Echinococcus multilocularis

A cross-sectional survey was conducted to estimate the prevalence of Echinococcus multilocularis and E. granulosus infections in domestic dogs and cats from Germany and other European countries. Faecal samples of 21,588 dogs and 10,650 cats routinely submitted to a private veterinary laboratory between June 2004 and June 2005 were examined using the ZnSO4–NaCl flotation method. Taeniid eggs were detected in

The influence of secondary Echinococcus multilocularis infection on reproduction and cellular immune response of mice was studied in BALB ⁄ c mice infected with 2000 E. multilocularis protoscoleces. Of the total infected mothers, 11AE7% did not give birth and 10% of uninfected ones did not deliver. Both, healthy and infected mothers, produced on average 6-7 offspring per litter. The changes in production of seral IFN-c, TNF and IL-10 did not significantly influence the course of gravidity. On the other hand, more intensive metacestode growth was observed after the delivery. This study confirmed the ability of host organism to adapt to severe damage caused by E. multilocularis, not only in normal conditions, but also during pregnancy.

The alimentary canals of 9 species of domesticated or wild animals were examined for the presence of E. multilocularis. The tapeworm was found in 9 red foxes (Vulpes vulpes) out of 155 examined (5.8%) and in 2 raccoon dogs (Nyctereutes procyonoides) of 25 examined (8.0%). Our studies proved that in Poland the raccoon dog can also be infected with E. multilocularis.

Following the collapse of the Soviet Union in 1991, there was an increase in the numbers of cases of human echinococcosis recorded throughout central Asia. Between 1991 and 2001 incidence rates of cystic echinococcosis (CE) increased by 4 fold or more. There also appeared to be increases in prevalence of CE in livestock and prevalences of Echinococcus granulosus reported in dogs. The increase in human echinococcosis was associated with changes in livestock husbandry, decline in veterinary public health services, increases in dog populations and increased poverty all of which served to promote transmission of E. granulosus. A few years after reports of increased transmission of E. granulosus, the first reports of E. multilocularis infection in dogs were recorded. Further studies indicated that in both Kazakhstan and Kyrgyzstan prevalences of up to 18% were present. Recently there has been a dramatic increase in the number of cases of human alveolar echinococcosis recorded in Kyrgyzstan with over 60 cases reported in 2011.

In Svalbard, Norway, the only intermediate host for Echinococcus multilocularis, the sibling vole, has restricted spatial distribution. A survey of feces from the main host, the arctic fox, showed that only the area occupied by the intermediate host is associated with increased risk for human infection.

A new cost-effective method using silicon dioxide-and guanidine isothiocyanate-containing buffers, after previous alkaline lysis, was established for the DNA extraction from taeniid eggs isolated from canine faeces. The purified DNA can be used to amplify the species-specific 12S mitochondrial DNA of Echinococcus multilocularis in direct and nested polymerase chain reaction in order to differentiate between E. multilocularis and Taenia spp.

Background: Localized concentrations of Echinococcus multilocularis eggs from feces of infected red fox (Vulpes vulpes) can create areas of higher transmission risk for rodent hosts and possibly also for humans; therefore, identification of these areas is important. However, in a low prevalence environment, such as Sweden, these areas could be easily overlooked. As part of a project investigating the role of different rodents in the epidemiology of E. multilocularis in Sweden, fox feces were collected seasonally from rodent trapping sites in two regions with known parasite status and in two regions with unknown parasite status, 2013-2015. The aim was to evaluate background contamination in rodent trapping sites from parasite eggs in these regions. To maximize the likelihood of finding fox feces positive for the parasite, fecal collection was focused in habitats with the assumed presence of suitable rodent intermediate hosts (i.e. targeted sampling). Parasite eggs were isolated from feces through sieving-flotation, and parasite species were then confirmed using PCR and sequencing. Results: Most samples were collected in the late winter/early spring and in open fields where both Arvicola amphibius and Microtus agrestis were captured. Fox feces positive for E. multilocularis (41/714) were found within 1-3 field collection sites within each of the four regions. The overall proportion of positive samples was low (≤5.4%) in three regions, but was significantly higher in one region (22.5%, P < 0.001). There was not a significant difference between seasons or years. Compared to previous national screenings, our sampling strategy identified multiple E. multilocularis positive feces in all four regions, including the two regions with previously unknown parasite status. Conclusions: These results further suggest that the distribution of E. multilocularis is highly aggregated in the environment and provide support for further development of a targeted sampling strategy. Our results show that it was possible to identify new areas of high contamination in low endemic environments. After further elaboration, such a strategy may be particularly useful for countries designing surveillance to document freedom from disease.

Background: Alveolar echinococcosis (AE) is a severe helminth disease affecting humans, which is caused by the fox tapeworm Echinococcus multilocularis. AE represents a serious public health issue in larger regions of China, Siberia, and other regions in Asia. In Europe, a significant increase in prevalence since the 1990s is not only affecting the historically documented endemic area north of the Alps but more recently also neighbouring regions previously not known to be endemic. The genetic diversity of the parasite population and respective distribution in Europe have now been investigated in view of generating a fine-tuned map of parasite variants occurring in Europe. This approach may serve as a model to study the parasite at a worldwide level.

Echinococcosis is one of the 17 neglected tropical diseases (NTDs) recognized by the World Health Organization. The two major species of medical importance areEchinococcus granulosusandEchinococcus multilocularis.E. granulosusaffects over 1 million people and is responsible for over $3 billion in expenses every year. In this minireview, we discuss aspects of the epidemiology, clinical manifestations, and diagnosis of cystic echinococcosis or cystic hydatid disease caused byE. granulosus.

Echinococcus multilocularis, an emerging zoonotic disease is extending its geographical distribution within the European Union (EU). At present, five member states including Ireland are considered free. Previous EU regulations on importing domestic pets allowed these countries to maintain national rules that required all dogs be treated with an anti-cestode compound before entry. The controls on the movement of pet animals within the EU were recently reviewed by the European Commission and it was decided that the five countries had to demonstrate freedom from E. multilocularis before they could continue with the mandatory tapeworm treatment.

Domesticated dogs are an important potential source of Echinococcus multilocularis infection in humans; therefore, new molecular approaches for the prevention of the parasite infection in dogs need to be developed. Here, we identified and characterized an immunogenic protein of the parasite by using a proteome-based approach. The total protein extracted from protoscoleces was subjected to two-dimensional Western blotting with sera from dogs experimentally infected with E. multilocularis. Two protein spots showed major reactivity to the sera from infected dogs. The N-terminal amino acid sequences of these spots were identical to the deduced amino acid sequence of the product of the putative hsp20 gene. RT-PCR and Western blot analyses revealed that the putative hsp20 gene and its products were expressed in almost all stages of the parasite life cycle. Furthermore, recombinant hsp20 showed specific reactivity to the sera from infected dogs, suggesting that this molecule may facilitate the development of a practical vaccine.

Alveolar echinococcosis (AE) is a severe zoonotic disease caused by the metacestode stage of Echinococcus multilocularis. The infection can have fatal consequences in humans if treatment is not provided, so early diagnosis is fundamental for initiating treatment and reducing morbidity and mortality. In addition, detection of the parasite in the definitive host plays a central role in epidemiological studies and surveillance programmes for control of AE. This review presents an overview of the present situation regarding the immunodiagnosis of E. multilocularis infection. Special attention is given to the description of the native, partially purified and recombinant antigens available currently for immunodiagnostic purposes. Recent advances in the primary serodiagnosis and follow-up of AE patients are highlighted, including the detection of specific cytokine profiles. Progress in the immunodiagnosis of intestinal E. multilocularis infection in definitive hosts, particularly the detection of excretory–secretory and integument products of the worm in faeces (copro-antigens) by ELISA, is also discussed.

A hybridization probe-based real-time multiplexnested PCR system was developed for the simultaneous detection of Echinococcus multilocularis and host species directly from faecal samples. Species identification was determined by melting curve analysis. Specificity was assessed by using DNA extracted from various cestodes (E.

The raccoon dog (Nyctereutes procyonoides) is an introduced species in Europe with a continually expanding range. Since the species is capable of affecting local ecosystems and is a vector for a number of severe zoonotic diseases, it is important to understand its food habits. Raccoon dog diet was studied in Estonia by examining the contents of 223 stomach samples collected during the coldest period of the year, August to March, in 2010-2012. The most frequently consumed food categories were anthropogenic plants (e.g. cereals, fruits; FO = 56.1%) and carrion (e.g. carcasses of artiodactyls and carnivores; FO = 48.4%). Carrion was also the only food category that was consumed significantly more frequently by raccoon dogs exhibiting symptoms of sarcoptic mange than by uninfected animals. Small mammals, which represent intermediate hosts for the zoonotic tapeworm Echinococcus multilocularis, were more commonly recorded in samples also containing anthropogenic plants than expected by ch...

To investigate echinococcosis in co-endemic regions, three polymerase chain reaction (PCR) assays based on the amplification of a fragment within the NADH dehydrogenase subunit 1 (ND1) mitochondrial gene were optimized for the detection of Echinococcus shiquicus, Echinococcus granulosus G1, and Echinococcus multilocularis DNA derived from parasite tissue or canid fecal samples. Specificity using parasite tissue-derived DNA was found to be 100% except for E. shiquicus primers that faintly detected E. equinus DNA. Sensitivity of the three assays for DNA detection was between 2 and 10 pg. Ethanol precipitation of negative PCR fecal samples was used to eliminate false negatives and served to increase sensitivity as exemplified by an increase in detection from 0% to 89% of E. shiquicus coproDNA using necropsy-positive fox samples.

The prevalence rates of Echinococcus multilocularis in foxes (n ¼ 5600) evaluated in several Austrian surveys conducted between 1991 and 2004 were analysed for spatial and temporal differences. Data from early studies (1993)(1994)(1995)(1996)(1997) in which the intestinal scraping technique (IST) was utilized were compared with data from recent (1999)(2000)(2001)(2002)(2003)(2004) investigations, which made use of the shaking in a vessel technique (SVT), and it was assessed whether or not the infection rates of Austrian foxes had increased between the investigated intervals. In total, data from 85 districts are presented and both the retrospective and recent data are available from 39 of these districts. A Bayesian hierarchical model of parasite prevalences is presented which (i) accounts for differences in the sensitivity of IST and SVT, (ii) incorporates spatial auto-correlation between neighbouring districts, (iii) investigates the possibility of a temporal shift in the infection status of foxes, and (iv) quantifies uncertainty at each level of the model. The national average prevalence rates in the mid-1990s and at the turn of the millennium were 2.4% (95% confidence intervals 1.1-4.8) and 3.9% (95% confidence intervals 1.5-8.4) respectively. Above average prevalence rates were observed in the western and the northern parts of the country. Evidence is also presented for a temporal augmentation of the prevalence rates in some districts in the northern and eastern parts of the country. These findings are in concordance with several investigations in other European states where both newly emerged areas and elevated levels of transmission in existing endemic areas have been found. None of the districts investigated here showed significant evidence of a drop in prevalence.

Gram-positive, catalase-negative, chain-forming, coccus-shaped organisms were isolated both from intraperitoneally grown vesicles of the fox tapeworm Echinococcus multilocularis and the oropharynges of laboratory-kept Mongolian jirds (Meriones unguiculatus). The strains displayed no haemolytic activity on Columbia sheep blood agar, pyrrolidonyl arylamidase activity was negative and the organisms reacted weakly with Lancefield group D antiserum. On the basis of phenotypic characteristics, the strains were tentatively identified as members of the genus Streptococcus. Comparative 16S rRNA gene sequencing studies confirmed their assignment to the genus Streptococcus and revealed that Streptococcus hyointestinalis DSM 20770 T was their closest phylogenetic neighbour (96.5 % sequence similarity). The levels of 16S rRNA gene sequence similarity between the isolates and representatives of species of the genus Streptococcus were only 95.7-96.2 %. On the basis of the phenotypic and molecular data presented, the isolates from Mongolian jirds represent a novel species of the genus Streptococcus, for which the name Streptococcus merionis sp. nov. is proposed. The type strain is WUE3771 T (5DSM 19192 T 5CCUG 54871 T ).

Artemisinin is an antimalarial sesquiterpene lactone that contains a 1,2,4-trioxane heterocycle. Dihydroartemisinin and artesunate demonstrated activity against Echinococcus multilocularis metacestodes in vitro but were not effective in a mouse model. In this study, the in vitro effects of a small library of synthetic ozonides (1,2,4-trioxolanes) were investigated. Initial compound screening against E. multilocularis metacestodes was performed at 20 M, and selected ozonides were further assessed in dose-response studies in metacestode cultures and mammalian cells. Transmission electron microscopy (TEM) was employed to characterise compound-induced structural alterations. At 20 M, the most potent ozonides (OZ401, OZ455, OZ491 and OZ494) led to death of ca. 60-100% of the parasites. Subsequent dose-response experiments demonstrated that OZ401, OZ455 and OZ491, which contain an aminopropylether substructure, were the most potent, with 50% inhibitory concentrations ranging from 11 M to 14 M. Cytotoxicity for these three ozonides, assessed in human foreskin fibroblasts, rat hepatoma cells and green monkey epithelial kidney (Vero) cells, was evident only at high concentrations. TEM demonstrated that OZ401 and OZ491 treatment induced considerable metabolic impairment in metacestodes at 1 day post exposure. At Day 3 post exposure, the germinal layer was severely distorted, although some intact cells were still visible, demonstrating that not all cell types in the parasite tissue were equally affected. Complete destruction of the germinal layer was noted at 5 days post exposure. Synthetic ozonides could represent interesting leads that will be further investigated in a suitable in vivo model of E. multilocularis infection.

Echinococcus granulosus antigen B fraction (AgB) was evaluated for its prognostic value in the serological follow-up of cystic echinococcosis (CE), compared to crude hydatid fluid (HF) as well as soluble somatic Echinococcus multilocularis antigen (Em). The sensitivity and specificity of enzyme-linked immunosorbent assays (ELISA) were compared examining 177 sera from patients with different clinical courses and outcome of CE and with other parasitic infections. AgB-ELISA in comparison to confirmed cystic echinococcosis has 96.4 % sensitivity and 97.2 % specificity, with 93.1 % positive predictive value and 98.6 % negative predictive value. The HF-ELISA was more sensitive than the AgB-ELISA, but its specificity was lower. Our results indicate that AgB-ELISA was more satisfactory for seroconfirmation of acute echinococcosis than HF-ELISA. Moreover, the AgB-ELISA has a potential key role in control measures implemented in patients undergoing surgery. In sera of patients more than 3 months after the treatment, no antibody response to antigen B was detected; however, with conventionally used HF-ELISA, they were still positive. The AgB is recommended for the confirmatory diagnosis. AgB-WB allocated low background with typical "triplet" bands at 8-12-16 kDa.

This study evaluated the impact of biological and environmental factors on the infection of red foxes (Vulpes vulpes) by Echinococcus multilocularis in an endemic area of north-east France. From January 2004 to April 2006, 127 foxes were examined for E. multilocularis and their stomach contents analysed. The effect of year, season, age, sex and urbanisation level on E. multilocularis presence was estimated using a General Linear Model (GLM) with logit link, (i.e. logistic regression). Urbanisation level was the only influencing factor, with a decreasing gradient from rural [54 %, CI 95 % (40-68)] to peri-urban [31 %, CI 95 % (15-52)] and urban area [4 %, CI 95 % (0.7-15)]. The consumption of Arvicola terrestris and Microtus sp., grassland species, the main presumed intermediate hosts of E. multilocularis,

Echinococcus multilocularis causes a rare but potentially lethal zoonotic infection in humans. This tapeworm is known to be endemic in foxes in several countries of Western and Central Europe. In Western Europe, the common vole (Microtus arvalis) and the water vole (Arvicola terrestris) are considered to be the most important intermediate host species of this cestode whereas the red fox is by far the most important final host. The purpose of this study was to provide data on the prevalences in Wallonia (Southern part of Belgium) both in the red fox and in different potential intermediate hosts.

SUMMARYEchinococcus species are important parasites of wildlife, domestic animals and people worldwide; however, little is known about the prevalence, intensity and genetic diversity of Echinococcus tapeworms in Canadian wildlife. Echinococcus tapeworms were harvested from the intestines of 42% of 93 wolves (Canis lupus) from five sampling regions in the Northwest Territories, Manitoba and Saskatchewan, and visually identified to genus level by microscopic examination. Genetic characterization was successful for tapeworms from 30 wolves, and identified both Echinococcus canadensis and Echinococcus multilocularis in all sampling locations. Mixed infections of E. canadensis/E. multilocularis, as well as the G8/G10 genotypes of E. canadensis were observed. These findings suggest that wolves may be an important definitive host for both parasite species in western Canada. This represents the first report of wolves naturally infected with E. multilocularis in North America, and of wolves ...

The parasitic tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a serious zoonotic infection present in Europe that can be fatal. The United Kingdom currently has E. multilocularis free status but the possibility of introduction exists, most likely via an imported or returning dog or other deliberately introduced animal that has not had anthelmintic treatment. We have developed a model to predict the probability of successfully eliminating a focal outbreak of E. multilocularis using a programme of anthelmintic bait distribution. We investigated three different potential control programmes, each with 36 monthly campaigns commencing five, ten or 15 years after disease introduction over an area of 2827km(2). We assumed equilibrium disease prevalence of 30%, 40% and 55% based on the range of values reported across Europe. However, for all of these scenarios, equilibrium had not been reached at five to 15 years after introduction and simulated local prevalence values were between 0.5% and 28%. We found that it is possible to eliminate the disease with a 38%-86% success rate if control is started five years after introduction, dropping to 0% to 56% if control is delayed until 15 years after introduction, depending upon the prevalence equilibrium. We have also estimated the costs involved in these programmes to be from €7 to €12 million (2013 prices).

ObjectiveTo determine the true community prevalence of human cystic (CE) and alveolar (AE) echinococcosis (hydatid disease) in a highly endemic region in Ningxia Hui, China, by detecting asymptomatic cases. Methods Using hospital records and “AE-risk” landscape patterns ...

Echinococcus multilocularis, the causative agent of alveolar echinococcosis (AE) in humans, has been found in 4.8% of 561 red foxes originating from various regions of Romania. Infected foxes were identified in 8 of 15 counties with average prevalence rates between 1.7% and 14.6%. In previous studies, E. multilocularis was not found in 535 foxes from three counties, but larval stages (metacestodes) were present in four species of rodents. Furthermore, AE was diagnosed in two patients. Experiences from other European regions indicate that several factors (such as increasing fox populations with higher parasite prevalences and urban cycles of the parasite) may result in an increased infection risk for humans.

We investigated the role of urban coyote feeding ecology in the transmission of Echinococcus multilocularis, the causative agent of Alveolar Echinococcosis in humans. As coyotes can play a main role in the maintenance of this zoonotic parasite within North American urban settings, such study can ultimately aid disease risk management. Between June 2012 and June 2013, we collected 251 coyote feces and conducted trapping of small mammals (n = 971) in five parks in the city of Calgary, Alberta, Canada. We investigated E. multilocularis epidemiology by assessing seasonal variations of coyote diet and the selective consumption of different rodent intermediate host species. Furthermore, accounting for small mammal digestibility and coyote defecation rates we estimated the number of small mammal preys ingested by coyote and consequently, coyote encounter rates with the parasite. Dominant food items included small mammals, fruit and vegetation, although hare and deer were seasonally relevant. The lowest frequency of occurrence per scat of small mammals was recorded in winter (39.4 %), when consumption of deer was highest (36.4 %). However, highest encounter rates (number of infected hosts predated/season) with E. multilocularis (95% CI: 1.0-22.4), combined with the lack of predation on non-competent small mammal species, suggest that winter is the critical season for transmission and control of this parasite. Within the small mammal assemblage, voles (Microtus pennsylvanicus and Myodes gapperi) were the selected preys of urban coyotes and likely played a key role for the maintenance of the urban sylvatic life-cycle of E. multilocularis in Calgary.

Background: Following the first finding of Echinococcus multilocularis in Sweden in 2011, 2985 red foxes (Vulpes vulpes) were analysed by the segmental sedimentation and counting technique. This is a labour intensive method and requires handling of the whole carcass of the fox, resulting in a costly analysis. In an effort to reduce the cost of labour and sample handling, an alternative method has been developed. The method is sensitive and partially automated for detection of E. multilocularis in faecal samples. The method has been used in the Swedish E. multilocularis monitoring program for 2012-2013 on more than 2000 faecal samples. Methods: We describe a new semi-automated magnetic capture probe DNA extraction method and real time hydrolysis probe polymerase chain reaction assay (MC-PCR) for the detection of E. multilocularis DNA in faecal samples from red fox. The diagnostic sensitivity was determined by validating the new method against the sedimentation and counting technique in fox samples collected in Switzerland where E. multilocularis is highly endemic. Results: Of 177 foxes analysed by the sedimentation and counting technique, E. multilocularis was detected in 93 animals. Eighty-two (88%, 95% C.I 79.8-93.9) of these were positive in the MC-PCR. In foxes with more than 100 worms, the MC-PCR was positive in 44 out of 46 (95.7%) cases. The two MC-PCR negative samples originated from foxes with only immature E. multilocularis worms. In foxes with 100 worms or less, (n = 47), 38 (80.9%) were positive in the MC-PCR. The diagnostic specificity of the MC-PCR was evaluated using fox scats collected within the Swedish screening. Of 2158 samples analysed, two were positive. This implies that the specificity is at least 99.9% (C.I. = 99.7-100). Conclusions: The MC-PCR proved to have a high sensitivity and a very high specificity. The test is partially automated but also possible to perform manually if desired. The test is well suited for nationwide E. multilocularis surveillance programs where sampling of fox scats is done to reduce the costs for sampling and where a test with a high sensitivity and a very high specificity is needed.

The tumour-like growth of larval Echinococcus multilocularis tissue (causing alveolar echinococcosis, AE) is directly linked to the nature/orientation of the periparasitic host immune-mediated processes. Parasite-mediated immune suppression is a hallmark triggering infection outcome in both chronic human and murine AE. So far, little is known about secondary systemic immune effects of this pathogen on other concomitant diseases, e.g. endogenous gut inflammation. We examined the influence of E. multilocularis infection on murine dextran sodium sulphate (DSS)induced colitis. At 3 months after E. multilocularis infection (chronic stage), the mice were challenged with 3% DSS in the drinking water for 5 days plus subsequently with tap water (alone) for another 4 days. After necropsy, fixed tissues/organs were sectioned and stained with haematoxylin & eosin for assessing inflammatory reactions. Cytokine levels were measured by flow cytometry and quantitative RT-PCR. Colitis severity was assessed (by board-certified veterinary pathologists) regarding (i) colon length, (ii) weight loss and (iii) a semi-quantitative score of morphological changes. The histopathological analysis of the colon showed a significant reduction of DSS-induced gut inflammation by concomitant E. multilocularis infection, which correlated with down-regulation of T helper type 1 (Th1)/Th17 T-cell responses in the colon tissue. Echinococcus multilocularis infection markedly reduced the severity of DSS-induced gut inflammation upon down-regulation of Th1/Th17 cytokine expression and attenuation of CD11b + cell activation. In conclusion, E. multilocularis infection remarkably reduces DSS-induced colitis in mice by attenuating Th1/Th17-mediated immune reactions.

Alveolar echinococcosis is a refractory disease caused by the metacestode stage of Echinococcus multilocularis. The life cycle of this parasite is maintained primarily between foxes and many species of rodents; thus, dogs are thought to be a minor definitive host except in some endemic areas. However, dogs are highly susceptible to E. multilocularis infection. Because of the close contact between dogs and humans, infection of dogs with this parasite can be an important risk to human health. Therefore, new measures and tools to control and prevent parasite transmission required. Using 2-dimensional electrophoresis followed by western blot (2D-WB) analysis, a large glycoprotein component of protoscoleces was identified based on reactivity to intestinal IgA in dogs experimentally infected with E. multilocularis. This component, designated SRf1, was purified by gel filtration using a Superose 6 column. Glycosylation analysis and immunostaining revealed that SRf1 could be distinguished from Em2, a major mucin-type antigen of E. multilocularis. Dogs (n = 6) were immunized intranasally with 500 mg of SRf1 with cholera toxin subunit B by using a spray syringe, and a booster was given orally using an enteric capsule containing 15 mg of the same antigen. As a result, dogs immunized with this antigen showed an 87.6% reduction in worm numbers compared to control dogs (n = 5) who received only PBS administration. A weak serum antibody response was observed in SRf1-immunized dogs, but there was no correlation between antibody response and worm number. We demonstrated for the first time that mucosal immunization using SRf1, a glycoprotein component newly isolated from E. multilocularis protoscoleces, induced a protection response to E. multilocularis infection in dogs. Thus, our data indicated that mucosal immunization using surface antigens will be an important tool to facilitate the development of practical vaccines for definitive hosts.

One tetraspanin, designated as E24, was cloned from a full-length enriched vector-capping cDNA library of Echinococcus multilocularis metacestode. The amino acid sequence and phylogenetic analysis suggested that E24 is a T24-like protein. The crucial, functional large extracellular loop (LEL) domain of E24 was expressed and characterized using a polyclonal antiserum by Western blot and immunohistochemistry. The results showed that anti-recombinant-E24 (anti-recE24) antibody can specifically recognize approximately 25 kDa recombinant protein and 25 kDa cyst-extracted antigen; the germinal layer of both the protoscolex-free and protoscolex-formed cysts were intensely labeled by immunofluorescent antibody. This study revealed that E24 is an antigenic, germinal layer-located protein of E. multilocularis metacestode, implying for its potential in diagnostic and vaccine development.

Alveolar echinococcosis is caused by infection with the larval stage of Echinococcus multilocularis. We recently identified a cDNA clone, designated as emy162, that encodes a putative secreted protein. EMY162 shares structural features with the EM95 antigen, which is a host-protective antigen. The amino acid sequence of EMY162 shows 31.4% identity to EM95 whereas these antigens are distinguishable with respect to their predicted secondary structure and antigenicity on Western blot analysis. RT-PCR analysis revealed that the gene expression of emy162 was significantly higher than that of em95 at each life-cycle stage. Recombinant EMY162 antigen induced a significant level of host-protection (74.3%) in experimental infection with E. multilocularis eggs in mice. Notably, recombinant EMY162 antigen showed significant reactivity to the sera from alveolar echinococcosis patients. These results may help in the development of a practical vaccine to reduce the level of alveolar echinococcosis in humans.

The Tibetan plateau of western China has been shown to have a very high prevalence of human cystic echinococcosis (CE) caused by Echinococcus granulosus and human alveolar echinococcosis (AE) caused by Echinococcus multilocularis. The domestic dog is suspected to be the primary definitive host for the transmission of both E. granulosus and E. multilocularis to humans in this locality. A purgation study of 371 dogs in Shiqu County, Sichuan Province during 2002-2003 resulted in an E. multilocularis prevalence of 12% and an E. granulosus prevalence of 8%. These crude prevalences were then adjusted, based on the known sensitivity of arecoline purgation for the detection of E. granulosus and a suggested sensitivity for the detection of E. multilocularis. In addition, it was assumed that some immature parasites of either species could be misidentified morphologically and wrongly assigned. This resulted in credible true prevalence intervals of between 13-33% for E. multilocularis and 8-19% for E. granulosus. Prevalences of other intestinal helminthes found on purgation were: Taenia spp. 31%, Dipylidium caninum 1%, and ascarids 8%. Risk factors associated with the acquisition of canine echinococcosis were evaluated based on responses to a questionnaire administered to dog owners. Male dogs were more likely to be infected with Echinococcus spp. than female dogs (P < 0.05) and dogs allowed to roam were more likely to be infected with E. multilocularis (P < 0.05).

A multiplex PCR system was developed to identify the carnivore origins of faeces collected in Hokkaido, Japan, for epidemiological studies on Echinococcus multilocularis. Primers were designed against the D-loop region of mitochondrial DNA. Two separate primer mixtures (mix 1, specific forward primers to fox, raccoon dog and dog, and a universal reverse primer [prH]; and mix 2, specific forward primers to cat, raccoon and weasels and prH) were used so that the PCR products (160 bp, fox and cat; 240 bp, raccoon dog and raccoon; and 330 bp, dog and weasel) were distinguished by size. The multiplex PCR exhibited no cross-reactivity between carnivore species and did not amplify DNA from rodent prey. When 270 field-collected faeces were examined, 250 showed single PCR products belonging to specific target sizes, suggesting successful carnivore identification for 92.6% of samples. Taeniid eggs were detected in 11.1% of samples and coproantigen in 30.4%; whereas the prevalences of taeniid eggs and coproantigen were 12.9% and 34.0% in fox faeces, and 0% and 26.3% in cat faeces, respectively. These results suggest that the prevalence in different target animals can be evaluated individually and precisely using multiplex PCR system.

Canine alveolar echinococcosis (AE) is a rare liver disease caused by the larval stage (metacestode) of E. multilocularis. Its diagnosis so far is rather difficult and invasive. Imaging techniques for the diagnosis have been described recently and in this study, the serological diagnosis of canine AE was evaluated in 30 dogs with confirmed AE using ELISA based on seven different antigens (E. multilocularis-antigens: Em2G11, EmII/3-10, protoscolex (EmP), excretory/secretory and adult integument (EmAd/I); E. granulosus antigens: hydatid fluid (EgHF) and antigen B (EgAgB)) and by the detection of circulating Em2G11-antigen. EmII/3-10 and Em2G11-based tests detected AE in 50 and 53%. The combined serological investigation for specific circulating Em2G11antigen and specific antibodies against this antigen showed a sensitivity of 77%. This strategy is useful in animals with large parasite masses and ascites. The highest sensitivities for AE were obtained by the EmAd/I and the EmP antigens (97 and 93% sensitivity) with high specificities in the control group of 76 dogs (100 and 98.7%, respectively). Heterologous E. granulosus antigens (EgHF and EgAgB) showed low sensitivities (43 and 50%, respectively) with high numbers of unspecific reactions (>16%) in the control group. In the follow-up of four dogs, the serological reaction patterns against the antigens Em2G11, EmP and EmAd/I showed some correlations with the development of the parasite mass. Intestinal infections cannot be differentiated from AE by most antigens (except EmII/3-10) and have to be excluded by the absence of coproantigens or DNA of E. multilocularis in faecal samples. # d Non-endemic area for E. multilocularis.