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Contents lists available at ScienceDirect

Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

Identification of novel Cryptosporidium species in aquarium fish

N. Zanguee a,b , J.A. Lymbery b , J. Lau c , A. Suzuki c , R. Yang c , J. Ng c , U. Ryan c,∗
a
Department of Fisheries, Faculty of Marine Natural Resources, Khoramshahr Marine Science and Technology, Khoramshahr, Khouzestan, Iran
b
Fish Health Unit, Centre for Fish and Fisheries Research, Murdoch University, Murdoch, Western Australia 6150, Australia
c
Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia

a r t i c l e i n f o a b s t r a c t

Article history: Little is known about the prevalence and genotypes of Cryptosporidium in fish. The present
Received 1 June 2010 study investigated the prevalence of Cryptosporidium species in 200 aquarium fish of 39
Received in revised form 29 July 2010
different species in Western Australia by PCR amplification at the 18S rRNA locus. A total
Accepted 10 August 2010
of 21 positives were detected by PCR (10.5% prevalence) from 13 different species of fish.
Nineteen of these isolates were successfully sequenced. Of these, 12 were similar or iden-
Keywords:
tical to previously described species/genotypes of Cryptosporidium, while the remaining
Cryptosporidium
seven isolates appeared to represent three novel species.
Ornamental fish
18S rRNA © 2010 Published by Elsevier B.V.
New species

1. Introduction guppy (Poecilia reticulate) (hereafter referred to as piscine

genotype 1) (Ryan et al., 2004), a freshwater angelfish
Currently little is known about the epidemiology, tax- (Pterophyllum scalare) (hereafter referred to piscine geno-
onomy, pathology and host specificity of Cryptosporidium type 2) (Murphy et al., 2009) and more recently C. parvum,
species infecting piscine hosts. There are two recog- C. xiaoi and pig genotype II were identified in whiting (Sil-
nised species of Cryptosporidium in fish: Cryptosporidium lago vittata) (Reid et al., 2010) and a novel Cryptosporidium
molnari in gilthead sea bream (Sparus aurata) and Euro- spp. was identified in sea mullet (Mugil cephalus) (hereafter
pean sea bass (Dicentrarchus labrax) and Cryptosporidium referred to as piscine genotype 3) (Reid et al., 2010).
scophthalmi in turbot (Psetta maxima, syn. Scophthal- The aim of the present study was to determine the
mus maximus) (Alvarez-Pellitero and Sitja-Bobadilla, 2002; prevalence of different species of Cryptosporidium in orna-
Alvarez-Pellitero et al., 2004). C. molnari primarily infects mental fish in Western Australia (WA).
the epithelium of the stomach and seldom the intestine
(Alvarez-Pellitero and Sitja-Bobadilla, 2002), whereas C. 2. Materials and methods
scophthalmi mainly infects the epithelium of the intes-
tine and very seldom the stomach (Alvarez-Pellitero et 2.1. Sample collection
al., 2004). Currently, genetic sequences are available
in GenBank for C. molnari (GenBank accession number A total of 200 ornamental fish from 39 different species
HM243547) but not C. scophthalmi. To date only three (see Table 1) were collected from local aquariums and pet
additional studies have generated genetic sequences from shops in metropolitan WA. These fish included both marine
piscine-derived Cryptosporidium spp.; an isolate from a and freshwater, and tropical and temperate species. On
arrival in the laboratory, fish were measured for length
and weight and dissected. Stomach and intestinal epithelial
∗ Corresponding author at: Division of Health Sciences, School of Vet-
cells were scraped off using a scalpel blade and placed into
erinary and Biomedical Sciences, Murdoch University, Murdoch, Western
Australia 6150, Australia. Tel.: +61 89360 2482; fax: +61 89310 414.
a 1.5 mL Eppendorf tube. Remaining stomach and intestinal
E-mail address: [email protected] (U. Ryan). tissue were stored separately in 10% formalin.

0304-4017/$ – see front matter © 2010 Published by Elsevier B.V.

doi:10.1016/j.vetpar.2010.08.006

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Table 1
Ornamental fish species sampled and tested for infection with Cryptosporidium during this study.

Common name Scientific name Freshwater/marine No. collected No. positive

Acei Pseudotropheus sp. Freshwater 3 0

Albino cory Corydoras paleatus Freshwater 4 0
Angelfish Pterophyllum scalare Freshwater 4 1
Azure damselfish Chrysiptera hemicyanea Marine 3 0
Molly Poecilia latipinna Freshwater 7 0
Balloon kissing gourami Helostoma temminckii Freshwater 4 0
Banded dwarf cichlid Apistogramma bitaeniata Freshwater 3 0
Black ghost Apteronotus albifrons 5 0
Black widow tetras Gymnocorymbus ternetzi Marine 5 0
Blue star leopard wrasse Macropharyngodon bipartitus Marine 5 0
Bristlenose catfish Ancistrus cirrhosus Freshwater 8 0
Bristle tooth tang Ctenochaetus tominiensis Marine 1 1
Butter bream Monodactylus Argenteus Marine 3 2
Madder seaperch Pseudanthias dispar Marine 2 1
Electric yellow Labidochromis caeruleus Freshwater 5 0
False gramma Pseudochromis paccagnellae Marine 1 1
Golden algae eater Crossocheilus aymonieri Freshwater 5 3
Goldfish Carassius auratus auratus Freshwater 7 0
Green acara/Green terror Acara rivulata Freshwater 2 0
Green chromis Chromis viridis Marine 13 2
Guppy Poecilia reticulata Freshwater 43 1
Hornet (Bumblee) cichlid Maylandia crabro Freshwater 1 0
Humbug damsel Dascyllus aruanus Marine 5 0
Kupang damsel Chrysiptera hemicyanea Marine 1 1
Moss green tiger barb Puntius tetrazona Freshwater 3 0
Neon tetra Paracheirodon innesi Freshwater 2 2
Orange anemone (clownfish) Amphiprion percula Marine 14 0
Oscar Astronotus ocellatus Freshwater 4 4
Red hi fin platy Xiphophorus maculatus Freshwater 1 0
Red melon discus Symphysodon discus Freshwater 2 0
Schwartz cory Corydoras schwartzi Freshwater 2 0
Silver gourami Trichogaster trichopterus Freshwater 2 0
Silver shark Balantiocheilos melanopterus Freshwater 9 0
Striped kuhli loach Pangio kuhlii Freshwater 1 0
True rummy nose tetra Hemigrammus bleheri Freshwater 10 0
Upside down cat fish Synodontis nigriventris Freshwater 2 1
Wedgetailed blue tang Paracanthurus hepatus Marine 1 1
Yellow tailed damsel Chrysiptera parasema Marine 2 0
Zebra fish Danio rerio Freshwater 5 0

Total 200 21

2.2. DNA extraction and PCR amplification extractions to determine if negative results were due to PCR
inhibition.
DNA was extracted from ∼250 mg of pooled intesti-
nal and stomach tissue scrapings from each fish sample 2.3. Sequence and phylogenetic analysis
using a Qiagen DNeasy tissue kit (Qiagen, Germany). DNA
was eluted in 50 ␮L of AE buffer to concentrate the DNA. Purified PCR products were sequenced using an ABI
All extracted DNA samples were stored at −20 ◦ C until PrismTM Dye Terminator cycle sequencing kit (Applied
required for screening. Biosystems, Foster City, CA) according to the man-
All samples were screened at the 18S rRNA locus ufacturer’s instructions with the exception that the
and positives were genotyped by sequencing. A two-step annealing temperature was raised to 58 ◦ C. Nucleotide
nested PCR protocol was used to amplify the 18S rDNA sequences were analyzed using Chromas lite version
gene of Cryptosporidium as previously described (Ryan et 2.0 (http://www.technelysium.com.au) and aligned with
al., 2003). For all isolates that were positive at the 18S reference genotypes from GenBank using Clustal W
locus by PCR, attempts were also made to amplify the actin (http://www.clustalw.genome.jp).
locus as previously described (Ng et al., 2006). PCR con- Phylogenetic trees were constructed using additional
tamination controls were used including negative controls isolates from GenBank. Distance estimation was con-
and separation of preparation and amplification areas. The ducted using TREECON (Van de Peer and De Wachter,
amplified DNA fragments from the secondary PCR products 1994), based on evolutionary distances calculated with the
were separated by gel electrophoresis and purified using Kimura’s distance and grouped using Neighbour-Joining.
the freeze-squeeze method (Ng et al., 2006). A spike anal- Parsimony analyses were conducted using MEGA version
ysis (addition of 0.5 ␮L of Cryptosporidium positive control 3.1 (MEGA3.1: Molecular Evolutionary Genetics Analysis
into each sample) was conducted on randomly selected software, Arizona State University, Tempe, AZ, USA). Boot-
Cryptosporidium negative samples from each group of DNA strap analyses were conducted using 1000 replicates to

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Fig. 1. Evolutionary relationships of Cryptosporidium piscine-derived isolates inferred by ML analysis of 18S rRNA sequences. Percentage support (>50%)
from 1000 pseudoreplicates from neighbor-joining, parsimony and ML analyses indicated at the left of the supported node (ns: not supported).

assess the reliability of inferred tree topologies. Maximum (n = 2), dispar anthias (n = 1), false gramma (n = 1), golden
Likelihood (ML) analyses were conducted using the pro- algae eater (n = 3), green chromis (n = 2), guppy (n = 1),
gram PhyML (Dereeper et al., 2008) and the reliability of the kupang damsel (n = 1), madder seaperch (1), neon tetra
inferred trees was assessed by the approximate likelihood (n = 2), oscar (n = 4), upside down cat fish (n = 1), a bristle
ratio test (aLRT) (Anisimova and Gascuel, 2006). tooth tang (n = 1) and a wedgetailed blue tang (n = 1). No
PCR inhibition was detected.
2.4. Calculation of prevalences
3.2. Identification of Cryptosporidium species in fish
Prevalences were expressed as percentage of positive
samples, with 95% confidence intervals calculated assum- Partial sequences were obtained for 19 of the 21 positive
ing a binomial distribution, using the software Quantitative isolates at the 18S rRNA locus. Unfortunately no isolates
Parasitology 3.0 (Rozsa et al., 2000). were successfully amplified at the actin locus despite
numerous attempts. Neighbour-joining, parsimony and ML
3. Results analysis of the 18S rRNA sequences from these 19 iso-
lates and a range of Cryptosporidium species and genotypes
3.1. Prevalence of Cryptosporidium in fish hosts obtained from GenBank produced similar results (Fig. 1-
ML tree shown). Twelve of the 19 isolates were similar
Of the 200 fish sampled during this study from 39 dif- or identical to previously described species/genotypes of
ferent species, 21 positives were detected in 13 different Cryptosporidium. Isolate 101 from a neon tetra was 100%
species by PCR (10.5% prevalence, with 95% CI 6.3–14.7) identical to piscine genotype 1 (GenBank accession no.
(Table 1). Infected hosts were angelfish (n = 1), butter bream AY524773). Isolates 13, 112 and 113, all from oscar fish and

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Percentage sequence difference at the 18S rRNA locus between new genotypes of Cryptosporidium found in ornamental fish in this study and other Cryptosporidium species and genotypes calculated using Kimura’s
isolate 110 from a neon tetra were genetically identical and

C. parvum
exhibited just one single nucleotide polymorphism (SNP)
from piscine genotype 2 (GenBank accession no. FJ769050).
Isolate 9 from a butter bream, 107 from an upsidedown

0
catfish, 127 from a wedgetailed blue tang, 144 from a mad-
der seaperch, 145 from a bristle tooth tang and 151 from

C. baileyi
a green chromas were 100% identical to each other and

7.3
99.2% identical (three SNPs) to C. molnari (GenBank acces-

0
sion number HM243547). Isolate 117 from a golden algae
eater had 8 SNPs from C. molnari and 9 SNPs from isolates

C. muris
9, 107, 127, 144, 145 and 151. The remaining seven isolates

9.2
12.1
0
represent three novel species/genotypes; piscine genotype
4 which comprised isolate 16 from a golden algae eater,
106 from a kupang damsel and 114 from an oscar (100%
identical); piscine genotype 5 which comprised isolates 95

(isolate 192)
genotype 6
from an angelfish and 129 from a butter bream and isolate

Piscine
121 from a golden algae eater. Isolates 95 and 129 were

15.4
15.6
16.4
100% identical to each other and had two SNPs from isolate

0
121; piscine genotype 6 which comprised isolate 192 from
a guppy. The genetic distance between these three novel
species/genotypes and other, previously described species

(isolates 95,
genotype 5
and genotypes of Cryptosporidium is shown in Table 2.

Piscine

129)

5.9
13.2
11.7
16.1
3.3. Nucleotide sequence accession numbers

0
The unique partial 18S rRNA sequences of the isolates
9, 16, 95, 101, 117, 121 and 192 have been deposited

(isolates 16,
in the GenBank database under the accession numbers

genotype 4

106, 114)
HM989832 to HM989837 and HM991857. Piscine

7.3
8.1
15.7

18.4
17.0
0
4. Discussion

In the present study, the overall prevalence of Cryp-

genotype 1
AY524773

tosporidium spp. in the ornamental fish was 10.5% (CI

Piscine

6.3–14.7). This was considerably higher than the preva-

13.4
12.5
12.7
15.2
13.8
15.9
0
lence of 0.8% (6/709) for Cryptosporidium reported in 709
cultured, wild marine and wild freshwater fishes in a recent
study (Reid et al., 2010). Other studies have reported preva-
FJ769050
genotype
Piscine

lences of up to 100%, mostly among juvenile fish (Landsberg

14.2

4.3
4.6
17.1
14.6
16.3
4.0

and Paperna, 1986; Sitjà-Bobadilla et al., 2005; Alvarez-

0
2

Pellitero et al., 2004, 2009; Murphy et al., 2009). The

higher prevalence of Cryptosporidium in ornamental fish
genotype 3

in the present study compared to the study by Reid et al.

GQ925452

(2010) may have been due to the crowded environment of

Piscine

5.7
12.6
5.6
6.5
7.3
17.4
13.6
18.2

the aquarium tanks and the frequent introduction of new

0

species; infectious diseases are often much more prevalent

in ornamental fish than in wild fish or captive aquaculture
C. molnari

fish (Burgess et al., 1999).

In the present study, a variety of new hosts were
12.9
10.6
8.8
12.8
10.5
12.3
14.1
13.2
16.7
0

identified for established piscine species/genotypes of

Cryptosporidium. Piscine genotype 1 (AY524773) was iden-
tified in a neon tetra and piscine genotype 2 (FJ769050)
Piscine genotype 3GQ925452

Piscine genotype 1AY524773

Piscine genotype 2 FJ769050

was identified in a neon tetra and oscar fish. C. molnari-like

genotypes were identified in a butter bream, a golden algae
eater, a green chromas, a madder seaperch, an upsidedown
Piscine genotype 4
Piscine genotype 5
Piscine genotype 6

catfish and a wedgetailed blue tang.

In addition to these extensions of host range for estab-
lished species/genotypes of Cryptosporidium, three new,
C. molnari

C. parvum
C. baileyi
C. muris

distinct piscine genotypes numbered 4–6, were identi-

distance.
Table 2

fied in angelfish, butter bream, golden algae eater, kupang

damsel, oscar and guppy (Fig. 1). A previous study by Reid

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et al. (2010), identified piscine genotype 3 in sea mullets. evolutionary timelines of fish-derived Cryptosporidium iso-
In the present study, this genotype was not identified and lates.
the isolates examined exhibited 5.6–12.6% genetic distance The pathogenesis of the Cryptosporidium species identi-
from piscine genotype 3. fied in aquarium fish in the present study is unknown. How-
Molecular and phylogenetic analysis of the 18S rRNA ever, previous studies on piscine genotype 2 (FJ769050),
locus supports the species status of these genotypes. which was identified in a hatchery, revealed that infected
The genetic distance between the piscine genotypes 4–6 fish exhibited variable levels of emaciation, poor growth
and other fish-derived species of Cryptosporidium was rates, swollen coelomic cavities, anorexia, listlessness and
4.0–13.4% and between the novel genotypes and other increased mortality (Murphy et al., 2009). In affected fish,
Cryptosporidium species and genotypes was between 10.5 large numbers of protozoa were identified both histo-
and 18.3%. This is considerably greater than the genetic dis- logically and ultrastructurally associated with the gastric
tance between most other species of Cryptosporidium. For mucosa. Piscine genotype 1 (AY524773) was associated
example, C. parvum and C. hominis differ by 0.6% at the 18S with high mortalities amongst guppies and was detected
locus and the genetic distance between C. parvum and all in the stomach, with oogonial and sporogonial stages
other intestinal species is 0.6% - 2.3%. Within Cryptosporid- observed deep within the epithelium, similar to C. molnari
ium taxonomy, if the genetic distance between a new (Ryan et al., 2004). All of the ornamental fish examined in
genotype and its closest relative is equal to or greater than the present study appeared unwell and exhibited a variety
the genetic distance between established Cryptosporidium of symptoms including emaciation, anorexia, listlessness,
species, then this is regarded as a valid criterion for claim- tail and/or fin rot and swollen coelomic cavities. Isolate 106
ing species status (Xiao et al., 2004). By this criterion, the from a kupang damsel (piscine genotype 4) had skin lesions
novel genotypes found in ornamental fish in this study and a bacterial infection, while three of the four oscar fish-
appear to be separate species. Unfortunately, we have not derived isolates (112, 113, 114) (piscine genotypes 2 and
yet been able to examine the morphological or life history 4) had concomitant bacterial and gill fluke infections.
characteristics of these genotypes. The genetic distance The pathogenesis of Cryptosporidium is an issue of
between C. molnari and the C. molnari-like genotypes was potentially much wider significance than its effects on
0.8–2.8% and therefore the C. molnari-like genotypes are companion aquarium fish and the ornamental fish indus-
also likely to be separate species. Genetic characterization try. A large number of freshwater ornamental fish species
at additional loci in the future will be important for clarifi- have been released, either accidentally or deliberately,
cation of their species status. into waterways throughout the world, where they have
Histological examination of intestinal/stomach tissue of established self-sustaining populations (Rahel, 2002). Pop-
infected ornamental fish was conducted, but due to rapid ulations of exotic ornamental fish may adversely affect the
autolysis of fish tissue were unable to confirm the presence native freshwater fish through competition, predation and
of Cryptosporidium spp. in either intestinal or stomach tis- the introduction of diseases (Arthington, 1991; Morgan et
sues. Some of the fish were dead for several hours prior al., 2004; Kennard et al., 2005; Lymbery et al., 2010). The
to being collected and some were obtained frozen from high prevalence of potentially pathogenic Cryptosporidium
suppliers, which would also contribute to the substantial found in ornamental fish in the present study may, if these
autolysis seen. fish are released, constitute a major threat to the highly
Phylogenetic analysis of 18S rRNA sequences indicated endemic and threatened freshwater fish fauna of Western
that all the piscine genotypes identified in the present study Australia.
and all piscine genotypes from previous studies (with the
exception of C. parvum, C. xiaoi and pig genotype II iden-
tified in whiting in a previous study by Reid et al., 2010), Acknowledgements
formed clades that were basal to all other Cryptosporidium
species and genotypes in the tree, suggesting that piscine We would like to thank Dr. Alvarez-Pellitero, Dr. Sitja-
Cryptosporidium isolates may be the most primitive of Cryp- Bobadilla and Dr Oswaldo Palenzuela at the Institute of
tosporidium species. Piscine genotype 1 (AY524773) was Aquaculture Torre de la Sal in Spain for the provision of the
previously referred to as C. molnari-like on the basis of C. molnari 18S rRNA sequence used in the present study.
detection in the stomach and presence of oogonial and
sporogonial stages deep within the epithelium, similar to C. References
molnari (Ryan et al., 2004). However, phylogenetic analy-
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species, Sparus aurata L. and Dicentrarchus labrax L. Int. J. Parasitol.
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