Accurate diagnosis is the primary tool to support authorities in regulatory actions to prevent, control and possibly
eradicate a disease outbreak. Such reliable diagnostic process usually requires the expertise of veterinary and
para-veterinary pathologists, able to evaluate all known factors simultaneously from clinical signs to
environmental conditions. In this context, there is an urgent need for scientific and environmental protection
communities to agree on research approach and conservation priorities. Therefore, all authors joining this
consensus work aim to use this growing body of the science-based clinical, diagnostics, and laboratory evidence
to support the hypotheses that P. nobilis MMEs involve a complex pathogenesis, caused by polymicrobial
infections in conjunction with abiotic factors. This approach will serve as the foundation for future studies aimed
at preserving endangered populations of native bivalves.

Page 149
11.1 Bivalve and crustacean diseases II

Pacific oysters in Danish waters – screening for pathogens

Lone Madsen1, Pernille Nielsen1, Camille Saurel2, Pedro S. Freitas2

1DTU Aqua (Technical University of Denmark, National Institute of Aquatic Resources), Denmark
2DTU Aqua, Nykøbing Mors, Denmark

[email protected]

Pacific oyster (Crassostrea gigas) is an invasive species in Denmark, but the proportion of Pacific oysters has
for the last decades increased in Danish waters and is now at a level, where it is of interest for the commercial
fisheries. In other countries with Pacific oyster stocks, diseases caused by oyster herpesvirus (OsHV-1) and
the bacterium Vibrio aestuarianus have resulted in mortalities in affected batches. In Denmark, the European
flat oyster (Ostrea edulis) in the Limfjorden is affected by Bonamia ostreae, a parasite found in the population
in this area for the last 8 years, and in the last few of those years also in connection with mortalities in some
of the affected batches. This parasite does not normally give rise to disease in Pacific oysters. Another
notifiable parasite Marteilia refringens has not been found in the flat oysters in the Limfjorden. The intention
was to screen Pacific oysters for the mentioned pathogens, and to investigate if Pacific oysters of the area are
carrying Bonamia parasites and thereby could spread the disease in the Limfjorden. Pacific oysters are also
found in larger and/or persistent populations in two other areas, the Wadden Sea and the Isefjord, and
samples from batches of Pacific oysters in these two areas were also investigated for the mentioned
pathogens.

Methodology
Molecular methods were used for screening of the oysters samples; a duplex Real Time PCR method for
Bonamia sp. and Marteilia refringens and Real Time PCR methods for OsHV-1 (EURL mollusc diseases SOP
2011) and Vibrio aesturianus (EURL mollusc diseases SOP, 2021), respectively.

Results
The screening is still ongoing, but until now, none of the pathogens has been found in Pacific oysters, neither
from the Wadden Sea, Isefjord nor Limfjorden. Pacific oyster samples taken at sites harbouring flat oysters
with Bonamia have not been found to be Bonamia-positive.

Conclusions
Currently, there is no indications of OsHV-1 or Vibrio aestuarianus in the Danish Pacific oyster populations.
Moreover, it does not seem that the Pacific oysters in the Limfjorden are drivers for Bonamia disease
outbreaks among flat oysters.

Page 150
11.1 Bivalve and crustacean diseases II

A survey of oyster pathogens in the compartments in the productions zones of the Ebro
Delta, Spain

Dolors Furones1, Margarita Fernandez1, Faiz Ur Rahman1,2, Noelia Carrasco1, Karl B. Andree1

1IRTA,Ctra. Poble Nou. Km 5.5, Spain

2Unitof Microbiology, Department of Basic Health Sciences, Faculty of Medicine and Health Sciences, IISPV,
Universitat Rovira i Virgili, Spain

[email protected]

In the Ebro Delta, recurrent mortalities are reported in oysters in springtime. In 2007, the C. gigas spat
mortalities increased drastically (80%) and have been occurring since in spring and autumn coinciding with
the water temperature reaching 16 °C. OsHV-1 μvar was first described in Ebro Delta in 2008. More recently,
detection of Vibrio spp. has been associated with mortalities among adult oysters.

Three-years of data are presented to assist the analysis of recurrent Pacific oyster mortalities (C. gigas)
related to OsHV-1 and bacterial pathogens Vibrio aestuarianus and Vibrio splendidus in the Ebro Delta, to
understand how different environmental drivers might be acting to increase mortality and to better
understand the epidemiology of these pathogens. Concurrently to bivalve sampling, water, sediment,
phytoplankton, zooplankton, biofilm, and micro invertebrates were collected following the harmonized
sampling approach.

Among the samples collected only during mortality events, 24% each of biofilm and invertebrate samples,
while a few of plankton samples were PCR positive for OsHV-1. All sediment samples were found to be
negative for OsHV-1.

Results obtained for Vibrio spp. were less consistent than those from OsHV-1. All biofilm samples analyzed
for V. aestuarianus were negative. In contrast, 40% of the samples were positive for V. splendidus coinciding
with the positives for OsHV-1 in spring and autumn. Among plankton samples, V. aestuarianus was detected
in one sample from May (1/7) and V. splendidus was detected in 5 samples out of 7 both from spring and
winter. From invertebrate samples, only V. splendidus was detected in April samples. All sediment samples
were negative for V. aestuarianus. V. splendidus was detected in 33.3% of the samples, mostly collected
during mortality events.

The results obtained demonstrated that testing environmental compartment samples during mortality
events was, to some degree, a proxy for the presence of OsHV-1 in oysters. The detection of OsHV-1 during
major mortality events tended to be positive only when mortality was above 30% but with low abundance.
This work provides an interesting set of preliminary results on Vibrio prevalence in C. gigas during recurrent
unexplained mortality episodes and, on their presence in different environmental compartments.

Funding
This project has received funding from the European Union’s Horizon 2020 research and innovation program
under the Marie Skłodowska-Curie Grant No. 713679; the Universitat Rovira i Virgili (URV); VIVALDI-
Preventing and mitigating farmed bivalve diseases (2016-2020) under H2020-EU.3.2, Grant Agreement ID:
678589; by the European Maritime and Fisheries Fund (EMFF) and the Directorate for Fisheries & Maritime
Affairs (Government of Catalonia); FEMP FANGAR 18-20 ARP029/18/00008; and by a 2017 INIA grant for
project EMERGER (E-RTA2015-00004-00-00).

Page 151
11.2 Fish and shellfish immunology II

Expression of putative Fc receptors in different immune cell types of the rainbow trout
Oncorhynchus mykiss

Jovana Majstorovic1,2, Jiří Kyslík1,2, Justin Tze Ho Chan1, Astrid Holzer1, Tomas Korytář1,3

1Instituteof Parasitology, Biology Centre of the Czech Academy of Sciences, Czech Republic
2Faculty of Science, University of South Bohemia, Czech Republic
3Faculty of Fisheries and Protection of Waters, University of South Bohemia, Czech Republic

[email protected]

In all vertebrates, including teleost fishes, adaptive and innate immune responses work synergistically to
protect the host. One key component interacting with both members of the innate and adaptive immune
systems is the antibody, which can target various antigens due to the high variability of the antigen receptors.
An important part of antibody-antigen complexes is the fragment crystallizable (Fc) region of the
immunoglobulin molecule. In mammals, receptors with affinity for these regions (Fc receptors) are found on
certain cells of the immune system, including B cells, myeloid cells, mast cells, and others, and enable
antibody-dependent effector functions. However, in bony fishes, their existence and the heterogeneity of
their expression on various cell types and different tissues remain unknown.

Methodology
In this study, we aimed to fill the gap in the available knowledge and elucidate the expression of selected Fc
receptors in different tissues and cell types of the rainbow trout. To this end, we have evaluated the
expression of FC receptors in the main systemic and mucosal immune organs and individual subpopulation
of blood cells, including the MACS sorted myeloid cells IgM+ B lymphocytes, thrombocytes and erythrocytes.

Results
The obtained data provided new insights on the possible involvement of studied Fc receptors in variety of
tissues including spleen, head kidney, trunk kidney, liver, gills, intestine, adipose tissue, muscle and heart.
Furthermore, we have observed high lineage specificity of FC receptor expression in sorted populations of
leukocytes. Unexpectedly, our data also identified unprecedented expression of FC receptors on
erythrocytes, suggesting their potential role in the FC receptor-mediated binging of Igs.

Conclusion
Overall, the data present first description of lineage specificity of Fc receptor expression in teleost fish and
highlight their specialized roles in the immune system allowing future studies of their effector roles on
individual leukocyte subsets.

Page 152
11.2 Fish and shellfish immunology II

Non-classical MHC I L and U lineage genes expression levels are modulated in response to
Piscine orthoreovirus subtype 1 infection in vivo in Atlantic salmon (Salmo salar)

Agata Teresa Gondek Wyrozemska1, Steingrim Svenning1, Maria K. Dahle1,2, Eva-Stina Edholm1

1 Norwegian College of Fishery Science, faculty of Biosciences, Fisheries & Economics, University of Tromsø,
The Arctic University of Norway, Norway
2 Department of Fish Health, Norwegian Veterinary Institute, Norway

[email protected]

Major histocompatibility Complex class I (MHC I) molecules play an essential role in immune response of all
jawed vertebrates. MHC I can be divided into two groups, classical and non-classical. While the structure and
functions of classical molecules are well known, and consist in presentation of pathogen-derived antigens to
CD8+ T cells, roles of non-classical molecules are in most cases yet unknown. Homologues of mammalian
non-classical genes do not occur in fish due to a variety of genomic events, such as genome replication in
Salmonids. In mammals and amphibians, functional analogues of certain non-classical MHC I genes have
been shown to be important in the differentiation and function of distinct subsets of unconventional T cells,
indicating that a similar mechanism exists in fish. To date, six non-classical MHC lineages (U, Z, S, L, P, and H)
have been identified in Atlantic salmon. The U lineage is represented by multiple non-classical molecules, as
well as a singular classical MHC I (Sasa-UBA). The other five lineages L, S, P, Z, and H are represented by non-
classical MHC I exclusively.

Methodology
Substantial number of live animals and animal derived tissues were used in the experiment. Housing and
husbandry of fish was held at Kårvika Research station – UiT. Animals were i.p. with Piscine orthoreovirus.
Control with PBS i.p. was included in the experimental setup. Tissue samples were collected at various time
points post infection. Methods used include RNA extraction from homogenised tissues, DNase treatment,
reverse transcription and quantitative PCR, as well as data archiving and analysis.

Results
In this study, we examine the modulation of non-classical MHC I L and U lineage genes, along with classical
Sasa-UBA, in response to a long-term (10 weeks) Piscine orthoreovirus subtype 1 (PRV-1) infection in vivo.
Transcription levels prove to be diverse for distinct non-classical genes with cases of overexpression and
tissue specificity. Moreover, the peak of expression is slightly different for distinct genes.

Conclusions
The results give an affirmation that non-classical L and U lineage genes take part in Atl. salmon immune
response to PRV-1 infection and are a base for further examination of functions.

Page 153
11.2 Fish and shellfish immunology II

Identification of novel and promising biomarkers of inflammation in Atlantic salmon by a

plasma proteomic approach

Hege Lund1, Baojian Sun1, Dino van Dissel2, Ingrid Mo1, Preben Boysen1, Hanne Haslene-Hox2

1Faculty of Veterinary Medicine, Norwegian University of Life Sciences (NMBU), Norway

2SINTEF AS, Department of Biotechnology and Nanomedicine, Norway

[email protected]

Monitoring fish welfare has become a central issue for the fast-growing aquaculture industry, and finding
proper biomarkers of stress, inflammation and infection are prerequisite in addition to existing methods.
In this study, a proteomic approach using mass spectrometry was applied to identify indicators of the acute
response in Atlantic salmon blood plasma by comparing Aeromonas salmonicida subsp. salmonicida infected
fish and non-infected controls.

The antimicrobial proteins cathelicidin (CATH), L-plastin (Plastin-2) and soluble toll-like receptor 5 (TLR5S)
were uniquely or mainly identified in the plasma of infected fish. In addition, five immune-related proteins
showed significantly increased expression in plasma of infected fish: haptoglobin, high affinity
immunoglobulin Fc gamma receptor I (FcγR1, CD64), leucine-rich alpha 2 glycoprotein (LRG1), complement
C4 (C4) and phospholipase A2 inhibitor 31 kDa subunit-like. On the other hand, various fibrinogen
components, and CD209 and CD44 antigen-like molecules decreased in infected fish. Selected biomarkers
were further verified by western blot analysis of plasma and real time PCR of spleen and liver, including
CATH1, CATH2 and L-plastin. A significant increase of L-plastin occurred as early as 24 hours after infection,
and a CATH2 increase was observed from 72 hours in plasma of infected fish. Real time PCR of selected genes
confirmed increased transcription of CATH1 and CATH2. However, L-plastin was not consistently induced in
liver and spleen.

To conclude, an unbiased proteomics approach was successfully applied to find proteins that were
differentially expressed between infected fish and controls. The current study provides valuable protein-
level evidence for the unreviewed salmon proteome. Such in-depth information obtained via LC-MS studies
will undoubtedly improve our understanding of fish immune responses. Following additional validation,
some of these molecules have the potential for utilization as new biomarkers in future monitoring of health
and welfare of farmed salmonids.

Funding
We thank Olav Mjaavatten and Even Birkeland at the Proteomics Unit of the University of Bergen (PROBE)
for performing mass spectrometry analysis, and Grethe Marie Johansen for laboratory assistance.
This study was funded by the Norwegian Seafood Research Fund (FHF, project nr 901462).

Page 154
11.2 Fish and shellfish immunology II

Innate immune-related gene expression profiles in European seabass (Dicentrarchus

labrax) bath challenged with Tenacibaculum maritimum
Inês Ferreira1,2,3,4, Paulo Santos1,2,5, Marina Machado1, Francisco Guardiola1,6, Ana do Vale3,4, Benjamín
Costas1,2
1CIIMAR, University of Porto, Portugal
2ICBAS, University of Porto, Portugal
3Fish Immunology and Vaccinology Group, IBMC, University of Porto, Portugal
4i3S, University of Porto, Portugal
5MARE, ESTM, Polytechnic Institute of Leiria, Portugal
6Department of Cell Biology and Histology, Faculty of Biology, Campus Regional de Excelencia Internacional

“Campus Mare Nostrum”, University of Murcia, Spain

[email protected]

One of the most devastating bacterial diseases, associated with high mortality and economic losses, of wild and
farmed marine fish is tenacibaculosis, caused by Gram-negative bacterium Tenacibaculum maritimum. The
development of effective strategies to prevent and treat tenacibaculosis requires knowledge about the host-
pathogen interactions occurring during infection. In this study, the expression of immune-related genes in
European seabass (Dicentrarchus labrax) in response to infection with Tenacibaculum maritimum was evaluated.

Methodology
For this purpose, a time-course trial was performed, in which groups of seabass (31.9 ± 6.9 g) were bath-
challenged for 2 h in aerated seawater with 5 x 105 CFU/mL-1 T. maritimum (challenged fish) or mock-challenged
with marine broth medium (mock-challenged fish). Non-challenged fish randomly selected from the groups
just before infection were used as controls (time 0). Following 4, 8, 24 and 48 h post-challenge, 8 fish from each
group were randomly selected, euthanized and head-kidney (HK), distal gut and skin collected for total RNA
extraction and subsequent cDNA synthesis. Gene expression was analysed by RT-qPCR and normalized with
40s and ef1b. To determine the severity of the challenge, a lethality trial was performed in parallel, using the
same bacterial inoculum/challenge protocol used for the time-course trial.

Results
Challenge with T. maritimum induced 40% mortality, whereas no mortality occurred after mock-challenge. An
up-regulation of il-1β gene expression was detected in the distal gut and HK at 8 h post-challenge, whereas il-
10 transcripts, were up-regulated at 8 h in the HK and at 24 h in skin and distal gut. The expression of mmp9
gene also increased at 8 and 24 h post-challenge in distal gut and skin, respectively. Moreover, cxcr4 and il-8
transcripts increased at 8 and 24 h in skin whilst in the HK the response was delayed for il-8, with an up-
regulation at 48 h for the challenged fish.

Conclusions
Preliminary data suggest that bath infection by T. maritimum is able to trigger a local immune response at
mucosal tissues (i.e. gut and skin), but also a systemic response (HK). Further work will be required to disclosure
the dynamics and timing established between the local and systemic immune responses.

Funding
This work is partially supported by project BE4AQUAHEALTH (16-02-05-FMP-0013), funded by MAR2020
Operational Programme through FEDER. I. Ferreira, A. do Vale and B. Costas benefited from grants by FCT
(SFRH/BD/147750/2019, L57/2016/CP1355/CT0010 and IF/00197/2015, respectively).

Page 155
11.2 Fish and shellfish immunology II

Poly Lactic-Co-Glycolic Acid (PLGA) microparticles deliver potential as adjuvants and drug
carriers in fish health – the acute immune response of common carp (Cyprinus carpio) to
PLGA particles

Tomas Korytář1,3, Ruth Tamara Montero2, Justin Tze Ho Chan1, Peter Podhorec3, Astrid Sibylle Holzer1

1Instituteof Parasitology, Biology Centre of the Czech Academy of Sciences, Czech Republic
2Friedrich-Loeffler-Institut,Federal Research Institute for Animal Health, Institute of Immunology, Germany
3Faculty of Fisheries, University of South Bohemia, Czech Republic

[email protected]

In mammals, as alternatives to sometimes toxic classical adjuvants such as Freund’s, PLGA particles safely
and effectively deliver pharmaceutical ingredients, with many applications of PLGA approved for clinical use
by the European Medicines Agency. These polymers are also being tested in fish species such as the common
carp (Cyprinus carpio), one of the top ten most produced species in aquaculture. However, in this
evolutionarily distant teleost species, we need to further study the reactivity of PLGA particles before
delivering any active substance. Not only do we need to differentiate the effects of PLGA from those of an
active component but different applications have opposite requirements - whereas immune activation and
particle uptake are desired in vaccine delivery, carriers of therapeutic drugs should remain inert.

Methodology
Microparticles were prepared by a method based on a standard w1/o/w2 principle. Either 10 mg or 0.1 mg
of PLGA particles were intraperitoneally injected into animals with peritoneal influx of leukocyte
subpopulations measured by flow cytometry. The phenotype of recruited cells was profiled by quantitative
polymerase chain reaction against select markers of inflammation or tolerance. We measured
dihydrorhodamine 123 oxidation as an indicator of reactive oxygen species (ROS) production, and observed
phagocytosis of PLGA particles via fluorescence and transmission electron microscopy.

Results
Injection of PLGA particles mimicked zymosan, a yeast-derived pyrogen, and recruited leukocytes in a dose-
dependent manner but with a delayed peak relative to the toll-like receptor 2 agonist. We observed signs of
inflammation including reactive oxygen species (ROS)-producing cells and upregulated expression of tnfa,
il1b and il6a, but these were resolved 96 hours after particle injection. In contrast, LPS-coated particles
mimicked zymosan in kinetics of leukocyte influx, and inflammation remained unresolved by the last
timepoint. Phagocytosis of the smallest particles was directly observed by fluorescence and electron
microscopy.

Conclusions
Our findings indicate that PLGA particles themselves are not harmful short-term. If coated with antigen, they
can serve as vaccine carriers and adjuvants in fishes but rational design of the particles and dose
management will help adapt them for specific purposes and for delivery of different types of drugs.

Page 156
11.2 Fish and shellfish immunology II

Experimental testing of a VLP-based vaccine against Viral Nervous Necrosis in European sea
bass (Dicentrarchus labrax)

Niels Lorenzen1, Sofie Barsøe1, Anna Toffan2, Niccolò Vendramin1, Francesco Pascoli2, Dagoberto
Sepúlveda1, Ansgar Stratmann3, Tobia Pretto2, Andrea Marsella2, Niels Jørgen Olesen1, Jakob Skov1, Kerstin
Skovgaard4, Niels Lorenzen1

1Technical University of Denmark, National Institute of Aquatic Resources (DTU AQUA), Denmark
2Istituto
Zooprofilattico Sperimentale delle Venezie (IZSVe), Italy
3W42 Biotechnology GmbH, Germany
4Technical University of Denmark, Department of Biotechnology and Biomedicine (DTU Bioengineering),

Denmark

[email protected]

European sea bass is one of the main cultured fish species in the Mediterranean. However, the production
is compromised by outbreaks of viral nervous necrosis (VNN), having major negative impact on both
mortality and growth. As part of the EU Horizon2020- funded project “Med Aid” (GA 727315) we have tested
an innovative virus-like particle (VLP) vaccine against VNN, developed in the earlier EU FP7 project
“Targetfish”. The VLP is based on expression of a recombinant viral capsid protein in transformed Pichia
pastoris. This presentation sum up the main results from vaccination trials including evaluation of the
mediated protection and vaccine-induced immune response.

Different vaccination-challenge experiments with European sea bass (5-22 g) have been carried out, the main
three being, (1) a “dose-response” experiment with different concentrations of VLP, (2) a formulation
experiment where the VLP was given with a w/o adjuvant, and (3) a “duration of immunity” experiment
where vaccinated fish were followed for almost 9 months and challenged at 3 and 7.5 months pv. The VLP
vaccine was administered by intraperitoneal injection of a 50 µl volume in all experiments and challenge was
performed by intramuscular (IM) injection or bath with RGNNV grown in SNN-1 or E11 cell cultures. Serum
samples from vaccinated fish were collected in all experiments and antibody reactivity was analyzed by ELISA
and serum neutralization. Tissue samples were collected to investigate the immune response to vaccination
at gene expression level.

The VLP vaccine induced a dose-dependent response of neutralizing antibodies, along with a high level of
specific antibodies detectable in ELISA from 27 days pv. (540 degree days (dd.)) until at least 9 months pv.
(~5000 dd.). The protection was also dose-dependent, with VLP doses as low as 1.25 µg/fish providing
significant protection, and doses of at least 20 µg/fish providing good protection in all experiments (RPS
ranging from 67.2 to 87.2). The protection persisted at least until 7.5 months post vaccination (~4500 dd.).
The results suggest that the vaccine has high potential as a prophylactic tool against VNN-related losses in
farmed sea bass.

Page 157
11.3.1 Diagnostics II

Detection and quantification of Saprolegnia parasitica in aquaculture environments in

Finland

Tiina Korkea-aho1, Satu Viljamaa-Dirks1, Maarit Moisio2, Christine Engblom3, Lotta Landor3, Tom Wiklund3

1Finnish Food Authority, Laboratory and Research Division, Veterinary Bacteriology and Pathology Unit,
Finland
2University of Eastern Finland, Department of Environmental and Biological Sciences, Finland
3Åbo Akademi University, Environmental and Marine Biology, BioCity, Finland

[email protected]

Saprolegniosis in fish is causing large economic losses in aquaculture worldwide and is also a threat to wild
salmonid populations. The taxonomic classification of Saprolegnia genus is constantly evolving, which has
also implications for the development of Saprolegnia species specific diagnostic tools. The objectives of this
study were to determine the most prevalent Saprolegnia species causing infections in Finnish fish farms and
to create a fast and reliable quantitative PCR (qPCR) assay to be used for the detection and quantification of
this Saprolegnia from fish and water samples in aquaculture environments.

Saprolegnia spp. were collected from 19 different fish farms in Finland, nuclear rDNA ITS region sequenced
and used to develop an ITS nucleotide sequence database. A qPCR primers and probe were designed based
on the database to be complement to the ITS region of Saprolegnia parasitica strain most often isolated from
salmonid fish. The qPCR assay was studied for sensitivity and specificity with the sequenced oomycete strains
and water samples from S. parasitica experimental challenge. Furthermore, water samples from the
experimental challenge were used to compare the qPCR with other microbiological detection methods. The
qPCR assay was also compared with microwell plate assay (MWP) to quantify Saprolegnia in a field study
using tank water samples from four different aquaculture facilities and from lake water samples.

The results showed S. parasitica to be the most prevalent oomycete found from salmonid fish (85% of the
isolates) at Finnish fish farms. In the water samples from experimental challenge, the sensitivity of qPCR
assay was 96%, while sensitivity with three microbiological methods used varied between 0 - 29%. No false-
positive S. parasitica results were detected with any of the methods. In the field-based water samples,
numbers of Saprolegnia spores quantified with the MWP correlated highly with Saprolegnia DNA amount.
The amount of Saprolegnia in water samples was the highest when Saprolegnia was detected also from the
fish. The possible applications of the developed qPCR assay to monitor Saprolegnia and usage in risk analysis
in aquaculture environments is discussed.

Funding
Part of the research was conducted with financial support from the European Maritime and Fisheries Fund
Operational Programme for Finland 2014–2020 (no. 87476).

Page 158
11.3.2 Emerging pathogenes

Comparative molecular characterization of novel and known piscine toti-like viruses

Aase B Mikalsen3, Liv Sandlund1, Sunil K Mor2, Vikash K Singh V2, Soumesh Padhi2, Nicholas BD Phelps2, Stian
Nylund1

1Pharmaq Analytiq, Norway

2University
of Minnesota, USA
3Norwegian University of Life Sciences, Norway

[email protected]

Piscine myocarditis virus (PMCV) is a virus well known to the Atlantic salmon farming industry as the cause
of cardiomyopathy syndrome. The virus has been characterized as a toti-like virus due to its homology to
viruses of Totiviridae of the genome encoded polymerase, but also genomic organization of capsid and
polymerase genes. Totiviridae has been well known as a family including viruses infecting unicellular
organisms like fungi and protozoa. In more recent years, viruses characterized as toti-like viruses, have been
found on primarily arthropods, but also in planarians and piscine species. All the toti-like viruses share
phylogenetic similarities to totiviruses; however their genome also includes sequences in either 5’ or 3’ ends
expected to relate to more advanced infection mechanisms in more advanced hosts. In this work, we have
used next generation sequencing (NGS) and discovered three new toti-like viruses in common carp and
bluegill from the USA and in lumpfish from Norway, named common carp toti-like virus 1 (CCTLV-1), bluegill
toti-like virus 1 (BGTLV-1) and Cyclopterus lumpus toti-like virus (CLuTLV), respectively. The genomes of these
viruses have been characterized and compared to the three previously known piscine toti-like viruses, PMCV
found in Atlantic salmon and the two from golden shiner, now named golden shiner toti-like virus 1 and 2
(GSTLV-1 and -2), and to totiviruses and other toti-like viruses.

We found that four of the piscine toti-like viruses had additional gene(s) in the 3’ end of the genome and
that these also clustered phylogenetically based on both capsid and RdRp-genes. This cluster constituted a
distant branch in Totiviridae, and we suggest this should be defined as a separate genus named Pistolvirus,
to reflect piscine toti-like viruses included. Two of the novel viruses shared characteristics with PMCV by
having one additional open reading frame (ORF3) with similar length, and although the similarity of amino
acid sequence between the encoded proteins was low, they all shared characteristics domains in the amino
acid sequence.

Main findings from this collaboration on piscine toti-like viruses will be presented and discussed.

Page 159
11.3.2 Emerging pathogenes

First report of Tenacibaculum piscium isolates recovered from outbreaks in Chilean

salmonids

Ruben Avendaño-Herrera1,2, Anne Berit Olsen3, Mónica Saldarriaga-Cordoba4, Duncan J Colquhoun5, Eric
Duchaud6, Rute Irgang2

[email protected]

1Universidad Andrés Bello, Chile

2Centro FONDAP Interdisciplinary Center for Aquaculture Research (INCAR), Chile
3Section of Research and Aquatic Biosecurity - Norwegian Veterinary Institute, Norway
4CIRENYS - Universidad Bernardo O'Higgins
5Fish Health Research Group - Norwegian Veterinary Institute
6Unité de Virologie et Immunologie Moléculaires - Université Paris-Saclay, UVSQ, INRA

Tenacibaculosis, an ulcer disease produced in marine fish, is the third most important bacterial infection
affecting the Chilean salmon industry. Tenacibaculum dicentrarchi, Tenacibaculum finnmarkense and
Tenacibaculum maritimum have been detected and confirmed as the cause of death of specimens of Atlantic
salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and/or coho salmon (Oncorhynchus kisutch)
farmed in different marine farms located in the Los Lagos, Aysén and Magallanes regions. Following MLSA, a
group of eight isolates (one from coho salmon, three from rainbow trout and four from Atlantic salmon),
clustered together with the recently described T. piscium TNO020T (Olsen et al. 2020), recovered from
Atlantic salmon and corkwing wrasse (Symphodus melops) in Norway.

The present study elucidated the taxonomic status of these Chilean isolates using biochemical, phenotypic
and whole-genome sequence-based analyses. Microbiological analysis demonstrated long rods presenting
tolerance to seawater ranges (30-100% seawater), negative tween hydrolysis, temperature growth range
(4-25 °C) and growth on blood agar supplemented with 1.5% NaCl (w/v) as described for T. piscium NO020T.
All Chilean isolates were found to be extremely closely related to TNO020T (98.70% > ID < 98.55%), ANI
values ≥ 97.88% and isDDH ≥ 88.60% when compared with the type strain TNO020T.

Genome sequences showed a number of interesting features and genes related to possible virulence factors
such as iron acquisition mechanisms and resistance to tetracycline and fluoroquinolones and Multidrug
resistance efflux pumps. Our results confirm the presence and first description of T. piscium isolates
recovered from outbreaks in Chilean salmonid, thus demonstrating that the geographical and host
distributions of this pathogen are much wider than Norway and Atlantic salmon.

Page 160
11.3.3 Nutrition and fish health

Immune gene expression analysis and growth performance in gilthead sea bream and
European sea bass fed bioactive peptides under normal and stress conditions

Volpe Enrico1, Errani Francesca1, Busti Serena1, Parma Luca1, Oterhals Åge2, Aspevik Tone2, Morsiani
Lorenzo1, Gatta Pier Paolo1, Bonaldo Alessio1, Ciulli Sara1

1Department of Veterinary Medical Sciences, University of Bologna Alma Mater Studiorum, Italy
2Nofima, Aquaculture division, Department of Nutrition and feed technology, Norway

[email protected]

The growth of intensive aquaculture leads to increased stressful conditions and health impairment.
Functional feed can contribute to a better growth and fish health boosting resistance to stress and infectious
disease. In particular, aquaculture processing by-products are a rich source of bioactive peptides, molecules
which can improve fish welfare and modulate immunity and inflammatory processes. The aim of this study
was to test immunomodulatory effect of bioactive peptides derived from Atlantic salmon (Salmo salar)
processing by-products in European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata)
reared under normal and after suboptimal condition.

Fish were fed over 58 days with three experimental diets containing different levels of bioactive peptides
(0% BP0, 5% BP5 and 10% BP10) in substitution to fish meal. After the end of the trial, fish were subjected
to suboptimal rearing conditions (temperature 30°C and 70% oxygen saturation) for 8 days. Growth, feed
efficiency parameters and immune response marker genes in liver (Ferritin, Hepcidin, C3) and intestine (IL-
1β, IL-8, IL-10, IFN-1α, Mx protein, TGF-β) were assessed at the end of the feed trial (T1) and after suboptimal
rearing conditions (T2). Data were analysed by a two-way ANOVA and, when significant, by post-hoc
comparison tests.

At the end of the trial no significant differences among diets were observed regarding growth performance
and survival rate in both species. The immune gene analysis showed a wide downregulation of all markers
after the stress condition period showing how stress may affect the ability of fish to express important
immune response factors. In particular, ANOVA analysis showed a statistical relationship between T1 and T2
for IL-8 (p=0.0021), Mx protein (p=0.0036), ferritin (p<0.0001) and C3 (p=0.021). Moreover, significant dose
and time effects were evidenced for IL-1β (p=0.0364). Deepening in the tested diets, fish fed BP5 diet showed
a downregulation of pro-inflammatory genes such as IL-1β and IL-8, and a significant upregulation of the
anti-inflammatory gene IL-10 (p=0.0012). Conversely, fish fed BP10 diet reported an upregulation of IL-1β
and IL-8.

These findings show that bioactive peptides derived from Atlantic salmon processing by-product could
modulate immune gene expression stressing the importance to calibrate their supplementation.

Funding
Novofeed EU Eranet - Marine Biotech project (604814).

Page 161
11.3.3 Nutrition and fish health

Effects of dietary silk nanoparticles on gut inflammation and intestinal absorption in skin
wounded gilthead seabream (Sparus aurata)

Nora Albaladejo Riad1 Cristobal Espinosa1, María Ángeles Esteban Abad1, Guzmán Carissimi2, F. G Díaz
Baños2, Gloria Víllora2

1Immunobiology for aquaculture group, Department of Cell Biology and Histology, Faculty of Biology,
University Of Murcia, Spain
2Department of Physical Chemistry, Faculty of Chemistry, University of Murcia, Spain

[email protected]

The Bombyx mori silkworm has been used in cooking, industry, home textiles and even medicine. The fibroin
is one of the two proteins present in their cocoons, and it shows high biocompatibility and biodegradability,
as well as low immunogenicity. The effects of dietary supplements of silk nanoparticles on the intestinal
barrier function in non-wounded and skin wounded gilthead seabream (Sparus aurata) were studied.

Thirty-six gilthead seabream were randomly distributed in 6 seawater aquaria. After the acclimation period,
fish in two aquaria fed one of the following experimental diets: 0 (control) 50 (SN1) and 100 (SN2) mg of silk
nanoparticles per kg of feed. After 30 days, 6 fish from each group were sampled. The remaining fish were
wounded on the right side, below the lateral line, with an 8 mm diameter punch. The fish were photographed
and returned to their tanks to continue feeding for an extra 7 days. Afterwards, all the fish were sampled,
and the wounds were photographed again. All sampled fish were weighed and measured and then sacrificed.
Blood and gut samples were collected. Peroxidase, protease, antiprotease and immunoglobulin M activities
were determined in serum. Results demonstrated that serum peroxidase activity was increased in wounded
fish respect to non-wounded ones.

Gut samples were processed for light microscopy and silk-nanoparticles dose-dependent increases were
observed in the number of goblet cells in wounded and non-wounded fishes. Other gut samples were
processed for gene expression by PCRrt. The expression profile of six pro-inflammatory cytokines (MPO,
CSF1R, IL-1β, IL-6, IL-8, and TNFα), two anti-inflammatory cytokines (IL-10 and TGFβ1), two mucins (MUC2,
and MUC18), two immune molecules (IGHT and LYZ), and three tight junction-associated proteins (ZO1,
tricellulin and occludin) was studied. The expression of anti-inflammatory genes and mucin-associated genes
was statistically significantly increased in a dose-dependent way, mainly in wounded fish. The present results
demonstrated the benefits of this nanoparticles to preserve fish gut integrity specially in presence of skin
wounds.

Page 162
12.1 Bivalve and crustacean diseases III

Confirmation of infection of native oysters (Ostrea edulis) with Bonamia ostreae at multiple
additional locations in Scotland

Silvia MC Soares, Rebecca E McIntosh, Amanda Walker, Eann Munro

Marine Scotland Science, Marine Laboratory, Scotland, UK

[email protected]

In recent years, there has been increasing interest in the restoration of wild native oyster (Ostrea edulis)
beds of varying scale with the aim of providing environmental services to and increasing the biodiversity of
the marine environment in Scotland. One of these projects, aiming to re-establish wild native oysters in the
east coast of Scotland (Site A), employed an intermediary site (Site B) to hold oysters in containment
aquarium based in Edinburgh, whilst a subsample was tested for the presence of specific pathogens. In
addition, shellfish were observed and cleaned to minimise the risk of transfer of invasive non-native species.

Methodology
Thirty native oysters from a commercial mollusc site (Site C), located on the west coast of Scotland, was
submitted for disease screening on 15 October 2019, prior to supply of a larger shipment of flat oysters to
the restoration site. No morbidity or mortality had been observed in the donor population prior to the
submission of oysters for disease screening. Molecular screening was conducted using a TaqMan real-time
PCR assay for the detection of Bonamia sp. with all positive samples confirmed as B. ostreae by conventional
PCR with sequencing targeting the small subunit (SSU) rDNA.

Results and Conclusions

Seven out of thirty oysters were identified as positive for Bonamia sp. on 21 October, based upon the qPCR
results, subsequently confirmed as B. ostreae with DNA sequence data on 24 October 2019.

Movement restrictions in the form of Initial Designation Notices (IDN) were served upon both the Site B and
Site C and follow up testing was conducted to confirm or rule out the presence of B. ostreae. Both upstream
(supplying) and downstream (supplied) contact tracing of the sites epidemiologically connected to Site C and
the Site B was performed with subsequent testing taking place. In addition, from the testing conducted as a
result of epidemiological information collected from contact tracing, the parasite was also confirmed to be
present in oysters located at the restoration site (Site A), and a shellfish hatchery located in the Orkney Isles.
Confirmed Designation Notices were served on the aforementioned areas/sites with protection zones
established around these areas.

Page 163
12.1 Bivalve and crustacean diseases III

Perspective on disseminated neoplasia in the eastern oyster Crassostrea virginica based on

analyses in Virginia from 1999-2020

Safi Lucia, Rita Crockett, Ryan Carnegie

Virginia Institute of Marine Science, USA

[email protected]

Disseminated neoplasia (DN) in marine bivalves is characterized by the uncontrolled proliferation of

abnormal cells circulating in the hemolymph. This disorder has received attention in the aquatic health
community with the observation of transmissible neoplastic host cell lineages underlying the condition in
Mya arenaria and Mytilus species in the USA and Europe, respectively. Claims of a similar condition emerging
in the quahog clam Mercenaria mercenaria in Massachusetts and Rhode Island, USA, have recently
intensified concerns about DN in the country. Against this backdrop, the question emerged as to the DN
status of the eastern oyster, Crassostrea virginica, about which little has been written.

We retrospectively evaluated two decades of diagnostic records to address the question of the DN status of
this key resource species. Histopathological diagnostic data from 1999-2020 for C. virginica, with a primary
focus on lower Chesapeake Bay in the eastern USA, was reviewed for all records of observation of DN. These
analyses represented two data sets. The first was a long-term monitoring program of 32 natural oyster reefs
evaluated annually every October, sampling extended to 4 x annually at six of the sites. The second
comprised analyses of aquacultured oysters, including seed, juveniles, and adults, with >100 such samples
evaluated annually in recent years. Where the condition was noted, slides were reevaluated to rate the
condition as focal, multifocal, to systemic. DN was occasionally detected in samples of wild oysters but was
not common, with mean annual prevalence in annual October samples ranging from 0.0-0.2% and a
maximum prevalence at any reef of 4.0%.

This represents a single positive case, presenting focal DN. DN has more frequently been observed in
aquacultured oysters, although still at low prevalence (3.96%). While 87.1% of the cases in aquaculture
samples were advanced and systemic, there is no evidence the condition causes substantial mortality. DN in
some cases was detected in several distinct aquacultured lines at the same site, evidence for an infectious
etiology, the nature of which remains to be resolved. Regardless of etiology, available data suggests that DN
should not presently be viewed as an emerging disease threat to eastern oysters.

Page 164
12.2 - Prophylaxis & treatment

Virulence factors of Anisakis pegreffii as potential drug therapy targets

Ivona Mladineo1, Nikola Palevich2, Vincenzo Carbone2, Željka Trumbić 3, Jerko Hrabar 4

1Institute of Parasitology, BC CAS, Czech Republic

2AgResearch Limited, Grasslands Research Centre, New Zealand
3University of Split, Croatia
4Institute of Oceanography and Fisheries, Split, Croatia

[email protected]

Human anisakiasis is a disease caused by infective larvae of the genus Anisakis spp. (Anisakidae, Nematoda),
eliciting gastric, intestinal, ectopic or gastro-allergic form, or eventually an asymptomatic form within the
Anisakis-seropositive population. Analysing differentially expressed genes (DEGs) after Illumina RNA-
sequencing of A. pegreffii third-stage infective larvae (L3) experimentally infecting rat (accidental host, model
for human infection) and fish (paratenic host), we identified transcripts that expressed the highest
upregulation common for L3 migrating through both hosts. From the initial 65 transcripts, deemed virulence
factors (VFs), we selected those with at least 2FC expression, discarded constitutive cuticle elements and
non-annotated transcripts, obtaining a list of several catalysts and transporters, already recognised as
excretory/secretory products.

These included five targets: cytosolic non-specific dipeptidase (CNDP2), leukotriene A-4 hydrolase (LKHA4),
aspartic protease 6 (ASP6), ATP-binding cassette sub-family B member 9 (ABCB9), and UDP-
glucuronosyltransferase (UGT3). We searched VFs against the Wormbase Parasite protein BLAST database,
obtained predicted proteomes of selected Nematoda (Clades III, V, IV, C, I) and Platyhelminthes (Clades
Monogenea, Trematoda, Cestoda, Rhabditophora), determined homologs of A. pegreffii VFs, and identified
single-copy orthologous groups (OGs) in selected proteomes (OrthoFinder v 2.5.2). Finally, we determined
VFs 3D structures and catalytic sites utilizing online modelling techniques and comparing our models to
structures co-crystalized with inhibitors or substrate analogues. Phylogenetic analyses inferred the presence
of four families (ABCB9s, ASP6s, LKHA4s and CNDP2s) in almost all Nematoda, Platyhelminthes and Metazoa
examined, and the lack of UGT3 in Trematoda and Cestoda.

All VFs showed high levels of duplication and widespread occurrence in closely related Toxocara canis,
Ascaris suum, Parascaris univalens and H. contortus, supporting their vital biological functions in nematodes.
VFs tertiary structure predictions and modelling analyses showed to be useful for the search of currently
available inhibitor molecules, being also applicable in a screening of broad-spectrum efficacy for all Clade III
and V nematodes examined.

Page 165
12.2 - Prophylaxis & treatment

Potential of microalgae as antimicrobial agents against the fish pathogen Yersinia ruckeri
and insight into their inhibitory molecular mode of action

Menanteau-Ledouble Simon, Nielsen Jeppe Lund

Aalborg University, Denmark

[email protected]

Several studies have suggested the potential benefits of algae as immunostimulatory and therapeutic agents.
However, the subject remains under-investigated and in particular, the potential mechanisms of action of
algae on microbial pathogens often remain to be elucidated.

Methodology
The effect of exposure to 8 different algae isolates (Haematococcus pluvialis, Arthrospira maxima, Chlorella
vulgaris, Tetradesmus obliquus, Scenedesmus acuminatus, Chlorella sorokiniana, as well as two isolates of
Arthrospira platensis) on the growth of Yersinia ruckeri isolate CSF007 was tested at nine different
concentrations (ranging from an equivalent of 4.9 µg COD/mL-1 to 38 mg COD/mL-1). Furthermore, the
effect of different mode of preparation for the algae were assessed and the effect of H. pluvialis (at 1.5 and
25 mg COD/mL-1) on the proteome of Y. ruckeri CSF007 was analysed by mass-spectrometry based on the
genome of Y. ruckeri.

Result
The results revealed that several isolates had the potential to completely inhibit bacterial growth at doses
down to 0.6 mg COD/mL-1 (C. sorokiniana). Following these observations, the bacterium was exposed to two
concentrations of the alga H. pluvialis and the proteomes were analysed by mass-spectrometry. In total,
more than 6026 proteins were identified. Among these, 23 proteins were found to be significantly up- (15,
n=4) and downregulated (8, n=4) in the bacteria exposed to the high dose of the treatment compared to the
control samples. These included several proteins associated with stress responses in Y. ruckeri (e.g. enolase,
ATP-dependent zinc metalloprotease; chaperone protein and Shikimate kinase). In addition, 8 proteins
associated with the cell membrane were found at statistically decreased levels (e.g. P pilus assembly protein,
Major outer membrane lipoprotein and the Periplasmic protein YqjC).

Conclusions
These results support previous forwarded hypothesis suggesting that exposure to H. pluvialis damages the
membrane integrity of the bacteria. Furthermore, this mechanism of action is consistent with what has been
described for several antimicrobial compounds of algae, including phenolic compounds and β-carotene
pigments, both previously isolated from H. pluvialis.

Page 166
12.2 - Prophylaxis & treatment

Effect of water hardness/alkalinity and humic substances on the toxicity and

bactericidal/fungistatic effect of peracetic acid: suggests for aquaculture practices

Dibo Liu1, Thomas Meinelt1, David L Straus2, Christopher Good3

1Leibniz-Institute
of Freshwater Ecology and Inland Fisheries, Germany
2U.S. Department of Agriculture, Agricultural Research Service, Harry K. Dupree-Stuttgart National
Aquaculture Research Center, USA
3The Conservation Fund Freshwater Institute, USA

[email protected]

The prophylactic use of peracetic acid (PAA)-based disinfectants is becoming more popular in aquaculture
due to rising concerns regarding sustainability, fish welfare and food safety. However, specific and effective
PAA dosing protocols have not been developed to guide the aquaculture industry under diverse production
conditions.

Methodology
In the present study, the effect of water hardness/alkalinity and humic substances (HS) on the toxicity of PAA
to zebrafish Danio rerio embryos and the efficacy of PAA against the in vitro growth of Yersinia ruckeri and
Saprolegnia parasitica was investigated. Meanwhile, the pH and oxidation-reduction-potential (ORP) values
were monitored at tested conditions.

Results and Conclusions

PAA concentrations that were safe to fish embryos demonstrated strong bactericidal, but limited fungistatic
properties. In higher hardness/alkalinity water, or when HS was supplemented, the same concentration of
PAA resulted in a smaller pH decrease and a weaker ORP increase, and showed lower toxicity and weaker
antimicrobial effects than in lower hardness/alkalinity waters. We suggest the determining factor of PAA
toxicity and its antimicrobial capacity was likely ORP. At low hardness/alkalinity conditions, strong pH
reduction (resulting in pH<5) was the dominant role in PAA toxicity to D. rerio embryos. In aquaculture
settings, therefore, lower PAA doses should be used under lower hardness/alkalinity conditions.
Supplementation of HS under lower hardness/alkalinity conditions can assist with reducing the risk to fish.
Finally, we determined that repeated PAA disinfection is necessary to achieve a sustained prophylaxis, and
we caution that counting colony forming units may underestimate the number of viable bacteria attached
on aggregates.

Page 167
12.2 - Prophylaxis & treatment

Surface decontamination of Nile tilapia fertilized eggs with a bronopol based biocide

Hoang Phan1, Khanittha Sang-Ngam1, Sasibha Jantrakajorn2, Janenuj Wongtavatchai3, Philippe Mahl1

1Virbac, Aquaculture Division, France & Vietnam

2Faculty of Veterinary Science, Prince of Songkla University, Thailand
3Faculty of Veterinary Science, Chulalongkorn University, Thailand

[email protected]

Nile tilapia (Oreochromis niloticus) is one of the most promising species for freshwater aquaculture in many
tropical and subtropical countries thus the intensive artificial egg incubation is developed to provide
uniformity of larvae for tilapia culture. However, intensive incubation technique of Nile tilapia eggs allows
favorable conditions for microbial proliferation which often leads to massive mortalities of fish larvae.

In this study, the exposure of tilapia eggs to Antizol which is a bronopol based biocide was to test its effect
on bacterial surface decontamination and larval survival rate of eggs. Immersion treatments were applied
on fertilized eggs at various Antizol doses of 20, 50, 100, 200 and 500 mg/L for 10, 20 and 30 min. Bacterial
numbers on Nile tilapia eggs were significantly reduced following the immersion treatments of Antizol at all
doses and immersion time. The most reduction in bacterial numbers (1.58 × 10 3 cfu/g of egg) was observed
in the maximum treatment dosage i.e. 500 mg/L for 30 min, but this was not significant when compared with
other treatments of lower doses, which showed a linear response when varying the doses.

Treatments using Antizol of 100 mg/L for 20-30 min and 200-500 mg/L for 10-30 min provided better larval
survival rates (89.33-94.67%) than those of <100 mg/L doses (p < 0.05). There were no significant differences
among treatments of 20 and 50 mg/L for 10-30 min, and negative controls (79.33-80.97%). Additional in vitro
test was assessed to evaluate the kinds of bacteria cultured from fertilized Nile tilapia eggs, which showed
that there were 22 bacterial isolates, including Aeromonas hydrophila (n=12), Staphylococcus aureus (n=6),
Escherichia coli (n=2) and Micrococcus spp. (n=2). In overall, Antizol is safe for fertilized fish eggs to be
disinfected from bacterial species present in incubation process in order that the biocide can enhance the
larval survival rate.

Page 168
12.2 - Prophylaxis & treatment

Exposure of Vibrio ordalii strain Vo-LM-18 to antimicrobials: changes in the production,

composition, and antimicrobial response of outer membrane vesicles

Macarena Echeverría-Bugueño1,2, Rute Irgang1,2, Ruben Avendaño-Herrera1,2,3

1 Universidad Andrés Bello, Facultad de Ciencias de la Vida, Chile

2Centro FONDAP INCAR, Universidad Andrés Bello, Chile
3 Centro de Investigación Marina Quintay, Universidad Andrés Bello, Chile

[email protected]

Vibriosis and the characteristic hemorrhagic septicemia of this important disease impact both brackish and
marine fish farms in Chile. The main etiological agent of vibriosis is Vibrio anguillarum, which is the most
widely distributed globally. However, other species such as Vibrio ordalii have also been involved in fish
diseases. Despite the economic impacts caused by V. ordalii infections, several facets of virulence
mechanisms remain unclear, including the production of outer membrane vesicles (OMVs).

Commercial antimicrobials with diverse mechanisms of action were tested against V. ordalii Vo-LM-18 to
determine (i) any changes in OMV production and composition, and (ii) if OMVs impact the response of the
bacterium to antimicrobial agents. Surface-enhanced Raman spectroscopy was used to determine the
composition of the cell-membrane surface of bacteria that survived antimicrobial exposure. Membrane-level
alterations, which would suggest a structural change in the released OMVs, were determined by SDS-PAGE
analysis. Furthermore, OMV concentrations were compared between bacteria cultured with and without the
antimicrobial agents to establish antimicrobial-specific differences in the release of OMVs.

Our research suggests important changes at the bacterial membrane level when V. ordalii strain Vo-LM-18
was exposed to antimicrobials, as confirmed by changes in the protein profile of the released OMVs. In
addition, a relationship was observed between the released OMVs and the degree of bacterial susceptibility
to the tested antimicrobials.

Funding
FONDAP INCAR 15110027 from Agencia Nacional de Investigación y Desarrollo de Chile (ANID) from Chilean
government.

Page 169
Poster Abstracts

A-Z by Surname of presenting author Page 170

016-P
VHSV and IHNV experimental infections in rainbow trout: evaluating mortality kinetics and
intra-host viral replication in single and co-infections

Miriam Abbadi, Andrea Marsella, Lorena Biasini, Francesco Pascoli, Alessandra Buratin, Anna Toffan

Istituto Zooprofilattico Sperimentale Delle Venezie, Italy

[email protected]

Infectious Haematopoietic Necrosis (IHN) and Viral Haemorrhagic Septicaemia (VHS) are two OIE-listed
viruses triggering systemic diseases in salmonid fish. Historically, VHS has been identified as the major issue
for Italian trout farms, causing higher mortality in comparison to IHN. The extensive circulation of IHNV and
VHSV in Italian trout farm has recently been demonstrated, and co-infection within the same farm or even
the same individual was observed. As a result, the Italian rainbow trout farming industry has progressively
experienced severe disease outbreaks and significant production losses.

This study aimed to investigate and compare the viral replication kinetics and the pathogenicity of IHNV and
VHSV in single or in co-infection in rainbow trout (O. mykiss). Hence, two Italian isolates were selected to
conduct in vivo trials. Rainbow trout juveniles (≃15 gr) were purchased from a Category I hatchery and were
divided into four experimental groups counting 34 fish each, housed in 80 L tanks, challenged with 10^4
TCID₅₀/ml of virus and monitored for 30 days. One group out of four included mock infected fish.

Data regarding daily mortality were recorded and survival probability was estimated by Kaplan-Meier curves.
Intra-host viral replication was determined with qRT-PCR assessed in spleen and head kidney of all dead
animals.

Experimental challenges confirmed the different pathogenic phenotype of IHNV and VHSV, as demonstrated
by cumulative mortality estimates ranging from 38.18% and 85.19%, respectively. Interestingly, our data
highlighted a higher mortality rate (92.45%) in co-infected fish probably due to a synergistic interaction of
the viruses. Data obtained through qRT-PCR analyses confirmed the presence of the viruses, singularly or in
combination, and quantified the viral load in each specimen. Notably, all co-infected dead fish resulted
positive for both viruses. Intra-host viral quantification results evidenced higher viral loads in spleen rather
than in head kidney in all co-infected dead animals. A positive correlation between viral replication and
mortality kinetics was also observed in each group.

Further analyses are required to better investigate the impact of VHSV and IHNV co-infection on rainbow
trout productions.

Page 171
063-P
Transcriptome analysis reveals a complex response to reassortant strain RGNNV/SJNNV in
sea bream larvae

Luca Peruzza1, Francesco Pascoli2, Giulia Dalla Rovere1, Rafaella Franch1, Serena Ferraresso1, Massimiliano
Babbucci1, Lorena Biasini2, Miriam Abbadi2, Valentina Panzarin2, Anna Toffan2, Luca Bargelloni1

1University of Padova, Department of Comparative Biomedicine and Food Science, Italy

2Istituto Zooprofilattico Sperimentale Delle Venezie, Italy

[email protected]

Gilthead sea bream is an important marine fish for Mediterranean aquaculture. This species has historically
been considered resistant to viral nervous necrosis, a devastating disease affecting Mediterranean
mariculture. However, with the recent appearance of RGNNV/SJNNV reassortant strains sea bream
hatcheries are seriously threatened, as these viruses are able to severely infect larvae and juveniles of this
species. While host response to betanodavirus in adult fish has widely been investigated, little is known
regarding the most sensitive early life stages.

This study reports the first time-course RNA-seq analysis on 21-day old sea bream larvae experimentally
infected with a RGNNV/SJNNV strain. Infected and mock samples were collected at 6h, 12h, 24h, and 48h
post infection (hpi). Four biological replicates, of five pooled larvae each, were analysed for each time point.

Results evidenced a large set of significantly regulated genes, especially at 6 hpi and 12 hpi. Particularly,
several heat shock protein encoding transcripts were up-regulated (e.g. hspa5, dnaj4, hspa9, hsc70), while
many immune genes were down-regulated (e.g. myd88 and irf5 at T06, pik3r1, stat3, jak1, il12b and il6st at
T12). A gene set enrichment analysis (GSEA) was applied to identify altered pathways/processes.
Interestingly, at 6 hpi and 24 hpi the autophagy pathway and the formation of peroxisomes were found to
be down-regulated. Finally, two custom “reactomes” were defined and used. The first reactome integrated
the transcriptomic response to betanodavirus in different fish species, while the second one included all
genes found to be stimulated either by interferon or by IFN and Chikungunya virus in zebrafish. In both
reactomes, genes showed predominant up-regulation at 6 hpi and 12 hpi and a general down-regulation at
24 hpi. Data obtained highlight a similarity in terms of response to betanodavirus between sea bream and
other fish species, while the down-regulation of IFN- and viral-stimulated pathways indicates a possible
interference of the virus against host response.

Overall, results confirm the hypothesis that the reassortant RGNNV/SJNNV, unlike the RGNNV strain, induces
transcriptional changes that lead to lethal damage in the early larval stages of a NNV-resistant species.

Page 172
108-P
Are the mysteries of mass mortality events (MME) within freshwater pearl mussels
(Margaritifera margaritifera) populations in Sweden associated with parasite infections

Anders Alfjorden1,2, Anders Alfjorden1,2, Ioana Onut Brännström2, Niklas Wengström3,4, Fabien Burki2

1NationalVeterinary Institute, Sweden

2Uppsala University, Sweden
3Swedish Anglers Association, Sweden
4Gothenburg University, Sweden

[email protected]

Margaritifera margaritifera, the freshwater pearl mussel (FPM, Europe) or eastern pearlshell (North
America) are only inhabiting running water courses (creeks or rivers) and has a high demand on the water
quality. Therefore, this specie is also used as an important ecosystem indicator and are as such monitored
on regular basis in many countries within northern Europe. In Sweden there is a regional program for
population surveillance focusing on this mussel, governed by regional counties where this specie is known
to exist, 16 out of 21 counties. Historical and ongoing declines have put a strain on the survival of many of
these populations and today approximately only one third of these populations are considered healthy with
signs of juvenile recruitments. Recent years of surveillance during 2011 to 2020 in collaboration with the
National Veterinary Institute SVA, have investigated the declining population by field servey combined with
postmortem investigation and molecular genetics to follow up on these mortalities.

In this presentation, methods for post-mortem investigations will be highlighted together with macroscopic
and microscopical findings from ongoing studies between 2018-2020. Lesions and associated pathology
detected in visceral organs primarily: gonads and digestive glands, will be visualized together with description
of the parasitic cell morphology associated with these eukaryotic parasites. Microscopic, ultrastructure and
PCR details will also be presented. These findings indicate a previously unknown parasitic agent related to
the apicomplexan group of parasites. Further investigations are ongoing to characterize and describe these
findings. This type of parasitic infection within the FPM host, have previously not been described within this
bivalve host. However other parasitic eukaryotes might still remain undetected. Further histopathological
investigations and increased interest for investigation of these bivalves may increase our knowledge about
the currently neglected host parasite interactions within the freshwater pearl mussels.

Page 173
080-P
Differential antiviral activity of Porphyridium cruentum polysaccharides against VHSV and
NNV infections

Carmen Alonso2, Geovanna Parra-Riofrio1, Patricia Moreno2, Roberto Teófilo Abdala-Diaz2, Julia Bejar2,
Esther Garcia-Rosado2, Eduardo Uribe-Tapia1

1Facultad de Ciencias del Mar, Universidad Católica del Norte, Chile

2Facultad de Ciencias, Universidad de Malaga, Spain

[email protected]

In addition to vaccination, the use of functional food can be an interesting approach to prevent viral disease
outbreaks in aquaculture facilities. In recent years, an increasing number of studies have suggested that
marine organisms and microorganisms can be a relevant source of substances with a putative use in
preventing viral diseases. In particular, the antiviral activity of polysaccharides has been widely described
against infections affecting higher vertebrates, suggesting that they can be good candidates to be used in
aquaculture. In this study, we evaluate the activity of Porphyridium cruentum polysaccharides, which have
been demonstrated to have important biological activities in mammals, such as antiviral or antioxidant
effects.

We have evaluated this substance against two different fish viruses:

(i) viral haemorrhagic septicaemia virus (VHSV, genotype I), an ssRNA enveloped virus, and (ii) nervous
necrosis virus (NNV, RGNNV genotype), a bisegmented ssRNA naked virus. Both viruses may affect a broad
range of fish species, causing massive mortality in aquaculture facilities at larval and juvenile stages. Two
different approaches have been used, since polysaccharides can block either viral-cell adsorption or any
other intracellular viral replication step: (i) treating cells (RTG-2 for VHSV and E-11 for NNV) with
polysaccharides before virus inoculation (adsorption assay) and (ii) adding polysaccharides on cells after viral
inoculation (post-adsorption assay). Viral multiplication in treated and non-treated cells has been measured
by viral genome quantification at different times post infection (p.i.).

The anti-VHSV activity of P. cruentum polysaccharides has been demonstrated, acting at both levels, blocking
viral adsorption to cellular receptor, and preventing intracellular viral replication. A significant decrease in
viral genome multiplication has been recorded at 24 and 36 h p.i. in RTG-2 cells treated with polysaccharides
compared to control untreated cells. On the contrary, no anti-NNV activity has been detected, demonstrating
a differential antiviral activity of P. cruentum polysaccharides.

Funding: This study has been supported by the projects AGL2017-84644-R, from MINECO/AEI/FEDER, UE,
and P18-RT-1067, from Junta de Andalucía (Regional Government). G.P. has been supported by ANID-PFCHA,
National Doctoral Scholarship, 2018-No. 21180059.

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Microencapsulation of the ssIL-8a peptide in chitosan for the control of bacterial pathogens
of importance for culture salmon

Nancy Alvarado1, Adolfo Guerra2, Nancy Alvarado1, Manuel Ahumada3, Paula Sepúlveda1

1Universidad Autónoma De Chile, Instituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería, El Llano
Subercaseaux 2801, Chile
2Universidad Mayor, Escuela de Biotecnología, Ciencia, Chile
3Universidad Mayor, Centro de Nanotecnolgia Aplicada, Facultad de Ciencias, Chile

[email protected]

In the salmon industry, the most important commercial species are Atlantic salmon, rainbow trout and Pacific
salmon. However, the intensive cultivation of these species exposes them to potential pathogens such as
Aeromonas salmonicida and Yersinia ruckeri, which cause large mortalities, bringing millions in losses to the
industry and an indiscriminate use of antibiotics. In this sense, the excessive use of antibiotics fails to control
the infections produced in salmonids, given the generation of resistant strains, so it is imperative to seek
new alternatives.

This is where antimicrobial peptides play an important role due to their characteristics such as broad-
spectrum antimicrobial activity, low cytotoxicity, and environmental compatibility. Recently, our research
group identified a peptide with potential antibacterial activity derived from the C-terminal end of Salmon IL-
8 (ssIL-8α). However, this peptide is susceptible to being degraded by proteases affecting its bioavailability
and action. In order to overcome these disadvantages, the ssIL-8α peptide was encapsulated in chitosan
microcapsules. This biopolymer is characterized by being biodegradable, biocompatible and soluble in
aqueous solution. Through microdilution and agar plate assays, the antibacterial activity of the ssIL-8α
peptide (10 to 30 µM), chitosan and encapsulated peptide against A. salmonicida and Y. ruckeri was
evaluated.

Additionally, for these same molecules, the cytotoxic and hemolytic activity was evaluated. The results
obtained showed that the peptides were capable of inhibiting the growth of 1 x 10-6 CFU/ml of A.
salmonicida over 20 µM, not so for Y. ruckeri where the peptide showed low activity. On the other hand,
chitosan showed low activity against these bacteria. In the case of the encapsulated peptide, an antibacterial
activity greater than that observed for chitosan is manifested, but less than that of the peptide. Both the
ssIL-8α peptide (5, 20 and 60 µM), as well as chitosan and the encapsulated peptide (the latter at 2.9 and 29
mg/ml) did not show cytotoxic or hemolytic activity. Studies of the encapsulation of antibacterial peptides
are necessary to be applied in cultured species of commercial importance for the control of pathogens.

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Evaluation of the effectiveness of a synthetic variant of hepcidin in the control of Salmon
Rickettsial Syndrome

Claudio Alvarez1, Claudio Alvarez1, Luis Mercado2, Fanny Guzman3, Paula Santana4

1Laboratorio de Fisiología y Genética Marina, Centro de Estudios Avanzados en Zonas Áridas, Chile
2Grupo de Marcadores Inmunológicos, Laboratorio de Genética e Inmunología Molecular, Instituto de
Biología, Pontificia Universidad Católica de Valparaíso, Chile
3Núcleo Biotecnología Curauma (NBC), Pontificia Universidad Católica de Valparaíso, Chile
4Facultad de Ingeniería, Instituto de Ciencias Químicas Aplicadas, Universidad Autónoma de Chile, Chile

[email protected]

Intensive fish farming carries the risk of acquiring infectious diseases by different pathogens. In chile, the
main salmonid bacterial pathogen is Piscirickettsia salmonis, which causes Salmon Rickettsial Syndrome
(abbreviated as SRS), generating annual losses exceed US$ 400 million. Therefore, the salmon industry
requires new compounds to control this pathogen. Antimicrobial peptides (AMP) have emerged as a family
of molecules with great potential for clinical use. Hepcidin is one of them, a 25-residue peptide with
physicochemical characteristics that allows it to have a broad spectrum of action against bacterial pathogens.

In this work, we generated a variant with only twenty residues by chemical synthesis, and the effectiveness
of this peptide in controlling SRS was evaluated in smolt size rainbow trout (Oncorhynchus mykiss) with 105-
120 g in body mass. The results show that the variant generated has a β-sheet structure, similar to 25-residue
hepcidin. Thirty days post-inoculation, the peptide significantly decreased mortality associated with SRS by
35% only when applied together with the pathogen. Therefore, new studies are required to analyze the use
of this molecule as a preventive method for SRS control in salmonids.

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Myxozoan host sensing and venom delivery in complex lifecycles

Benjamin Americus1, Tamar Lotan2, Jerri L. Bartholomew1, Stephen D. Atkinson1

1Oregon State University, USA

2University of Haifa, Israel

[email protected]

The myxozoans are an anciently derived clade of Cnidaria, related to jellyfish and sea anemones. They are
microscopic parasites that utilize vertebrate (fish) and invertebrate hosts and alternate between two life
stages: a fish-infective stage and an invertebrate-infective stage. In adaptations to parasitism, myxozoans
have reduced genomes and morphology relative to their free-living relatives. However, they have retained
nematocyst stinging cells that characterize phylum Cnidaria. Rather than using these for predation or
defense, myxozoans discharge nematocysts to attach to hosts in the initial step of infection. In this work, we
examine two features of nematocyst function in Myxozoa: host-sensing and venom delivery.

We compare venom and sensory genes from model free-living cnidarians to transcriptomes from the
myxozoans Ceratonova shasta and Tetracapsuloides bryosalmonae. For both myxozoans, we examined
transcriptomes from infections in both fish and invertebrate hosts. This allows us to compare genes between
life-stages, between species, and between free-living and parasitic cnidarians. We examine phylogenetic
relationships, domain presence/absence, predicted structures, and time series expression within infected
fish.

T. bryosalmonae and C. shasta have retained the sensory devices of free-living cnidarians including voltage-
gated ion channels and mechanosensors. We identified different isoforms expressed by each myxozoan life-
stage, suggesting adaptation to hosts. For both C. shasta and T. bryosalmonae, sensory genes identified in
infected invertebrates are more similar to those from free-living cnidarians than sensory genes isolated from
infected fish. In C. shasta, we describe 10 venom-like genes present in both life stages but not in T.
bryosalmonae. In C. shasta-infected fish, these venom-like genes are expressed asynchronously from known
nematocyst-specific genes.

Myxozoans inherited many of the sensory and venom genes of their free-living relatives but have adapted
them for parasitism. We found the sensory devices are relatively unchanged at a genetic level despite very
different nematocyst morphology. The venom repertoire in Myxozoa appears highly reduced but may have
acquired new function in host-immune invasion outside of nematocysts.

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Histopathological staging of the granulomatous inflammation against histozoic metazoan
parasites in mullets (Osteichthyes: Mugilidae)

Elisabetta Antuofermo1, Francesc Padrós2, Paolo Merella1, Marino Prearo3, Marina Antonella Sanna1, Fabio
Marino4, Giovanni Pietro Burrai1, Elisabetta Antuofermo1

1Department Of Veterinary Medicine, University of Sassari, Italy

2Fish Diseases Diagnostic Service, Facultat de Veterinaria, Universitat Autonoma de Barcelona, Spain
3Fish Disease Laboratory, State Veterinary Institute of Piedmont, Italy
4Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina,

Italy

[email protected]

Histozoic parasites, eliciting a chronic granulomatous inflammatory response, represent a common finding
in fish. However, the development and evolutive stages of granuloma in naturally infected fish caused by
histozoic parasites are scarcely investigated. Mullets (Osteichthyes: Mugilidae) represent a suitable model
for the study of parasites-induced granulomas as they are widely spread euryhaline fish species harboring
different classes of parasites. Two hundred and thirty-nine mullets were collected from 4 Sardinian lagoons
and visceral organs were examined for the presence of trematode metacercariae (TM) and myxozoan
plasmodia (MP). Also, culture tests were carried out on an isolation medium and in Stonebrink medium tubes
to isolate bacteria and mycobacteria, respectively.

Samples were formalin-fixed, submitted for histopathological evaluation, and stained with hematoxylin and
eosin. Selected 75 inflammatory lesions were classified into three granuloma developmental stages
according to histopathological features. Sections were stained with Masson’s Trichrome and Giemsa for the
evaluation of collagen and mast cells (MCs) and submitted to immunohistochemistry for the detection of
epithelioid cells (ECs) by the anti-CKAE1/AE3 antibody and fibroblasts by the anti-vimentin antibody. Slides
were analyzed with an open-source image processing program (https://imagej.net/ImageJ) and obtained
data were submitted to statistical analysis (Stata 11.2 software, StataCorp LP). Granulomas associated with
TM and MP were classified into three developmental stages: 1) pre-granuloma stage, characterized by intact
encysted parasite with no or mild inflammatory reaction; 2) intermediate stage, with partially degenerated
parasites, necrosis, and a moderate number of ECs; 3) late stage, with a necrotic core and no detectable
parasite with a high number of ECs and fibroblasts and collagen. Mast cells were mostly detected in
intermediate and late-stage granulomas associated with fibroblasts.

The identified histological patterns represent reliable tools to staging the granulomatous inflammatory
response associated with histozoic parasites and provide an effort in the knowledge of the inflammatory
response in different host-parasite systems.

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The genome of the salmonid parasite Ceratonova shasta (Cnidaria: Myxozoa) illustrates
structural and functional streamlining of parasitism

Stephen Atkinson1, Gema Alama-Bermejo2, Ed Davis1, Eli Meyer1, Shawn T. O'Neil1, Sascha L. Hallett1,
Jerri L. Bartholomew1

1Oregon State University, USA

2Biology Center of the Czech Academy of Sciences, Czech Republic

[email protected]

At least a fifth of Phylum Cnidaria are obligate parasites – the Myxozoa – which can cause diseases in wild
and cultured fish. Ceratonova shasta is an economically and ecologically significant myxozoan parasite of
salmonids in the Pacific Northwest of North America. We characterized the C. shasta genome to inform
multiple studies of this pathogen and to better understand structural and functional differences between
parasitic and free-living Cnidaria.

C. shasta myxospores and developmental stages were sampled from laboratory-infected rainbow trout
(Oncorhynchus mykiss). Living parasite stages were stained with DAPI and MitoTracker for microscopy, and
with propidium iodide for flow cytometry. Karyotypes were prepared from pre-sporogonic stages using
standard cell methods. Genomic DNA was sequenced on a single lane of Illumina HiSeq2000, 150bp paired-
end. We developed novel bioinformatics pipelines to remove host contamination by comparison with host
references. Genome reads were filtered, then de novo assembled using SPAdes, Velvet, Platinus and CLCbio;
Transposable elements were analyzed using Red and DFAM. Proteins were predicted using SNAP, assessed
with MAKER, and contigs mapped to our existing C. shasta transcriptome (Alama-Bermejo et al., 2020).

The C. shasta genome is smaller and structurally more compact that free-living Cnidaria (FL): it is organized
into 3 chromosomes (compared with 14-15 in FL), with most cells diploid (occasionally triploid). Diploidy is
consistent with asexual myxozoan proliferation in the vertebrate host. The genome was sequenced at high
coverage (300x), but assembly was a challenge due to intra-sample genetic variation (representative of the
metapopulation of parasites existing in the host) and a lack of structural information. SPAdes gave the best
assembly at 101Mbp (compared with 135Mbp by flow cytometry). The genome is thus half the size of the
smallest FL species, but consistent with other Myxozoa (23-180Mbp). We predicted 8,400 proteins with
highest confidence (again, comparable with other Myxozoa but fewer than FL species). Functional
mitochondria were observed, and canonical genes recovered in the SPAdes assembly. These results currently
are being augmented with PacBio long-read sequence data to better resolve C. shasta genome structure.

Page 179
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Weissella tructae sp. nov. an emerging pathogen for farmed rainbow trout (Oncorhynchus
mykiss)

Ruben Avendaño-Herrera4,5, Felipe Luis Pereira1,2, Fernanda Alves Dorella1, Jésica Castrejón-Najera3, Dr.
César Ortega3, Rute Irgang4,5, Henrique C. Pereira Figueiredo1

1AQUAVET, Federal University of Minas Gerais, Brazil

2IFC,Federal Institute of Santa Catarina, Brazil
3lCIESA-FMVZ, Universidad Autónoma del Estado de México, Mexico
4Universidad Andrés Bello, Chile
5FONDAP Interdisciplinary Center for Aquaculture Research (INCAR), Chile

[email protected]

Weissella ceti, an α-haemolytic at 37 oC bacterium, is recognized as the etiological agent of the so-called
“weissellosis”, an emerging disease that affect commercial farming of rainbow trout in China, Brazil, USA,
South Africa, Japan, Mexico, Canadá, Colombia and Peru. Fish infected (weight > 250 g) showed extensive
ocular lesions with hemorrhage and also in anal region, intestine and petechiae on liver and stomach
hydrocele. Remarkable differences have been observed between rainbow trout isolates with the W. ceti type
strain CECT 7719T isolated from beaked whale (Mesoplodon bidens).

This study elucidates the taxonomic position of isolates recovered from diseased rainbow trout and whale
within the genus Weissella. Two isolates, one from Brazil (WS08) and another from Mexico (W-1) recovered
in 2008 and 2015, respectively were included in this study. The identity between 16S rRNA genes varied from
98.98% (CECT 7719T versus WS08) to 100% (W-1 and WS08). Phenotypic assays were also conducted, and
W. ceti type strain showed divergence in hydrolysis of aesculin, D-mannose and potassium gluconate and
hydrolysis of Hippurate. Moreover trout-pathogenic WS08 and W-1 showed weak growth at 5 oC whereas
no growth was observed for the CECT 7719T. Major fatty acids (>10% total fatty acids) presented by the two
isolates were summed feature 8 (C18:1 ω7c/C18:1 ω6c), summed feature 3 (C16:1 ω6c/C16:1ω7c) and
C16:0. Digital DNA-DNA hybridization pairwise analyses showed a score of 98.7% between Brazilian WS08
and Mexican W-1 and 24.4% against W. ceti CECT 7719T.

The results of phylogenetic and phenotypic analyses enabled the differentiation of the W. ceti CECT 7719T
type strain from other trout-pathogenic isolates. Weissella tructae sp. nov. is proposed as a novel species
and W-1T as type strain.

Page 180
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Infectious pancreatic necrosis virus genogroup 6 - prevalence and susceptibility in
landlocked Swedish salmonids

Charlotte Axén1, Niels-Jørgen Olesen2, Dr Jacob G. Schmidt2, Mikael Leijon3, Arianna Comin4

1Dept of Animal Health and Antimicrobial Strategies, Section for Fish, National Veterinary Institute, Sweden
2DTU Aqua, Unit for Fish and Shellfish Diseases, Sweden
3Dept of Microbiology, National Veterinary Institute, Sweden
4Dept of Disease Control and Epidemology, Section for Epidemiological methods, National Veterinary

Institute, Sweden

[email protected]

Infectious pancreatic necrosis (IPN) is a viral disease in salmonids, mainly causing disease in juveniles. Seven
genogroups are known. Sweden is considered free from IPNV in the inland zone and has additional
guarantees/national measures against the virus. Vänern is Sweden’s largest lake, harboring two land-locked
strains (Klarälven, Gullspångsälven) of Atlantic salmon. These, as well as land locked sea trout from the same
rivers, are restocked. All broodstock females are sampled for virus and BKD post spawning. In 2016, one pool
(Gullspångsälven trout) was positive for IPNV genogroup 6. Few published sequences were available and no
information about virulence present. The roe was destroyed. Based on population and sample size and the
chance of finding one positive animal, it was estimated that the prevalence could be as high as 5.5% in Vänern
salmonids.

Further work has been done to investigate prevalence and virulence. In 2017-2020 a total of 151 pools,
containing in total 907-970 salmon or trout from Vänern have been investigated for IPNV. None of the pools
have contained virus. The prevalence estimate has been lowered to a maximum of 0.2-0.5%.

An experimental trial was performed at DTU-Aqua, Denmark. Rainbow trout (RT), Klarälven salmon (AS) and
Gullspångsälven trout (ST) were challenged by bath immersion at start feeding. Treatments were negative
controls (NC), Swedish IPNV6 and “Rindsholm” IPNV5. Treatments were run in duplicates. Fish were
monitored for 33 days post infection. All dead or moribund fish during the period were sampled. In addition,
five fish were sampled day 12 p.i. and all fish were sampled day 33 p.i. Analysis was done by cell cultivation
on BF-2 cells with confirmation by ELISA. IPNV5 was isolated to a higher degree than IPNV6 from RT and ST.
AS was less sensitive to both viruses. In RT mortality was significantly higher in IPNV5 than in IPNV6 groups.
Unexplained mortality occurred in both NC and infected AS and ST. We state that IPNV6 is less virulent than
IPNV5, and IPNV6 did not add to the mortality in AS and BT, indicating that IPNV6 caused minor mortality in
all three fish species.

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Carrageenans from red seaweed show antiviral activity against viral haemorrhagic
septicaemia virus (VHSV)

Isabel Bandín1, Milena Álvarez Viñas2, Dolores Torres2, Herminia Domínguez2

1Instituto
de Acuicultura, Departamento de Microbiología y Parasitología, Universidade de Santiago de
Compostela, Spain
2Departamento de Ingeniería Química, Facultad de Ciencias de Ourense, Universidade de Vigo, Spain

[email protected]

Viral haemorrhagic septicaemia virus (VHSV), the causative agent of viral haemorrhagic septicaemia (VHS),
is known as a significant pathogen of farmed rainbow but also causes disease in several marine fish species.
VHSV (genus Novirhabdovirus, F. Rhabodviridae) is an enveloped virus with a ssRNA genome encoding five
structural proteins. Among these proteins a transmembrane glycoprotein G is responsible for attachment to
the cell membrane and entry into the cell. At present no vaccines are commercially available for VHS
prevention and therefore effective antivirals are urgently needed to control the viral spread and the
development of the disease.

In this study, we have assessed the ant-VHSV activity of carrageenans obtained from the red seaweed
Chondrus crispus. Carrageenans are sulfated polysaccharides which have demonstrated to have antiviral
activity against enveloped human and veterinary pathogenic viruses as Herpes simplex virus, Dengue virus
or Rabies virus. Because carrageenans extraction conditions may affect their properties, a chemical-free
extraction process based only on pressurized hot water was used (heating up to temperatures in the range
120-200 oC during non-isothermal operation). After cooling, the suspension was separated by filtration and
the carrageenan was precipitated with ethanol (1.5:1, v/w ratio) and freeze dried. In an initial test, four
carrageenan samples (AH120, 140, 160 and 200) showed a percentage of VHSV inhibition (PI) >60% and they
were chosen for further analysis. A time-course study was performed to analyse the inhibitory effect at
different steps of the viral cycle. Carrageenans were added to EPC cells simultaneously with VHSV or at
different time intervals post virus inoculation. Pre-treatment of EPC cells was also assayed to study the
prophylactic effect of carrageenans.

In all experiments, treated and untreated VHSV was quantified by plaque formation and TCID50 assay as well
as by RT-qPCR. No protective effect on EPC cell monolayers against VHSV infection was observed. However,
a high inhibitory effect was shown when AH120 and AH200 were added to cells together with the virus,
indicating that carrageenans can block viral adsorption to EPC cells probably by binding to the G protein.

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Inactivated vaccines against reassortant betanodavirus infection in Senegalese sole

Yulema Valero, Isabel Bandín, José G Olveira, Carmen López-Vázquez, Carlos P Dopazo

Instituto de Acuicultura, Departamento de Microbiología y Parasitología.Universidade de Santiago de

Compostela, Spain

[email protected]

Nervous necrosis virus (NNV; Genus Betanodavirus, Family Nodaviridae), the causative agent of viral
encephalopathy and retinopathy (VER), is of the most threatening viruses for marine fish worldwide. NNV
isolates are classified in four genotypes: barfin flounder -, red-spotted - virus striped jack and tiger puffer
nervous necrosis virus (BFNNV, RGNNV, SJNNV and TPNNV, respectively). In addition, reassortants between
RGNNV and SJNNV genotypes have been isolated in Southern Europe. Senegalese sole is a promising fish
species in Mediterranean aquaculture but also highly susceptible to NNV infections. As matter of fact, one
of the main constraints for its intensive farming is its high susceptibility to reassortant RGNNV/SJNNV;
(referring to the RNA1/RNA2 of the donors) strains. Thus, the aim of this work was to assess the protection
conferred by two inactivated vaccines and the elicited immune response in juveniles of Senegalese sole after
vaccination and challenge.

A RGNNV/SJNNV reassortant strain (Ss160) highly pathogenic for sole was employed to design two
inactivated vaccines (using either formalin or binary ethylenimine, BEI). Juvenile sole individuals (2.9 ± 0.1g
body weight) were intraperitoneally vaccinated with every formulation independently at different
concentrations (105 or 107 TCID50/mL) and the antibody response and the expression profiles of immune-
related genes was assessed in head-kidney. After 1 month vaccinated fish were bath challenged with Ss160
(105 TCID50/mL) for 3 h and survival upon NNV infection, viral load and immunostimulation were recorded.

Our results showed the anti-NNV antibodies synthesis in BEI-vaccinated fish whilst not in those that were
injected with the formalin-inactivated vaccine. Moreover, only high BEI concentration up-regulated immune-
related genes in head-kidney. After NNV challenge, fish survival was improved specially with the high BEI
concentration formulation which also caused a significant decrease of the viral load in sole brain.
Interestingly, this vaccine was the only one which maintained the expression of immune-related genes in
challenged fish.

The consistence in the results obtained with BEI inactivation prompt us to think that reassortant strain Ss160
inactivated with BEI has the potential to develop vaccines which generate robust immune response in sole.
However, further experimental approaches are needed to improve this promising vaccine.

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Histopathological changes in the brain of European seabass (Dicentrarchus labrax)
experimentally infected with Nervous Necrosis Virus (NNV)

Teresa Baptista1, Mariana Vaz1, Damiana Pires1, Pedro Pires1, Ricardo Passos1, Beatriz Carmo1, Paulo Santos2

1Polytecnhic of Leiria, Portugal

2Centro Interdisciplinar de Investigação Marinha e Ambiental, Portugal

[email protected]

Nervous Necrosis Virus (NNV) is the etiological agent of Viral Encephalopathy and Retinopathy (VER), also
known as Viral Nervous Necrosis. Of all the genotypes of this virus, the RGNNV (Red-spotted Grouper
Nervous Necrosis Virus) genotype is the most widely distributed in Southern Europe and causes high
outbreaks of mortality in aquaculture, especially in larvae and juveniles of different species with high
economic importance. This study aims to observe changes in the brain of European seabass (Dicentrarchus
labrax), over a time course infection with NNV.

Methodology
The time course study was carried out at CETEMARES facilities (MARE- Polytechnic of Leiria, Portugal), using
seabass (31.25 ± 7.22 g), which were challenged by intramuscular injection (IM) with 106 TCID50/fish of NNV
(RGNNV genotype). For histopathological analyses, the brain was collected from two fish per aquarium,
before (at 0 h) and post-challenge (at 5 and 6 d post-challenge). The organs were fixed in 10% formalin for
24 hours and then transferred to 70% ethanol until processing. For process and sectioning, standard
histological techniques were used, and the sections (5 µm) were stained with Hematoxylin-Eosin. An optical
microscope (Leica DM2000 LED) was used to observe the sections, and a digital camera (Leica MC 170 HD)
was used to capture images.

Results
The histopathological analyses of this study revealed significant changes between 0 h and the other sampling
time points, with vacuolization and necrosis of the brain tissue. These changes coincide with the moment
when symptoms of the disease were observed, namely spiral swimming, whirling, horizontal looping and
exophthalmia.

Conclusions
The brain is considered to be one of the target organs in which rapid replication of the virus occurs, causing
significant damage. The lesions observed in this study demonstrated that NNV has a neurotropism with
replication in the places where the tissues are most sensitive, particularly in the mesencephalon (optic
tectum) and the metencephalon (cerebellum).

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Whole genome sequencing of Ostreid herpesvirus 1 microvar using a nanopore sequencing
approach

Frederico Batista1, Tim Bean2, David Ryder1, Ronny Van Aerle1, Chantelle Hooper1, Richard Paley1, David
Stone1

1Centre for Environment, Fisheries and Aquaculture Science (Cefas), UK

2The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Scotland, UK

[email protected]

Ostreid herpesvirus 1 (OsHV-1) has been detected in association with high mortality rates of different bivalve
mollusc species worldwide. Since the characterization of OsHV-1 reference type in the 1990s, different
variants of this virus have been described. This is the case of OsHV-1 microvariants that have emerged in
several European countries, Australia and New Zealand causing mass mortalities of Crassostrea gigas
juveniles. The detection and identification of OsHV variant(s) and genotype(s) involved in these mortality
events is of paramount importance to implement measures to mitigate the impact and spread of OsHV-1.
The information provided by sequencing the whole-genome of OsHV-1 is key to identify emergent
genotypes, shed light on virus evolution, and potentially infer virulence. The assembly of OsHV-1 whole
genomes from technologies producing short sequences (<500 bp; e.g. Illumina sequencing) is technically
challenging due to the complexity of OsHV-1. The recently developed Oxford Nanopore technologies (ONT)
methods allow sequencing of long DNA fragments or amplicons, which can be used for whole-genome
sequencing at a reduced cost.

In this project, we sequenced and assembled the OsHV-1 genome using ONT and Illumina sequencing and
compared the completeness and quality of the resulting assembled genomes. DNA was extracted from
Crassostrea gigas juveniles experimentally infected with OsHV-1 microvar isolated in the UK. OsHV-1 genome
was sequenced using Illumina Miseq (NexteraXT library prep and V3600 sequencing cartridge) and using an
ONT R9 flow cell (FLO-MIN106) in a MK1C device following library preparation by ligation protocol SQK-
LSK109. Quality-trimmed sequences were assembled using a selection of bioinformatics tools and the quality
of the resulting genome sequences assessed and compared.

The near full-length genome of OsHV-1 microvar was obtained using both Illumina and Nanopore sequencing
approaches. The results of the genome assemblies carried out using different pipelines is presented and
limitations and advantages of the approaches used are discussed. We expect that this approach will
contribute to improve current knowledge on diagnostics, surveillance, transmission and virulence of OsHV-
1, which will help to provide insights to implement measures to mitigate the impact of this virus to the
molluscan aquaculture industry.

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Microalgal extracts against cyprinid herpesvirus 3

Anna Maria Becker1, Stefanie Fritzsche1, Patrik Blenk1, Jürgen Christian2, Kathrin Castiglione1

1 Institute of Bioprocess Engineering, Friedrich-Alexander University Erlangen-Nuremberg, Germany

2 Bavarian Health and Food Safety Authority, Institute for Animal Health II, Germany

[email protected]

Microalgae often stand out for their high biodiversity as well as their associated large number of potent
bioactives. Therefore, they are interesting candidates as possible sources of antiviral substances, e.g. against
cyprinid herpesvirus 3 (CyHV-3). Although this virus leads to high mortalities in aquacultures, there is no
treatment available yet. Hence, ethanolic extracts produced with accelerated solvent extraction from six
microalgal species (Arthrospira platensis, Chlamydomonas reinhardtii, Chlorella kessleri, Haematococcus
pluvialis, Nostoc punctiforme and Scenedesmus obliquus) were examined in this study for inhibitory effects
on viral replication. An inhibition of the in vitro replication of CyHV 3 in common carp brain cells could be
confirmed for all six species, with the greatest effect for the C. reinhardtii and H. pluvialis extracts.

At still non-cytotoxic concentrations viral DNA replication was reduced by over 3 orders of magnitude (> 99.9
%) each compared to the untreated replication controls, while the virus titers were at or even below the limit
of detection. When pre-incubating cells and virus with C. reinhardtii and especially H. pluvialis extracts before
inoculation, the reduction of viral DNA and virus titer was even stronger. Based on these results, an
intervention in the initial replication steps like viral adsorption or membrane fusion is assumed. Moreover,
a protection mechanism preventing the production of viral proteins and the assembly of mature virions is
also possible. All in all, the results show that microalgae are a very promising source of natural antiviral
substances against CyHV-3.

Funding: This work was funded by the German Federal Ministry of Food and Agriculture (BMEL) through the
Federal Office of Agriculture and Food (BLE), grant number 2815HS010.

Page 186
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Pathogenicity of different betanodavirus RGNNV/SJNNV reassortant strains in European
sea bass

Lorena Biasini, Anna Toffan, Andrea Marsella, Miriam Abbadi, Alessandra Buratin, Francesco Pascoli

Istituto Zooprofilattico Sperimentale delle Venezie, Italy

[email protected]

European sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) represent about the 96% of the
total farming production in Mediterranean aquaculture. Outbreaks of viral encephalo-retinopathy (VER)
represent one of the main infectious threats for these species. Betanodavirus, the causative agent of VER, is
a small non-enveloped virus with a bisegmented and positive-sense ssRNA genome. The RGNNV genotype is
the most widely spread in Southern Europe, while the SJNNV genotype has been rarely detected. The
existence of natural reassortants between the RGNNV and SJNNV genotypes has been demonstrated, the
reassortant RGNNV/SJNNV strains being the most common.

This study aimed to evaluate the pathogenicity of different RGNNV/SJNNV strains in juvenile sea bass. Nine
reassortants isolated over the past years (2005-2018) in European outbreaks were selected. About 50 sea
bass per group were intramuscularly injected with 0.1 ml of viral inoculum (TCID₅₀/ml ranged between
10^5.80 and 10^7.55) kept at 25 °C and monitored for 28 days. This study also included: i) fish infected with
a RGNNV strain known to be highly pathogenic in sea bass; ii) two groups of fish infected with SJNNV strains
with low pathogenicity in sea bass, and iii) mock infected fish. Dead fish were confirmed all positive by virus
isolation. At the end of the challenge, five brains from survivor fish were analysed by RT-qPCR and the
presence of the virus confirmed in all the samples. The VNNV/D.labrax/I/132/Apr2005 strain showed a
similar cumulative mortality to the one induced by the highly pathogenic RGNNV strain, while the
VNNV/S.aurata/GR/292-1,2/Aug2009 and VNNV/D.labrax/GR/292-7,8/Aug2009 strains both showed
intermediate pathogenicity. Cumulative mortality of the remaining strains was low and comparable to the
one induced by betanodavirus strains belonging to the SJNNV genotype.

In conclusion, data confirmed the susceptibility of sea bass to the RGNNV/SJNNV genotype with strain-
dependent mortality. The positivity found in the brains of all surviving subjects, although asymptomatic, is
however relevant, as it suggests a probable carrier role of survivors towards the sea bream with which the
sea bass is often simultaneously raised with. Results of ongoing analyses will shed light on the correlation
between genetic virulence markers and pathogenicity of reassortant strains.

Page 187
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Isolation of Clostridium perfringens from starving juvenile sturgeons with severe enteritis

Ginevra Brocca1, Ginevra Brocca1, Samuele Zamparo2, Tobia Pretto3, Alessandro Calore1, Matteo Cornaggia3,
Marilena Boscarato3, Eleonora Fiocchi3, Luana Cortinovis3, Romy Lucon Xiccato3, Ranieri Verin1, Francesco
Quaglio1

1University of Padova, Italy

2Azienda Agricola Troticoltura Erede Rossi Silvio, Italy
3Istituto Zooprofilattico Sperimentale delle Venezie, Italy

[email protected]

In November 2020, a mortality episode in juvenile sturgeons occurred in a hatchery in Northern Italy,
associated with severe abdomen dilatation and peculiar behavioral alteration, characterized by upside-down
surface swimming.

Juveniles (4-5 months old, average weight ± 25g) of Siberian and Russian sturgeons (Acipenser baerii and A.
gueldenstaedtii) and hybrid sturgeons GUBA (A. gueldenstaedtii x A. baerii), were promiscuously maintained
in concrete hatchery tanks supplied by well water (15 °C, O₂ 7 ppm, CO₂ 12 ppm), and fed at 0.4% of body
weight per day. The outbreak resulted in cumulative mortality of 25%.

Thirty moribund sturgeons were collected, euthanized with an overdose of MS-222, and underwent necropsy
followed by histological, bacteriological, and virological investigations.
Main macroscopic findings included diffuse and severe bloating of gastrointestinal tracts due to foamy
contents, with severe thinning and stretching of the intestinal wall, absence of visceral fat, and small size of
the hepatopancreas.

Histology revealed variable degrees of attenuation, sloughing, and necrosis of the intestinal epithelium,
associated with bacterial aggregates, and hepatocellular degeneration.
All specimens were negative for Herpesviruses by WSSK-1 cell culture, and Iridovirus (AcIV-E) and
Betanodavirus by molecular methods. Bacteriological examination revealed Plesiomonas shigelloides and
Cetobacterium somerae in a subset of intestines, while other organs yielded negative results. Based on recent
literature, an intestinal dysmicrobism was suspected, and anaerobic gram+ bacteria were investigated.

Clostridium perfringens was isolated from the intestines, and specific PCRs identified the toxinotype A and
the β2 toxin gene. The quantity of food was raised to 1.5% of body weight, and after 5 days the abnormal
behavior and mortality ceased. The analyses were repeated after 12 weeks from the food increase and
showed no alterations including negative results for C. perfringens isolation. Therefore, we hypothesize that
underfeeding may have led to an imbalance in intestinal microbiota, favoring the C. perfringens infection,
which was successfully controlled through management improvement.

The importance of intestinal microbiota in sturgeons, of which the genera Clostridium is one of the main
components, has recently been highlighted and, although this will need further validation, the increased diet
in this case was successful and possibly restored the microbiota.

Page 188
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Stunning of large fish species – challenges and solutions

Gabi Bröcker1, Verena Jung-Schroers1, Karina Retter1, Matthias Kempkes2, Matthias Lüpke2, Inga Wilk3,
Agnes Baumer4, Hermann Seifert2, Dieter Steinhagen1

1University of Veterinary Medicine Hannover, Fish Disease Research Unit, Germany

2Institute for General Radiology and Medical Physics, University of Veterinary Medicine Hannover, Germany
3Institute of Animal Welfare and Animal Husbandry, Federal Research Institute for Animal Health, Germany
4Hessian State Laboratory, Germany

[email protected]

In Germany, stunning of animals in general is regulated in a directive and special requirements for stunning
of fish are given. For all fish species, stunning by percussion or electric current is allowed. Due to anatomical
and physiological peculiarities, both methods are problematic for the stunning of large fish species, like
sturgeons, African catfish or arapaima. The skull anatomy of some of these species hinders rapid percussion
stunning and especially in very large individuals, anaesthesia is achieved only, when the skull is hit with
considerable force. In African catfish, it is known that a satisfactory stunning cannot be achieved with
conventional electric stunning devices, but that high current densities are required, which pose a threat to
occupational safety.

Methodology
As alternative stunning methods, the use of a penetrating and non-penetrating captive bolt devices will be
tested for stunning of different species of sturgeons, African catfish, and additional large fish species. For
positioning of both devices, the identification of the location of the brain based on external anatomical
structures of the skulls is important. This was investigated in four different sturgeon species, in African catfish
and in arapaima. The positional relationship of external structures to the location of the brain was visualized
during dissection of slaughtered fish and by using X-ray-, CT- and MRT-images as well as three-dimensional
computer models.

Results
The position of the brain could be exactly determined by external anatomical structures and the points for
placing the captive bolt devices were defined for each species. The position of the brains varied between all
examined fish species. The determination of the correct position for the captive bolt devices are therefore
crucial for an effective stunning in each species.

Conclusions
The use of especially a non-penetrating captive bolt device seemed to be user- and animal-friendly. However,
a thorough anatomical examination of the skull regarding external features and the position of the brain is
needed, even in closely related fish, to find a suitable position for the captive bold stunning intervention.

Page 189
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Detection of Francisella halioticida in archived samples of blue mussels (Mytilus edulis) in
the UK

Irene Cano Cejas1, John P Bignell1, Georgia M Ward1,2, Chantelle Hooper1, Matthew Green1, Stuart Ross1,
Frederico M Batista1

1Cefas, UK
2Department of Life Sciences, The Natural History Museum, UK

[email protected]

Francisella halioticida is an emerging pathogen affecting a number of mollusc species. This bacterium was
first characterised associated with a mass mortality event of the giant abalone, Haliotis gigantea, in Japan in
2010. Since then, F. halioticida has been recorded causing mortality in Yesso scallops in Canada in 2017, and
in Japan in 2018 and 2019. More recently, F. halioticida has been reported to be associated with mussel
(Mytilus spp.) mortalities in France in 2020.

In the last decade, sporadic declines in the population of wild blue mussels (Mytilus edulis) in the Tamar
estuary (UK) have been recorded; however plausible causative agent(s) were not determined. In 2013, a
survey of blue mussels on the riverbed showed histopathological changes in the digestive gland in 7% of the
mussels sampled, consisting of basophilic intracellular microcolonies (IMCs) of bacteria associated with
inflammatory infiltrates. A short region of 16S rRNA was amplified and sequenced from these samples.
Operational Taxonomic Units (OTUs) analysis showed a high proportion of reads with nucleotide similarity
to Francisella sp. and Anaplasma sp., however identification to the species level was not possible.

In the present study, we used nanopore sequencing to generate a consensus sequence of 1456 bp of a 16S
rRNA gene. Sequences showed nucleotide similarity between 99.93 and 100% to published sequences of F.
halioticida. Additionally, a high proportion of the sequences obtained showed ca. 84% nucleotide sequence
similarity to the genus Anaplasma, suggesting a novel group of intracellular organisms affecting mussels.

In situ bybridisation, using a 415 bp digoxigenin probe generated with PCR primers specific for F. halioticida
(Kamaishi et al., 2010), confirmed the presence of F. halioticida on the IMC lesions and in haemocytes.

This is the first confirmation of the presence of F. halioticida in the UK. Archived mussel samples from 2016-
2019 are being re-analysed for the presence of F. halioticida, and a dedicated survey is planned for
September 2021 to elucidate the prevalence of this bacterium within the mussel populations. The
pathogenicity of the Anaplasma-like bacteria to molluscs and its role in F. halioticida infections are currently
unknown.

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Evaluation of real-time loop-mediated isothermal amplification (LAMP) assays for the fast
detection of Bonamia ostreae and B. exitiosa in oysters

Irene Cano Cejas1, Chantelle Hooper1, Abigail Parker1, Jennifer C Nascimento-Schulze1,2

1Cefas, UK
2College of Life and Environmental Sciences, UK

[email protected]

Infection with Bonamia ostreae and B. exitiosa, causative agents of bonamiasis, are OIE-listed diseases
impacting worldwide oyster production. These haplosporidian parasites infect haemocytes of several oyster
species and induce physiological disorders. Clinical signs are not pathogen-specific and outbreaks often result
in mass mortalities. Therefore, rapid and easy-to-use field diagnostic tests are desirable for border inspection
posts, surveillance programmes, and outbreak control.

In this study we designed fluorescence real-time loop-mediated isothermal amplification (LAMP) assays for
the detection of B. ostreae and B. exitiosa. Several LAMP assays were designed on available sequences for
the 18S and ITS1 of the ribosomal RNA gene array, and the actin 1 gene. Among those, the assays targetting
actin 1 showed faster detection and greater species specificity. The optimal isothermal temperature for both
LAMP assays was established at 67 °C, allowing the detection of the parasite DNA in clinical samples in under
20 minutes. The annealing temperature for the LAMP products was 89.3 °C (±0.7) for B. ostreae and 87.9 °C
(±0.5) for B. exitiosa.

The analytical limit of detection (LOD) of the B. ostreae LAMP assay was 106 copies at 16:30 ± 2:10 (min:sec)
and 103 copies at 26:45 ± 2:15 for a recombinant plasmid containing the LAMP region. 102 copies was
detected only in half of the runs, with an average of 27:20 ± 6:30.
The LAMP assay for B. ostreae was further tested on 84 swabs taken from gill and mantle of European flat
oysters (Ostrea edulis) collected during an experimental challenge. Positive detection of the parasite DNA in
swabs was observed under 30 minutes from clinical cases. The assay's performance was compared with an
OIE-approved SYBR Green real-time PCR, showing an agreement of 85% among diagnostic tests.

The LOD for the B. exitiosa LAMP assay and its performance with field samples is currently being tested.

These promising preliminary results, followed by further optimization of the tests, will allow for its
deployment as a point of care test for disease investigations and surveillance of wild stocks.

Page 191
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Skin mucosal parameters during Koi herpesvirus shedding in common carp, Cyprinus carpio

Irene Cano Cejas, Richard Paley

Cefas, UK

[email protected]

Koi herpesvirus (KHV), or cyprinid herpesvirus 3 (CyHV-3), is the causative agent of Koi herpesvirus disease
(KHVD), a reportable disease to the OIE affecting Koi and common carp. KHVD outbreaks are highly
influenced by water temperature. The likelihood of transmission and development of viremia typically occurs
when the water temperature is between 16–25 °C, with mortality rates reaching 90% of the infected
population. Infection at lower temperatures often results in asymptomatic and reduced pathology, with no
mortalities recorded below 13 °C. Despite mathematical models and field data suggesting that KHVD
outbreaks could be controlled by careful management of water temperature in an aquaculture setup, studies
on KHV-host response at non-permissive temperatures are scarce.
In the present study, common carp was infected with KHV at either a non-permissive temperature (12 °C) or
permissive temperatures (17 and 22 °C). After an initial infection, survivors were subjected to temperature
increase to 22 °C. The survival rate varied from 100% in fish initially exposed at 12 °C, to 20% at 17 °C and 0%
at 22 °C. Viral shedding episodes lasted for up to 29 days (493 DD) for fish clinically infected at 17 °C, and up
to 57 days (684 DD) for asymptomatic fish infected and held at 12 °C.

To study the dynamics of viral shedding and host response, the gene expression of mucosal lysozyme (lysG),
complement 3 (c3), immunoglobulin M (IgMsec) and immunoglobulin Z2 (IgZ2) were measured for up to 900
degree days (DD) in skin swabs from common carp exposed to KHV.

The present study showed that the host response to KHV infection at the mucosal site was similar at the
three different temperatures analysed, except for lysozyme which was inhibited at low temperature. The
gene expression patterns suggested that KHV replication might subvert the complement system, which
coupled with a weak antibody response at the mucosal site, favours long periods of viral shedding. The fact
that a high survival rate after heat stress is recorded when the primary infection takes place during non-
permissible temperatures might allow for fish movements into KHV-positive sites, where water temperature
management and interventions to minimise viral shedding are possible.

Page 192
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Disease and prevalence of the endoparasite Perkinsus affecting the Mediterranean mussels
(Mytilus galloprovincialis) in the Italian waters of Campania (Italy)

Francesca Carella, Francesco Bolinesi, Olga Mangoni, Gionata De Vico

University Of Naples Federico II, Italy

[email protected]

Mussel (Mytilus galloprovincialis) production is an important sector of Campania region aquaculture,

occupies a significant part of territory (FAO, 2009). During the last twenty years, many different pathogens
have been reported on mussel’s population of the area. Perkinsus parasites are known to affect a wide range
of bivalve species. In the Mediterranean basin, three different Perkinsus species have been identified in
commercial bivalves: P. olseni, P. mediterraneus and P. andrewsi but, to date, global records of Perkinsus sp.
in mussels are nearly absent. Routine histopathological studies performed on Mediterranean mussel M.
galloprovincialis showed the presence of morphological features typical of the protozoan parasite Perkinsus
in two areas of Campania form March 2019 to November 2020.

The pathogen displayed the typical spherical cells with “signet ring” appearance, observed primarily at gonad
and digestive tissue, and elicited multiple inflammatory capsules of different size. Parasite prevalence was
26% (8/30) in Spring 2019, 40% in Spring 2020 (8/20), 16% in Winter 2020 (5/30). In order to define the water
column productivity, seawater samples were collected at discrete depth, to define the concentration of total
phytoplankton biomass and main functional groups in the area, with a maximum of 0.8 μg/L of chl a observed
near the bottom, and a net dominance of diatoms. Sequences of the ribosomal internal transcribed spacer
region (ITS1, 5.8S and ITS2) showed a similarity of 96% with P. olseni of Campania to those found in New
Zealand, Australia and Uruguay in bivalves like Pitar rostrata, Astrovenus sp. and Haliotis levigata and
surprisingly divergent from other P. olseni described in bivalves in Europe (Italy, France, Spain). On the other
hand, obtained sequences of non-transcribed spacer (NTS) gene compared to those available in gene bank
showed 87% similarity to Perkinsus isolates in Australia and New Zealand.

Additional field surveys are urgently needed to identify pathogen geographic distribution and potential
pathogenicity on the local mussel population. Intensification of bivalve culture and changes in environmental
conditions could favour disease progression. In addition, mussel’s movement could increase the risk of
introduction of this Perkinsus sp. in other areas.

Page 193
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Evaluation of immune response after LCDV-Sa infection in DNA-vaccinated gilthead
seabream

Dolores Castro, Rocío Leiva-Rebollo, Juan Gémez-Mata, Juan José Borrego, Alejandro Manuel Labella

Universidad de Málaga, Spain

[email protected]

The immune-related gene expression in vaccinated gilthead seabream after Lymphocystis Disease Virus 3
(LCDV-Sa) infection was analysed by using an OpenArray® platform based on TaqMan quantitative PCR. The
DNA vaccine used in this study (pcDNA-MCP) encodes the viral major capsid protein (MCP) and confers
protection against LCDV-Sa infection in juvenile gilthead seabream.

Gilthead seabream juveniles were distributed into four experimental groups and intramuscularly injected
with the vaccine (vaccinated group), the empty-plasmid (mock-vaccinated group), or PBS (control groups).
Thirty days after vaccination, vaccinated and mock-vaccinated fish, as well as one of the control groups, were
injected intraperitoneally with LCDV-Sa (10⁶ TCID₅₀/fish). Samples of head-kidney (HK) from 6 fish were
individually collected 1 and 3 days post-infection (dpi). The relative expression levels of 49 genes related to
the immune response, 4 reference genes (ef1α, actβ, rps18, and ub), and 3 viral genes (mcp, mmp, and rpb)
were analysed using an OpenArray. Samples from the non-infected control group were collected at the same
time points and used as calibrator.

The number of genes differentially expressed (DEG) in HK at 1 dpi was higher in vaccinated fish compared
with both mock-vaccinated and non-vaccinated animals. Furthermore, 94% of those DEG were
downregulated in vaccinated fish, whereas 80 and 75% of DEG showed downregulation in mock-vaccinated
and non-vaccinated fish, respectively. At 3 dpi, most DEG were upregulated, and the differences in their
number among groups were minimized. The recombination-activating gene 1 (rag1), a mediator of
development of B and T lymphocytes, was the only gene upregulated in HK samples at 1 dpi. This gene was
also upregulated in non-vaccinated animals but at 3 dpi. In contrast, early mx induction was observed in non-
vaccinated animals (upregulation of mx2 at 1 dpi) in comparison to vaccinated seabreams (upregulation of
mx1 and mx2 at 3 dpi). The results that will be discussed could evidence the role of the DNA vaccine as
regulator of the primary lymphoid tissues (HK) in gilthead seabream against LCDV-Sa infection, through
downregulation of inflammation related-genes, early upregulation of rag1, and a later expression of
interferon stimulated genes.

Page 194
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Interpreting cathepsin proteases from transcriptome of Anisakis pegreffii

Anand Chakroborty1, Jerko Hrabar2, Željka Trumbić3, Ivona Mladineo1

1BiologyCentre, PARU, Czech Academy of Sciences, Czech Republic

2Laboratoryof Aquaculture, Institute of Oceanography and Fisheries, Croatia
3Department of Marine Studies, University of Split, Croatia

[email protected]

Anisakis pegreffii is a nematode that parasitizes marine cetaceans as definitive hosts, accidentally infecting
humans for a limited time span. Although the knowledge of disease mediators during anisakiasis in humans
is incomprehensive, we can close the gaps by applying what is known from other helminth infections.
Cathepsins are key virulence factors in disease progression and synchronized expression of these proteolytic
enzymes is considered a hallmark of parasitosis. Figuring as potential disease biomarkers, they are also
important subjects in drug or vaccine targets investigation.

The functional insights of these proteases vary during life cycle of parasites in context of infection type,
eclosion, feeding and immune evasion. Cathepsins are clusters of proteolytic enzymes of lysosomal origin
divided into three classes – cysteine, serine and aspartic proteases. Cysteine cathepsins occur in multiple
forms as cathepsin B-like (B, O, C, and X or Z), cathepsin L-like (L, V, K, S, and H), cathepsin F-like (W and F),
and placenta-specific cathepsins (cathepsins J/P, M, Q, R, 3, 6, 7 and 8) that are exclusively expressed in
rat/mouse placenta. Biochemically, cathepsins A and G have been distinguished as serine proteases, whereas
D and E belong to aspartic endopeptidases. Through in silico data-mining of A. pegreffii infective third-stage
larvae (L3) transcriptomes, we have identified multiple signatures of cathepsin A carboxypeptidases and
cathepsin D. Apart from these, L3 express a vast array of cysteine proteases. In addition to cathepsin B, F and
L, the presence of putative cathepsin Z and W has also been validated. A. pegreffii presents at least four
distinct clades of cathepsin B and L proteolytic enzymes, and a unique cathepsin F having 51% identity to
human orthologue. In general, A. pegreffii cysteine cathepsin orthologues share maximum identity to
Toxocara canis rather than free-living model Caenorhabditis elegans. Putative A. pegreffii cathepsin B1 has
duplet histidine conserved in occluding loop, thus suggesting both effective exopeptidase and endopeptidase
activities on specific substrates.

Interestingly, the three other putative cathepsin B clades (B2, B3, B4) show to be non-canonical, expressing
shorter occluding loops. Such atypical proteases could open interesting dimensions in understanding
nematode biology and scoping for drug discovery.

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Detection of Red-spotted grouper nervous necrosis virus (RGNNV) in shrimp and squid of
the Mediterranean Sea

Elena Chaves-Pozo, Carolina Johnstone, Montse Pérez, Marta Arizcun, Cristina García-Ruíz, Cristina
Ciércoles, Elena Chaves-Pozo

Instituto Español de Oceanografía, Spain

[email protected]

The quest for sustainable fisheries and procurement of food supply has increased aquaculture production
up to the world fisheries capture. Viral encephalopathy and retinopathy (VER), also known as viral nervous
necrosis (VNN), is caused by nervous necrosis virus (NNV) and results in high mortality of larvae and juveniles
of continuous increasing amount of fish species. The disease causes vacuolation and necrosis of the central
nervous system.

The virus has a nude capsid protecting a bipartite RNA genome that consists of positive stranded molecules
RNA1 and RNA2. Four NNV genotype strains distributed worldwide are discriminated according to sequence
homology of the capsid protein encoded by RNA2. Preventive treatments prioritize the RGNNV (Redspotted
grouper nervous necrosis virus) genotype that has the highest optimum temperature for replication and the
broadest range of susceptible species. A flow of NNV between wild and cultured fish had been demonstrated,
and reservoirs of NNV have been reported in invertebrates, raising concern on the spreading of NNV in the
mariculture industry through contaminated food. The present study aimed to contribute in the surveillance
of reservoirs of NNV in invertebrates of the unexplored western Mediterranean Sea.

We report the detection of the RGNNV strain in two species of squid (Alloteuthis media and Abralia veranyi),
and in one shrimp (Plesionika heterocarpus) collected in 2015 in the Alboran Sea. According to RNA2
sequences obtained from invertebrates and reported to date in the Mediterranean Sea, the strain RGNNV is
predominant in this semi-enclosed sea. Our results suggest that RGNNV distribution is apparently
independent of host species and ecosystem, and similar between invertebrates and fish species that feed on
invertebrates, calling for an increase in surveillance of NNV reservoirs in the wild.

Page 196
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Microplastic and an associated metal contaminant (Palladium) impair the immune
response against pathogenic bacteria of the marine bivalve Mytilus galloprovincialis

Elena Chaves-Pozo, Emmanuel Gafo, Marina Albentosa, Juan Santos-Echeandía, Elena Chaves-Pozo

Instituto Español De Oceanografía, Spain

[email protected]

Microplastics (MPs) are widely distributed in marine environments and have been reported to cause harmful
physiological effects in marine bivalves including immune modulation. While Mytilus galloprovincialis is a
model species in environmental monitoring studies, little is known regarding the effects of MPs and
palladium (Pd), an emerging contaminant, on the immune functioning of this species. In this study, gills and
gland samples of specimens of M. galloprovincialis which were exposed to the same particle concentration
(2.5 x 10 6 μm /mL) of Control (microalgae), MP (virgin microplastics) and MP-Pd (Pd spiked microplastics)
once an hour during 4 hours. Thus, mussels were exposed to 6095 ng of Pd/mussel. Samples analyzed were
collected after 4 and 24 hours of exposure and after 24, 48 and 144 hours of depuration. Several innate
immune activities (lysozymes, peroxidase, protease, antiprotease, and bactericidal activities) were analyzed.

Our data demonstrated that after 24 hours of exposure to MP, a decrease in lysozymes and peroxidase
activities occurred, but not in the bactericidal activity which increased. However, when MP-Pd was used, an
increase in all these activities was observed compare to MP levels group. These data suggest that when MP
are spiked with Pd, the latter compound might induce an inflammatory process that will results in higher
levels of most of all the immune activities analyzed. In the gland, however, most of the activity levels were
decreased upon MP-Pd treatment compared to MP levels at different time point of exposure depending on
the activity.

Interestingly, after 24 hours of removing the pollutants from the water, most of the activity levels in both
tissues, gills and gland, were recovered to control levels, but not the bactericidal activity. Considering this
impairment of the bactericidal activity against possible pathogenic bacteria, a potential threat to mussels
population in a polluted scenario is highly plausible.

Page 197
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Risk factors linked to the onset of Nodular Gill Disease (NGD) in rainbow trout farms in
North-eastern Italy

Alessia Cocco1, Marica Toson1, Alberto Perolo2, Claudia Casarotto1, Ginevra Brocca2, Ranieri Verin2,
Francesco Quaglio2, Manuela Dalla Pozza1, Laura Bille1

1Istituto Zooprofilattico Sperimentale delle Venezie, Italy

2 Department of Comparative Biomedicine and Food Science, University of Padova, Italy

[email protected]

Nodular gill disease (NGD) is an emerging disease caused by amoebae affecting freshwater salmonids. It can
cause high mortality and severe economic losses, in particular in rainbow trout (Oncorhynchus mykiss) farms.
In Italy, most of the production of this species is found in the North-eastern regions. Therefore, an area in
this part of the Country was selected to perform an epidemiological study with the aim of identifying the risk
factors related to the onset of the disease.

The survey was carried out between 2017 and 2019 and involved all the rainbow trout farms located in the
Autonomous Province of Trento (North-eastern Italy). We administered a questionnaire to the 50 farmers,
requesting information on the health status of the farms, their structural and management features, the
applied biosecurity measures and the proximity and hydrological connection to other farms.

Data were stored in a dedicated Access database, validated and analysed through statistical software STATA
12.1 and SAS 9.4. Univariate statistics were used to summarize the data characteristics. Afterwards, about
30 variables were selected as possible risk factors for NGD onset and analysed through bivariate analysis
(Chi-square or Fisher exact test for categorical variables, Wilcoxon/Mann-Whitney nonparametric test for
two independent samples when quantitative variables were considered). A significant association (p-
value<0.05) with NGD was detected for 6 variables. A logistic regression analysis with stepwise selection was
then performed on these variables.

The data analysis revealed that 42% of the farms experienced at least one episode of NGD in the previous 5
years. Furthermore, the reported cases increased during the study period, particularly in winter. The
identified risk factors for the onset of NGD were the presence of other diseases in the farm in recent years
(OR=17.496; 95% IC=2.746; 111.454), such as viral haemorrhagic septicaemia (VHS), infectious
haematopoietic necrosis (IHN) and enteric redmouth disease (ERM), and having farms in the 5 km upstream
(OR=24.679; 95% IC=2.885; 211.116).

The results show that the disease is widespread among rainbow trout farms in the selected study area and
underline the relevance of the exposure via water to amoebae and lack of biosecurity measures for the
onset of the disease.

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The first evidence of the monogenean fluke Zeuxapta seriolae on a greater amberjack
(Seriola dumerili) farmed in the Adriatic Sea

Slavica Colak1, Tomislav Šarić1, Relja Beck2, Matko Kolega1, Danijel Mejdandžić1, Tomislav Šarić3

1 Cromaris d.d., Croatia

2 Croatian Veterinary Institute, Croatia
3 University of Zadar, Croatia

[email protected]

Diversification of products in aquaculture, such as introduction of new species in farming, is one of the
strategic determinants of the development of aquaculture in Croatia and the EU. One of the new species
playing an increasingly important role in aquaculture is the greater amberjack (Seriola dumerili) due to its
excellent market potential, rapid growth, large final mass, and collected positive experiences of farming
individuals caught from nature (fattening of greater amberjack).

Among the various pathogens recorded in the farming of greater amberjack in sea cages, one of the most
important/pathogenic is the gill monogenean parasite Zeuxapta seriolae, which causes considerable losses.
Here we present the first evidence of the gill parasite Z. seriolae on the greater amberjack farmed in the
Adriatic Sea.

During the parasitological examination of greater amberjack farmed in sea cages on a farm located in the
central part of the Croatian Adriatic coast, the gill arches of the ten fish were removed. Examination of the
gill arches under a magnifying glass revealed the presence of the flukes, which we assumed to be Z. seriolae.
The parasites were removed from gills and stored in 95% alcohol solution for further molecular analyzes.
DNA was extracted with commercial Blood and tissue kit, amplified with conventional PCR, sequenced in
both directions and assembled. BLAST analysis of sequences of internal transcribed spacer 2 (ITS-2) region
from four individual flukes revealed 98.38% similarity to Z. seriolae from the gills of mulloway (Argyrosomus
japonicus) cultured in Australia.

Best to our knowledge current finding represents the first one in the Adriatic Sea of a new pathogen able to
cause economic losses in greater amberjack farming.

Page 199
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Improving SUstainability and PERformance of aquaculture farming system: breeding for
lactococcosis resistance in rainbow TROUT (SUPERTROUT)

Silvia Colussi1, Marino Prearo1, Ilhan Altinok2, Konstantina Bitchava3, Lucio Fariano4, Ana Isabel Vela Alonso5,
Donatella Volpatti6, Pier Luigi Acutis1

1 Istituto Zooprofilattico Sperimentale Piemonte, Liguria E Valle D'aosta, Italy

2 Karadeniz Technical University, Turkey
3 ELGO-DEMETER, Greece
4 Azienda Agricola Canali Cavour, Italy
5 VISAVET-Universidad Complutense de Madrid, Spain
6 Università degli Studi di Udine, Italy

[email protected]

Aquaculture represents the winning strategy to face the depletion of fish resources due to the constant
growth of world population. Small-scale farming systems are common in the Mediterranean area and trout
farming is suitable for this purpose, efficiently exploiting surface and underground water resources. Italy,
Turkey, Spain and Greece are at the top of the list of the main producers of rainbow trout (Oncorhynchus
mykiss).

The increase in the demand for aquaculture products brings with it the need to make production systems
more efficient and sustainable: this entails a management and technological improvement, including the
development of vaccines, the reproductive aspect and the genetic selection of characters related both to
productive traits and to disease resistance.

SUPERTROUT has been designed to face infectious diseases, a major concern in aquaculture farming systems,
using novel approaches, and improving at the same time environmental sustainability and profitability of
small-scale farming system. In particular, Lactococcus garvieae is reported to be responsible for significant
economic losses in aquaculture worldwide: the loss due to this infection is 10-60% of the total rainbow trout
production; mortality increasing are reported when water temperature exceeds 15 °C. This is a critical issue
for the countries bordering the Mediterranean Sea where the temperate climate, associated with the global
warming, in recent years, favoured the durability and diffusion of lactococcosis outbreaks.

The overall objective of SUPERTROUT is to improve sustainability and profitability of small-scale farming
system facing lactococcosis in rainbow trout applying innovative strategies based on: exploitation of natural
genetic resistance of trout through marker assisted selection; development of recombinant vaccine to be
administered by immersion; improvement of reproductive performances exploiting trout genetic features.

Using this approach costs related to disease control will be reduced, increasing profitability, while
sustainability will be enhanced reducing environmental contamination due to antibiotic treatment and
reducing antibiotic resistant strains.

Page 200
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Antimicrobial peptides: an insight on their efficacy against major viral and bacterial fish
pathogens

Luana Cortinovis1, Romy Lucon Xiccato1, Luana Cortinovis1, Eleonora Fiocchi1, Rosita Quartesan1, Jane
Budai1, Francesco Montesi1, Anna Toffan1, Tobia Pretto1, Fernando Porcelli2, Francesco Buonocore2

1Istituto Zooprofilattico Sperimentale delle Venezie, Italy

2University of Tuscia, Italy

The increasing outbreaks of viral and bacterial diseases and the risk of antimicrobial resistance in aquaculture
have prompted the need to look for new molecules able to replace or support the use of conventional drugs.
Antimicrobial peptides (AMPs) represent an interesting field of investigation, showing a broad spectrum of
action against viruses, bacteria, fungi and parasites while playing a key-role in the innate immune system.

Chionodracine (Cnd), Trematocine (Tmc) and a Cnd-derived mutant peptide (Cnd-m3) are AMPs obtained
from two Antarctic fish species (Chionodraco hamatus and Trematomus bernacchii). In this study, their
antimicrobial activity has been investigated against major fish pathogens.

In order to identify an appropriate working concentration range, cytotoxicity level of the peptides was
assessed in fish cell lines (EPC and E-11).

Virological investigations focused on Betanodavirus, Viral Haemorrhagic Septicemia Virus (VHSV) and Spring
Viraemia of Carp Virus (SVCV). The lowest cytotoxic concentration of each peptide was mixed with different
titers of each virus, with and without overnight incubation at 4 °C, and subsequently inoculated on cells
monolayer. Tmc reduced VHS and SVCV activity in cell-lines only after overnight incubation with the virus.
Transmission electron microscopy highlighted that viral particles had been damaged after overnight
incubation with Tmc, suggesting a direct effect on the virus integrity. Conversely, Cnd and Cnd-m3 displayed
no activity against the investigated viruses.

To explore the bactericide and bacteriostatic activity of these peptides, their minimal inhibitory (MIC) and
minimal bactericidal (MBC) concentrations were determined against five bacterial species affecting fresh and
saltwater fish (Gram+ and Gram-). For both MIC and MBC determination, growth media, incubation
temperature and time derived from CLSI guidelines, whereas AMPs concentrations followed previous
cytotoxicity-test results. In all the tested species the most active peptide turned out to be Cnd-m3, since the
two other molecules showed MIC and MBC values above the cytotoxic concentrations.

Further studies will be necessary to better characterize AMPs activity and to evaluate their possible field
applications; nevertheless, our preliminary results support their antimicrobial role and their possible
relevance as novel drugs in aquaculture.

Page 201
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Exposure to nanoplastics increases the susceptibility to nodavirus (nnv) infection

Alberto Cuesta, Carmen González-Fernández, Maria A. Esteban

Immunobiology for Aquaculture Group, Department of Cell Biology and Histology, Faculty of Biology,
University of Murcia, Spain

[email protected]

Nowadays, nanoplastics (NPs) are the most abundant nanomaterials on the market. Recently, its presence
in the marine environment has been evidenced. NPs have been revealed to cause different negative
physiological effects on marine organisms, including reproduction, behaviour or oxidative stress. However,
the impact of NPs on disease resistance is unknown.

The Nervous Necrosis Virus (NNV) is recognized to affect many fish species producing viral encephalopathy
and retinopathy disease, which causes great losses in marine aquaculture. Thus, the aim of this study was to
assess if the exposure to nanoplastics increases the susceptibility to NNV both in vitro and in vivo. For that
purpose, a brain gilthead seabream (Sparus aurata) cell line (SaB-1) was exposed to functionalized
polystyrene NPs (PS-NH2, PS-COOH, PS-Plain) and then infected by NNV.

In addition, European sea bass (Dicentrarchus labrax) juveniles were injected with the same NPs and then
infected with NNV. In both cases, we observed that the exposure to NPs altered the resistance of the cell line
or sea bass specimens to NNV infections. These results highlighted that the presence of NPs can impact the
infection process of NNV and fish resistance to the disease posing an additional risk to marine organisms.
Further studies are needed to elucidate the impact of NPs in the infection process in fish.

Page 202
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Antimicrobial peptide (AMP)-encoding plasmids show little immunomodulatory and
nodavirus-protective actions in European sea bass juveniles

Alberto Cuesta, Laura Cervera, Carmen González-Fernández, Daniela Cano, Miguel-Angel García-Álvarez,
Maria-Angeles Esteban

Immunobiology for Aquaculture Group, Department of Cell Biology and Histology, Faculty of Biology,
University of Murcia, Spain

[email protected]

Nodavirus (NNV) causes the viral encephalopathy and retinopathy in fish. This is an important virus in the
Mediterranean area, which affects, among other fish species, to European sea bass (Dicentrarchus labrax)
causing great mortalities in larvae and juvenile stages; therefore, with great economic impact on the
aquaculture industry. Antimicrobial peptides (AMPs) are low molecular weight peptides, normally cationic
and amphipathic, that interact with a wide range of pathogens causing its death. In teleost, many AMPs have
been described to date but their role as antiviral agents has been very scarcely evaluated. Apart from the
direct viral lytic activity, their use as prophylactic agents, probably through their immunomodulatory
potential, has been not evaluated in fish.

Therefore, in this work, the main aim was to determine if the intramuscular injection of AMP-encoding
plasmids was immunomodulatory and protective against NNV infections in the European sea bass. Thus,
plasmids encoding the sea bass AMPs dicentracin (DIC), beta-defensin (DB), hepcidin (HAMP) or NK-lysin
(NKL) were generated and intramuscularly injected into sea bass juveniles. In addition, after 3 days of
treatment fish were infected by NNV. Fish muscle and head-kidney tissues were sampled to assess the
plasmid transcription as well as the expression of immune-related genes. In addition, immune-related genes
and viral transcription was also evaluated in the brain after infection. Clinical signs of disease and mortalities
were also recorded. Our results show that AMP-plasmid encoded genes were transcribed in the muscle
accordingly but the regulation of the expression of immune-related genes was slightly affected. In addition,
NNV-induced clinical signs or mortality were slightly affected by the plasmids encoding sea bass AMPs.

To conclude, our study suggests that the administration of AMPs through coding plasmids to European sea
bass has very low immunomodulatory and preventive NNV-disease actions. More studies should be carried
out to ascertain if AMPs could be used as a prophylactic method for NNV infection and the forms and routes
of administration.

Page 203
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Immunological response of gilthead seabream during an outbreak of the parasite
Cryptocaryon irritans

Alberto Cuesta1, Cervera1,2, Carmen González-Fernández1, Marta Arizcun2, Alberto Cuesta1, Elena Chaves-
Pozo2

1Immunobiology for Aquaculture Group, Department of Cell Biology and Histology, Faculty of Biology,
University of Murcia, Spain
2Oceanographic Center of Murcia, Spanish Institute of Oceanography (IEO), Spain

[email protected]

Cryptocaryon irritans, a holotrichous ciliate protozoan, causes “marine white spot disease” and is frequent
in commercial marine fish farms. It is considered to cause one of the most devastating parasitic diseases in
aquaculture producing significant economic losses. This parasitosis mainly causes respiratory distress,
disruption of lamellae structure, mucus hyperproduction and skin damage. C. irritans has low host-specificity
and one of its susceptible species is gilthead seabream, which has an enormous importance in the
aquaculture industry in the Mediterranean area.

In this work, we aimed to evaluate different parameters of the immune response of gilthead seabream during
a natural outbreak with C. irritans. Diagnosis was carried out by visualization of the parasite in the gills, along
with the presence of compatible disease signs. Two days after the appearance of fish mortalities due to C.
irritans, fish were sampled. Serum was isolated and innate humoral immune responses including peroxidase,
bactericidal, natural haemolytic complement or lysozyme activities analyzed. Gills and head-kidney were
obtained to evaluate immune-relevant gene transcription by real-time PCR and for microscopical studies.
The genes studied code for inflammatory proteins, leukocyte markers or antimicrobial peptides. Our
Histopathological data confirmed the existence of the parasite in the gills and a heavy disruption of gills
morphology. In serum, however, only the bactericidal activity was modified upon infection. The most altered
genes were those related to inflammation.

This work is a first approach to determine the immune response triggered by this parasite in gilthead
seabream and further research should be done to clarify the parasite-host interactions.

Page 204
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NK-lysin peptides partially protect against nodavirus infection improving viral
encephalopathy and retinopathy pathological condition

Alberto Cuesta1, Yulema Valero1,2, Carmen González-Fernández1, Constanza Cárdenas3, Fanny Guzmán3,
Rosa León4

1Immunobiology for Aquaculture Group, Department of Cell Biology and Histology, Faculty of Biology,
University of Murcia, Spain
2Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidade de Santiago de

Comgpostela, Campus Vida, Spain

3Núcleo Biotecnología Curauma (NBC), Pontificia Universidad Católica de Valparaíso, Spain
4Laboratorio de Bioquímica, Facultad de Ciencias Experimentales, Campus de Excelencia Internacional del

Mar (CEIMAR), Universidad de Huelva, Spain

[email protected]

The different strategies in the search of effective tools against fish viruses has been mainly focused on the
vaccines’ development or the exploitation of the direct antiviral activity of synthetic and natural compounds.
Although antimicrobial peptides (AMPs) have been widely studied against bacteria, they also show a potent
antiviral and immunomodulatory role. NK-lysin is a human granulysin orthologue that exert a double
function. It acts as an AMP and a direct effector in the cell-mediated cytotoxic response. This molecule is
suggested as a crucial molecule involved in the defence upon nodavirus (NNV), an epizootic virus that causes
serious problems in welfare and health status in Asian and Mediterranean farmed fish. After demonstrating
the direct antiviral activity of different NK-lysin derived peptides (NKLPs) against NNV in vitro, we aimed to
evaluate their potential use as a prophylactic treatment for European sea bass (Dicentrarchus labrax), one
of the most susceptible cultured-fish species to NNV.

In this work, we intramuscularly injected synthetic antiviral NKLPs (3 derived form sea bass NKL and other
form tongue sole NKL) to healthy juvenile European sea bass specimens and studied the immunomodulation
elicited in the head-kidney. After 72 hours, we in vivo infected the treated fish with NNV and
immunomodulation was analysed after 3 days of infection. Survival and signs of disease were also recorded.
Our results showed that NKLPs produced very little up-regulation in the gene expression though some NKLPs
did for T- and B-cell and macrophage markers up to 24 hours post-treatment. After NNV infection, sea bass-
derived NKLPs ameliorated the clinical sings of infection while all the tested NKLPs showed protection in the
mortalities, ranging from low to high relative protection.

To conclude, synthetic NKLPs corresponding to different NK-lysin regions, diverse amino acid composition
and structure, produced little immunomodulation in European sea bass involving markers of B and T
lymphocytes and macrophages. Interestingly, pre-treatment with NKLPs triggered the improvement in the
signs of disease and survival upon NNV infection. However, further studies are needed to fully understand
the mechanisms induced to ameliorate the NNV disease.

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Shi drum (Umbrina cirrosa) juveniles are susceptible to all Nodavirus genotypes

Alberto Cuesta1, Elena Chaves-Pozo2, Marta Arizcun2

1Immunobiology for Aquaculture Group, Department of Cell Biology and Histology, Faculty of Biology,
University of Murcia, Spain
2Oceanographic Center of Murcia, Spanish Institute of Oceanography (IEO), Spain

[email protected]

Nervous necrosis virus (NNV; family Nodaviridae, genus Betanodavirus) is the causative agent of viral
retinopathy and encephalopathy (VER) disease, which mainly affects the larval and juvenile stages of fish.
The virus has an acute lethal effect in larval stages and juveniles. According to the RNA2 sequence, NNV are
mainly divided into four genotypes: RGNNV, SJNNV, BFNNV and TPNNV.

The shi drum (Umbrina cirrosa) is a serious candidate for the diversification of Mediterranean aquaculture.
Although aspects related to nutrition or reproduction have been elucidated others related to pathology or
immunity have been poorly studied. In this regard, the shi drum is a susceptible species for betanodavirus
(RGNNV) infection, as several natural outbreaks have been reported. In order to expand the actual
knowledge and understand the shi drum-NNV interactions we evaluated whether this fish species is
susceptible to all the NNV genotypes.

Our data demonstrate that the laboratory infections with all the NNV genotypes produced clinical signs of
the VER disease and mortalities in shi drum juveniles. Interestingly, clinical signs and histopathological lesions
in the brain and retina were different depending on the genotype used. Finally, viral capsid protein was
immunodetected in the brain and retina from all infected fish whilst infective particles were only recovered
from RGNNV-, BFNNV- and TPNNV-infected specimens.

In conclusion, this work demonstrates that shi drum juveniles are susceptible to all four genotypes of NNV
and represent the first step in studying host–NNV interactions and immune responses in this species, which
should be further characterized.

Page 206
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RGNNV and SJNNV reassortants produce mortality and replicate in gilthead seabream
larvae

Alberto Cuesta1, Miguel Angel García-Álvarez1, Laura Cervera1, Elena Chaves-Pozo2

1Immunobiology for Aquaculture Group, Department of Cell Biology and Histology, Faculty of Biology,
University of Murcia, Spain
2Oceanographic Center of Murcia, Spanish Institute of Oceanography (IEO), Spain

[email protected]

Nervous Necrosis Virus (NNV) is one of the most challenging pathogens for aquaculture development
nowadays, mainly affecting marine teleost fish of major interest to the aquaculture industry and causing
great economic losses. NNV consist in four genotypes, which seem to have a tropism for certain teleost fish
species. Among them, gilthead seabream (Sparus aurata) has been considered as a non-susceptible species
to the disease produced by traditional NNV genotypes. However, there are some evidences that indicate
seabream is able to develop the disease in the presence of certain reassortant strains of NNV, called
RGNNV/SJNNV, which possesses the RNA1 segment of the RGNNV genotype and the RNA2 segment of the
SJNNV genotype, which may cause a new threat to aquaculture.

Therefore, the main objective of this study was to evaluate the susceptibility of gilthead seabream larvae to
the reassortant strains RGNNV/SJNNV and SJNNV/RGNNV. For this purpose, larvae were exposed to 104
TCDI50/mL in triplicate tanks with the reassortant strains. Samples of 5 individual larvae were collected at
different days post-infection and used for gene expression and infective NNV isolation. Our data show that
both reassortants produced mortalities, although the RGNNV/SJNNV was the one which produced the
highest mortality and viral gene transcription, which significantly increased from 1 to 7 days post-infection.

In conclusion, our study demonstrate that seabream larvae are susceptible to both RGNNV/SJNNV and
SJNNV/RGNNV reassortants under laboratory conditions. Further studies should be performed to
understand the pathogenicity of the NNV reassortant strains to prevent and control future outbreaks in
aquaculture farms.

Page 207
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Survey on parasite fauna of the dusky grouper (Epinephelus marginatus, Lowe 1834) caught
off the Sicilian coasts, south of Italy

Giovanni De Benedetto1, Ivan Corti2, Fabio Marino3, Gabriella Gaglio1

1Dept. of Veterinary Sciences, University of Messina, Italy

2Agenzia di Tutela della Salute dell’Insubria, Italy
3Dept. of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Italy

[email protected]

From 2018 to 2020, 70 internal organs and gills of dusky groupers were collected from different fish markets
or seized during official checks in Southern Italy.

The aim of this survey was to investigate parasite fauna of dusky grouper, a species of high economic value
but poorly studied from a parasitological point of view. Fish were identified and weight and total length
recorded during sampling. External examination was performed to investigate the presence of ectoparasites
on the gills and skin. All internal organs were inspected for helminth and gross lesion presence with the aid
of a stereomicroscope. Parasites collected were stored in 70% ethanol until morphological identification.

Examined specimens were further divided according to the capture period into two groups namely warmer
months (N= 23), and colder months (N= 47). At the same time, the specimens were again divided into 4 size
classes (juvenile, medium, sub-adult and adult). For each parasite species, epidemiological indices were
calculated. Pearson’s chi-square was applied to evaluate differences in parasite infections between warmer
and colder months (P< 0.005).
More than half (67.2%) of specimens were infected with one or more parasite species. The most abundant
species was Prosorhynchus caudovatus (42.9%, MA 5.32, MI 7.9), followed by Podocotyle temensis (28.6%,
MA 1.14, MI 1.70), Didymodiclinus sp. (18.6%, MA 1.10, MI 1.63), Philometra jordanoi (5.7%, MA 0.40, MI
0.19), Hemipera sp., Pseudoempleurosoma sp., Megalocotyle hexacantha (1.4%, MA 0.01, MI 0.02) and
Anisakis type II larvae (5.7%, MA 0.21, MI 0.31).

Statistical evaluation showed a dynamic infection level. In particular, P. caudovatus and P. temensis were
mostly found in colder months. Higher prevalence of infection of P. jordanoi and Contracaecum sp. was found
in warmer months. Biometric data including weight and TL of E. marginatus investigated were positively
correlated with the parasitic load of P. jordanoi and Didymodiclinus sp.

The different prevalence of parasite infection found between size classes, and warmer and colder months is
probably related to diet, which is characterized by mollusks that are intermediate hosts for parasite species
found.

This investigation improves current knowledge on the parasitic fauna of E. marginatus, inhabiting
Mediterranean coasts.

Page 208
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Concurrent infections with two species of Philometra spp. in the eyes and gonads of greater
amberjack Seriola dumerili from the Canary Islands

Carolina de Sales-Ribeiro1, Natalia García-Álvarez2, Pilar Foronda3, Jordi Miquel4, Fernando Real2, Antonio
Fernández1, María José Caballero1

1Division of Veterinary Histology and Pathology, University Institute for Animal Health and Food Safety
(IUSA), Veterinary School, Universidad Las Palmas de Gran Canaria, Spain
2Division of Infectious Diseases and Ichthyopathology, University Institute for Animal Health and Food Safety

(IUSA), Veterinary School, Universidad de Las Palmas de Gran Canaria, Spain

3Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna,

Spain
4Department of Biology, Health and Environment, Faculty of Farmacy and Food Sciences, Universitat de

Barcelona, Spain

[email protected]

Family Philometridae represents one of the most important groups of dracunculoid nematodes affecting fish.
With a worldwide distribution, these parasites are present in both freshwater and saltwater environments
with an ever increasing number of species being identified. However, their role as a potential pathogens is
still unclear.
Here, we detail the presence of philometrid parasites in greater amberjacks Seriola dumerili captured in the
Northeast Atlantic Ocean, along the coasts of the Canary Islands.
Philometra spp. were found in 30% of the greater amberjacks necropsied (n=70). They were present in the
gonads (38%, 29% in the ovary and 10% in the testis), retrobulbar region (33%) or in both the gonads and
retrobulbar region (24%).
Grossly, in the retrobulbar tissues, often entangled, there were numerous threadlike, orange to light brown,
soft, measuring up to 150 mm in length and 1 mm in diameter nematodes. In the gonads, affecting mostly
the ovaries, there were either individualized or small groups or threadlike nematodes with narrowed pointed
ends, dark red to dark brown, firm with a smooth cuticle, measuring up to 250 mm and 2 mm in diameter.
Histologically, philometrids collected from both sites also showed dissimilar morphological features. In the
retrobulbar tissues, they showed a very thin to almost absent cuticle and a pseudocoelom containing uterus
with numerous eggs. In turn, in the gonads, philometrids presented a thicker and strongly eosinophilic cuticle
and, in the pseudocoelom, the digestive tract was displaced by uterus filled with numerous eggs and
developed larvae. In both sites, inflammatory reaction was minimal to absent.
These parasites are compatible with the genus Philometra. Although, given their distinct morphological
characteristics and locations, we believe these represent two different species. Morphological and molecular
studies are currently being conducted to identify both species.
To our knowledge, this is the first description of concurrent infections with two species of Philometra spp. in
the eye and gonads of greater amberjack from the Canary Islands.

Page 209
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CD38 defines a subset of B cells in rainbow trout kidney with high IgM secreting capacities

Patricia Díaz-Rosales1, Diana Martín1, Pedro Perdiguero1, Esther Morel1, Irene Soleto1, J Germán Herranz-
Jusdado1, Luis A Ramón1, Tiehui Wang2, Carolina Tafalla1

1Animal Health Research Center (CISA-INIA, CSIC), Spain

2Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, UK

[email protected]

CD38 is a multifunctional molecule that functions both as a transmembrane signaling receptor and as an
ectoenzyme with important roles in cell adhesion, calcium regulation and signal transduction. Within the B
cell linage, CD38 is expressed in diverse murine B cell subsets, with highest levels in innate B cell
subpopulations such as marginal zone (MZ) B cells or B1 cells. In humans, however, CD38 is transiently
expressed on early lymphocyte precursors, is lost on mature B cells and is consistently expressed on
terminally differentiated plasma cells.

In the present work, we have identified two homologues of mammalian CD38 in rainbow trout
(Oncorhynchus mykiss), designating them as CD38A and CD38B. Although constitutively transcribed
throughout different tissues in homeostasis, both CD38A and CD38B mRNA levels were significantly up-
regulated in head kidney in response to a viral infection. In this organ, through the generation of a specific
monoclonal antibody (mAb) against this CD38A, the presence of CD38A+ populations among IgM+ B cells
and IgM- leukocytes was investigated through flow cytometry. Interestingly, the percentage of IgM+CD38A+
B cells increased in response to an in vitro stimulation with inactivated Aeromonas salmonicida.
Furthermore, IgM+CD38+ B cells had an increased IgM secreting capacity than that of cells lacking CD38A on
the cell surface, also showing increased transcription levels of genes associated with B cell differentiation.
This study contributes to further understanding B cell differentiation process in teleosts, and provides us
with novel tools to identify novel B cell subsets in these species.

Page 210
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In vitro assessment of antagonistic activities of isolates from gilthead seabream (Sparus
aurata) gastrointestinal tract fed microalgae supplemented diet

Marta Domínguez Maqueda1, I. M Cerezo1, Jorge García-Márquez1, Francisco J Alarcón2, Juan Martos-
Sitcha3, Juan Miguel Mancera3, Miguel Ángel Moriñigo1, Maria Carmen Balebona1

1Departamento de Microbiología, Universidad de Málaga, Spain

2Departamento de Biología y Geología, CEIMAR-Universidad Almería, Spain
3Departamento de Biología, Instituto Universitario de Investigación Marina (INMAR), Spain

[email protected]

The use of probiotics has emerged as a sustainable alternative to antibiotics in the control of infectious
diseases, favouring fish health management, growth performance and feed utilisation, among others. On the
other hand, microalgae represent an interesting source of nutrients and functional ingredients for
aquafeeds. However, their digestibility is often limited by the presence of anti-nutritional factors or absence
of appropriate enzymatic activities in the fish gastrointestinal tract (GIT). The aims of the present work were
to isolate potential probiotics from the GIT of healthy gilthead seabream (Sparus aurata) fed with a diet
containing 25% microalgae and characterize their antagonism against fish pathogens.

Altogether, 117 strains were isolated from juvenile seabream (146.8 ± 16.4 g) and firstly screened for
hydrolytic enzyme activities (proteolytic, collagenolytic, lipolytic, amylolytic, phytase, tannase and cellulose
hydrolysis). Results showed that 48%, 41%, 77% and 30% of isolates were able to hydrolyse protein, lipids,
collagen and starch, respectively. Moreover, 46%, 8% and 57% of isolates exhibited the ability to degrade
phytate, tannins and cellulose, respectively.

Based on these results, a total of 32 isolates were selected for inhibitory activity against several fish
pathogens assessment. Inhibition against Aeromonas hydrophila and Vibrio anguillarum was detected in 38
% of the isolates, whilst 44 % and 47% inhibited Photobacterium damselae subsp. damselae and P. damselae
subsp. piscicida, respectively. Finally, different inhibition abilities were detected in the isolates when tested
against Tenacibaculum species. Thus, 56% inhibited Tenacibaculum maritimum; 63% T. soleae and 22% T.
gallaecium. Overall, results showed that three strains display ability to hydrolyse 4 of the assayed substrates
and produce inhibition against 8 fish pathogens, and two strains are capable to hydrolyse 5 substrates and
inhibit 8 fish pathogens. Therefore, selected strains show potential characteristics that make them suitable
to be considered for further characterization as potential probiotics in gilthead seabream aquaculture and
microalgae-supplemented aquafeeds.

Page 211
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Identification of TonB-dependent outer membrane transporters in Tenacibaculum
maritimum

Pilar Escribano, Miguel Balado, Manuel L Lemos, Beatriz Magariños

University of Santiago De Compostela, Spain

[email protected]

Tenacibaculum maritimum is the etiological agent of tenacibaculosis, a common fish disease that causes
great economic losses in aquaculture facilities around the world. A key feature which enables pathogenic
bacteria to survive within the vertebrate host is the production of siderophores and the synthesis of their
cognate transport systems, which are crucial in overcoming the nonspecific defence mechanisms of the host
and allow bacterial multiplication. However, little is known about the iron transport pathways in the genus
Tenacibaculum.

Previous studies suggested that T. maritimum possesses at least two different systems of iron acquisition:
one involving the synthesis of siderophores and another one that allows the utilization of heme groups as
iron sources. The complete genome sequence of T. maritimum provided insights into virulence mechanisms
and genes encoding iron uptake systems were identified. A siderophore biosynthesis gene cluster was found
and it seems to be involved in the synthesis of bisucaberin, a siderophore of the macrocyclic hydroxamate
class. This cluster also contains two genes that would encode TonB-dependent transporters (TBDT).
However, at least 10 more putative TBDT receptors were identified in the genome of T. maritimum.

In the present work we tried to identify and characterize these genes using a proteomic approach.
Representative strains of this species were examined. The presence of iron-regulated proteins was tested by
growing each T. maritimum strain under iron-supplemented or iron-restricted conditions. Outer membrane
proteins were obtained and separated in SDS-polyacrylamide gel electrophoresis. Protein bands obtained
were identified by MALDI-TOF peptide mass fingerprinting (PMF). Our results show that 4 of these candidate
TBDTs (Protein ID: WP_10021155.1, WP_100210529.1, WP_100211875.1, WP_024742127.1) are expressed
only under iron deficiency, being one of them (WP_100210529.1) located in the neighborhood of the
bisucaberine locus. The identification of the possible siderophore receptors in T. maritimum and the analysis
of the factors that regulate their expression will allow to gain an insight into the pathogenesis of fish
tenacibaculosis and for the development of new vaccines to prevent this infection.

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Genetic diversity of Saprolegnia spp. (Oomycota) isolated from freshwater farmed fish and
their eggs in Hungary

Edit Eszterbauer, Tímea Hardy, Noémi Erdei, Viktória Verebélyi, Győző László Kaján

Veterinary Medical Research Institute, Hungary

[email protected]

The Saprolegnia spp. (Oomycota: Saprolegniales) also called water moulds, are endemic in most freshwater
habitat worldwide. Parasitic Saprolegnia spp. cause remarkable losses in fish farms every year, mainly by
damaging fish eggs, but also fish fry and adult fish. In the course of a comprehensive research project, we
intend to gain a global picture of the nature and environmental preference of the pathogen Saprolegnia
species in fish hatcheries. Over 200 Saprolegnia strains were isolated from trout and carp farms in Hungary.
The internal transcribed spacer regions (ITS 1 & 2) and the 5.8S ribosomal RNA gene in between were used
for species identification.

The phylogenetic trees were reconstructed using maximum likelihood and Bayesian inference methods with
the software RAxML and MrBayes, respectively. Saprolegnia parasitica, S. ferax and S. australis were the
most abundant species isolated from infected fish individuals and eggs. In the water of the brood house
outflow, S. torulosa and S. hypogyna were detected occasionally, whereas S. anisospore and S. salmonis were
isolated from a few affected fish. The geographic distribution and pathogenicity of the strains were compared
considering the phylogenetic relationships. Although clear clustering was observable among the isolates of
similar origin, concerning phylogenetic reconstruction, the resolution of ITS seems to be sufficient for
species-level diversification only. Our findings suggest that further, most likely protein-coding genes are
required for a more in-depth analysis of the genetic relationships and their influencing factors.

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Microsporidia in European smelt (Osmerus eperlanus) from the River Thames

Georgina Fauconier1, Alice Bayness1, Chris Williams2

1Environment Agency, National Fisheries Laboratory, UK

2Brunel University London, UK

[email protected]

Osmerus eperlanus (European smelt) is a small diadromous fish species found in coastal waters of northern
Europe. The UK (UK) populations have suffered significant declines since the 1800s; however, populations
are slowly recovering in some large estuaries due to water quality management and habitat conservation
practises. Commercial fisheries for human consumption no longer exist in the UK; however, two small
fisheries operate to supply bait for recreational angling. European smelt is noted by the EU as an important
environmental indicator for ‘Good Ecological Status’ under the Water Framework Directive (WFD) and is
protected by national environmental legislation.

In the UK, there has been a resurgence of interest in smelt, to better inform management practices for this
understudied species. We present a potential new threat to recovering UK smelt populations; a
microsporidian parasite recorded in juvenile smelt in the River Thames estuary. In 2018, over 80% of the
juvenile smelt sampled in the Thames catchment were infected by this microsporidian. Infected fish
exhibited major deformities and pronounced abdominal swellings as a consequence of numerous xenomas
in the body cavity. Heavily infected fish were considered unlikely to survive the infection, with the potential
loss of a large proportion of the year class. We describe the parasite in UK smelt for the first time, using
traditional histopathological methods, as well as innovative x-ray microtomography (micro-CT) techniques
to visualise the phenotype of parasite infections in-situ and in 3D. We were able to determine the molecular
identification to species level using polymerase chain reaction (PCR) and DNA sequencing to target and
amplify rRNA genes specific to microsporidia.

Page 214
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Immune responses after vaccination and infection with salmonid alphavirus in rainbow
trout

Uwe Fischer1, Kimberly Veenstra1, Rodrigo Belmonte2, Kjartan Hodneland2, Edwige Quillet3, Kota Takehana4

1Friedrich-Loeffler-Institut,
Germany
2MSD Animal Health Innovation, France
3IGABI, INRA, AgroParisTech, Université Paris-Saclay, France
4Nagano Prefectural Fisheries Experimental Station, Japan

[email protected]

Salmonid alphavirus (SAV) is the causative agent of pancreas disease, which poses a significant threat to the
production of salmonids. There are a number of commercially available vaccines, however there is limited
information on how these vaccines confer protection. As inactivated vaccines against viral diseases often
have limited efficacy they can only be optimised if we have sufficient knowledge about induced immune
mechanisms. Thus, in our study we examined fundamental cell-mediated and humoral immune responses
after vaccination of rainbow trout with an oil-adjuvanted inactivated vaccine against SAV and after infection
with SAV. This included cell-mediated cytotoxicity, specific antibody response, changes in immune cell
populations, and immune gene expression. These parameters were compared to pathology and virus loads.
Our results hint that different protective mechanisms are being triggered by infection compared to
vaccination. While vaccination alone did not provoke strong cytotoxic or humoral responses subsequent
challenge of vaccinated fish resulted in significantly stronger and faster cell-mediated cytotoxicity against
virus infected cells. When compared to control fish that were challenged, only the vaccinated and challenged
fish had a reduced viral burden and pathology scores. Our observations suggest that immune memory is
induced by the vaccine, but that the testing of a vaccine on its capacity to induce cell-mediated cytotoxicity
will still require the additional trigger of a challenge.

Page 215
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Eleven years report of the microsporidian Steinhausia mytilovum (Field, 1924) affecting the
Mediterranean mussel (M. galloprovincialis) along the coastline of Campania region (Italy):
disease, epidemiology, and phylogeny

Vincenzo Gaita, Francesca Carella, Grazia Villari, Gionata De Vico, Francesca Carella

University Of Naples Federico II, Italy

[email protected]

Microsporidia are a spore-forming, obligate, intracellular parasites infecting all major taxa in all
environments.

Field of research has focused on terrestrial hosts, from insects to important humans’ parasites. Of the 187
genera described, almost half are known to infect aquatic organisms, mainly teleost and crustaceans. In
mollusks main descriptions are related to one genus (Steinhausia sp.) reported in bivalves’ and gastropods,
and to an undetermined genus affecting the digestive tissue of Queen scallop (Aequipected tubercularis).
About Steinhausia, the species S. mytilovum and S. ovicola affect bivalves belong to the families Mytilidae
and Ostreidae, with a Steinhausia-like parasite being described in the cockle Cerastoderma edule.

Major species descriptions include detailed microscopic and ultrastructural images, but no information is
present about pathogen phylogeny. In this study we report the constant occurrence of the microsporidian
parasite S. mytilovum affecting the population of mussel, M. galloprovincialis, in natural beds and farms
along Campania region coastline (Italy). A survey conducted on M. galloprovincialis in 13 mussel farms and
1 natural bed was conducted since 2009 with a total of 1800 examined animals. 9 out 13 of the farms resulted
affected, with a prevalence ranging from 2-30% of the females, depending on the areas and the seasons.

The parasite develops at oocyte level within a sporophorous vesicle where it produces a spore in a number
of one, three per cell, at cytoplasm and at nuclear level. S. mytilovum elicited an infiltrative (24,8%) or a
strong capsular inflammatory response (43.4%) at gonadal follicles and surrounding vesicular connective
tissue, in some case accompanied by gonadal atresia (24.8%), eventually associated to loss of gonadal
architecture. In other cases (7%) no reaction was observed. Microsporidian systematics currently turn
around five taxonomic clades, identified by SSU ribosomal gene sequence data. Neighbor Joining of the 18S
assigned S. mytilovum in a separated branch within the Clade V, defined as the Class Terresporidia, with
closest genetic relationship (84% identity) to an undermined invertebrate’s ovarian microsporidian. The
constant presence, prevalence and severity of S. mytilovum in the area may indirectly reflect
immunocompetence at individual, population and ecosystems levels and need to be clarified.

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A time-course study on the host responses induced at different time points post PMCV
infection

Amr A A Gamil1, Aase Mikalsen1, Øystein Evensen1, Sunil Mor2, Vikash Singh2

1Norwegian University Of Life Science, Faculty of Veterinary Medicine,Norway

2University of Minnesota, College of Veterinary Medicine, USA

[email protected]

Piscine myocarditis virus is a toti-like virus that cause cardiomyopathy syndrome (CMS) in Atlantic salmon.
Currently, CMS is one of the main disease challenges facing the salmon industry and continues to have
significant economic losses. Many aspects of the virus-host interaction are still not known including the
pathogenesis and the host responses generated at different phases of infection.

In the present study Atlantic salmon post smolts were infected with heart homogenate prepared from PMCV
infected fish and the hearts were collected at 6, 8 and 10 weeks post infection (p.i.). RNA was extracted from
the hearts collected from infected as well as from control fish. Next generation sequencing was used to
evaluate the changes observed in the transcriptome at the different times p.i. In addition, a histopathological
evaluation was also performed. Different transcriptomic profiles were observed at the different times post
infections with the late times points (i.e. 8 and 10 weeks) being more similar. The list of genes that were
differentially expressed included amongst others immune response related genes such as the MHC I & II,
STAT proteins, type I interferon related genes as well as genes involved in the proteolytic process and antigen
processing and presentation. The findings of the study will be presented and discussed.

Page 217
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Senegalese sole immune response against betanodavirus recombinants harboring
modifications in the 3’ terminal region of the RNA1

Juan Gémez Mata1, Alejandro Manuel Labella Vera1, Sandra Souto Pereira2, Isabel Bandín Matos3, Juan Jose
Borrego García1, Esther García Rosado1

1UniversidadDe Málaga, Instituto de Biotecnología y Desarrollo Azul, IBYDA, Spain

2UniversityParis-Saclay, INRAE, UVSQ, VIM, France
3Universidade de Santiago de Compostela, Instituto de Acuicultura, Spain

[email protected]

The nervous necrosis virus (NNV) is the etiological agent of the viral nervous necrosis (VNN), a disease
affecting a high number of fish species worldwide. NNV genome is composed of two segments RNA1 and
RNA2, encoding the RNA-dependent RNA polymerase and the capsid protein, respectively. NNV has been
classified into four species: striped jack nervous necrosis virus (SJNNV), tiger puffer nervous necrosis virus
(TPNNV), red-spotted grouper nervous necrosis virus (RGNNV), and barfin flounder nervous necrosis virus
(BFNNV). Furthermore, reassortants between RGNNV and SJNNV have been reported, such as wt160 isolated
from Senegalese sole (Solea senegalensis), which presents a RGNNV-RNA1 and a SJNNV-RNA2 type
segments, and causes 100% mortality in this fish species. This isolate exhibited differences in the 3’ non-
coding region (NCR) of both genomic segments when compared to the reference strains of each genotype.

In this study, the effect on virulence of the substitutions observed in the 3’NCR of the wt160-RNA1 has been
evaluated, by the development of two recombinants harbouring mutations at position 3073 and 3093, which
make the wt160-RNA1 similar to the reference RGNNV. Moreover, immune response of Senegalese sole
against the infection with these recombinants compared to the wild-type, has been evaluated using an 112-
OpenArray®.

The infection with the recombinants r3073 and r3093 decreased the mortality to 29.3% and 25.3%,
respectively. Furthermore, the number of differential expressed genes (DEGs) was higher at 3 days than at 2
days post-inoculation (p.i.), after the infection with the three viruses, being the number of DEG quite similar
among viruses. Significant differences between DEG fold changes after infection with the mutants and the
wt160 will be discussed. It should be highlighted that at 2 days p.i., the interferon stimulated gene (ISG) gig1
was not expressed after the infection with r3073 and r3093. However, at 3 days p.i. this gene was expressed
at the highest level after the infection with the three viruses. Moreover, the infection with the wt160 induced
the down-regulation of the genes gilt (related to antigen processing and presentation) and magel2 (related
to protein ubiquitination process), which was not observed after infections with r3073.

Page 218
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Anisakis spp. in European hake (Merluccius merluccius) – a risk to Portuguese consumers?

Olwen Golden1, Andreia J R Caldeira1,2, Maria João Santos1,3

1Porto University - CIIMAR, Portugal

2Goias State University, Brazil
3Porto University - FCUP, Portugal

[email protected]

Portugal has one of the highest levels of fish consumption in the world and the European hake is one of the
most popular fish in Portugal. Although there is not a tradition of consuming raw or undercooked fish in
Portugal, these products are becoming increasingly popular, and common cooking methods such as grilling
do not always reach sufficient temperatures to ensure death of parasite larvae, such as Anisakis spp. These
are nematode parasites of aquatic vertebrates.

As, the high prevalence of zoonotic parasites in this fish represents a high risk of infection to consumers, this
work evaluated forty-five European hake (Merluccius merluccius) caught in Atlantic waters off the north
coast of Portugal to determine infection levels of Anisakis species. The UV-Press method was used to detect
stage-three larvae (L3). A total of 473 Anisakis L3 were found, with a prevalence of 95.6% (95% CI 89.5-100%),
intensity (mean ± SD, range) of 11.3 ± 9.7 (1-41), density of 0.05 ± 0.04 (0-0.16) worms/muscle weight in
grams and density of 0.54 ± 0.5 (0-2.53) worms/viscera weight in grams. The fish were split in to two groups,
of equal number and a significantly higher number of larvae were found in the viscera and the muscle of the
larger fish (Mann-Whitney U test, Z <-2.21, and P<0.03). However, the muscle density values were not
significantly different (Mann-Whitney test, Z =-0.07, and P=0.95). The fish analysed had a mean ± SD length
of 31.6 ± 3.7 cm and weight of 212.6 ± 85.7 g.

The consumption of cooked fish containing this parasite could result in allergic anisakiosis in previously
sensitized individuals. And, although there are only few reports of anisakiosis in Portugal, studies in other
countries, such as Spain, a neighbouring country, have highlighted that this is a highly under-reported disease
due to the non-specific symptoms and lack of awareness of the disease. Our findings form a basis from which
we can establish a more accurate estimate of the risk posed to Portuguese consumers from the consumption
of hake and highlight a need for consumer education about this parasite.

Page 219
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Anisakis spp. and its potential risks to human health, an assessment among consumers

Olwen Golden1, Andreia J. R Caldeira1,2, Carla P P Alves3, Maria João Santos1,4

1Porto University - CIIMAR, Portugal

2Goias State University, Brazil
3Brasilia University, Brazil
4Porto University - FCUP, Portugal

[email protected]

Anisakiasis is a zoonosis resulting from the accidental ingestion of viable Anisakis spp. larvae in raw or
undercooked fishery products. Due to the growth of international trade and the popularization of food
consumed without cooking, this parasitosis deserves extra attention. This study evaluated the level of
knowledge that 746 individuals have about Anisakis spp. and its potential health risks, from a questionnaire
that was circulated online. Most respondents are aged between 30 and 40 years (55%) and are female (72%).
About 67% have graduate degrees, 39% of which are in the area of Biological Sciences and Health.

Most respondents (86%) cited “transmission of parasites” as a risk associated with the consumption of raw
fish. Most respondents do not know Anisakis spp. (66%) nor the methods of preventing worms (79%). Of
those who affirm to know the methods of prevention, the majority cited "cooking thoroughly" and "freezing"
as the most important. Only 7% of people have avoided buying or eating fish due to the presence of worms.
The most cited fish avoided were cod (29%), anchovies (26%), salmon or trout (22%). Of the interviewees,
35% would be willing to pay between € 1 and € 2.5 in addition to the original value of the product, for some
type of treatment to ensure Anisakis-free fish.

Respectively 13% and 25% of people would stop buying or would not pay an extra price for the treated fish
and the main reason given was that they should not have to pay extra to have access to safe food. It is clear,
therefore, that a significant number of participants have little or no knowledge about Anisakis spp., its
potential dangers to human health and methods to avoid it. We should stress that most of our respondents
had a graduate degree. Thus, it is evident that programs aimed at health education, aiming to inform
consumers about how to manage the risks of infection are fundamental for the formulation of health
promotion programs and for planning effective strategies that encourage and strengthen safe behaviours.

Page 220
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First trials initiating spore formation of the myxozoan Sphaerospora molnari in the gills of
common carp (Cyprinus carpio)

Urvashi Goswami1, Joana Pimentel-Santos2, Astrid Sibylle Holzer2

1Institute Of Veterinary Medical Research, Centre for Agricultural Research, Hungary

2Biology Centre of the Czech Academy of Sciences, Czech Republic

[email protected]

Sphaerospora molnari is a myxozoan parasite causing skin and gill sphaerosporosis in common carp (Cyprinus
carpio) in central Europe. The host laboratory has recently established the first continuous in vivo model
system for myxozoan presporogonic stages, using proliferative blood stages of S. molnari. More recently,
interests in spore-forming stages in the gills have grown, but we never detected spores in occasional
screenings in our laboratory system. In this project, we set out to study gill invasion and spore formation of
S. molnari in common carp.

We performed two infection experiments, a natural infection in S. molnari-enzooic river water, and an IP
injection of infective blood stages, as commonly performed in the laboratory model. Fish were sampled from
28 days post infection (dpi), since peak numbers in the gills are known to occur 35 dpi. Fish were sampled
every two days and quantification of S. molnari numbers and their minicollagen expression (indicator of
spore formation) was performed, while observing fresh gill smears. Sera were collected for specific antibody
screening using ELISA. Gill perfusions were performed in heavily infected fish to guarantee clearance of the
vascular system and observation of histozoic stages, rather than blood stages, in the target organ.
Immunofluorescence was performed to visualize S. molnari in the gill tissues.

All fish sera tested positive for S. molnari antibodies, though levels were low in natural infections. We
observed irregularities in the gills between 28 and 38 dpi, which were characterized by the invasion of
transparent stages into the epithelia of the gills of naturally infected fish. Unfortunately, these did not match
qPCR results and may represent stages of another pathogen (possibly Sphaerothecum destruens). Several
samples of gills showed low qPCR values (Cts ranging from 21-28) after perfusion, indicating the presence of
histozoic stages. However, qPCR values did not indicate important levels of minicollagen-expression,
indicating that spore maturation didn’t take place. Immunofluorescence showed the presence of pluricellular
S. molnari stages in the epithelia.

Future experiments will focus on investigating if triggers originating from the environment (temperature),
the host (acquired immunity), or the parasite itself (quantity) are required to initiate spore formation.

Funding: EAFP Smal Grant Scheme to UG

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The occurrence of the imazalil fungicide in aquatic environment and experimental setup for
assessing its effect on the physiology of sturgeon juveniles

Iulia Grecu1, Lorena Dediu1, Angelica Docan1, Blandine Davail2

1University "Dunarea de Jos" of Galati/Faculty of Food Science and Engineering/MoRAS Research Center,
Romania
2University of Bordeaux, UMR EPOC 5805 CNRS, France

[email protected]

Sturgeons are scientifically considered an interesting and valuable group of fish due to their large
evolutionary age and diadromous migratory life that make them important environmental indicators in
research the quality of the marine and freshwater habitats. These interlinked transitions between those two
kinds of environment, as well as the benthic specialization during their life cycle, offer important arguments
in using them for analysing the ecosystems' overall health. Moreover, because some of the sturgeon species
have reached an endangered position regarding their conservation status, an increased concern to know
more about the environmental pollution that affect their habitats becomes mandatory. So, studies
concerning the toxicity of the chemicals originated from various anthropogenic sources are continuously
expanding to provide data on adverse effects. Among pesticides, the imazalil fungicide has been identified
as a contaminant of emerging concern, with limited studies related its quantification in aquatic ecosystems
(surface water, sediments and biota). In Europe, the presence of imazalil has been reported ranging from
low ng/L to several μg/L in areas of intensive agricultural activity situated along the large rivers’ borders, like
in Danube River basin which is still contains important spawning habitats for at least 4 sturgeon species.
Despite their protected status, those sturgeons’ species are still affected due to the degradation of suitable
habitat by pollution.

This paper summarizes recorded data on environmental imazalil concentrations found in aquatic
ecosystems, values necessaries to set-up an experimental in vivo study in the aim to assess the toxicological
effects of such concentrations on sturgeons’ physiology. The biological model selected was the sterlet
(Acipenser ruthenus), in juvenile stage, originated from a local fish farm, dietary exposed to imazalil for 21
days. The experimental design and the procedures for physiological investigations regarding the sterlet
ecotoxicological response to this pollutant are presented, too.

Funding: This work was supported by the project AUF-ECO_RI_SRI_2021_16_IMASTUR.

Page 222
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Effect of a managed river flow on a waterborne parasite and its annelid and salmonid hosts

Sascha Hallett, Julie Alexander, Ryan Craig, Stephen Atkinson, Benjamin Americus, Richard Holt, Jerri
Bartholomew

1Oregon State University, USA

[email protected]

Juvenile salmonids in the Pacific Northwest of North America are exposed to the myxozoan parasite
Ceratonova shasta, which can cause enteronecrosis and is associated with fish population-level impacts in
the Klamath River. Waterborne stages cycle through obligate annelid and salmonid hosts. Parasite
abundance increases each Spring, concurrent with the emergence of fry and infection severity depends upon
parasite dose, water temperature and discharge. The Klamath River is regulated, with a series of dams. One
management action to mitigate fish disease is to release upstream reservoir water below the dams.
Ecosystem impact will depend on the magnitude, duration and timing of a flow event, but may include
dilution of infectious stages, removal of annelids, and promotion of juvenile salmonid outmigration. In Spring
2020, in response to high disease risk, a ‘surface flushing flow’ was executed. Discharge increased 4.4 fold
for 24 hours then ramped back down in daily steps to the baseline. To evaluate the impact of this flow event
on fish disease, we monitored parasite levels in river water samples, infection in sentinel fish and infection
in annelids before, during and after the release.

Methodology
Commencing 4 days prior to the flow increase and continuing several days post flow, 24-hour composite
river water samples were collected at two river sites and the amount of C. shasta quantified by qPCR. Forty
Chinook salmon (Oncorhynchus tshawytscha) were held at the same sites for 72 hours before and after the
flow release (April 18-21 & May 2-5), then monitored for 60 days at 13 °C for clinical disease signs. Benthic
annelids were sampled at 6 sites before and after.

Results
The density of waterborne spores decreased by an order of magnitude during the flow event and remained
reduced after discharge returned to baseline. Fish mortality was lower in both groups of Chinook salmon
after the flow (2.5 & 15% pre-flow, no mortality post-flow). Densities of infected annelids were lower post-
flow at upstream sites but were slightly higher at downstream sites.

Conclusions
These findings suggest that this Spring flow event reduced disease risk for native salmonids in the Klamath
River by lowering spore levels.

Page 223
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Induction of apoptosis in resistant strain goldfish after cyprinid herpesvirus 2 experimental
infection

Natsuho Hayashi1, Makoto Shirato1, Keisuke Yoshii1, Mayuri Nakajima1, Mikio Tanaka2, Shungo Minami2,
Goshi Kato1, Takashi Sakamoto1, Motohiko Sano1

1Tokyo University of Marine Science And Technology, Japan

2Saitama Fisheries Research Institute, Japan

[email protected]

Herpesviral hematopoietic necrosis (HVHN) caused by cyprinid herpesvirus 2 (CyHV-2), has affected the
commercial production of goldfish Carassius auratus and gibelio carp C. auratus gibelio. We have established
a goldfish strain of Kurodemekin variety resistant to HVHN and developed a SNP marker linked to the
phenotype: C/T for resistant and C/C for susceptible fish. Our previous gene expression study using different
varieties inferred that the resistant strain can induce apoptosis to survive in CyHV-2 infection. In this study,
to elucidate the disease resistant mechanisms, we analyzed differences in induction of apoptosis between
the both SNP type fish bred in 1 by 1 cross with C/T male and C/C female.

Methodology
We conducted infection experiment using 1 by 1 cross siblings (theoretically C/T:C/C=1:1) challenged by
immersion with CyHV-2 Sat-1 isolate at a concentration of 10^0.8 TCID50/mL for 2 h. The fish were reared
at 24.5 °C with running water. Ten fish were randomly collected at 0, 3, 5 days post infection (dpi), and the
trunk kidney and caudal fin were sampled. Extracted DNA from the caudal fin was used for SNP typing in
HRM analysis. Formalin-fixed trunk kidney was processed for paraffin sections followed by staining with a
commercial apoptosis TUNEL detection kit (MBL). RNA and DNA extracted from the trunk kidney were
subjected to gene expression analysis of apoptosis related genes (Caspase9, Caspase7 and Caspase3) and
virus DNA quantification by qPCR, respectively.

Results
Dead fish appeared since 5 dpi and the cumulative mortality rate reached 50%. The concordance rates
between phenotype and SNP type were 100% and 90% in surviving and dead fish, respectively. Virus DNA
loads in the both SNP type fish increased from 3 dpi through 5 dpi. At 3 and 5 dpi, apoptotic cells were
observed in the sections of C/T fish, but not of C/C fish in TUNEL staining. Gene expression analysis showed
that the expression level of Caspase9, Caspase7 and Caspase3 in C/T fish were significantly higher than those
in C/C fish.

Conclusions
The results suggest that induction of apoptosis of the infected cells is involved in resistant mechanism in C/T
goldfish resistant against HVHN.

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Monitoring of salmon health in the river Tornionjoki in 2020

Riikka Holopainen1, Charlotte Axén2, Marjukka Rask1, David Persson2, Niccolò Vendramin3, Tine Moesgaard
Ibur3, Argelia Cuenca3, Tuija Kantala1, Mervi Rokka1, Joachim Sturve4, Riina Huusko5, Atso Romakkaniemi5,
Satu Viljamaa-Dirks1

1FinnishFood Authority, Finland

2National Veterinary Institute, Sweden
3National Institute of Aquatic Resources, Technical University of Denmark, Denmak
4University of Gothenburg, Sweden
5Natural Resources Institute, UK

[email protected]

Weak and dying salmon have been observed in several Baltic rivers in recent years and the severity of the
health problems has varied depending on the year and the river. River Tornionjoki (Torneälven in Swedish)
runs on the border between northern Finland and Sweden and is one of the most important spawning rivers
of Atlantic salmon in the world. Increased mortality in salmon has been seen in the river since 2014, but
extensive laboratory testing has not offered any clear explanation for it. In 2019, dead salmon were reported
more frequently from the Tornionjoki than from any other Baltic Sea river. Additionally, signs of the
weakened upstream migration of salmon have been observed.

To monitor the salmon health status in the Tornionjoki, collection of samples was organized in 2020. The
sampling was conducted at the estuary and ca. 60 km upstream of the river on both the Finnish and Swedish
side of the border. The aim on the Finnish side was to sample salmon, which had haemorrhagic or necrotic
skin lesions. The aim on the Swedish side was to sample 80 salmon randomly. Sixty-six fish were caught at
the estuary, 39 in the Swedish archipelago just west of the estuary, 47 upstream on the Finnish side and 29
upstream on the Swedish side. Six fish were received from the river Kemijoki as reference samples and 9 fish
were received from the public from the Tornionjoki and Bothnian Bay. Three of the 66 fish from the
Tornionjoki estuary had major redness in the ventral skin area.

The Finnish fish were sampled for bacteriological, virological, histopathological and thiamine analyses. The
Swedish fish were sampled for histopathology, metabolomics, thyreoid status and thiamine. In samples
collected in Finland, no known fish pathogens that would cause the symptoms were detected using standard
diagnostic methods. Additionally, no signs of thiamine deficiency were discovered in the fish studied.
Selected samples were analyzed for a broader range of fish pathogens in National Institute of Aquatic
Resources, Denmark. At the time of writing, thiamine analyses, metabolomics and thyreoid analysis of the
Swedish samples are ongoing.

Page 225
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Preliminary evaluation of a commercial immunostimulant in Sparus aurata experimentally
infected with Vibrio anguillarum

Carmelo Laria1, Serena Savoca1, Sabrina Natale1, Fabiano Capparucci1, Ivan Corti2, Birkir Thor Bragason3,
Giovanni De Benedetto4, Alessia Giannetto1, Giovanni Lanteri1

1Dept. of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Italy
2Agenzia di Tutela della Salute dell’Insubria (CO), Italy
3Institute for Experimental Pathology, University of Iceland, Iceland
4Dept. of Veterinary Sciences, University of Messina, Italy

[email protected]

An acute phase response (APR) was experimentally induced in Sparus aurata (Linnaeus, 1758) by
intraperitoneal (IP) injection of Vibrio anguillarum, to evaluate the immunostimulant activity of the Imoviral®
complex (CRISTALFARMA ) in Sparus aurata, through the study of the expression of genes involved in the
APR. Imoviral® is a blend of exclusively natural extracts, such as uncaria (Uncaria tomentosa), shiitake
(Lentinula edodes), beta-glucan and black-currant (Ribes nigrum), whose immunostimulant and analgesic
properties have already been demonstrated. One hundred specimens each weighing ~15 grams, obtained
from a fish farm, were used and divided into 5 experimental groups (in duplicate). After the feeding period,
an experimental IP infection with V. anguillarum was performed and the APR evaluated at different time
points i.e., 1, 24, 72 and 168 hours post infection.

The study of IL-1b, TNF-a, defensin, and hepcidin gene expression were evaluated through RT-qPCR and
compared to control groups. Fish that were fed a diet supplemented with Imoviral® showed a significant and
faster immune response than those not fed Imoviral®. This study opens up the possibility for further studies
analysing the expression of more genes related to the immune response given that the data from this study
support the use of Imoviral® as an interesting immunostimulation for farmed gilthead sea bream and as a
valid alternative to prophylactic and therapeutic plans based on vaccines and synthetic antibiotics. More
studies related to the immune response should be performed using different doses of Imoviral® to evaluate
possible protective effects against other fish diseases.

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An immersion challenge for infectious salmon anaemia virus (ISAV) in Atlantic salmon fry
for testing resistance phenotypes in selective breeding

Claire Joiner1, Ross Houston3, Ólafur Kristjánsson2, Alan Tinch2, Richard Paley1

1Cefas, UK
2Benchmark Genetics, UK
3The Roslin Institute, UK

[email protected]

Highly polymorphic region (HPR)-deleted infectious salmon anaemia virus (ISAV) has caused severe disease
in marine farmed Atlantic salmon around the world and is reportable to the World Organization for Animal
Health (OIE). Disease outbreaks in Atlantic salmon are mainly reported in seawater cages, and only a few
cases have been reported in the freshwater stage. To facilitate testing of resistance phenotypes in selective
breeding programs we established an experimental immersion challenge model for ISAV in 0.5 g freshwater
Atlantic salmon fry. 264 fish were challenged per tank, comprised of equal numbers of fish from 4 families
with predicted high levels of genetic resistance and 4 families with predicted lower levels of genetic
resistance (based on breeding values calculated from seawater challenge data from parental siblings).
Challenge was by static immersion for 4 h with one of 2 isolates of ISAV (Norwegian isolate Glesvaer 2/90
and Scottish isolate 390/98) at either 10³ or 10⁵ TCID₅₀/ml in duplicate tanks. Both isolates were highly
virulent with consistent tank replication.

First mortalities commenced between 4 and 11 days post exposure, peaking on day 12-16 for the high doses
of both isolates. The high doses gave a strong challenge resulting in 97-100% cumulative mortality. The low
doses gave a much slower mortality event, a peak in mortality was not observed in either isolate. Cumulative
mortality reached 14-36% with mortalities expected to continue beyond 35 days post exposure. Mortality
rates (cumulative % or time to death) were not significantly different between the predicted high and low
genetic resistance groups. A proportion of mortalities, moribund fish and survivors were examined by cell
culture, RT-qPCR and histopathology. The fry challenge model will be used in future studies to assess
resistance phenotype in both selected and genome edited salmon fry and compared to response in seawater
stages. Strong correlation between life stages would be advantageous in terms of welfare, cost and
environmental considerations for future testing. Equally, a lack of correlation is potentially informative on
mechanisms of resistance. Further evaluation of this correlation is therefore required to assess the utility of
the fry challenge model for assessing genetic resistance.

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Comparative study on antigen persistence and immunoprotective efficacy of intramuscular
and intraperitoneal injections of squalene-adjuvanted viral hemorrhagic septicaemia virus
vaccine in olive flounder (Paralichthys olivaceus)

Sung Ju Jung, Showkat Ahmad Dar, Sajal Kole, Professor Sung Ju Jung

Department of Aqualife Medicine, South Korea

[email protected]

The profitability of the olive flounder (Paralichthys olivaceus) aquaculture industry in Korea depends on high
production and maintenance of flesh quality, as consumers prefer to eat raw flounders from aquaria and
relish the raw muscles as ‘sashimi’. For sustaining high production, easy-to-deliver and efficient vaccination
strategies against serious pathogens, such as viral hemorrhagic septicemia virus (VHSV), is very important as
it cause considerable losses to the industry. Whereas, a safe and non-invasive vaccine formulation that is
free from unacceptable side-effects and does not devalue the fish is needed to maintain flesh quality.

We previously developed a squalene-adjuvanted VHSV vaccine that conferred moderate to high protection
in flounder, without causing any side effects when administered through the intraperitoneal (IP) injection
route. However, farmers often demand intramuscular (IM) injection vaccines as they are relatively easy to
administer in small fishes. Therefore, we administered the developed vaccine via IP and IM routes and
investigated the safety and persistency of the vaccine at the injection site. In addition, we conducted a
comparative analysis of vaccine efficacy and serum antibody response.

The clinical and histological observation of the IM and IP groups showed that our vaccine remained
persistence at the injection sites for 10-17 weeks post vaccination (wpv), without causing any adverse effects
to the fish. The relative percentage of survival was 100% and 71.4% for the IP group and 88.9% and 92.3%
for the IM group at 3 and 17 wpv, respectively. Thus, considering the persistency period (24 wpv) and both
short and long-term efficacy of our vaccine, the present study offers an option to flounder farmers in
selecting either IM or IP delivery strategy according to their cultured fish size and harvesting schedule – IM
vaccination for small-sized fish and IP vaccination for table-sized fish.

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Mass production of manipulated hagfish antibodies to apply as a passive immune vaccine
against acute hepatopancreatic necrosis disease in shrimp

Tae Sung Jung1,3, Jaesung Kim3, Kim Thompson2, Tae Sung Jung1,3

1Gyeongsang National University, South Korea

2Moredun Research Institute, UK
3Earwyntech Co Ltd, Australia

[email protected]

In jawed vertebrates, B-cell receptors (BCRs) and T-cell receptors (TCRs) play a pivotal role in the antigen
recognition step of adaptive immunity, which is characterized by antigen-specific interactions and memory.
On the other hand, the existence of BCR, TCR or major histocompatibility complex (MHC) molecules were
not identified in agnathans until the middle of 2004. Lampreys and hagfish (the extant jawless vertebrates)
utilize variable lymphocyte receptors (VLRs) for antigen recognition. VLRs, which are structurally unrelated
to BCRs or TCRs, are members of the leucine-rich repeat (LRR) protein family.

The VLR, composed of leucine-rich repeats, is a mediator of the humoral immune response in lamprey and
hagfish. The production of circulating Ag-specific VLRBs in response to antigens demonstrated that VLRBs
are capable of performing agglutinating and neutralizing activities. In a previous study, we proved that the
modified VRLs were able to reduce shrimp mortality by recognizing and neutralizing the effect of the Vibro
parahaemolyticus binary toxins that induce the pathogenesis of Acute Hepatopancreatic Necrosis Disease in
the shrimp. We therefore now aim to mass produce the modified VLRs to test their application in a field
study.

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Cultivation and identification of flavobacteria in diagnostics

Verena Jung-Schroers1, Julia Bauer1, Felix Teitge1, Peter Steinbauer2, Marcus Zielasko2, Dieter Steinhagen1

1University of Veterinary Medicine Hannover, Fish Disease Research Unit, Germany

2Bavarian Fish Health Service, Germany

[email protected]

Flavobacteria are ubiquitous in the aquatic environment and can regularly be isolated from the gills of
clinically healthy fish. Some species within the genus Flavobacterium are known as fish pathogens and
reliable methods for detection and identification of these bacteria are necessary in fish diagnostics.

Methodology
117 isolates of Flavobacterium spp. were examined. The isolates were cultivated on blood agar, Anacker-
Ordal agar, modified Anacker-Ordal agar, TYES agar and Shie agar at 15 °C and 20 °C for 14 days. Additionally,
110 isolates of Flavobacteria spp. were identified by using MALDI-TOF MS and by sequencing a 750 bp long
part of the 16S rRNA gene.

Results
It could be shown that at 15 °C the isolates grew fastest on TYES agar and the highest number of isolates
could be cultured on Shie agar. At 20 °C the isolates grew fastest on modified Anacker-Ordal agar and on
Shie agar and the highest number of isolates could be cultured on modified Anacker-Ordal agar. Especially
isolates of F. psychrophilum and F. columnare were growing fastest and best on modified Anacker-Ordal agar
at both temperatures. The classical Anacker-Ordal agar was most specific for Flavobacteria, but the modified
Anacker-Ordal agar showed a high specificity for these bacteria as well. Sequencing of the 16S rRNA gene
resulted in 24 different species and a secure identification on species level was possible. By using MALDI-TOF
MS ten different species could be identified. The results were secure on genus level and for potential
pathogenic species, like F. psychrophilum and F. columnare also on species level. For all but one isolates the
identification on genus level was consistent with both methods and for 62 isolates the results corresponded
also on species level.

Conclusions
The use of the modified Anacker-Ordal ager and an identification by sequencing the 16S rRNA gene can be
recommended for the examination of Flavobacteria in diagnostics.

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Spinal damages in eels

Verena Jung-Schroers1, Felix Teitge1, Julia Bauer1, Anne Miebach1, Vanessa Guddorf2, Erik Fladung3, Janek
Simon3, Dieter Steinhagen1

1University of Veterinary Medicine Hannover, Fish Disease Research Unit, Germany

2University of Veterinary Medicine Hannover, Clinic for small mammals, reptiles and birds, Germany
3Institute of Inland Fisheries, Germany

[email protected]

Eels are katadrome migratory fish that are migrating downstream the rivers to reach their spawning areas in
the sea. During their way, they are exposed to numerous risks, like passages through hydroelectric power
plants. Because of their body shape and length as well as their swimming behavior and their preference to
swim with the main water current, eels are more prone to be injured by a turbine passage than other fish
species. Injuries might result in higher mortalities or in reduced swimming abilities. For the preservation of
the European eel, it is existential to enable their migration into the sea for reproduction since artificial
propagation of the species is still not possible.

Methodology
To examine possible spinal damages 220 eels were caught in the river Weser near the city Hameln in Lower
Saxony, Germany. Upstream several hydropower plants are located but not directly in the fishing area. The
animals were anaesthetized, x-rayed and evaluated for spinal damages.

Results
Only one eel showed an external damage of the spine, all other eels were clinically healthy and showed no
swimming abnormalities. By x-raying, changes of the spinal column could be observed in 41 eels (19% of the
examined eels). In 24 animals several damages were seen. The detected injuries of the spinal columns were
mostly severe, and the defects ranged from compression and fractures of the vertebral bodies,
displacements of the vertebral bodies against each other and tears of the spinous processes. These defects
occurred mainly in the last third of the body.

Conclusions
Most likely the damages of the spinal cord were caused by the passage through hydropower plants located
above Hameln. External examinations of eels are not sufficient to evaluate possible injuries caused by the
passages through hydropower plants and examination by x-ray should always be included. Although in most
of the eels no changes in swimming behavior due to the existing damage to the spines were seen, in the 41
affected fish, considerable impairments and increased losses are to be expected in the further migration of
to the spawning grounds in the Sargasso Sea.

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Biochemical and non-specific immune responses towards chronic infection of
tenuiproboscis keralensis in lutjanus argentimaculatus (Forsskal, 1775)

Pinky Kaur, Pillaru K Asokan, NK Sanil

Fish Health Section, Marine Biotechnology Division, ICAR-Central Marine Fisheries Research Institute, India

[email protected]

Preliminary studies revealed the presence of heavy Tenuiproboscis sp. infections in Lutjanus
argentimaculatus (red snapper) from the southwest coast of India. Considering the influence of serum
biochemical indices is deliberate as a key tool to understand the fish health status, thus the present study
was aimed to study the influence of acanthocephalan infection on 13 relevant serum biochemical and non-
specific immune parameters, namely glucose, protein, albumin, globulin, A/G ratio, cholesterol, triglycerides,
serum glutamate oxaloacetate transaminase (SGOT/AST), serum glutamate pyruvate transaminase
(SGPT/ALT), acid phosphatase (ACP), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) were
evaluated spectrophotometrically, for each collected sample using a commercial kit, and qualitative assay of
superoxide anion production (ROS) were carried out.

Forty-six wild samples of L. argentimaculatus were screened during May 2018 – February 2020. The
prevalence observed for T. keralensis was 95% and 2-35 parasites/host. The above mentioned 13 serum
biochemical and non-specific immune parameters were evaluated individually for each collected infected
and uninfected sample. The variation observed in various parameters are serum glucose vary from 11.94-
490.38 mg/dl (109.15 ± 106.24), total protein 2.36-10.51 g/dl (5.93 ± 1.90), globulin 0.98-6.26 g/dl (3.92 ±
1.59), A/G ratio 0.191-1.65 (0.58 ± 0.38), albumin 0.4-4.3 g/dl (2.00 ± 0.96), cholesterol 63.54-210.78 mg/dl
(142.42 ± 41.61), triglyceride 50.00-623.25 mg/dl (299.7 ± 190.01), SGPT 23.8-435.33 U/L (172.76 ± 126.48),
SGOT 2.8-286.34 U/L (143.61 ± 108.86), alkaline phosphatase 88.12-1872.7 U/L (804.14 ± 594.56), acid
phosphatase 20.25-142.5 U/L (65.22 ± 36.94) and LDH 80.95-536.69 U/L (536.69 ± 594.75). Superoxide anion
production (ROS) showed variation from 0.685 to 4.032 (2.154 ± 1.37). Pearson correlation among the serum
variables with parasite numbers recovered from individual infected hosts showed that the glucose, ROS and
LHP are on a negative scale while other serum attributes are positively correlated with parasite numbers. In
conclusion, as the chronic infection by T. keralensis provoking insights on serum and non-specific immune
response in wild mangrove red snapper based on modulation of these parameters.

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Occurrence of parasites and diseases in the southern Russia fish under nowdays
conditions

Anna Kazarnikova

Federal Research Centre The Southern Scientific Centre of The Russian Academy of Sciences, Russia

[email protected]

In this study, the occurrence of parasites and diseases in herring Alosa immaculate (Clupeidae), pike perch
Sander lucioperca (Percidae), ram Rutilus rutilus (Cyprinidae), carp Cyprinus carpio (Cyprinidae), crucian carp
Carassius gibelio (Cyprinidae), bream Abramis brama (Cyprinidae), sirman goby Ponticola syrman (Gobiidae),
sand goby Neogobius fluviatilis (Gobiidae), round goby N. melanostomum (Gobiidae) was investigated from
spring 2019 till autumn 2020. From 2009 to 2015, the average salinity for the Azov Sea increased from 11 to
12.8‰, and for the Taganrog Bay - to 6.4‰.

During the study, a total of 1207 fish specimens were examined for parasites at the Don River delta and
eastern part of the Taganrog Bay of the Azov Sea. Based on the studies carried out, 33 species of parasitic
organisms belonging to 9 classes were identified in representatives of the Azov fish fauna: Microsporea,
Myxosporidia, Peritricha, Monogenea, Trematoda, Cestoda, Nematoda, Bivalvia, Crustacea. Among the
recorded invasions with indicators of prevalence (P) 60-100%, and with different levels of infection, the
trematodes Pseudopentagramma simmetricum, a marine species that came with herring from the Black Sea
(P = 66.7%, 51.6 ± 26.06 parasites per fish (PPF), 27.5 ± 19.03 abundence index (AI). This is followed by the
invasion of bream by the euryhaline species Dactylogyrus wunderi (P=80.0%, PPF=7.8 ± 1.48, AI=6.2 ± 1.32).
Carp by the freshwater species D. anchoratus (P= 60%, PPF=10.4 ± 2.68, AI=6.1 ± 2.04). And round goby
freshwater species Eustrongylides excisus y (P=100.0%; PPF= 4.0 ± 1.74; AI = 3-5).

Parasitic diseases that harm the host's body (fish), caused by the beetles of this family, have been registered.
Ligulidae (Ligula intestinalis and Digramma interrupta) as well as potentially dangerous for animals and
humans - representatives of the family. Heterophyidae (Apophallus donicus, Cryptocotyle concavum, C.
lingua), fam. Anisakidae (Hysterothylacium aduncum) and family Eustrongylidae (Eustrongylides excisus l.).
This study adding the knowledge of geographical distribution and host range of parasite species in changing
ecological conditions – increased salinity, changed stocks of zooplankton and zoobenthos, reduction of
anadromous and semi-anadromous fish numbers.

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Fish parasites with potential hazard for animal and human health in Southern Russia

Anna Kazarnikova

Federal Research Centre The Southern Scientific Centre of The Russian Academy of Sciences, Russia

[email protected]

The constantly changing ecological situation influences both the species diversity and the number of
parasites. From 2009 to 2015, the average salinity for the Azov Sea increased from 11 to 12.8‰, and for the
Taganrog Bay - to 6. ‰. The reasons for the increase in salinity lie in the intrasecular climate cyclicity (30, 60
years), a decrease in river runoff into the Sea of Azov since 1952, and an increase in the frequency of years
with anomalously low river runoff. In this study, the occurrence of potentially dangerous for animals and
humans health parasites in herring Alosa immaculate (Clupeidae), pike perch Sander lucioperca (Percidae),
ram Rutilus rutilus (Cyprinidae), carp Cyprinus carpio (Cyprinidae), crucian carp Carassius gibelio
(Cyprinidae), bream Abramis brama (Cyprinidae), sirman goby Ponticola syrman (Gobiidae), sand goby
Neogobius fluviatilis (Gobiidae), round goby N. melanostomum (Gobiidae) was investigated from spring 2019
till autumn 2020 at the River Don delta and eastern part of the Taganrog bay of the Azov Sea.

During the study, a total of 1207 fish specimens were examined for parasites. The main indexes of infection
– prevalence (P), intensity (I), abundance (A) were measured. Based on the studies carried out, 3 species of
helminth parasites were attributed to potential pathogens of animal and human helminthiasis according to
the literature data. There are Paracaenogonimus ovatus (Trematoda: Cyathocotilidae) from bream (P=30-
62.5%, I=0.35-2.9 spec./1 g of muscle tissue), Hysterothylacium aduncum (Nematoda: Raphidascarididae)
from herring (P=33.3%, I=63.4 ± 24.04, A=21.1 ± 4.13), Eustrongylides excisus (Nematoda: Dioctoophymidae)
from body cavity and at internal organs of pike perch (P=26.7%, I=1.2 ± 0.25, A=0.3 ± 0.12) sirman goby (P=40-
53.3%, I=9.0 ± 2.49, A=3.0 ± 1.44), sand goby (P=26.7-33.3%, I=1.5 ± 0.5, A=0.2 ± 0.55) and round goby
(P=46.7-100%, I=4.0 ± 1.74, A=2.3 ± 0.75). The high level of ecological plasticity of parasites, their ability to
use different hosts was proved and the results were compared with other literature data. This study adding
the knowledge of geographical distribution and host range of parasite species with potential hazard for
animal and human health in changing ecological conditions – increased salinity, heterogeneity of
zooplankton and zoobenthos stocks, reduction of anadromous and semi-anadromous fish.

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Susceptibility of viral haemorrhagic septicaemia virus (vhsv) genotypes i, ii, iii, iva and ivb
in olive flounder (Paralichthys olivaceus), rainbow trout (Oncorhynchus mykiss) and
zebrafish (danio rerio)

Hyoung Jun Kim1, Niels Olesen2, MS Kim3

1NationalInstitute of Fisheries Science, South Korea

2NationalInstitute of Aquatic Resources, Denmark
3Sejong University, South Korea

[email protected]

Viral haemorrhagic septicaemia (VHS) is a serious viral disease in farmed olive flounder, rainbow trout as
well as a broad range of wild freshwater and marine species. VHS virus (VHSV) is classified into four
genotypes, I to IV. Olive flounder farms in Korea are infected with Genotype IV only. With the increasing
trade of aquatic animals the risk of introduction of other genotypes in Korea is increasing. We therefore
analyzed the susceptibility of olive flounder, rainbow trout and zebrafish to infection with VHSV genotypes
I, II, III, IVa and IVb as a preliminary risk analysis.

Methodology
Olive flounder (3-4 g), Rainbow trout (3-4 g), and zebrafish were acclimated for 2 weeks in 50 L tanks at 15
°C. The fish were randomly divided into 6 groups, 2 x 20 fish per group in duplicate tanks. The fish were each
intramuscularly injected with 103 TCID50 of the 5 VHSV genotypes, the negative control group was injected
with L-15 culture medium. The cumulative mortalities were recorded daily for 4 weeks post-injection.

Results
In case of the VHSV genotype I isolate, the cumulative mortalities were 64%, 94%, 71% in olive flounder,
rainbow trout and zebrafish, respectively. In case of the VHSV genotype II isolate, no mortality was observed
in any of the three fish species. In case of the VHSV genotype III isolate, the mortalities were 100%, 11%, and
63% in the 3 fish species, respectively. In case of VHSV genotype IVa isolate, the mortalities were 100%, 28%,
and 94%, and in case of the VHSV IVb isolate, the mortalities were 100%, 0% and 75%. Viral RNA was detected
in all dead fish tested.

Conclusions
VHSV genotype Ia is highly pathogenic to all three fish species by IM injection. VHSV genotype III, IVa and IVb
is highly pathogenic to olive flounder and zebrafish but not to rainbow trout. Genotype II, however, is not
pathogenic to any of three fish species. The mortalities were strongly related both to the VHSV genotype and
to fish species.

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Cell susceptibility of 81 vhsv isolates in three different cell lines

Se Ryun Kwon1, ALF Alencar2, Hyoung Jun Kim4, Niels Olesen2

1Sunmoon University, South Korea

2National Institute of Aquatic Resources, Denmark
3National Fisheries Research and Development Institute, South Korea
4National Institute of Fisheries Science, South Korea

[email protected]

Phylogenetic analyses of viral haemorrhagic septicaemia virus (VHSV) have identified 4 main genotypes.
Genotype I is subdivided into Ia to If and genotype Ia are majorly responsible for outbreaks in European
rainbow trout farms. Genotype Ib, II and III from marine fish species are low virulent to rainbow trout.
Genotype IV can be subdivided into IVa to IVd. The mortality caused by VHS varies among infected fish
species and VHSV isolates from marine fish are considered low virulent to rainbow trout. Currently, the
association between the virulence in vivo and in vitro is still unclear. This study aims to determine the
susceptibility of different fish cell lines to VHSV isolates belonging in different genotypes.

Methodology
Eighty-one VHSV isolates representing all known genotypes and subtypes were tested for their infectivity in
cell cultures. BF-2, EPC and RTG-2 cell lines were seeded in 96-well microplates. Ten-fold serially diluted VHSV
isolates were inoculated on each cell lines and viral titer was determined by assessing TCID50/ml.

Results
All isolates grew well in the BF-2 cell line, while viral titers in EPC and RTG-2 cell lines showed different
patterns according to genotype. Isolates from rainbow trout showed no titer change or higher titer in RTG-2
cell line as well as BF-2 cell line, and the same or slightly lower titer in EPC cell line. Genotype Ib and II isolates
from other fish species showed very low titers or no proliferation in EPC cell line as well as RTG-2 cell line.
Genotype IIIb isolates showed no difference in viral titer between BF-2 and EPC cell lines, but lower viral
infectivity titers in RTG-2 cell line. Most isolates of genotype IV showed no difference in viral titer between
BF-2 and EPC cell lines, while showed lower viral titer in RTG-2 cell.

Conclusions
It was found that VHSV isolates, which are low pathogenic to rainbow trout, have a significantly lower titer
in RTG-2 cell line. Our results indicate a close relationship between genotype, virulence to rainbow trout and
cell susceptibility. This relationship might be used for assessment of the virulence of VHSV to rainbow trout.

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Disseminated phaeohyphomycosis in beluga sturgeon (Huso huso)

Luciana Mandrioli1, Esteban Soto2, Zeinab Yazdi2, Enrico Volpe1, Francesca Errani1, Sara Ciulli1, AC Camus3

1Department of Veterinary Sciences, University of Bologna, Italy

2Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, USA
3Department of Pathology, College of Veterinary Medicine, University of Georgia, USA

[email protected]

Phaeohyphomycoses are fungal infections caused by pigmented (melanized) yeasts or molds. Systemic
phaeohyphomycosis has been reported in captive and farmed fish, amphibians, and other cold-blooded
vertebrates, as well as crustaceans. In fish, infections often result in severe tissue necrosis, frequently
associated with angioinvasion and resultant infarction. The genera Exophiala, Ochroconis/Verruconis,
Phoma, and Veronaea contain well-recognized pathogens that cause cutaneous, subcutaneous, and systemic
phaeohyphomycosis under varying husbandry conditions in a broad range of fish hosts. Among cultured
sturgeon species, systemic phaeohyphomycosis caused by Veronaea botryosa has been reported in Siberian
sturgeon (Acipenser baerii) and white sturgeon (A. transmontanus).

Regarded as the most important infectious disease affecting the North American caviar industry, the disease
has not been reported in European sturgeon culture. A captive juvenile beluga sturgeon (Huso huso) farmed
for restocking purpose in Italy presented severe arching of the body (U-shaped) and multifocal reddening
and erosions of ventral skin. Full necropsy, conducted a few hours after death, revealed grey-pale gills, sero-
hemorrhagic celomic effusion, a dark-brown softened liver, and a light-brown gastro-intestinal tract devoid
of ingesta. Histological findings were characterized by disseminated pigmented mycosis associated with
tissue necrosis. Pigmented, bronze hyphae had 3-5 µm (up to 10 µm), thin, parallel walls, infrequent
septation, and acute angle branching. Hyphae were detected within vessels and the gills, colonized the
intestinal serosa from the celomic cavity, massively destroyed the hepatic parenchyma, and caused extensive
necrosis of renal hematopoietic tissue.

As hyphal morphology in situ is unreliable for identification purposes, an attempt to identify the fungal agent
was made using laser capture microdissection performed on formalin-fixed paraffin embedded tissue
sections. Histological fields containing fungal hyphae were excised, total DNA extracted, and PCR conducted
using V. botryosa specific and generic fungal primers following whole genome amplification. Although V.
botryosa infection is suspected, PCR failed to amplify the corresponding fungal sequences likely due to
formalin-associated degradation of DNA. While most mycoses in poikilotherms are considered
“opportunistic”, the emergence of fungal pathogens such as V. botryosa in the sturgeon farming, and the
lack of approved therapeutants and prophylactic measures against fungal diseases in fish warrants future
investigation.

Page 237
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Safety and efficacy of DNA vaccines against VHS and IHN under controlled and field
conditions

Andrea Marsella1, Francesco Pascoli1, Alessandra Buratin1, Tobia Pretto1, Lorena Biasini1, Miriam Abbadi1,
Luana Cortinovis1, Amedeo Manfrin1, Jesper Skou Rasmussen2, Dagoberto Sepulveda2, Niccolò Vendramin2,
Niels Lorenzen2, Anna Toffan1

1Istituto Zooprofilattico Sperimentale delle Venezie, Italy

2Technical University of Denmark, Denmark

[email protected]

Viral Hemorrhagic Septicemia (VHS) and Infectious Hematopoietic Necrosis (IHN) have a great impact on the
Italian rainbow trout farming industry and the availability of vaccines is a key point in controlling and reducing
their effect on productions. Since the late ‘90s, DNA vaccines against both diseases have been designed and
tested through in vivo challenges under controlled conditions, providing promising results. Furthermore, a
field testing in Denmark suggested that a VHS DNA vaccine gave protection also under earth-pond farming
conditions, but few data are available on the efficacy of DNA vaccines in intensive raceways trout farming.
Italian VHSV and IHNV glycoprotein gene sequences have been used to design two DNA vaccines tailored
towards current virus variants causing disease in rainbow trout farms in Italy. An experimental trial was first
conducted at the IZSVe Experimental Aquarium in order to obtain efficacy and safety baseline data. Ten-
gram rainbow trout were treated with different doses of the vaccines, injected singularly and combined. Fish
were bath challenged after 60 days with two recently isolated Italian VHSV and IHNV singularly and in
combination. One (1) micrograms/fish resulted the best performing dose.

One Category I hatchery supplied 15,000 rainbow trouts weighing approximately 8 grams that were then
divided into three experimental groups: one injected with PBS (Group 1), one injected with 1 micrograms/fish
of VHS vaccine (Group 2) and the last group treated with 1 micrograms/fish of each plasmid (Group 3). Fish
were moved to the VHS/IHN infected facility 60 days post vaccination (dpv). Mortality began 7 days post
transfer with different trends in the three groups. At 30 days post transfer, mortality resulted to be 10.5 %
in Group 1, 3.3% in Group 2 and 1.0% in Group 3, with a RPS of 69.3% and 90.5% provided by VHS and
VHS/IHN vaccine respectively. Vaccinated trout are going to be monitored for zootechnical performances
evaluation until they reach the commercial size.

DNA vaccines confirmed to be safe for injected fish and effectively reduced the impact of VHS and IHN in the
field.

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Morphological and biological characterization of Mycolicibacterium salmoniphilum
cultured at low temperatures

Megumi Matsumoto1, Naoki Kabeya1, Yutaka Haga1, Shuichi Satoh1, Motohiko Sano1, Goshi Kato1

1Tokyo University of Marine Science And Technology, Japan

[email protected]

Mycobacteriosis caused by Mycobacterium spp. gives economic damages to the world aquaculture industry.
Mycobacterium spp. enter a state of dormancy in an adverse environment such as anaerobic condition,
nutritional starvation and low pH condition. The dormant bacteria lack the pathogenicity due to its ability to
survive in host macrophages, but resuscitated bacteria regain pathogenicity. In our preliminary experiment,
mortality of Mycobacterium sp.-challenged fish paused during winter season and restarted in the next spring
season. These data suggest that Mycobacterium sp. was alive in the dormant state under the low water
temperature, and then resuscitated along with the increase of water temperature. In the present study, we
investigated biological and morphological changes of Mycolicibacterium salmoniphilum during incubation at
low temperatures to identify dormant state.

Methodology
Growth curve, antimicrobial resistance, morphological changes and mycolic acid profiles were investigated
to identify the dormant state. M. salmoniphilum were incubated at 5 ℃, 10 ℃, 15 ℃, 20 ℃ and 25 ℃ for 7
days. Antimicrobial resistance of the bacteria cultured at each temperature was evaluated by minimum
bactericidal concentration (MBC) for amikacin, clarithromycin and ciprofloxacin. Morphological changes and
mycolic acid profiles were examined by a transmission electron microscopy and a gas-chromatography/mass
spectrometry (GC/MS) analysis, respectively.

Results and Conclusions

The bacteria cultured at 20 oC and 25 oC showed the fastest growth, low MBC value to amikacin (7.8 μg/ml),
clarithromycin (3.9 μg/ml) and ciprofloxacin (<0.9 μg/ml). The morphology was long rod shapes with thick
cell walls. The bacterium was not grown at 5 ℃ and showed the highest MBC than the other groups. Short
rod or round shapes with thin cell wall was observed and mycolic acids were almost undetected in the
bacteria cultured at 5 ℃. These features observed in the bacteria incubated at 5 ℃ correspond with features
for a dormant state bacterium, indicating that M. salmoniphilum entered dormant state in 5 ℃ incubation.
Changes in water temperature would be one of the factors to induce dormancy and resuscitation in a fish
pathogen, M. salmoniphilum.

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Tackling aquacultures disease problem: do early life conditions hold the key to future food
security?

Rob McFarling1,2, Lyndsay Christie2,3, Ellen Blaker2, Ronny van Aerle2, Eduarda Santos1, Ioanna Katsiadaki2

1University of Exeter, UK
2Centre for Environment, Fisheries and Aquaculture Science (Cefas), UK
3University of Bath, UK

[email protected]

Despite advancements in treatment and prevention, disease remains a major issue in aquaculture with
implications for sustainability, welfare and food security. Genetic selection, often bolstered by in-depth
genomic analysis, has proved successful at combating diseases in several cases, but manipulation of the
epigenome in the context of disease prevention has seldom been explored. The propensity for vertebrate
epigenomes to be manipulated by environmental stimuli, especially during critical windows of development,
provides a potential opportunity for phenotypic selection by altering epigenetic regulation of transcription.
We previously observed an elevated heat shock protein (hsp) transcriptional response to temperature stress
in rainbow trout alevins previously exposed to heat shock during embryo development. Here we hypothesise
that exposure to heat shock during critical windows of early embryo development also has the potential to
alter disease related mortality by modifying responsiveness to a pathogen via the manipulation of the
epigenome and its regulation of gene expression.

To test this hypothesis, we exposed rainbow trout (Oncorhynchus mykiss) embryos to a temperature shock
during early development. At the fry stage these fish were infected with bacterial pathogen Yersinia ruckeri,
the causative agent of enteric redmouth disease (ERM), via bath exposure. Survival was monitored for 16
days and samples were taken at 19, 43 and 67 hours post infection for molecular analysis. Developmental
exposure to temperature shock had no significant effect on survival of fish exposed to Y .ruckeri. There was
no significant difference in mortality during the course of the disease challenge, and no difference in overall
survival between temperature-shocked fish and controls following exposure to the bacteria. This suggests
that the developmental treatment applied here does not lead to long term epigenetic alterations that are
capable of affecting the responsiveness of rainbow trout to Y .ruckeri in a positive or negative manner.

We plan to conduct transcriptional analysis to determine whether there were any differences in immune
response between temperature-shocked fish and controls, to gain an insight into any alterations in the
regulation of gene transcription caused by the temperature shock. Additional studies are required to fully
determine whether temperature manipulations hold potential for disease management in aquaculture.

Page 240
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Influence of genetic adaption of rainbow trout (Oncorhynchus Mykiss) fed with alternative
protein sources based on arthrospira platensis and hermetia illucens on intestinal health
and animal welfare

Anne-Carina Miebach1, Julia Bauer1, Mikolaj Adamek1, Carsten Dietz2, Jakob Gährken3, Stephan Wessels3,
Verena Jung-Schroers1, Dieter Steinhagen1

1Fish
Disease Research Unit, University of Veterinary Medicine Hannover, Germany
2AnimalNutrition Physiology, Georg-August-Universität, Germany
3Aquaculture and Water Ecology, Georg-August-Universität, Germany

[email protected]

Due to an intense feeding of fishmeal to carnivorous fish species, not only sustainably managed and
overfished fish stocks have to face a growing problem. Therefore, novel sources of protein substituting
fishmeal in fish feed are of increasing interest in aquaculture.

However, the use of plant-based protein sources in the diet of carnivorous fish is in controversial discussion.
Previous studies have shown that the replacement of fishmeal in the diets of carnivorous fish species is
frequently associated with several issues related to food conversion, growth rate, fish health and welfare
effects.

The current study, as part of the cooperative project “Sustainable Trout Aquaculture Intensification” aims at
making use of the genetic variability of rainbow trout (Oncorhynchus mykiss) in order to gain new insights
into the adaptability to innovative raw materials (Arthrospira platensis, Hermetia illucens) with special
attention to the question of how the replacement of fish meal by Arthospira platensis or Hermetia illucens
in fish feed affects the intestinal health and animal welfare.

The results showed that the stress response, investigated by analysing the concentration of heat shock
protein 70 in liver samples, proved to be at very low values for all groups. A chronic stress exposure due to
the substitution could thus be excluded.

Growth was particularly efficient in a commercial line 9 and regional line 8, whereby the genetic background
was playing a decisive role. Despite macroscopical and microscopical morphological changes in the anterior
intestine of tested feeding groups, no negative influence on growth could be detected.

Altogether, no inflammatory reactions were detected in the intestines of the animals, despite the Arthrospira
fed groups showed partially significant higher gene expressions than fish from other feedings. Also, a
significantly higher expression of occludin in the posterior intestine of the Arthrospira groups with respect
to the epithelial barrier was noted.

Therefore, the current study revealed that the substitution with Arthrospira platensis or Hermetia illucens to
the fishmeal portion of rainbow trout diets did not negatively affect fish health and animal welfare.

Page 241
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Effect of dietary fumonisin on the immune response in Rainbow Trout (Oncorhynchus
mykiss)

Hana Minářová1,2, Ľubomír Pojezdal1, Miroslava Palíková2,3, Jan Mareš3, Karolína Hlavová1, Kamil Šťastný1,
Martin Faldyna1, Helena Modrá3

1Veterinary
Research Institute, Czech Republic
2University
of Veterinary Sciences Brno, Czech Republic
3Mendel University in Brno, Czech Republic

[email protected]

The effects of mycotoxins have been investigated in different animal species; however, more information
about their impact on the fish immune response is still required. Fumonisins, produced by several Fusarium
species, represent the most common mycotoxins in plant meals. They can cause major health problems in
fish, including both immunostimulation and immunosuppression, and their influence needs to be examined,
especially in the main aquaculture species.

Methodology
Fumonisins were administered to rainbow trout (Oncorhynchus mykiss) in feed for a period of 10 weeks. At
week 6, some of the fish from the fumonisin group and from the control group were vaccinated against
Yersinia ruckeri. Samples of the head kidney were taken after euthanasia of the fish at weeks 3 and 10, and
non-specific mitogen-driven as well as specific antigen-driven lymphocyte proliferation assay was
performed. At weeks 9 and 10, specific antibodies were also determined by an in-house ELISA.

Results
With non-specific stimulation, the fish from both fumonisin groups showed a significant increase in
proliferative activity after vaccination, indicating a pro-inflammatory immune reaction. The results were
similar in the non-vaccinated control. Significantly lower proliferation levels were observed in the vaccinated
compared to the non-vaccinated fish from the control group. With specific stimulation, significantly higher
values were detected in the vaccinated fish from the fumonisin group compared to the vaccinated control.
At week 9, levels of specific antibodies were significantly increased in the vaccinated fumonisin group
compared to the non-vaccinated fish. However, at week 10, the control fish showed similar results with even
higher values. The enhanced immune reaction, which occurred very quickly in the vaccinated fish from the
fumonisin group, may be indicative of pro-inflammatory changes.

Conclusions
A significant effect of fumonisins on the immune response of rainbow trout was observed. Alterations in
lymphocyte proliferative activity and in the production of specific antibodies confirm the negative impact of
these toxins on fish health.

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Is the proteomic profile of sea lice, Lepeophtheirus salmonis shaped by its host?

Janina Costa1, Janina Costa1, Philip Steele1, Katie Poulter2, Kim D. Thompson1, Alasdair Nisbet1, James E.
Bron3, Kevin McLean2, Sean Monaghan3

1Moredun Research Institute, Pentlands Science Park, UK

2Proteomics Facilities, Moredun Research Institute, Pentlands Science Park, UK
3Institute of Aquaculture, Faculty of Natural Sciences, University of Stirling, UK

[email protected]

One of the major health limitations for growth and sustainability of the Atlantic salmon aquaculture industry
is infection by sea lice, Lepeophtheirus salmonis (Krøyer, 1837). The economic impact of this parasite disease
is >£700M p.a. for the global industry. With increasing parasite resistance to available veterinary drug
treatments, stricter controls on discharge and an urgent need for more weapons in the integrated pest
management (IPM) arsenal, there is increased pressure for research into development of a sea louse vaccine.
One approach to developing a sea lice vaccine is through the extraction and characterisation of sea lice
proteins, and in particular proteases, including assessment of their antigenic properties. A transcriptomic
study suggested that the expression of virulence-associated sea lice proteins is host-dependent. The aim of
this study was to assess whether protein extracts from sea lice infecting Atlantic salmon (Salmo salar L.) and
rainbow trout (Oncorhynchus mykiss, (Walbaum, 1792)) present different proteomic profiles.

Parasites collected from both Atlantic salmon and rainbow trout, were homogenised and extracted proteins
were enriched for thiol groups using a thiol-sepharose column, with the goal of enriching for cysteine
proteases. Extracts of thiol-sepharose binding proteins (SLTSBP) were analysed using liquid chromatography-
electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), ProteinScape2 was used for data mining
and Blast2go for functional analysis of the identified proteins.

The majority of the sea lice SLTSBPs identified appear to be independent of the host fish species from which
the lice were obtained, with 13 identified proteins common to sea lice from both Atlantic salmon and
rainbow trout and only 2 and 3 proteins unique to Atlantic salmon and rainbow trout, respectively. More
detailed analysis comparing the protein profiles of the sea lice is on-going, the final results of which will be
presented, together with the implications this may have with regards to antigen mining for sea louse vaccine
development for cultured Atlantic salmon and rainbow trout.

Page 243
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Could acipenser iridovirus european (aciv-e) be an emerging threat to the conservation of
the Adriatic sturgeon (Acipenser naccarii)?

Davide Mugetti1, Claudio Pedron2, Paolo Pastorino1, Mattia Tomasoni1, Vasco Menconi1, Giuseppe Esposito1,
Alessandro Dondo1, Marino Prearo1

1The Veterinay Medical Research Institute for Piemonte, Liguria and Valle d'Aosta, Italy
2DVM, Itlay

[email protected]

In recent years, iridovirosis caused by Acipenser Iridovirus European (AcIV-E) is an emerging problem
concerning Acipenseridae farms. This virosis is highlighted above all in specimen of russian sturgeon
(Acipenser gueldenstaedtii), which seems to be the most sensitive species. Despite this, problems related to
AcIV-E are also reported in other sturgeons species, including siberian sturgeon (A. baerii), sterlet (A.
ruthenus), starry sturgeon (A. stellatus), beluga (Huso huso) and Adriatic sturgeon (A. naccarii). For the latter
species, in addition to the economic aspect, the importance from the conservation point of view must be
considered.

For these reasons, samples of A. naccarii were analyzed to evaluate AcIV-E presence. For each fish examined,
a portion of gills was taken. DNA was extracted from these samples by silica columns and a real-time PCR
was performed to evaluate the presence of the viral major capsid protein (MCP). Anatomopathological,
parasitological and bacteriological analyses were also performed to evaluate the symptoms and to exclude
the presence of other pathogens.
A total of 47 farmed adriatic sturgeons were analyzed in the period 2019-2021. Of them, 2 (p=4.3%) tested
positive for biomolecular analysis to detect the presence of AcIV-E. Other pathogens were excluded following
the performed tests.

The presence of AcIV-E in A. naccarii is already known, as previous studies have made it possible to highlight
this virosis on the Adriatic sturgeon. Considering that this species is often bred together with other species
of sturgeon susceptible to this infection (especially the Russian sturgeon) it is not surprising the confirmation
of the presence of this pathogen in farmed Adriatic sturgeon. The importance of this report lies in the fact
that A. naccarii is included in the International Union for Conservation of Nature (IUCN) Red List of
Threatened Species as critically endangered with continuously decreasing stock. As restocking programs are
doing, constant monitoring of this pathogen is essential to avoid introducing an additional factor in nature
that could further damage the Adriatic sturgeon. For this reason, our data represent an initial monitoring
that must be implemented with future samplings

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Revealing Myxobolus diversity from the Brazilian Amazon: two novel species parasitising
the gills of Semaprochilodus insignis

Maria Isabel Müller, Juliana Naldoni, Edson Aparecido Adriano

University of São Paulo - UNIFESP, Brazil

[email protected]

Myxozoans are cnidarian endoparasites that infect mainly fish, with some species causing important
diseases. As parasite diversity is expected to mirror host diversity, our study takes place in the biodiverse
Amazon Basin, that harbours approximately 5000 fish species. Among the fish that are economically
important for extractive fishing and farming are species of the Prochilodontidae family. The present study
reveals two species of Myxobolus infecting the gills of Semaprochilodus insignis, caught in the Tapajós river,
Pará State, Brazil. This prochilodontid species is one of the most consumed freshwater fish species in the
Brazilian Amazon.

Methodology
For this study, we analysed morphology, ultrastructure, small subunit ribosomal DNA sequencing data, and
performed a phylogenetic tree.

Results
Two morphotypes of Myxobolus were found co-infecting the gills in five Semaprochilodus insignis specimens.
Myxobolus sp. 1 located in the gill arch had oval shaped myxospores, measuring 8.0 ± 0.4 µm in length, 5.6
± 0.4 µm in width, and 3.8 ± 0.5 µm in thickness. Two equal polar capsules measuring 2.9 ± 0.3 µm in length
and 1.5 ± 0.2 µm in width. Polar tubules had 4–5 turns. Myxobolus sp. 2 located in the gill filament had round
shaped myxospores, measuring 13.9 ± 0.5 µm in length, 12.3 ± 0.5 µm in width, and 6.9 ± 0.6 µm in thickness.
Two equal polar capsules measuring 5.5 ± 0.5 µm in length and 2.3- 3.0 ± 0.3 µm in width. Polar tubules had
4–5 turns. Plasmodia of Myxobolus sp. 1 developed in the connective tissue of the gill arch and were
surrounded by collagen fibers. Plasmodial membrane presented pinocytotic channels. Generative cells,
immature, and mature myxospores were observed in the plasmodia. Phylogenetic analysis showed
Myxobolus sp. 1 and Myxobolus sp. 2 grouped with species parasites of prochilodontids fish. Myxobolus sp.
1 cluster as sister taxon to M. porofilus and Myxobolus sp. 2 grouped as sister species to the branch formed
by M. curimatae and M. prochilodus.

Conclusions
The combination of morphological, molecular and biological data revealed two undescribed Myxobolus
species infecting the Amazonian prochilodontid Semaprochilodus insignis.

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An unknown echinocephalus species (Nematoda: Gnathostomidae) infecting the
amazonian freshwater stingray Potamotrygon motoro

Maria Isabel Müller1, Edson Aparecido Adriano1, Lincoln Corrêa2

1University of São Paulo - UNIFESP, Brazil

2Universidade Federal do Oeste do Pará - UFOPA, Brazil

[email protected]

The Potamotrygonidae is a family in the class Chondrichthyes exclusively from freshwater with members
distributed in most river basins of South America that have drainage to the Caribbean Sea and Atlantic Ocean.
This study reports an unknown nematode species of the genus Echinocephalus found in the spiral intestine
of Potamotrygon motoro in the Tapajós river, Amazon basin, Brazil.

Methodology
Morphology and phylogeny, based on ribosomal gene 18S rDNA analyses, were carried out aiming the
identification of the nematode species.

Results
Twenty-three stingrays of the species P. motoro were examined and one was positive for these nematodes.
The measurement of females (n=5) nematodes were 55–85 mm in length and 0.9–1.4 mm in width and males
(n=4) were 55–66 mm in length and 0.6–0.9 mm in width. Phylogeny confirmed the monophyly of the
Gnathostomidae, which comprised species from Gnathostoma spp. and Echinocephalus spp. The
Echinocephalus species found in the intestine of P. motoro appears in a well-supported branch among the
Echinocephalus species, and related to E. pseudouncinatus, E. overstreeti, Echinocephalus sp. 1 and
Echinocephalus sp. 2, retrieved from GenBank.

Conclusions
The morphological, molecular, and phylogenetic analysis revealed that the Echinocephalus species found in
the intestine of Amazonian P. motoro is an undescribed nematode of the Gnathostomatidae family.

Page 246
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Distribution of anisakid nematodes in the muscle tissue of cod (Gadus morhua) from the
Norwegian Sea

Katarzyna Nadolna-Ałtyn1, Magdalena Podolska1, Joanna Pawlak1, Beata Szostakowska2

1National Marine Fisheries Research Institute, Poland

2Medical University of Gdansk, Poland

[email protected]

Atlantic cod (Gadus morhua) is one of most important commercial fish species on the world. Consumers
prefer its mild flavour and dense, flaky white flesh. However, the occurrence of potentially zoonotic
nematodes of the genera Anisakis and Pseudoterranova in cod muscle tissue arouse the food safety and
human health concerns. The aim of our studies was to explore the presence, intensity of infection and
distribution of the nematodes of the different genera of Anisakidae in the muscle tissue of G. morhua from
the Norwegian Sea. Samples were collected during commercial surveys in March 2017 in fishing areas FAO
IIa1 (n = 50) and FAO IIa2 (n = 56). After ichthyological analysis, the unskinned flesh of each fish was divided
into three parts – anterior ventral (belly flaps), dorsal fillet and caudal fillet – and examined using a white-
light transilluminator.

All parasites found were collected and identified to the genus level and subsample was identified using
molecular methods. Higher prevalence of infection with Anisakis than with Pseudoterranova in the
musculature of cod from both fishing areas was found. In FAO IIa1, a lower prevalence of infection with
Pseudoterranova was recorded (14%) than in FAO IIa2 (~39%), whereas the opposite was found with Anisakis
(88% and ~55%, respectively). The two parasite genera were distributed differently in cod muscle tissue:
most Anisakis larvae were present in the belly flaps (predominantly the left side), while Pseudoterranova
spp. were dispersed with descending frequency in belly flaps, dorsal fillet and caudal fillet.

Page 247
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Liver of cod (Gadus morhua) from the Norwegian Sea infected with anisakid nematodes

Katarzyna Nadolna-Ałtyn1, Magdalena Podolska1, Joanna Pawlak1, Beata Szostakowska2

1National Marine Fisheries Research Institute, Poland

2Medical University of Gdansk, Poland

[email protected]

Atlantic cod (Gadus morhua) belong to the most important commercial fish species on the world, not only
because of its flesh, but also liver (used for consumption or production of liver oil). However, the occurrence
of potentially zoonotic nematodes in that organ arouse the food safety and human health concerns. The aim
of our studies was to explore the presence, intensity of infection and distribution of the nematodes of the
different genera of Anisakidae in the liver of G. morhua from the Norwegian Sea.

Cod from two fishing areas : FAO IIa1 (n = 36) and FAO IIa2 (n = 52) were investigated for the presence of
parasites in the liver. After digestion in artificial gastric juice all parasites found were collected and identified
to the genus level. The subsample of parasites was identified using molecular methods. In FAO IIa1 94.4% of
livers were infected with Anisakidae nematodes, while in FAO IIa2 – 86.5%. Identification on the basis of
anatomo-morphological features of nematodes revealed the presence of representatives of Contracaecum,
Anisakis and Pseudoterranova genera. Molecular identification of nematodes (gene cox 2 analysis) revealed
the presence of Pseudoterranova bulbosa in both sampling areas.

Page 248
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Parasitic nematodes in the liver of cod (Gadus morhua) from Swedish and Polish waters of
the Baltic Sea

Katarzyna Nadolna-Ałtyn1, Magdalena Podolska1, Michele Casini2, Joanna Pawlak1

1National Marine Fisheries Research Institute, Poland

2Swedish University of Agricultural Sciences, Sweden

[email protected]

Cod, Gadus morhua, important for Baltic Sea fisheries, is one of the fish species that is most heavily infected
with nematodes. Increasing level of cod’s liver parasitic infection has been previously reported in Danish and
Polish waters. Conversely, there is a lack of knowledge about cod’s liver infection level in Swedish fishing
areas. The aim of our study was to evaluate the prevalence, intensity of infection and species composition
of nematodes in the liver of cod caught in Swedish waters and compare it with Polish data. Samples of cod
were collected during bottom trawl surveys in Swedish and Polish waters (February and November 2015;
February and November 2016; November 2017).

During this period, in total 1793 livers of cod from Swedish and 2210 from Polish waters were analysed for
the presence of parasites. Livers were digested in artificial stomach juice. All parasites were collected and
identified on the basis of anatomo-morphological features. Subsample of parasites was identified using
molecular methods. The most abundant nematode parasite was Contracaecum osculatum. Molecular
identification of nematode parasites confirmed also the presence of Pseudoterranova decipiens, Anisakis
simplex and Hysterothylacium aduncum. Generalized linear models (GLMs) were applied to analyse the
prevalence and intensity of cod infection with C. osculatum. Year, quarter and area of sampling as well as
sex and total body length of fish were statistically significant. The increasing prevalence of infection in cod
livers was observed from southern (Polish waters) to northern areas of sampling (Swedish waters
surrounding Gotland island). High level of cod’s liver infection with C. osculatum could be the result of the
increasing number of the grey seal Halichoerus grypus, which is one of the most important final hosts in the
life cycle of this parasite species in the Baltic Sea.

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Microbiological studies of Baltic Sea herring Clupea harengus – pilot studies

Katarzyna Nadolna-Ałtyn1, Agnieszka Pękala-Safińska2

1National Marine Fisheries Research Institute, Poland

2National Veterinary Research Institute, Poland

[email protected]

Baltic herring Clupea harengus, belong to the most important fish species in the Baltic sea due to the both
economic and ecological point of view. It is a target for fisheries: used for human consumption. It is also
important prey in the diet of piscivorous fish and sea mammals. Due to their role, there is a concern of health
status of that fish. The microbiological studies of that fish are neglected, while bacteria might be a source of
many fish borne diseases. The aim of our studies was to describe the microbiota of Baltic herring, caught in
the southern Baltic Sea.

In total 30 fish without external symptoms of diseases were sampled in 3 areas of Polish sea waters in autumn
2020. Samples from fish with urcels were also collected. Standard ichthyological analyses of each fish were
performed. The samples for microbiological analysis were collected from the kidney of each fish in sterile
way. In case of fish with urcels, the microbiological samples were taken also from the urcels.The bacterial
strains were cultivated and determined. The following pathogenic bacterial strains have been detected:
Pseudomonas, Shewanella, Acinetobacter, Aeromonas. Due to our best knowledge it is the first attempt to
describe the microbiota of Baltic herring from Polish waters, the southern Baltic Sea.

Page 250
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Muscle tissue parasites and diet of European perch Perca fluviatilis from estuary section of
the Nogat River - a tributary of the Vistula Lagoon, Poland

Katarzyna Nadolna-Ałtyn, Joanna Pawlak, Ryszard Kornijów

1National Marine Fisheries Research Institute, Poland

[email protected]

European perch (Perca fluviatilis) in the Vistula Lagoon belong to the most important fish species for fisheries
and ecosystem function. It is also eagerly chosen by consumers. During recent years there were reports from
the anglers about the presence of parasites in the muscle tissue of these fish. That arouse concern of
consumers health. The aim of our studies was to describe the parasite composition and diet of European
perch from the Vistula lagoon. Fish were caught by an angler in the estuary section of one of the lagoon's
tributaries - the Nogat River in June 2020 and transported to the laboratory for further analysis. During
standard ichthyological analyses of 42 fish the viscera and muscle tissue were inspected for the presence of
parasites.

The unskinned fillets were digested in artificial gastric juice to revel the presence of parasites. During visual
inspection two infected fish have been detected. Digestion revealed more infected fish. The prevalence of
infection with nematode Camallanus lacustris was 21.43%, and intensity of infection 1-6 parasites/fish. To
determine the diet composition of European perch, the stomach content of each perch was analyzed.
Stomachs of 81% of fish were empty. The dominant prey was Gammarus sp. Previous studies focused on the
parasite fauna of European perch from the Vistula Lagoon were conducted between 1994 and 1997 and the
infection level was on much lower level (prevalence 3.5 % and intensity of infection 1-2 parasites/fish).

Page 251
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Spatial patterns in infection of sprat with nematode parasites from Kiel Bight to the central
Baltic Sea

Katarzyna Nadolna-Ałtyn1, Bastian Huwer2, Uwe Krumme3, Richard Klinger4, Jane Behrens2

1National Marine Fisheries Research Institute, Poland

2National Institute of Aquatic Resources, Technical University of Denmark (DTU Aqua), Denmark
3Thünen Institute of Baltic Sea Fisheries, Germany
4Institute of Marine Ecosystem and Fishery Science, University of Hamburg, Germany

[email protected]

Sprat (Sprattus sprattus) is a key fish species in the Baltic Sea. It is important for the fisheries both for
industrial and consumption purposes, and is at the same time one of the most important fish species in the
pelagic food webs of the Baltic Sea, where it is a major prey of piscivorous predators such as cod, salmon and
marine mammals. However, sprat may not only be a source of energy and nutrients, but infected fish might
also be a source of parasitic infection for their consumers.

Preliminary studies in Danish and Polish waters indicated that sprat was infected with larval stages of the
anisakid nematode Contracaecum osculatum, a parasite that is also heavily infesting cod (Gadus morhua)
and grey seals (Halichoerus grypus) in the Baltic. However, these local studies were limited in spatial
coverage. As previous studies on C. osculatum in Baltic cod found increasing infection from the western to
the central Baltic, the aim of the present study was to investigate the current spatial differences in prevalence
and abundance of anisakid nematodes in sprat on a broader scale. Sprat samples (whole fish) were collected
in five different areas on a west-east transect from Kiel Bight to the southern Gotland Basin during surveys
in first quarter 2020, and after standard ichthyological analyses fish were visually inspected for the presence
of nematodes.

We present and discuss the results in relation to previous findings, where increasing levels of infection with
nematode parasites, especially with C. osculatum, have been observed during the latest decade in many
Baltic Sea fish species, concurrent with an increase in abundance of grey seals, the final host in the life cycle
of C. osculatum.

Page 252
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MALDI TOF MS - a useful tool in diagnostic fish bacteriology

Hanne Nilsen, Snorre Gulla, Bjørn Spilsberg, Anne Berit Olsen, Duncan Colquhoun

Norwegian Veterinary Institute, Norway

[email protected]

Identification of fish pathogenic bacteria is crucial for correct diagnosis and subsequent disease control in
aquaculture. Bacterial pathogens of cold-water fish grow slowly, are commonly biochemically unreactive and
may be difficult to identify with classical diagnostic techniques. MALDI-TOF MS (Matrix-Assisted Laser
Desorption/ionization Time-Of-Flight Mass- Spectrometry) essentially generates a protein fingerprint for the
investigated isolate, which is then compared to reference fingerprints stored in a local database.

With this method, identification of bacteria to the genus-, species-, and for some pathogens genomovariant-
level, can be done in few minutes at relatively low cost. The generation of MALDI-TOF profiles specific for
selected fish pathogens and their validation using sequencing based methods, will be presented.

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Molecular epidemiological analysis revealing dynamics of infectious hematopoietic
necrosis virus in facility

Aoi Nonaka1, Tsuboi Gosuke1, Suguru Hirabe1, Wei Chang1, Tomohiro Takeuchi2, Kouta Takehana2, Shigeru
Ogawa2, Eisuke Nakamura3, Hajime Matsuyama3, Shiro Itoi4, Goshi Kato1, Motohiko Sano1

1Tokyo University Of Marine Science And Technology, Japan

2Nagano Prefectural Fisheries Experimental Station, Japan
3Fuji Trout Hatchery, Shizuoka Prefectural Research Institute of Fishery and Ocean, Japan
4Nihon University, Japan

[email protected]

Infectious hematopoietic necrosis (IHN) has caused significant losses salmonid aquaculture in Japan.
Increasing virulence of the causative virus, IHNV, is a current issue. Dynamics of IHNV in facilities, which is
the fundamental information required to elucidate the mechanism of increasing virulence, is not fully
understood. In this study, we attempted molecular epidemiological investigation on IHNV isolates in facilities
using virus genome sequencing.

Methodology
IHNV was isolated on EPC cells originated from ovarian fluid or seminal plasma of matured broodstock (3-
year-old: October and December, 2019) and internal organs of dead fry of an IHN case (May to June, 2019)
in two prefectural institutes conducting rainbow trout breeding in Nagano and Shizuoka Prefecture.
Extracted virus genome RNA were subjected to RT-PCR followed by sequencing virus G and N protein genes
(total 1,085 bp), and consequent phylogenetic analysis was conducted to examine relationship of the
isolates.

Results
The phylogenetic tree of IHNV isolates from dead fry (n=19) in the Nagano Institute showed the case was
caused by multiple virus strains. Since water supply was mixed with wastewater of two trout farms in
upstream, it was likely that viruses were supplied from there at certain concentrations as an infection source
and infected the fry simultaneously. In addition, the isolates from broodstock (n=17) and dead fry were
placed differently in phylogenetic tree, suggesting reactivated virus in the broodstock might be the same
virus in primary infection when the fish was at fry stage. At the Shizuoka Institute, viruses from dead fish
(n=25) in experimental infection using wastewater from growth ponds were plotted at similar clades but at
two different genetic lineages in phylogenetic tree as well as those from broodstock(n=20). Contrastingly,
the isolates from dead fry (n=25) were almost identical. Although rearing water of the fry pond was mainly
supplied from hatchery, there were fish along the supply water channel. It is possible that limited viruses at
low concentrations were supplied, probably from fish in the channel, as an infection source and spread
among the fry. Thus, the epidemiological analysis can be useful to reveal IHNV dynamics through breeding
cycle in facility.

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Are sea bass (Dicentrarchus labrax) susceptible to infection with VHSV and IHNV?

Niels Olesen, Jacob Schmidt, Niccolò Vendramin, Argelia Cuenca

National Institute For Aquatic Resources, DTU Aqua. Technical University of Denmark, Denmark

[email protected]

VHSV and IHNV are the most threatening viral pathogens for the European trout industry. One of the most
intriguing characteristics of VHSV is its ability to cross species boundaries, not only to cause sporadic
infection, but also to create stable intra-species transmission in novel fish species. Indeed, VHSV has been
found in more than 90 different fish species in freshwater and marine environments. Although the ability of
VHSV to infect sea bass, another very important European aquaculture species, has been suggested,
infections dynamics remains only poorly described at best. Although only salmonids are listed as susceptible
to IHNV, cases of cross-species transmission to non-salmonid fish have been reported.
Assessment of the susceptibility of sea bass to infection with VHSV and IHNV, and whether this species should
be included in the list of susceptible species given in the OIE Aquatic Code and in the EU legislation, is of high
relevance for the European aquaculture.

Methodology
We performed two different infection trials. In the first trial, susceptibility to infection with VHSV genotypes
Ia, Ie and III and IHNV genotype E was assessed. These isolates were selected based on high pathogenicity to
rainbow trout (Ia and E), reported isolation from sea bass in Turkey (Ie), or ability to infect a number of
marine fish species (genotype III). The trial included 40 sea bass of 3 g in triplicate tanks. Half were IP injected
shedders and half were cohabitants. In the second trial only the VHSV isolates were included in order to
corroborate the susceptibility by an immersion trial.

Results
Results of the first trial show no effect of infection by VHSV genotype III and IHNV E in the survival rate of
the IP injected fish, whereas fish injected with VHSV subtypes Ia and Ie showed signs similar to those
observed for VHSV infection in rainbow trout, i.e. a general darkening and petechial bleedings with 52% and
18% survival, respectively. Neither survival reduction nor virus replication was detected in any of the
cohabitants.

Conclusions
Sea bass do not fulfil the requirements for being susceptible to VHS and IHN.

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Effect of temperature on transfer of midichloria-like organism (MLO) and development of
Red Mark Syndrome (RMS) in rainbow trout (Oncorhynchus mykiss)

Massimo Orioles1, Jacob Gunther Schmidt2, Elena Saccà1, Marco Galeotti1

1Udine University, Department of Agricultural Food Environmental and Animal Sciences (DI4A), Italy
2National Institute of Aquatic Resources (DTU aqua), Technical University of Denmark, Denmark

[email protected]

Red mark syndrome (RMS) affects farmed rainbow trout worldwide. No aetiological agent has been
unequivocally identified for RMS, although MLO (Midichloria-like organism) seems an increasingly most
probable candidate.

RMS is generally associated with water temperatures below 16 °C. However, since experiments have
demonstrated a long incubation phase of the disease, and since water temperature on farms can fluctuate
substantially during this time, the real effect of temperature on RMS transfer and symptom development
remains elusive. At DTU a number of experiments have been conducted involving transfer of RMS, but all at
12 °C. The authors set up an experiment to investigate the transfer of MLO and the development of disease
at three different temperatures.

Methodology
We tested the effect of 3 temperatures (12 °, 15.5 ° 19 oC) on the transfer of MLO from RMS-affected fish to
naïve SPF cohabitants, and the development of skin pathology. The trial was carried out in duplicate 180 L
tanks and included one negative control tank per temperature.
To each tank was added 4 seeder fish and 18 SPF fish. The fish were followed for 12 weeks, during which
samples were taken from 4 fish from each tank on three occasions. Samples consisted of skin samples for
MLO detection by qPCR and full-thickness lesion skin and control skin samples for histology. Lesions were
classified macroscopically on the fish and microscopically on H&E-stained sections by a scoring system
recently proposed by the authors.

Results
At 12 °C the development of skin lesions peaked in severity at 12 weeks post-cohabitation. At 15.5 °C the
skin pathology was most severe at 8 weeks, and 4 weeks later skin lesions were in the healing phase or
healing completely resolved. At 19 °C only three fish developed mild macroscopical and microscopical skin
lesions, which had completely healed at 12 weeks.
The amount of detected MLO in affected fish measured by PCR reflected the observed pathology.

Conclusions
The results show that RMS develops only in mild form and in few fish at 19 °C and at 15.5 °C the disease
symptoms disappear more quickly compared to 12 °C.

Page 256
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Development and application of a sensitive droplet digital PCR (ddPCR) for the detection of
rickettsia-like organism on rainbow trouts (Oncorhynchus mykiss) affected by Red Mark
Syndrome (RMS)

Massimo Orioles1, Michela Bulfoni2, Elena Saccà1, Daniela Cesselli2,3, Jacob Gunther Schmidt4, Marco
Galeotti1

1University of Udine, Department of Agricultural, Food, Environmental and Animal Sciences, Italy
2University of Udine, Department of Human Medicine, Italy
3Udine University Human Hospital, Italy
4National Institute of Aquatic Resources (DTU aqua), Technical University of Denmark, Denmark

[email protected]

Red mark syndrome (RMS) affects farmed rainbow trout worldwide. No aetiological agent has been
unequivocally identified for RMS, although RLO (Rickettsia-like organism) seems an increasingly more
probable candidate. The RLO presence is usually detected by qPCR, but for relative quantification a
housekeeping gene is required, while the absolute quantification need the construction of a standard curve,
through cloning in vectors and multiplication in bacteria. Droplet digital PCR (ddPCR) is a sensitive, accurate
and absolute quantitation method, relatively simple and quick to perform. In this study, we established a
ddPCR method to detect and quantify RLO DNA and compared the sensitivity and specificity with qPCR.
Spleen samples from rainbow trout (Oncorhynchus mykiss) affected by RMS and from healthy fish were
obtained from an experimental infection trial at DTU aqua (Denmark). The same primers and probe were
used in both ddPCR and qPCR reactions to amplify the RLO DNA. The presence/absence analysis of RLO and
the analysis of standard curve were performed by qPCR.

We defined the better probe concentration and annealing temperature for ddPCR. Specificity of ddPCR was
verified against the DNA of 4 different bacterial species affecting fish. The sensitivity of qPCR and ddPCR was
compared using serially diluted samples. We explored the feasibility of ddPCR to detect RLO on 109 fish
spleen samples and compared the accuracy of the data obtained by qPCR.

The optimal probe concentration and annealing temperature were established at 250 nM and 64.5 °C,
respectively. The specificity of ddPCR for RLO DNA was confirmed by the absence of signal for bacteria
species different from RLO. The sensitivity of ddPCR was higher compared to qPCR (detection limit: 2.5 copy
number for ddPCR vs. 20 copy number for qPCR). The slope of the standard curve and the PCR efficiency
value were in the appropriate range (Y = -3.341X + 41.466; R2 = 0.989; efficiency = 99.2%). On examined
samples, the positive detection rate of ddPCR was 5.62% higher than that of qPCR.

We established a ddPCR method for the detection and absolute quantification of the RLO associated with
RMS with a higher level of sensitivity compared with qPCR.

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Verrucous epidermal hyperplasia on pectoral fin of a Wels catfish (Silurus glanis, Linnaeus
1758)

Massimo Orioles1, Samuele Zamparo2, Marco Galeotti1, Donatella Volpatti1, Francesca Errani2, Enrico Volpe2,
Sara Ciulli2, Luciana Mandrioli2

1Udine University, Department of Agricultural, Food, Environmental and Animal Sciences, Italy
2Bologna University, Department of Veterinary Medicine Sciences, Italy

[email protected]

The Wels catfish (Silurus glanis) is a commercially important Eurasian species that originally evolved in Asia
before expanding its range to the west. It possesses the attributes of a species well adapted to introductions
outside its native range.

The majority of disease reports in this species, especially dealing with viral and bacterial infections, are
related to aquaculture or experimental conditions.

In 2019 a Wels catfish, kept in a 1k liters tank in a public aquarium of Northern Italy, showed a superficial
translucent discrete, soft, raised cutaneous verrucous growth on the right pectoral fin. This slow-growing,
irregularly spherical, sessile mass was attached to fin, not ulcerated and measured 0.5 cm of diameter.

The tank is supplied by an artesian well water with temperature of approximately 12 °C. The Wels catfish
shares the tank with a Southern Pike (Esox cisalpinus) and a Black bass (Micropterus salmoides). The fish was
anesthetized with MS-222 and an excisional biopsy was performed.

Skin mass was routinely processed for histology; anti-Proliferating Cell Nuclear Antigen (PCNA) antibody was
employed to assess the proliferative activity of epidermal cells. Molecular analyses were carried out to detect
viral pathogens by extracting DNA from the skin mass. Several PCR-based protocols were applied to the
extracted DNA in order to detect viral genome belonging to viruses with fibroblastic tropism or associated
with epithelial proliferation such as herpesviruses, ranaviruses and adenoviruses.

The verrucous growth was well circumscribed, unencapsulated, densely cellular and composed by 12 to 20
layers of densely packed epithelial cells, forming papillomatous structures interdigitating with the lower
dermis. The epithelial cells had prominent nuclei and frequent double nucleoli; cytoplasms showed frequent
vacuoles and mitotic figures were rarely observed (ranging from 0 to 1 per HPF). Goblet cells were scarce in
the most central part of the epidermal cells proliferation area. The type of stroma was vascular, composed
by septa of connective tissue containing florid capillaries and small foci of necrosis. No bacteria were noticed.

PCNA expression analysis revealed variable positivity in different areas of the growth. A diagnosis of
epidermal hyperplasia, possibly related to environmental and abiotic stressors, was made.

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The gill pathobiome of carp edema virus (CEV)-positive common carp

Miroslava Palikova1,2, Lubomír Pojezdal3, Lenka Dávidová-Geržová1, Věra Nováková1, Ivana Papežíková1,2,
Hana Minářová1,3, Iva Dyková1,2

1UniversityOf Veterinary Sciences Brno, Czech Republic

2Mendel University in Brno, Czech Republic
3Veterinary Research Institute, Czech Republic

[email protected]

The approaches to understanding fish diseases have changed over time. The demands on the knowledge of
the relations between the host and the members of microbial communities of the aquatic environment in
real ecological contexts are increasing. Related to this is the need to distinguish between
symbionts/commensals, potential and actual pathogens, including an assessment of their significance in so-
called co-infections.

Methodology
Two adult common carp (Cyprinus carpio L.) from Sedlo pond (South Moravia, Czech Republic) with high fish
mortality occurring at the water temperature of 21 °C were collected for detailed examination using standard
diagnostic methods supplemented by isolation procedures and eDNA targeting microorganisms other than
bacteria and viruses.

Results
The above mentioned methods allowed us to detect carp edema virus (CEV) in two carp and also in the
ectoparasite Argulus foliaceus collected from carp skin. In addition to Flavobacteria (found to be associated
with gill lesions also in previous studies), we found that free-living eukaryotes colonizing gills (amoebae,
ciliates) and a temporary parasite (Ichthyobodo) may also substantially contribute to the alterations of gill
structure and function either directly due to more (Ichthyobodo) or less firm attachment (amoebae) to the
gill epithelium or indirectly, being associated with bacteria as their hosts. High-throughput sequencing
targeted on bacteria in the gill tissue and cytoplasm of isolated eukaryotes revealed bacterial assembly rich
in families and genera, within which Flavobacterium predominated.

Conclusions
This study supports the assumptions of previous authors about the copathogen role of Flavobacterium spp.
in the carp edema virus disease (CEVD) development, integrates this knowledge with newly recognized
eukaryotic copathogens into gill pathobiome responsible for irreversible CEVD gill lesions, and draws
attention to pathobiome-host and member-to-member interaction issues to be clarified taking advantage of
genetic, cellular and molecular tools now available.

Page 259
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Seasonal changes of haematological and immunological parameters in rainbow trout
(Oncorhynchus mykiss) reared under different conditions

Ivana Papežíková1,2, Zdeněk Hanák1, Miroslava Palíková1,2, Hana Minářová1,3, Jan Mareš2

1Veterinary University in Brno, Czech Republic

2Mendel University in Brno, Czech Republic
3Veterinary Research Institute, Czech Republic

[email protected]

Fish physiology is known to be affected by rearing conditions, which can be largely regulated in indoor
aquaculture systems, but the possibilities of regulation are limited in outdoor systems. This work was aimed
at testing seasonality of haematological and immunological parameters in rainbow trout and their
comparison in fish reared in an outdoor pond and in an indoor flow-through system.

Methodology
Fish were sampled during the whole year in 2-3 month intervals. Whole blood from ten fish randomly
selected from each system was used for chemiluminescence assay of opsonized zymosan-activated
respiratory burst of phagocytes and for determination of white blood cell counts and differential cell count.
Rest of the blood was centrifuged and the obtained plasma was used for chemiluminescence assay of
bacteriolytic complement activity and for measurement of total immunoglobulins. Skin mucus was sampled
by scraping skin surface by a dull scalpel blade and used for determination of lysozyme activity by radial
diffusion assay.

Results
Marked seasonal differences in most parameters were seen in fish from both rearing systems. The most
significant changes were found in lysozyme and complement activity and in the number of phagocytic cells.
Effect of rearing conditions was seen mainly in respiratory burst of phagocytes. In summer, faster reaction
of phagocytes to opsonized zymosan was observed in fish reared in the outdoor pond compared to fish
reared in the indoor system.

Conclusions
Selected haematological and immunological parameters were more affected by the season than by rearing
conditions. More marked seasonal changes were seen in fish reared in the outdoor pond than in the indoor
basin.

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An unusual isolation of carnobacteria from the eyes of healthy salmonids from high-
mountain lakes: a trojan-horse effect?

Paolo Pastorino1, Silvia Colussi1, Elisabetta Pizzul2, Katia Varello1, Vasco Menconi1, Marco Bertoli2, Davide
Mugetti1, Mattia Tomasoni1, Giuseppe Esposito1, Alessandro Dondo1, Elena Bozzetta1, Pier Luigi Acutis1,
Marino Prearo1

1The Veterinary Medical Research Institute for Piemonte, Liguria and Valle d’Aosta, Italy
2Department of Life Sciences, University of Trieste, Italy

[email protected]

Carnobacterium maltaromaticum and C. divergens have been associated with infections in fish. In a previous
study, carnobacteria were isolated from the eyes of healthy wild salmonids from a high-mountain lake. To
better understand these findings, salmonids were captured from three high-mountain lakes (northwest Italy)
and subjected to bacteriological and histological examination.

During August 2019, specimens of brook trout (Salvelinus fontinalis) were captured from Upper Balma Lake
(n=25) and Lower Balma Lake (n=57); 46 specimens of brown trout (Salmo trutta) were captured from Rouen
Lake. Liver, kidney, spleen, brain and eyes were collected and immediately fixed in formalin for histological
examination. Kidney, brain, and eyes from each fish were subjected to bacteriological examination. Twenty-
five isolates identified as C. maltaromaticum and 9 identified as C. divergens, according to phenotypic and
biochemical characterization, were subjected to molecular characterization. PCR amplification was
performed using the primers for the intergenic spacer region (ISR) of the 16S and 23S of rRNA genes and the
pisA precursor gene for the piscicolin 126 protein. Terrestrial insects collected from the lake shoreline and
the stomach contents were also screened for the presence of carnobacteria since salmonids feed mainly on
terrestrial insects, which are considered possible vectors for carnobacteria that might catabolize the
exoskeleton chitin.

Although all fish were healthy as confirmed by the absence of internal and external lesions at necropsy and
histological analysis, 8.7% (Lower Balma Lake), 24% (Upper Balma Lake), and 32.6% (Rouen Lake) were
positive for carnobacteria colonization of the eyes. A Trojan-horse effect was hypothesized to explain
carnobacteria isolation in the eye. This immune-escaping macrophage-mediated mechanism has been
identified in other Gram-positive bacteria. Carnobacterium maltaromaticum strains were found to display
genotypic heterogeneity and a low percentage of pisA positive amplification. All insects tested negative for
carnobacteria, but as a small number of samples were analyzed, their role as possible vectors of infection
cannot be excluded.

Features of geomorphology, geographic isolation, and microbiota common to the three lakes are thought to
be possibly related to our findings. Further research will be carried out also to test the hypothesis of the
present study.

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Occurrence of Eustrongylides spp. in Perca fluviatilis from Lake Maggiore (northwest Italy)

Paolo Pastorino, Vasco Menconi, Mattia Tomasoni, Davide Mugetti, Marino Prearo

The Veterinary Medical Research Institute for Piemonte, Liguria and Valle d’Aosta, Italy

[email protected]

The genus Eustrongylides includes nematodes having heteroxenous life cycles that infect several fish species
and ichthyophagous birds of freshwater ecosystems. Nematodes ascribable to the genus Eustrongylides are
potentially zoonotic, and infection occurs after the consumption of raw or undercooked fish. In Italy the
occurrence of Eustrongylides spp. was previously described in fish species (Perca fluviatilis, Atherina boyeri,
Micropterus salmoides and Lepomis gibbosus). The main aim of this study is to report the occurrence of
Eustrongylides spp. in Perca fluviatilis from Lake Maggiore (northwest Italy).
In the two-year period 2020–2021, with the collaboration of local professional fishermen, a total of 100 perch
were examined for the occurrence of Eustrongylides spp. from Lake Maggiore. Parasitological examination
was performed according to European’s regulations (Regulation EC No. 2074/2005; EFSA 2010).

The prevalence of infestation was 3% and the mean intensity of infestation ranged from 1 to 3; the mean
abundance was 0.05.

This study reports the occurrence of Eustrongylides spp. in an Italian subalpine lake and improves our
knowledge about the distribution of this parasite. Furthermore, it’s important to highlight that our results
show an expansion of the geographic distribution of Eustrongylides in Italy. The expansion of geographical
range of E. excisus could be associated to the increased cormorant population, both in term of number and
area during last years in northern Italy, where it is mostly concentrated around large lakes. Perca fluviatilis is
a commercial relevant fish species and important for recreational fishing in European lake systems, Italy
included.

During last years the question about the risk of fish borne zoonotic parasites have call the attention of
scientific community and sanitary authorities. This survey demonstrated the necessity of communication
between veterinarians, professional fishermen and sanitary authority about this emergent zoonotic issue.

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Changes in European eel infection with Anguillicola crassus in Polish waters 2014 - 2020

Joanna Pawlak, Katarzyna Nadolna-Ałtyn, Tomasz Nermer, Łukasz Giedrojć, Piotr Pankowski

National Marine Fisheries Research Institute, Poland

[email protected]

The swim bladder nematode Anguillicola crassus is a common parasite of European eels (Anguilla anguilla).
The aim of the studies was to check the level of eel infection in the Polish EEZ, southern Baltic Sea. Fish has
been examined each year between 2014 and 2020. Parasitological analysis focused on the presence of
nematode A. crassus has been performed (the total number of investigated eels was 2819). The total number
of found parasites was 15681.

The correlation between infection intensity and host length, Fulton condition factor, age of the fish, area and
time of sampling have been analyzed. The prevalence and intensity of infection have been calculated.
Intensity of infection varied from 1 to 95 parasites per fish. We observe decreasing value of the mean
prevalence of A. crassus infection: from 72-73% in 2014-2015 to 53% in 2019-2020. Mean prevalence of
infection reported previously from Polish waters in 2000-2002 was 73.6-76.2%.

Page 263
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Liver nematode parasites and diet of shorthorn sculpin (Myoxocephalus scorpius) from
Polish waters

Joanna Pawlak1, Katarzyna Nadolna-Ałtyn1, Marcin Kuciński2

1National Marine Fisheries Research Institute, Poland

2Department of Marine Biology and Ecology, Institute of Oceanography, University of Gdansk, Poland

[email protected]

The studies focused on the shorthorn sculpin (Myoxocephalus scorpius) from the Baltic Sea are of little
interest, especially in terms of parasitological aspects. Shorthorn sculpin characterizes by a relatively
sedentary lifestyle, and therefore has been previously used as a model species for assessment of the
accumulation rate of the sealworm population in local waters. To date, presence of the nematodes in the
muscle tissue of shorthorn sculpin and Baltic cod (Gadus morhua) have been studied in fish collected along
the Swedish coast a few years ago, but no attention was paid to nematodes observed on or in the intestines
or livers. In general, sculpin were less infected than cod, taking into account the abundance and prevalence
of parasites.

The current level of cod infection with Anisakidae nematodes in Polish waters is well known, but no studies
focused on shorthorn sculpin were conducted in the southern Baltic Sea. In case of predatory fish the main
way of its infection with nematode parasites is via eating the infected preys. To explore and better
understand the dynamics of parasite spreading in environment the studies should include wide variety of
species that compose local ecosystem, including their trophic characteristics. The aim of our study was to
assess the presence of Anisakidae nematodes in the livers as well as diet composition of shorthorn sculpin
from north-west Polish waters. Samples have been collected during survey in November 2020. Standard
ichthyological analyses of 37 fish were performed onboard and livers were frozen for further parasitological
investigation. Thawed livers were digested in artificial digestive juice. All parasites were collected and
identified on the base of anatomo-morphological features. Contracaecum sp. nematode parasites have been
detected in 13.5% of investigated fish. Diet composition was studied on the basis of stomach content
analysis. Among food items the most abundant were Crangon crangon, Bylgides sarsi and Gammarus sp. All
found preys were parasitologicaly inspected for the presence of nematodes.

Due to our best knowledge, the present study is the first attempt to describe the current status of
parasitological infection with Anisakidae nematodes and diet of shorthorn sculpin in Polish waters.

Page 264
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Anisakidae nematodes and diet of salmon (Salmo salar) from Polish waters

Joanna Pawlak, Katarzyna Nadolna-Ałtyn, MSc Adam Lejk

National Marine Fisheries Research Institute, Poland

[email protected]

Atlantic salmon (Salmo salar) is at the top of the trophic pyramid in the Baltic Sea, in areas without sea
mammals. It is also important species for Baltic sea fisheries, willingly chosen by consumers. However, the
occurrence of potentially zoonotic nematodes arouse the food safety and human health concerns. The level
of Baltic salmon infection with Anisakidae parasites is unknown. The high level cod (Gadus morhua) (also
piscivorous, predatory fish) infection with these zoonotic parasites is recently observed in the Baltic Sea area.
Diet of fish is not only the source of nutrients, but may show the way of infection with parasites. Therefor
the aim of our studies was to check the presence of Anisakidae nematodes in the livers and diet of Baltic
salmon from Polish sea waters.

Samples have been collected during 2020. Standard ichthyological analyses of 120 fish were performed and
livers were frozen for further parasitological investigation. Thawed livers were digested in artificial digestive
juice. All parasites were collected and identified on the basis of anatomo-morphological features. A
subsample of parasites have been identified using molecular methods. Contracaecum osculatum nematode
parasites have been detected in 13.33% of investigated fish. Diet composition was studied on the basis of
stomach content analysis. Among food items the most abundant were fish: sprat Sprattus sprattus and three-
spined stickleback Gasterosteus aculeatus, while invertebrates were represented only by Mysis mixta. Baltic
Sea sprat have been previously found infected with Contracaecum osculatum, therefor it is probably the
main source of salmon infection with that nematode parasite.

Page 265
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Assessing the spread of renibacterium salmonarium between farmed and wild fish in
Sweden

David Persson, Anna Aspán, Paulina Hysing, Eva Jansson, Ludvig Orsén, Hampus Hällbom, Charlotte Axén

National Veterinary Institute (SVA), Sweden

[email protected]

Bacterial Kidney Disease (BKD) can be a devastating bacterial infection within salmonid aquaculture
throughout the world. It is caused by the gram-positive intracellular bacterium Renibacterium salmonarium
and is spread both horizontally and vertically. Disease signs include external ulcerations and blisters and
internal signs such as organ swelling and, and spleen, granulomas, petechiae and ascites. BKD accounts for a
significant loss of income in aquacultures due to the high mortality rate. In addition, uncontrolled spread in
aquacultures may threaten the survival of wild fish populations.

The aim of our study was to investigate the prevalence of BKD in wild salmonids caught in waters where net
pen farming occurs. four rivers with a recent history of BKD positive farms during mandatory controls were
selected. Furthermore, we evaluated the use of eDNA as potential sample for surveillance and monitoring
for ongoing infections. In total 1058 fish were sampled from four different river systems and out of them 52
(4.9%) were positive for BKD by antigen-ELISA. Surprisingly, these fish where not evenly distributed between
the four river systems, but 50 were caught in the same river (Ljungan). This accounts for an alarmingly high
rate of 17% BKD+ samples in wild salmonids this area. This number is far above what was expected and
clearly show the risk with an open farming system and the importance of effective health monitoring
programs to avoid an uncontrolled spread of the disease.

The use of eDNA for monitoring BKD is somewhat difficult to evaluate. Few of the water samples analyzed
were positive for BKD (2 of 38) and those where not collected where the positive fish were identified. In
addition to water, sediment samples were collected and analyzed in a net pen farm that recently slaughtered
all BKD+ fish. This method looks more promising as 5 out of 5 samples where positive for BKD. Thus, sediment
samples may be valuable for monitoring potential ongoing BKD infections in farms, without the need of
sacrificing valuable fish.

Page 266
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Combined application of a nutraceutical formulation for growth performance of larvae and
survival rate of white leg shrimp juveniles challenged by acute hepatopancreatic necrosis
disease

Hoang Phan1, Phuong Viet Do1, Tao Tai Chau2, Philippe Mahl1

1Virbac, Aquaculture Division, France and Vietnam

2College of Aquaculture and Fisheries, Can Tho University, Vietnam

[email protected]

Shrimp at early stages such as larva and juvenile are sensitive and require a high level of diverse nutrients
for their growth and survival. Vitamins and immunostimulants are essential but need to be supplemented
onto aquaculture feed as its pelleting process, particularly small size feed, introduces high heat which may
degrade those essentials. This study was to evaluate the effect of Nutrimix – a nutraceutical formulation via
a combined application strategy i.e. immersion and oral on white leg shrimp (Litopenaeus vannamei) larvae
and juveniles to improve health, growth, survival rate when challenged with Vibrio parahaemolyticus. Phase
1: three treatments of 5 g/m3/day, 3 g/m3/day and without supplement (control) were performed at 30 ppt
salinity on nauplii from Zoea-1 to PL-12 stage at the density of 200,000 nau/m3 for 18 days.

Phase 2: shrimp from phase 1 were divided into halves following their respective treatments, 1,000 shrimp
individuals of each half entered this phase with/without Nutrimix (15 g/kg of feed) supplemented onto feed
pellets (6 groups in total, including the control) for the next 30 days (from PL-12 to PL-42), and with the
salinity of 15 ppt. Phase 3: AHPND immersion challenge was applied on 30 shrimp individuals of each
experimental group with Vibrio parahaemolyticus at 1.9 x10^4 CFU/mL for 14-day post-challenge follow-up
of mortality. All phases and experimental groups were performed in triplicates with environmental
parameters were recorded in the suitable ranges for shrimp culture. Growth in length and survival of PL-12
was best in treatment supplemented with 5 g and 3 g Nutrimix/m3/day (p > 0.05) which were statistically
different from the control treatment (p < 0.05). Growth in weight and survival of PL-42 was significantly
higher in treatments with the Nutrimix supplementation. Shrimp with 5 g Nutrimix/m3/day (phase 1) plus
15 g Nutrimix/m3/day (phase 2) were with the significantly lowest mortality rate among the AHPND
challenge treatments. In overall, a combination of immersion and oral Nutrimix supplementation showed
better growth performance and survival rate of shrimp at sensitive cases.

Page 267
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Long term trends in infection with Contracaecum osculatum in cod Gadus morhua from the
southern Baltic Sea and its impact on condition and mortality of the host

Magdalena Podolska, Katarzyna Nadolna-Ałtyn, Jan Horbowy

National Marine Fisheries Research Institute, Poland

[email protected]

Cod (Gadus morhua) taken from the southern Baltic Sea between 2011–2020 were visually examined for the
presence of anisakid nematodes in the liver. Parasitological analysis revealed the presence of nematodes
belonging to the genera Contracaecum (most abundant genus), Anisakis, Pseudoterranova and
Hysterothylacium. A subsample of Contracaecum larvae was subjected to DNA extraction, followed by
species identification by sequence analysis of the ITS-1 region of rDNA. Parasites were identified as
Contracaecum osculatum s.s. Generalized linear models (GLMs) were applied to analyze the prevalence and
intensity of cod infection as dependent on year, quarter, sampling area and biological parameters of fish and
to investigate the association between the body condition factor and the intensity of infection.

The prevalence of infection with C. osculatum larvae is much higher compared with previous studies
undertaken over the past few decades. Remarkable increase in the infection level of cod was reported in
2011 and there was a further increasing trend in subsequent years (2012-2020). Infection dispersed to the
entire area of the southern Baltic, reaching ~90% in eastern Baltic (Gdansk Basin). Both the prevalence and
intensity of infection increased with fish length up to 70–80 cm but then began to decline. The decline may
be attributed to the increasing mortality of large and heavily infected cod. The body condition of cod
decreased significantly with an increasing numbers of parasites in the liver. Condition of heavily infected fish
was up to 20% lower than that of uninfected individuals. Elevated infection of cod is associated with an
increase in numbers of the grey seal (Halichoerus grypus) in the Baltic Sea, which are final hosts for C.
osculatum.

Page 268
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Carp edema virus and koi herpesvirus were the most prevalent viral pathogens of the Czech
common carp (Cyprinus carpio) in 2020

Ľubomír Pojezdal1, Hana Minářová1,2, Miroslava Palíková2,3, Jitka Motlová1, Stanislava Reschová1, Veronika
Piačková4

1Veterinary Research Institute, Czech Republic

2University of Veterinary Sciences Brno, Czech Republic
3Mendel University in Brno, Czech Republic
4University of South Bohemia in České Budějovice, Czech Republic

[email protected]

Common carp (Cyprinus carpio) is the most important fish species farmed in the Czech Republic, with the
annual production of circa 20 000 tonnes. Infectious diseases decrease the profitability of the carp
aquaculture, with a notifiable disease – koi herpesvirus (KHV) disease, and an emerging disease – carp edema
virus (CEV) disease, causing the largest losses in the last few years.

Methodology
Samples were obtained by the State Veterinary Administration during KHV surveillance program (556
samples from 96 locations) or directly from common carp farmers or koi carp breeders who reported clinical
signs of a disease (81 samples from 28 locations). The gill and internal organ tissue was pooled, homogenised
and the total DNA and viral RNA were extracted. Presence of KHV, CEV and carp sprivivirus (SVCV) was
confirmed using real-time PCR. Partial P4a gene sequences of detected CEV isolates were obtained via Sanger
sequencing.

Results
In total, four locations were confirmed KHV positive and five ponds were depopulated, with 252.5 tonnes of
fish euthanized due to control measures. CEV was diagnosed in six locations, of which four represent the
common carp farmers and two koi carp breeders. Clinical signs in these cases varied from asymptomatic (two
locations) to koi-sleepy-disease-like symptoms in both of the koi breeders to a 100% mortality rate in one of
the common carp farms. Phylogenetically, two of the established CEV genogroups were detected, namely
genogroup I in common carp and genogroup IIa in koi. SVCV was detected in one location, without the
presence of any clinical or patho-anatomical signs. None of the samples showed co-infection of viral
pathogens.

Conclusions
Viral diseases still pose a threat to the carp aquaculture in the Czech Republic. Besides the direct losses via
mortalities and decreased weight gains, mandatory control measures against the spread of KHV decrease
the profitability even further.

Page 269
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First record of Clinostomum complanatum (Trematoda: Digenea) in French perch (Perca
fluviatilis) from the Doubs (French Jura)

Françoise Pozet4, Eloïse Rochat1,2, Pierre Marle3, Isabel Blasco-Costa1,2

1UiTThe Arctic University Of Norway, Norway

2The Natural History museum of Geneva, Switzerland
3University of Geneva, Switzerland
4Departmental Analysis Laboratory of Jura, France

[email protected]

The natural distribution of Clinostomum complanatum (Rudolphi, 1814) in Europe is restricted to the Danube
basin and the North-East of Italy. This parasite mainly aims for the fish-eating bird and occasionally reptile,
amphibian and mammal. As a mammal, human can be infected with this parasite by eating raw fish and
develop a zoonosis: the Halzoun syndrome. In this study, we report important infections by the invasive
trematode Clinostomum complanatum in thirty-three wild perches (Perca fluviatilis L.) of the Doubs River
(France) in Western Europe. A total of 184 encysted Clinostomum complanatum specimens were collected,
reaching a prevalence of 97%.

No correlations were observed between fish traits such as standard length, weight, age and sex, and the
number of parasites. However, our results showed that the distribution of Clinostomum complanatum cysts
in the body of fish is not random. The opercula, buccal and branchial cavities, muscles along the lateral line
and the caudal and dorsal muscles were more infected than other parts. The occurrence of this parasite,
recorded for the first time in France, presents a possible risk for consumers of the neighbouring Lemanic
region (Switzerland) where Perca fluviatilis is a traditional dish cooked or raw, and already parasitized by
another zoonotic parasite.

Page 270
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First report of streptococcus iniae in Adriatic sturgeon (Acipenser naccarii) farmed in north
Italy

Marino Prearo1, Claudio Pedron2, Silvia Colussi1, Davide Mugetti1, Vasco Menconi1, Katia Varello1, Elena
Bozzetta1, Mattia Tomasoni1, Elisabetta Antuofermo3, Marta Polinas3, Alessandro Dondo1, Pier Luigi Acutis1,
Paolo Pastorino1

1TheVeterinary Medical Research Institute for Piemonte, Liguria and Valle d’Aosta, Italy
2DVM, Italy
3Department of Veterinary Medicine, University of Sassari, Italy

[email protected]

Streptococcus iniae has emerged as an important fish pathogen over the past few decades causing high losses
in aquaculture farms all over the world. Several fish species have been documented to be infected by S. iniae,
sturgeons (Acipenser spp.) included. This report describes for the first-time pathological findings in one
outbreaks of S. iniae infection in Adriatic sturgeon (Acipenser naccarii) farmed in north Italy.

Low mortality was recorded in a batch of A. naccarii (total lenght: 92.6 ± 4.2 cm; weight: 3860 ± 32 g). Non-
specific clinical signs were reported. Four specimens were sampled and destined to anatomopathological
examination. Kidney and brain were sampled by loop and immediately streaked onto Columbia blood Agar
(Liofilchem, Italy). Plates were incubated at 22 °C for 72 h. Obtained colonies were then sub-cultured for
phenotypic analysis. Then, molecular identification through 16S rRNA amplification and gyrB gene
sequencing was conducted. Bacteria identification was also performed by matrix-assisted laser desorption
ionization-time of flight mass spectrometry (MALDI-TOF MS) on a VITEK MS system (bioMérieux, France).
Liver, kidney, spleen and gut were collected and fixed in formalin for histological examination.

Gross pathology was characterized mainly by splenomegaly and intestinal congestion in all analyzed
specimens. Microscopically, multifocal parenchymal necrotic foci were detected in spleen and liver, with
haemorrhagic areas and vasculitis. Furthermore, gut showed severe necrotizing enteritis and gills mild
mononuclear multifocal infiltrate. Phenotypic identification revealed that isolates from brain were
nonmotile, Gram-positive cocci that displayed negative catalase and oxidase activities. MALDI-TOF MS, 16S
rRNA and gyrB gene sequencing confirmed infection of the sturgeons with S. iniae.

This is the first report of streptococcosis in Acipenser naccarii. Also, it represents the first record of S. iniae
in Italy. This diagnostic case highlights the emergence of streptococcosis for fish farms in Italy.

Page 271
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Mortalities of wild Anguilla associated with Anguillid herpesvirus 1 (AngHV-1) - evidence of
disease in all freshwater life stages of eels

John Price, Hannah Bradley, Amy Reading, Chris Williams

Environment Agency National Fisheries Laboratory, UK

[email protected]

Anguillid herpesvirus (AngHV-1) is an important viral pathogen of cultured eels, but relatively little
information exists on mortalities in wild populations. In particular, there are few reports of disease in
different life stages of eel, poor understanding of the triggers for outbreaks and some uncertainty over the
clinical and histopathological characteristics of infection. Here we present detail of 16 cases of AngHV-1
disease in wild Anguilla anguilla from wild freshwater fisheries in England spanning the last decade.

Methodology
Reports of eel mortalities were investigated as part of routine monitoring activities by the Environment
Agency. Samples were subjected to a detailed laboratory examinations involving clinical and
histopathological assessments; parasitology, bacteriology and virology screening; environmental
assessments of water quality and algal communities; morphological assessments of eel life stage and a
review of fishery management practices.

Results
All mortality events involved eel-specific losses reported between July and October with water temperatures
between 17.0 and 22.5 oC. These involved all life stages of eels including glass eels, elvers, yellow eels and
silver eels categorised morphologically up to migrating stage V. Consistent pathological changes included
lethargy, haemorrhaging of the fins and mouth, mottling of the skin and gill necrosis. Histopathological
changes were characterised by pronounced necrotic changes, haemorrhage and cellular inflammation of the
gills, kidney, skin, liver and spleen. The presence and role of parasitic, bacterial and viral co-infections are
presented. These outbreaks support the role of both environmental and anthropogenic triggers for Ang-HV1
disease, including barriers to riverine migration, water quality including algal blooms and the physiological
stressors of silvering in eels unable to escape landlocked waters.

Conclusions
These disease events detail the impact of Ang-HV1 in all freshwater life stages of eels, the potential for
outbreaks during the long and stressful life histories of these fish and importance of integrated management
approaches to aid recovery of this critically endangered species.

Page 272
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Are our fish well? Animal welfare in the midst of expectations and reality - “National Animal
Welfare Monitoring” NaTiMon

Karina Retter1, Karina Retter1, Felix Teitge1, Verena Jung-Schroers1, Vincent Lugert2, Stefan Reiser2, Dieter
Steinhagen1

1University Of Veterinary Medicine Hanover, Fish Disease Research Unit, Germany

2Thünen Institute of Fisheries Ecology, Germany

[email protected]

Animal welfare in livestock is an important and extremely controversial discussed issue. However, the
available information is very limited. In order to objectify the discussion and to gain an overview of the status
and the development of the welfare in farmed livestock the German Federal Ministry of Food and Agriculture
initiated a collaborative research project. In addition to terrestrial livestock, the project aims to create
prerequisites for a national animal welfare monitoring for the time being in rainbow trout and carp farms.
Rather than assessing an individual farm or setting new standards such a monitoring should enable a
nationwide status assessment. This could give valuable insights into welfare status and development,
uncover areas were improvement is possible and validate political measures taken.

Methodology
In a literature study potential indicators that are described to be suitable for the assessment of animal
welfare in rainbow trout and carp farms were compiled. During a variety of participation formats involving
various stakeholder groups, these indicators were evaluated with regard to validity, practicability and
reliability for a national animal welfare monitoring. Together with fish farmers and other professionals from
the aquaculture sector, veterinarians, government representatives, NGOs, and fisheries associations the
whole set of indicators was narrowed down to a selection that will be tested on a small number of rainbow
trout and carp farms. These anonymously collected data will help back up the previous assumptions and
identify the most suitable and practical indicators for a monitoring. Subsequently, the resulting list of
indicators identified as most suitable will be defined and tested on a larger scale by visiting a greater number
of farms throughout Germany.

Results and Conclusions

In the literature study about 165 animal-related, resource-related and management-related fish welfare
indicators were identified. Assisted by fish farmers and other stakeholders 65 indicators were pre-selected
and rated to be most suitable and practical. Divided into groups regarding the farm operational level, a
population level and individual fish level these 65 indicators are now being tested to verify their validity,
practicability and reliability in the field.

Page 273
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Development of a live attenuated vaccine candidate against herpesviral hematopoietic
necrosis of goldfish

Hiroaki Saito, Takumi Okamura, Mr. Tomoya Shibata, Goshi Kato, Motohiko Sano

Tokyo University of Marine Science And Technology, Japan

[email protected]

Herpesviral hematopoietic necrosis causes huge economic loss in goldfish Carassius auratus and Prussian
carp C. auratus gibelio farming. To develop a live attenuated vaccine as a preventive measure against the
disease, the causative virus, cyprinid herpesvirus 2 (CyHV-2) was attenuated to obtain vaccine candidates.

Methodology
CyHV-2 Sat-1 isolate, which was routinely propagated in goldfish cell line RyuF-2, was serially passed in CFS
cell line derived from Ginbuna C. auratus langsdorfii, which showed susceptibility in experimental infection.
The viruses passed in CFS were tested for pathogenicity in goldfish by immersion and whole genome
sequencing was performed to identify genome mutations. The virus was further passed several times in KF-
1 cell line from koi being a non-natural host. In vaccine trial, the viruses passed in CFS and KF-1 were
inoculated in goldfish by immersion and subsequently challenged with virulent Sat-1 to estimate protective
efficacy. Since reversion to virulence of live attenuated vaccine is a major concern, a candidate was passed
in RyuF-2 5 times and tested for pathogenicity in goldfish. Furthermore, as an in vivo test for virulence
reversion, a candidate was passed through goldfish by intraperitoneal injection 6 times. In the course of in
vivo passage, abnormality and mortality of the inoculated fish were observed and the virus DNA copy number
in the kidney and caudal fin were measured by qPCR.

Results
The viruses passed up to 20 times in CFS had non-synonymous changes in some genes and caused no
mortality in goldfish, but some fish showed hemorrhage on the skin. The virus passed 7 or 10 times in CFS
were further subcultured 5 or 8 times in KF-1 to generate more attenuated virus (abbreviated as P7-P5, P7-
P8, P10-P5 and P10-P8). Relative percentage survival of P7-P5, P7-P8, P10-P5 and P10-P8 were 80%, 90%,
90% and 50% in vaccine trial, respectively. P7-P8 which was selected for further reversion tests showed no
signs of virulence reversion in vitro and in vivo. Virus DNA copy number in the fish did not increase up to 6
passages. These results indicate that P7-P8 can be a promising candidate of live vaccine against the disease.

Page 274
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Protection of Atlantic lumpfish (Cyclopterus lumpus) by two autogenous vaccines against
Pasteurella sp.

Almudena Sanchez-Matamoros, Patricia Pedrola, Rosa Maria Merino

Laboratorios Hipra, S.A, Spain

[email protected]

With the increased use of lumpfish for delousing purposes, health management and disease control have
become issues of central importance on the farms. The most commonly isolated pathogenic agents are Vibrio
spp., atypical Aeromonas salmonicida and Pasteurella sp. Whilst preventive products against Vibrio and
Aeromonas are commercially available for lumpfish, there are none for pasteurellosis. Therefore, the
incidence of Pasteurella sp. outbreaks in farmed lumpsuckers in Norway and the Faroe Islands has steadily
increased in recent years, causing significant welfare issues. This study aims to evaluate the efficacy of 2
commercial autogenous vaccines for the protection of lumpfish against Pasteurella sp.

A blinded and controlled trial was conducted in the experimental facilities of the AquaBioTech Group. In
total, 225 lumpfish (13.7±3.4 g) were vaccinated with 0.05 ml of an oil-based injection vaccine (ICTHIOVAC
LUMPUS 5), containing inactivated Pasteurella sp., Pseudomonas anguilliseptica, Moritella viscosa,
Aeromonas salmonicida atypical III and Vibrio anguillarum (VG1), 0.05 ml of a monovalent oil-based injection
vaccine (AVAC PEC Pasteurella) against Pasteurella sp. (VG2) or 0.05ml of PBS (control group [CG]). The
intraperitoneal challenge was carried out 28 days after vaccination in duplicate using a virulent homologous
isolate. Vaccine efficacy was assessed based on the cumulative mortality rate and the relative percentage
survival (RPS). The post-challenge observation period lasted 21 days (dpc).

Any adverse effects after the vaccination were recorded. The results obtained after the challenge show that
the cumulative mortality rate was significantly lower in VG1 and VG2 than in CG. Mortality peak occurred
between 9 and 11 dpc, reaching a rate of 89.7 ± 4.9% in CG. However, the mortality rates in vaccinated fish
were between 45.7 ± 1.3% and 43.3 ± 4.7% in VG1 and VG2, respectively. Likewise, the RPS values obtained
were 49.0% in VG1 and 51.7% in VG2.

All in all, these results demonstrate that ICTHIOVAC LUMPUS 5 and AVAC PEC Pasteurella are effective in
conferring a degree of immunity against a virulent Pasteurella sp. isolate, showing that autogenous vaccines
could be a quick and cost-effective tool to reduce the mortality caused by Pasteurella sp. in lumpfish in the
absence of any other available preventive method.

Page 275
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Comparative analysis of ssIL8α peptide antibacterial activity in strains of Aeromonas
salmonicida from Chile and Perú

Paula Santana1, Claudio Álvarez2, Nancy Alvarado1, Nieves Sandoval3, Fanny Guzmán4

1Institutode Ciencias Químicas Aplicadas, Universidad Autónoma De Chile, Chile

2Fisiologíay Genética Marina (FIGEMA), Centro de Estudios Avanzados en Zonas Áridas (CEAZA) , Chile
3Facultad de Medicina Veterinaria, Universidad Nacional Mayor de San Marcos, Perú
4Núcleo de Biotecnología Curauma, Pontificia Universidad Católica de Valparaíso, Chile

[email protected]

The trout farming industry has positioned itself as one of the most important industrial areas in Chile and
Perú, from an economic and social point of view. However, its explosive expansion together with climatic
changes and high water pollution has facilitated the appearance of infectious diseases typical of its intensive
production. In this context, rainbow trout farming in both countries shares the recurrent presence of
pathogenic bacteria such as Aeromonas salmonicida.

The constant presence of these pathogens has forced the salmonid industry in both countries to increase the
use of antibiotics. It is therefore a necessity for this sector to study control alternatives that allow to stop the
proliferation of bacteria in farming cages and at the same time reduce the use of antibiotics. To date,
antimicrobial peptides (AMPs) derived from salmonids have been identified and characterized as a potential
alternative. These including hepcidins, cathelicidins and, more recently, peptides derived from IL-8 (ssIL-8α).
The latter have been shown to have antibacterial activity against strains that affect the Chilean salmon
farming. Therefore, in this work we evaluated the antibacterial activity of ssIL-8α peptide against A.
salmonicida strains from Chile and Perú. For this purpose, ssIL-8α was synthesized and its mode of action
against A. salmonicida was characterized by fluorescence (FM) and scanning electron microscopy (SEM).

Its antibacterial activity was evaluated at concentrations between 5 to 30 μM using microdilution plate
assay. The FM showed that ssIL-8α was accumulated on the surface of the cells and in the cytoplasm. By
SEM, bacterial cells treated with ssIL-8α showed formation of multiple bleb-like structures on the surface of
A. salmonicida. Throught antibacterial assay was observed that ssIL-8α has antibacterial activity at 20 and
30 μM against the Chilean strain; however, against the Peruvian strain, 100% activity was observed under 10
μM of peptide. This results evidencing that this peptide was capable of permeabilizing the bacterial
membranes and interact with cytoplasmic components.

Further research is necessary to elucidate the differences in the activity of the peptide against these two
strains, so to determine the potential of these peptides as a new antimicrobial agents to combat bacterial
infections.

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Genome organization of Sphaerospora molnari (Myxozoa), parasite of common carp

Anezka Santolikova1,2, Joana Pimentel-Santos1, Pimentel-Santos1, Magda Zrzava2,3, Frantisek Marec3, Astrid
Holzer1

1Institute of Parasitology, Biology Center of the Czech Academy of Sciences, Czech Republic
2Faculty of Sciences, University of South Bohemia, Czech Republic
3Institute of Entomology, Biology Center of the Czech Academy of Sciences, Czech Republic

[email protected]

Myxozoa are a parasitic group of extremely derived Cnidaria. Their life cycles consist of parasitic stages
alternating between vertebrate and invertebrate hosts, and transmitted by spores. Throughout their rapid
evolution, Myxozoa have lost most of their body tissues as well as multitudes of genes which are present in
free living Cnidaria. Although some myxozoan genomes have been already sequenced and assembled, the
knowledge of genome organization into chromosomes is noticeably lacking.

As a first step in parallel to genome sequencing, we aimed at developing protocols for the visualization of
chromosomes of the myxozoan S. molnari and target genes within them. Blood stages of S. molnari
maintained in a continuous laboratory fish-to-fish infection, were isolated from carp (Cyprinus carpio) using
ion exchange DEAE cellulose columns. Isolated parasites were consecutively treated with 0.01% colchicine
and 0.56% KCl and fixed in methanol: acetic acid (3:1). Parasite cells were then spread in 60% acetic acid on
superfrost slides. Fluorescence in situ hybridization was performed using a general metazoan telomeric
probe (TTAGGG)n and an 18S probe. Slides for detecting the 18S coding region were pretreated with RNase
A. In situ hybridization signals were visualized with Cy3-streptavidin. Slides were stained and mounted with
a medium containing DAPI. Metaphase chromosomes were observed and photographed on a confocal
microscope (Olympus FV3000). We demonstrate that S. molnari blood stages contain dividing cells, identified
as secondary cells undergoing mitotic division. We were able to arrest their development in metaphase and
to visualize chromosomes arranged in pairs.

From the pictures obtained so far, we conclude that S. molnari has seven chromosome pairs (2n=14), in
contrast to Ceratonova shasta, which has only three (2n=6). The chromosomes of S. molnari are very small
(approximately 0.6 µm) and similar in length. They are probably metacentric and submetacentric, although
it is difficult to distinguish the centromere position. We optimized fluorescence in situ hybridization protocols
which is essential for studies aiming at localizing specific genes. Our results are useful for more detailed
studies of S. molnari genetic information as well as for comparison with other species of Myxozoa and
Cnidaria.

Page 277
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Ectoparasites from Gilthead seabream, Sparus aurata, and European seabass,
Dicentrarchus labrax and their microhabitat preference

Ana Isabel Azevedo1,2, Luis Filipe Rangel1, Ricardo Severino1, Maria João Santos1,3

1Porto University - CIIMAR, Portugal

2Aveiro University, Portugal
3Porto University - FCUP, Portugal

[email protected]

The present work analyse the specific distribution of ectoparasites in cultured gilthead seabream and
European seabass. Monogeneans Diplectanum aequans, Sparicotyle chrysophrii and Furnestinia echeneis,
and the copepod Caligus minimus already reported in previous works and also found in our study, have been
frequently associated to fish mortalities, decreased growth rates and overall restricting aquaculture
productivity. Moreover, the interspecific relationships between parasite species were also tested, as well as
host-parasite parameters, such as weight/length in correlation with infection levels.

A total of 52 gilthead seabream and 20 European seabass came from a fish farm in Ria de Alvor in the South
of Portugal. Their gills were examined for parasites, and 48 potential infection sites were assigned: the four
gill arches on each side of the fish (left or right), each divided into three segments (distal, medial, proximal),
as well as anterior and posterior sides (hemibranchs). The data was statistically analyzed to test whether
each parasite species had a preference for any specific site within the gills, or to test interspecific or host-
parasite relationships.

Results showed high prevalence and abundance values for all parasite species (>94%) except S. chrysophrii
(40%), and that all 4 species generally prefer the anterior hemibranch. However, only L. echeneneis and D.
aequans showed a statistically significant preference for the medial segment of the branchial arch, with only
D. aequans significantly preferring a specific holobranch (the first, followed by the second holobranch).
Higher volumes of water pass through the first and second holobranchs than over the third and fourth, which
may explain why more parasites are commonly found in the first and second holobranchs. In terms of host
parasite-parameters, parasites were found in higher abundance in larger fish, probably because they provide
a larger surface area for parasite attachment or because they have been exposed to parasites for longer. No
interspecific relationship between species was found.

Page 278
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Investigations of thermal tolerances of bivalve species

Mohammad Nasif Sarowar1, Tiago Hori2, Mark Braceland1

1The Center For Aquaculture Technologies, Canada

2Atlantic Aqua Farms Ltd, Canada

[email protected]

Bivalve aquaculture is a continuously expanding industry, with global increases in tonnage of multiple species
in the last 20 years. Culture of bivalve species is highly sustainable and has a comparatively low carbon
footprint (lb/lb) to other livestock industries. However, our understanding of optimal growth conditions, and
effects of environmental stressors is limited. For instance, mortalities associated with warm water
conditions, and empirical data demonstrating such are and projected to increase in severity there is
significant interest in the industries sectors to understand means to mitigate risks to stocks.

Methodology
In this study, blue mussel (M. edulis), Eastern oyster (C. virginica), and Pacific oyster (M. gigas) from multiple
geographical locations were subjected to temperature variations acutely and chronically. Mortalities were
tracked in oyster and mussel stocks to determine risk thresholds. In addition, byssal thread strength was
used as a measure of robustness (and indication of when fall off may occur).

Results and Conclusions

Significant differences between thermal tolerances of the three species were identified in the study. In
addition, origin of spat within a species had an impact on these tolerances. Interestingly, a clear impact on
byssal thread strength was also demonstrated when temperature was increased, and a temperature
threshold determined for when knock off may increase and thus management practices should be altered.
Furthermore, a high degree of heterogeneity of thermal tolerance in all species and stock origins highlight
an opportunity to increase thermal scope of stocks through breeding. Further work on molecular and
genotype markers of increased thermal tolerance will be an important step in the advancement of the
industry.

Page 279
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Lethal infection of Atlantic puffins (Fratercula arctica) by Diplostomum baeri, transmitted
by feeding infected European minnows (Phoxinus phoxinus)

Heike Schmidt-Posthaus1, Inês Berenguer Veiga2, Leonore Küchler2, Regula Hirschi1, Stefan Hoby3

1Centre For Fish and Wildlife Health, Switzerland

2Institute
of Animal Pathology, Switzerland
3Berne Animal Park, Switzerland

[email protected]

Conservation of endangered animals is one major task of zoological institutions. Husbandry and breeding of
Atlantic puffins (Fratercula arctica) in captivity is difficult, and only few zoos worldwide keep it successfully.
In 2019, all puffin offspring of the puffin colony from the Berne Animal Park, with ages ranging between 5
and 45 days of age, deceased within 7 days' time. These chicks were fed with wild-caught European minnows
(Phoxinus phoxinus) starting from the second day of life. The dead puffin chicks were sent to the Institute of
Animal Pathology, University of Bern and the minnows were sent to the Centre for Fish and Wildlife Health,
University of Bern, for pathological and microbiological analyses.

Main pathological finding in the deceased puffin chicks was a multifocal, heterophilic and granulomatous
enteritis with intralesional adult trematodes and eggs, while metacercariae surrounded by few necrotic cells
and scattered macrophages were found in the brain and spinal cord of the minnows. Microbiological analysis
of both the puffin chicks and fish was unremarkable. Diplostomum baeri DNA could be identified from
formalin fixed paraffin embedded tissue from all the puffin chicks and from the minnows following PCR and
sequencing. This report illustrates the importance of a previous intensive health check of the feeding fish,
especially in valuable and rare animals kept in captivity.

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First detection of Aeromonas salmonicida from Liza carinata in Egypt

Mohamed Shaalan1,3, Asmaa K Al-Mokaddem1, Dalia A Abdel-Moneam1, Reham A Ibrahim2, Mona Saleh3

1Facultyof Veterinary Medicine, Cairo University, Egypt

2National Institute of Oceanography and Fisheries (NIOF) , Egypt
3University of Veterinary Medicine Vienna, Austria

[email protected]

Aeromonas salmonicida is an important fish pathogen, which was first reported in salmonid fish. Later, it was
isolated from different fish species. The bacteria is incriminated in septicemic diseases and high mortalities
in affected fish.

Methodology
Fish samples were cultured on blood agar plates and the isolates were identified by biochemical, molecular
and histopathological methods. Biochemical identification of the isolates was conducted using API 20 NE kits
and the molecular identification was performed using universal primer for 16S rRNA gene of Aeromonas
species. Moreover, the virulent genes of aerolysin-like protein (act) and serine protease (ser) were detected
in the isolates.

Results
In this study, A. salmonicida was isolated from Liza carinata. Culturing on blood agar produced beta hemolysis
and API NE test revealed the identity of A. salmonicida. Universal 16S rRNA gene of Aeromonas species, (act)
and (ser) genes were detected (amplicons lengths at 461, 232, and 211 bp, respectively). The sequencing
analysis of isolated bacteria showed high identity with A. salmonicida listed in the GenBank database.
Histopathological examination showed the presence of bacterial colonies in gills, kidney and liver. In kidney,
the renal tubular epithelium was necrosed, while liver showed congestion of hepatic veins and necrosis of
hepatocytes.

Conclusions
To the best of our knowledge, this is the first report of A. salmonicida isolation from Mugilidae.

Page 281
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Emerging monogenean infections in farmed meagre Argyrosomus regius (Asso, 1801)

Perla Tedesco1, Matko Kolega2, Slavica Čolak2, Andrea Gustinelli1, Francesco Quaglio3, Danijel Mejdandžić2,
Vicko Baranović2, Monica Caffara1, Renata Baric2, Maria Letizia Fioravanti1

1University Of Bologna, Italy

2CROMARIS d.d., Croatia
3University of Padova, Italy

[email protected]

Monogenean outbreaks threaten the health of farmed fish stocks and severely affect aquaculture
production. The meagre Argyrosomus regius is a promising species for diversification in Mediterranean fish
farming: in the wild, this species is known to host a variety of ectoparasites, however reports of infection
and pathology in farmed fish are rather infrequent. The present investigation reports the occurrence of an
outbreak of monogenean infection in A. regius broodstock, with identification of the parasite species
involved and associated histopathological findings.

Methodology
During a mortality outbreak registered in a meagre broodstock batch, parasites were isolated from gills and
skin and preserved in 70% ethanol and 10% buffered formalin. Gill samples were also fixed in 10% buffered
formalin for histology. The parasites were subjected to morphological analysis in light and scanning electron
microscopy, and to molecular analysis amplifying and sequencing the complete ITS and partial 28S rDNA.

Results and Conclusions

Two monogenean species were identified, based on their morphology: the calceostomatid Ktariella
polyorchis, reported for the first time in farmed A. regius, and the diplectanid Diplectanum sciaenae, already
reported from A. regius broodstock in Spain. K. polyorchis were mainly found on the skin, mainly along the
dorsal part of the body and tail, and in the oral cavity, while D. sciaenae were recovered from the gills. At
histological level, the presence of D. scienae was associated to severe gill damage with diffuse epithelial
hyperplasia and sloughing off in the lamellae, focal necrosis, inflammatory infiltration, hyperemia,
telangiectasias and hemorrhages. Histological lesions were similar to those observed in previous reports and
confirmed D. scienae as health-threatening parasite of meagre.

Our findings suggest a role of both K. polyorchis and D. sciaenae as pathogenic parasites of A. regius farmed
in the Mediterranean area, although at the moment they have been reported only from broodstock. In
addition, we provide the first sequence data for these two monogenean species, as a further diagnostic tool
for their correct taxonomical identification in possible future outbreaks.

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Fighting the parasitic challenge in modern fish farms: vacination against Ichthyopthirius
multifiliis

Felix Teitge1, Verena Jung-Schroers1, Christina Loy2, Dennis Kallert2, Marcus Zilasko3, Gregor Schmidt3,
Helmut Wedekind3, Dieter Steinhagen1

1UniversityOf Veterinary Medicine Hannover, Fishdisease Research Unit, Germany

2Kallert& Loy GbR, Germany
3Bavarian State Research Center for Agriculture – Institute of Fisheries, Germany

[email protected]

Combating parasitic infections, and especially Ichthyophthirius multifiliis, is a major challenge for European
aquaculture. In trout, where warmer water temperatures and lower water availability weakens the fish,
infections can lead to substantial losses. Due to drug legislation, treatment in farms is merely impossible, as
of today environmental disinfection is the last resort in battling infections with Ichthyophthirius multifiliis.
Besides the economic impact, the lack of an effective treatment against Ichthyophthirius multifiliis poses an
ethical dilemma. In this study, amongst other approaches, vaccination protocols against the parasite were
tested to control parasitic infections and to produce healthy, ecological sustainable fish.

Methodology
As fish develop immunity following infection with Ichthyophthirius multifiliis, resulting in both specific and
nonspecific immune responses, different preparations of Ichthyophthirius multifiliis were used in vaccination
trials. On top of that preparations of the ciliate Tetrahymena sp. were tested for their ability to induce cross-
immunity. In addition to living theronts, formalin inactivated theronts and isolated surface antigen of
Ichthyophthirius multifiliis were used as well as surface antigen preparations of Tetrahymena sp. The living
parasite stages were administered intraperitoneally while the other preparations were administered via a
bath treatment. In case of the fish vaccinated by bath treatment, it was examined whether the immunity
would be stronger after enhancing the antigen intake by inducing micro-lesions in the skin with fine needles
or by the use of ultrasound to irritate the mucosa directly bevor vaccinating compared to a simple bath
vaccination. After a period of 20 days (360 day-degrees) in which the immunity could build up, the fish were
challenged with theronts harvested in a lab cycle. The parasitic burden of the fish was evaluated after 7 day
post infection.

Results and Conclusions

A cross-immunity of Tetrahymena and Ichthyophthirius multifiliis was not sufficient to protect the fish
whereas specific preparation and application methods with Ichthyophthirius multifiliis had a positive impact.

Vaccination can be part of the solution. However, a major challenge remains unresolved: The fast, easy,
cheap and controlled production of large numbers of theronts in the laboratory for the preparation of
vaccines remains difficult and needs to be investigated further.

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lluminating the planktonic stages of salmon lice: a novel method using a unique
fluorescence signal for overcoming the needle in the haystack problem and enumeration a
rare copepod in zooplankton assemblages

Cameron Thompson1, James Bron2, Samantha Bui3, Sussie Dalvin1, Mark John Fordyce5, Gunnvør á Norði4,
Rasmus Skern-Mauritzen1

1Institute Of Marine Research, Norway

2University of Stirling, Scotland
3Institute of Marine Research, Norway
4Fiskaaling - Aquaculture Research Station of the Faroes, Faroe Islands
5Marine Scotland Science, Scotland

[email protected]

The salmon louse, Lepeophtheirus salmonis (Krøyer 1837) is an obligate ectoparasite of salmonids and the
principal environmental challenge constraining Atlantic salmon (Salmo salar) aquaculture. Prior to
attachment to its host the louse develops through several free-living larval stages that are relatively rare in
comparison to other copepods in the water column. In a typical mixed zooplankton sample, it may take a
worker over 4 hours to sort through 50,000 non-target copepods before finding a single planktonic salmon
louse. Thus, monitoring of planktonic salmon louse abundance and parameterization of key life history traits
has been hindered by labor intensive and error prone quantification using traditional light microscopy.

Addressing that obstacle, we have developed a novel method for the rapid enumeration of planktonic salmon
lice using a unique fluorescence signal. We investigated the fluorescence profiles of L. salmonis and non-
target copepods with Excitation Emission Matrices (200 - 600 nm) and fluorescence microscopy. Using
excitation wavelengths of 470 ± 40 nm and emission wavelengths of 525 ± 50 nm, we showed that after 90
days of formalin storage salmon lice have a mean fluorescence intensity that is 2.4 times greater than non-
target copepods. A 7-day heat treatment of 42° C in formalin increased the difference between salmon louse
copepodids and non-target copepods to a factor of 3.6, eliminating the need for prolonged storage.
Differences in the fluorescence signal and endogenous fluorophores were investigated and found to be
reliable with respect to variation in sea lice species, age, stage, and host fish origin. A ring test was then
conducted by taking standard zooplankton samples from multiple sites and times, spiking them with a known
number of lice, and having multiple participants enumerated them in blind trials using the fluorescence aided
method.

It typically took participants less than 30 minutes to process a sample with a mean accuracy of 74.4% to
82.1% depending on who was counting. In its current form, the method using fluorescence aided microscopy
is a reliable means of rapidly enumerating salmon louse larvae in plankton samples. Nevertheless, there are
multiple ways in which it could be improved with the end goal being automated detection.

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Flow cytometry-based analysis of blood leukocyte populations, for detection of
inflammatory processes in barramundi

Jaison Titus1,2, Sagar Nayak1, Benyamin Rosental2, Dina Zilberg1

1The French Associates Institute for Agriculture and Biotechnology for Drylands, The Jacob Blaustein
Institutes for Desert Research, Ben-Gurion University of the Negev, Israel
2The Shraga Segal, Department of Microbiology, Immunology, and Genetics, Faculty of Health Sciences,

Regenerative Medicine and Stem Cell Research Center, Ben-Gurion University of the Negev, Israel

[email protected]

Bacterial infections are a major contributor to losses in aquaculture production. Diagnosis of these bacterial
diseases is based on sacrificing fish and isolating the infectious agent from the internal organs. Infections
lead to an inflammatory response in the host that is characterized by changes in the profile and activity of
leukocytes. We hypothesize that the profile of circulating peripheral blood leukocytes and markers of their
activity, such as ROS production and lysosomal activity would be subjected to change, following an event of
bacterial infection in fish.

The research included the development of a flow cytometry-based methodology for analyzing blood
leukocytes in barramundi (Lates calcarifer), followed by a comparison of healthy and infected fish with a
common bacterial disease, Vibrio harveyi. The initial step of the analysis relies on the effective separation of
erythrocytes from leukocytes. Taking this into account, two methods were tested: 1) based on hypotonic
lysis of RBCs, and 2) a stain-based differentiation using Carboxyfluorescein succinimidyl ester (CFSE).
Significant differences were observed between fish infected with an LD50 dose and control fish. Changes in
blood leukocytes included an increase in the proportion of monocytes, while lymphocytes decreased in
infected fish. Furthermore, cell size and granularity of the different leukocyte populations changed over time
in infected fish as compared to controls. Infection with a lower dose, 1:3 of LD50, resulted in a change in the
observed differences between the control and infected fish when compared to the LD50 responses.
Differences, in this case, included a larger granulocyte size in the infected group along with elevated
expression of ROS in all leukocyte’s populations.

In this study, leukocyte isolation based on CFSE staining has been found to be an optimal approach for
analyzing inflammatory responses with flow cytometry. Non-lethal analysis of inflammation allows to
conduct repeated sampling of each fish over time, which is beneficial given the large variability among
individual fish.

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Case of Mycobacterium marinum infection in European seabass (Dicentrarchus labrax),
with emphasis on the possible entry routes of the pathogen

Mattia Tomasoni, Davide Mugetti, Katia Varello, Paolo Pastorino, Vasco Menconi, Alessandro Dondo, Elena
Bozzetta, Marino Prearo

The Veterinay Medical Research Institute for Piemonte, Liguria and Valle d'Aosta, Italy

[email protected]

Mycobacterium marinum is the best known alcohol-acid resistant bacillus in the onset of fish
mycobacteriosis. The main clinical signs of the pathology are skin erosions and granulomas at the level of
parenchymatous organs. M. marinum is reported in several farmed marine species, including European
seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata). The most connected problems to
mycobacteriosis in fish farms are the appearance of symptoms after long periods, the difficulty of eradicatig
the bacterium from surfaces and the lack of antibiotic treatments. It is therefore important to promptly
identify the possible sources of infection to limit the losses associated with the disease.

Following this approach, a farm in North-eastern Italy send to our laboratory batches of European seabass
showing skin erosions, emaciation and nodules in the internal organs. The fish were subjected to necropsy,
parasitological, cultural and virological examinations, at the end of which the cause of the losses was
attributed to M. marinum. To understand the possible source of entry of the mycobacterium, the new
batches of fish entering, plus water and tanks sediment were analyzed by coltural and molecular methods.

The analyzes carried out made it possible to highlight the presence of M. marinum in the sediment of the
tanks. Conversely, the new purchased fish and the waters were negative. Molecular analysis by PCR and
Sanger sequencing of the 65 KDa heat shock protein gene allowed to highlight a 100% identity with the
strains previously isolated in the same farm.

This study illustrates a comprehensive approach for the identification of a pathogen and its possible entry
routes into a marine fish farm. Although it has been possible to identify a possible source of accumulation of
M. marinum in the sediment, it is not possible to establish whether this is the matrix that caused the infection
or the fish were already infected, thus contaminating the environment. For a disease such as fish
mycobacteriosis, in which there are no treatments currently available, it is essential to adopt an approach
that considers all possible sources of infection, combining good zootechnical hygiene practices and constant
health survey for the disease management.

Page 286
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Detection of crayfish plague Aphanomyces astaci genotype D in northern noble crayfish

Satu Viljamaa-dirks, Sirpa Heinikainen, Tiina Korkea-aho

Finnish Food Authority, Finland

[email protected]

Crayfish plague is a destructive disease of European endemic freshwater crayfish including noble crayfish
(Astacus astacus). However, the original host species from North America can carry the plague with little
consequences. In the last decades, the crayfish plague epizooties in Finland have been studied in detail and
were caused by the genotypes As (A) or Ps1 (B).

Genotype As was only connected with noble crayfish. Genotype Ps1 caused acute disease episodes in noble
crayfish but was also found regularly from the invasive species signal crayfish (Pacifastacus leniusculus).
Signal crayfish have replaced the noble crayfish in all the main waterways in southern Finland. Additionally,
a few reports of the Chinese mitten crab (Eriocheir sinensis) are made mainly from the brackish water coastal
area of the Baltic Sea. Chinese mitten crabs are able to carry the crayfish plague agent, but their disease
status in Finland has not been studied and little is known of their interaction with the different A. astaci
genotypes. Kemijoki river in the North suffered the main mortality event in the productive populations of
noble crayfish in the years 2010-2011 due to the crayfish plague genotype As.

Only occasional crayfish have been encountered in the river since. However, in the river mouth under the
first power plant dam, a small population survived until 2020, when crayfish plague struck again.

A dozen crayfish was caught accidentally in fish nets and delivered to the laboratory, all dead or moribund
on arrival. Surprisingly, A. astaci genotype Pc (D) was detected. Genotype D has been associated with the
Louisiana red swamp crayfish (Procambarus clarkii). It has been found in southern European countries,
mainly in Spain with wild populations of red swamp crayfish, never in northern Europe. Another reservoir of
this genotype is the aquarium trade while many of the colorful Procambarus species are held as pets. Possible
infection routes and follow up of the affected noble crayfish population in 2021 are discussed.

Page 287
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Looking for cockle Cerastoderma edule protein markers of resistance against marteiliosis

Antonio Villalba, Asunción Cao, David Iglesias, María J Carballal

Centro de Investigacións Mariñas (CIMA), Spain

[email protected]

The infection with the protistan Marteilia cochillia causes huge cockle Cerastoderma edule mortality in the
southern rias of Galicia (NW Spain) since 2012. Stating ways to minimise losses due to marteiliosis became
peremptory; the production of marteiliosis-resistant cockle strains through selective breeding was
considered a promising strategy. In this context, the search for cockle molecular markers of marteiliosis
resistance was addressed through proteomic and genomic/genetic approaches. The proteomic approach is
reported in this poster.

The experimental design involved comparing the proteome of cockles before being exposed in the field to a
marteiliosis outbreak with the proteome of survivors after the outbreak, assuming that some of the proteins
differentially expressed in the survivors could be crucial to survive under marteiliosis pressure. The shellfish
bed of Lombos do Ulla (ria of Arousa) was chosen for the field work; previous studies had shown that
marteiliosis outbreaks started there every summer affecting the newly recruited cockles and resulting in
death of almost the whole recruited cohort by the next autumn or winter. Forty five newly recruited cockles
were collected from that bed in July 2018 and their soft tissues were processed for proteomic analysis.
Additionally, the marteiliosis dynamics in the recruited cohort was monitored by estimating monthly the
prevalence and intensity of marteiliosis and the mortality rate. In July 2019, 45 surviving cockles of that
cohort were collected from the bed, a half of their meat was processed for proteomic analysis while the
other half was analysed to confirm the absence of marteiliosis. Proteomic profiles of the cockles collected
before and after the outbreak were compared using a shotgun approach through liquid chromatography
coupled to mass spectrometry. Qualitative comparison allowed the identification of 93 proteins that were
found exclusively before the outbreak, 101 proteins exclusively found in survivors and 271 proteins in both
situations. Quantitative comparison allowed the identification of 45 proteins that were significantly down-
regulated and eight significantly up-regulated in the surviving cockles. The eight significantly up-regulated
proteins in the survivors have been selected as candidate markers of resistance to marteiliosis; they need to
be validated as true markers of marteiliosis resistance.

Page 288
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Field experience with vaccination for control of PD caused by SAV2 in Mid-Norway

Aoife Westgård2, Erika Kunickiene2, Paul J Midtlyng1

1Norwegian University of Life Sciences, Faculty of Veterinary Medicine, Norway

2Aqua Kompetanse AS, Norway

[email protected]

Since 2017, SAV-2 infection among sea farmed Atlantic salmon has been present in the Northern coastal area
of Trøndelag county, Mid-Norway, from which the data from this study originate.

Mandatory measures to limit spread of the infection towards the north have been monthly targeted
surveillance of fish from each site by quantitative RT-PCR, and mandatory vaccination of all smolts
transferred to sea. Using data from surveillance activities carried out by a medium-size salmon farming
company, supplemented by public information on suspicion or detection of SAV genome and clinically
apparent PD, we describe the field epidemiology of PD in in the regiont, suggesting ting that the spread has
been successfully halted, and that territorial clearance of the infection may be within reach.

The control program incurs substantial costs for the regional farmers, for the benefit of those operating
further north. Our field data further suggest that the use of some PD vaccines in some (but highly variable)
cases has led to increased downgrading at harvest. Further details from the field data, and examples on
benefit-cost assessment of the applied tactics of controlling SAV2 infection will be presented.

Page 289
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Detection of an acanthocephalan parasite from the genus Pomphorhynchus in Symphodus
tinca (Linnaeus, 1758) in Turkey

Eda Yardımcı1, Emre Turgay1, Terje Marken Steinum2, Süheyla Karataş1

1Istanbul University, Faculty of Aquatic Sciences, Department of Aquaculture and Fish Diseases, Turkey
2Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, Turkey

[email protected]

The East Atlantic peacock wrasse (Symphodus tinca) is native to coastal regions of Spain, Morocco, the
Mediterranean and the Black Sea. In contrast to cultured fish species, the knowledge about parasites of this
wrasse is very limited. Individuals (n=57) were collected using a gillnet from the north Aegean Sea over a
one-year period starting in October of 2017. The wrasses were transported to the laboratory in fiberglass
tanks and kept alive until parasitological examination.

Tissue samples were fixed in hot formalin (4%) to preserve morphometric properties and in buffered formalin
(10%) for histopathology examination but also in RNAlater for nucleic acid extraction and molecular
identification of detected parasitic worms by PCR. For the molecular identification, both a partial 18S rRNA
gene amplicon and the amplified internal transcribed spacer (ITS) region were sequenced.

Based on obtained sequence data, the observed worms could be identified as acanthocephalans belonging
to the genus Pomphorhynchus. They are intestinal parasites of several marine and freshwater fishes that
penetrate the intestinal wall and cause major damage to the digestive system. Histopathological examination
showed that the parasites were covered by a cuticle layer (cyst) and revealed varying degrees of
inflammation and damage to surrounding intestinal tissues. Our results suggest a seasonal variation in the
prevalence of parasitic worms ranging from 13% in summer to 85% in winter. This is the first report on
acanthocephalan infection in Symphodus tinca from Turkey.

Page 290
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Metazoan parasites found in combers (Serranus cabrilla (Linnaeus, 1758)) caught from the
North Aegean Sea in Turkey

Eda Yardımcı1, Emre Turgay1, Terje Marken Steinum2, Süheyla Karataş1,

1Istanbul University, Faculty of Aquatic Sciences, Department of Aquaculture and Fish Diseases, Turkey
2Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, Turkey

[email protected]

The comber (Serranus cabrilla) is a demersal fish that is native to the Mediterranean, the Red Sea, the Black
Sea and the eastern Atlantic Ocean with a habitat related to seagrass meadows and rocky bottoms down to
500 m. This study surveys metazoan parasites of the comber caught from the northern Aegean Sea in Turkey
over a one-year period starting from October 2017. Combers (n=52) were caught using gillnets, transported
to the laboratory in fiberglass tanks and kept alive until parasitological examination. During the dissection,
the body cavity, all internal organs, gills, eyes, skin, and fins were examined.

The observed parasites were fixed in hot formalin (4%) for determination of the morphometric
characteristics and examined further under the light microscopy. Two species of digenetic trematodes
(Ectenurus sp. and Helicometra fasciata), two species of nematodes (Hysterothylacium fabri and Philometra
serranellicabrillae), Ceratothoa oestroides and praniza larvae from Isopoda were observed. Our results
suggest that the variety of parasites and their total prevalence (≥47%) increases in autumn and in spring.

Page 291
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Susceptibilities of three kinds of hybrids between crucian carp and common carp, Carassius
cuvieri x Cyprinus carpio, Carassius buergeri grandoculis x Cyprinus carpio and Carassius
buergeri subsp.1 x Cyprinus carpio to cyprinid herpesvirus 3 (CyHV-3)

Kei Yuasa, Takafumi Ito

Fisheries Technology Institute, Japan Fisheries Research And Education Agency, Japan

[email protected]

Susceptible host species to cyprinid herpesvirus 3 (CyHV-3), commonly known as koi herpesvirus (KHV) are
common carp and its varieties, subspecies and hybrids (OIE), however, there are only a few papers showing
experimental data for susceptibilities of the hybrids to KHV. In this presentation, susceptibilities of three
kinds of hybrids between common carp, Cyprinus carpio and three species/subspecies of crucian carp,
Carassius cuvieri (Cc), Carassius buergeri grandoculis (Cbg) and Carassius buergeri subsp.1 (Cbs) to KHV were
evaluated with experimental infection.

Three kinds of hybrids (40 or 30 fish in each hybrid) were exposed to a dilution of KHV (10⁵.⁸ TICD50/10 L)
for an hour and reared in aquarium supplied by the free-flowing groundwater at 20 °C for 44 days. At 14 and
44 days post viral exposure (dpe), a part of the gills (approximately 20 mg in wet tissue weight) of 10 fish in
each hybrid was excised using anatomical scissors as biopsy sample and used for quantitative analysis of KHV
by a real-time PCR. Additionally, each 10 naïve koi carp fingerlings were cohabitated with 3 kinds of hybrids
between 2 dpe and 28 dpe to observe viral transmission from infected hybrids to healthy carp by detecting
KHV DNA from carp with the real-time PCR.

Cumulative mortalities of Cc, Cbg and CBs hybrids were 60, 0 and 0%, respectively. The gills of dead Cc hybrid
included 5.45x10³ to 3.81x10⁵ copies/20 mg gill tissues in KHV DNA number. KHV was detected from 3 and 4
fish in Cc hybrid, 4 and 3 fish in Cbg hybrid, and 1 and 0 fish in Cbs, at 14 and 44 dpe, respectively. Numbers
of KHV DNA copies/20 mg gill tissues in carp cohabited with Cg, Cbg and Cbs were 9.15x10⁷ to 1.66x10⁸,
2.44x10³ to 1.35x10⁴ and 0, respectively. These results indicate that Cc hybrids showed higher sensitivity to
KHV than other two hybrids. More importantly, infected Cbg hybrid transmitted the virus to naïve carp
without any mortality and clinical sign.

In conclusion, hybrid between carp and crucian carp are sensitive to KHV or can be carrier of the virus.

Page 292
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First report of bacteriophages against Weissella ceti isolates from rainbow trout in Peru

Jefferson Yunis Aguinaga, Violeta Flores-Dominick

Imarpe, Perú

[email protected]

Lytic bacteriophages against Weissella ceti, designated WC1, were isolated from infected rainbow trouts’
internal organs.

W. ceti is a recently emergent pathogen reported in Peru that causes up to 80% mortality in rainbow trout
farms in Puno, Peru and in intensive culture around the world.

In order to evaluate if the bacteriophage WC1 was viable, physical and chemical parameters were set to
evaluate its thermal stability, different pH ranges, sensitivity to chloroform and one-step growth curve.

The WC1 phage showed higher titers at pH 5 to pH 8. It also showed high titers at temperatures ranging from
15 to 20 °C, but its viability decreased at 40 °C and in pH <5. Exposure to chloroform reduced WC1 phage
viability by 15%. The one-step curve showed a 60 minute latency period and the burst size was 150 PFU / per
infective center. These characteristics show WC1’s potential use for phage therapy. This is the first research
in Peru of a lytic bacteriophage from the emerging pathogen W. ceti.

Page 293
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First report of Weissella ceti associated with mortalities in farmed rainbow trout
(Oncorhynchus mykiss) in Peru

Jefferson Yunis Aguinaga, Marco Medina, Carla Fernandez, Giovanna Sotil, Jefferson Yunis Aguinaga, Violeta
Flores Dominick

Imarpe, Perú

[email protected]

Mortalities of up to 60% farmed rainbow trout (Oncorhynchus mykiss) were recorded in six floating cages
farms in the Lake Titicaca during the wet season (December to April) of 2018. Fish presented uni or bilateral
exophthalmia, ocular hemorrhages and melanosis. Necropsy showed severe hemorrhage in swim bladder,
brain, liver and muscle, splenomegaly, and gut congestion. Histology revealed retrobulbar hemorrhage with
macrophage infiltration, multifocal necrosis in several tissues, meningitis, splenitis, epicarditis, and
congestion. After microbiological culture in blood agar, flat and circular colonies with smooth edges and a
zone of α-hemolysis were observed. The presence of Weissella ceti was confirmed through biochemical and
molecular analysis (16S rRNA gene sequence analysis), without differences between Peruvian isolates.
Worldwide, W. ceti isolates from rainbow trout were similar using 16S rRNA gene sequence, however,
showed discrepancies between those isolates from different hosts. Finally, Koch´s postulate was confirmed
with experimental infections using rainbow trouts (~9 cm) infected intraperitoneally (0.1 mL of 104 CFU).
After 21 days, clinical signs and lesions similar to the recorded in natural outbreaks were observed,
cumulative mortality rate was 90% and bacteria was successfully recovered from both moribund and survivor
fish. This is the first report and description of weissellosis caused by Weissella ceti isolated from rainbow
trout cultured in Lake Titicaca, Peru and it is the first report of the primary isolation of this bacterium on TSA
and TYES media instead of TSAB medium.

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Isolation of Aeromonas spp. from Amazonian fish cultured in Peru

Jefferson Yunis Aguinaga1, Marco Medina1, Jefferson Yunis Aguinaga1, Giovanna Sotil Caycho1, Gino
Rengifo2, Mery Vazques2, Germán Murriera Morey2, Carla Fernández Espinel1, Violeta Flores Dominick1

1Instituto Del Mar Del Perú, Perú

2Instituto de Investigaciones de la Amazonía Peruana, Perú

[email protected]

Aeromonas hydrophila, A. jansei, among other species have been shown to be pathogenic to Amazonian fish.
The bacterial genus is widely distributed and there is currently concern about its capacity to cause important
losses in aquaculture environments in tropical areas. Therefore, the aim was to isolate and identify bacteria
of the genus Aeromonas from captive Amazonian fish with symptoms suggestive of infectious diseases. In a
sample taken in the region Loreto, Peru, captive fish, cultured in aquariums and fish ponds, were collected.

The species collected were: Colossoma macropomum (gamitana), Arapaima gigas (paiche), Brycon sp.
(sabalo), Otocynclus sp. (otocynclus), Myleus schomburgkii (black band), Trachelyopterus galeatus (bride),
Apistogram sp. (apistogram) and Calophysus macropterus (mota). Aseptically, the internal organs were
collected, homogenizer, and streaked on base agar selective for Pseudomonads and Aeromonads (GSP)
supplemented with 100′000 IU/l Penicillin G, tryptone soybean (TSA) and TSA supplemented with
defibrinated ram blood (TSAB). Plates were incubated at 28 °C for 24 - 48 h. Yellow colonies surrounded by
yellow zones in GSP agar were selected for further analysis. Fourteen isolates were selected were Gram-
negative, catalase, and oxidase positive.

Selected colonies showed to be beta and gamma hemolytic on TSAB. Commercial API 20 E and conventional
biochemical tests were used to identify bacterial isolates. The API 20 E profiles identified all 14 isolates within
the Aeromonas hydrophila/caviae/sobria complex, however, with different percent of identification
(between 85 and 99%). Molecular analysis using the 16 S rRNA gene sequences also confirmed that the
fourteen bacterial isolates belong to the genus Aeromonas sp. Therefore, bacteria of the genus Aeromonas
are possibly involved in infectious processes in Amazonian fish in captivity in the Loreto region, Peru.
Pathogenicity studies to satisfy Koch's postulates will be important to determine their potential to cause
mortalities in species of aquaculture importance.

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