Italian wolves (Canis lupus italicus Altobello, 1921) and molecular detection of taeniids in the Foreste Casentinesi National Park, Northern Italian Apennines

After centuries of massive decline, the recovery of the wolf (Canis lupus italicus) in Italy is a typical conservation success story. To learn more about the possible role of parasites in the wolves' individual and population health and conservation we used non-invasive molecular approaches on fecal samples to identify individual wolves, pack membership, and the taeniids present, some of which are zoonotic. A total of 130 specimens belonging to 54 wolves from eight packs were collected and examined. Taeniid eggs were isolated using a sieving/flotation technique, and the species level was identified by PCR (gene target: 12S rRNA and nad1). Taeniid prevalence was 40.7% for Taenia hydatigena, 22.2% for T. krabbei, 1.8% for T. polyachanta and 5.5% for Echinococcus granulosus. The prevalence of E. granulosus is discussed. Our results show that the taeniid fauna found in wolves from the Foreste Casentinesi National Park is comparable to that described for other domestic and wild Italian canids and provides insights into the wolves' diet and their relationship with the environment

Latent class models for Echinococcus multilocularis diagnosis in foxes in Switzerland in the absence of a gold standard

Background: In Europe the principal definitive host for Echinococcus multilocularis, causing alveolar echinococcosis in humans, is the red fox (Vulpes vulpes). Obtaining reliable estimates of the prevalence of E. multilocularis andrelevant risk factors for infection in foxes can be difficult if diagnostic tests with unknown test accuracies are used. Latent-class analysis can be used to obtain estimates of diagnostic test sensitivities and specificities in the absence of a perfect gold standard. Samples from 300 foxes in Switzerland were assessed by four different diagnostic tests including necropsy followed by sedimentation and counting technique (SCT), an egg-PCR, a monoclonal and a polyclonal copro-antigen ELISA. Information on sex, age and presence of other cestode species was assessed as potential covariates in the Bayesian latent class models. Different Bayesian latent-class models were run, considering dichotomized test results and, additionally, continuous readings resulting in empirical ROC curves. Results: The model without covariates estimated a true parasite prevalence of 59.5% (95% CI: 43.1–66.4%). SCT, assuming a specificity of 100%, performed best among the four tests with a sensitivity of 88.5% (95% CI: 82.7–93.4%). The egg-PCR showed a specificity of 93.4% (95% CI: 87.3–99.1%), although its sensitivity of 54.8% was found moderately low (95% CI: 48.5–61.0%). Relatively higher sensitivity (63.2%, 95% CI: 55.3–70.8%) and specificity (70.0%, 95% CI: 60.1–79.4%) were estimated for the monoclonal ELISA compared to the polyclonal ELISA with a sensitivity and specificity of 56.0% (95% CI: 48.0–63.9%) and 65.9% (95% CI: 55.8–75.6%), respectively. In the Bayesian models, adult foxes were found to be less likely infected than juveniles. Foxes with a concomitant cestode infection had double the odds of an E. multilocularis infection. ROC curves following a Bayesian approach enabled the empirical determination of the best cut-off point. While varying the cut-offs of both ELISAs, sensitivity and specificity of the egg-PCR and SCT remained constant in the Bayesian latent class models. Conclusions: Adoption of a Bayesian latent class approach helps to overcome the absence of a perfectly accurate diagnostic test and gives a more reliable indication of the test performance and the impact of covariates on the prevalence adjusted for diagnostic uncertainty

In vivo viability of Echinococcus multilocularis eggs in a rodent model after different thermo-treatments

Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a serious and emerging zoonotic disease in many parts of the northern hemisphere. Humans but also primates and other accidental hosts can acquire the infection by the ingestion of eggs excreted by the carnivore definitive hosts, e.g. after hand contact with egg-contaminated environments or by consumption of contaminated food or beverages. The goal of this study was to develop a sensitive in vivo method to determine the viability of E. multilocularis eggs and to establish suitable conditions (optimal temperature, exposure time and humidity) for their (prophylactic) inactivation. The sensitivity of a rodent model was evaluated and, conclusively, C57Bl/6 mice were most susceptible to subcutaneous inoculation of small numbers of sodium hypochlorite-resistant oncospheres, even more than to oral inoculation of mature eggs. In the second part of the study, various combinations of exposure temperature (between 45 °C and 80 °C), times (between 30 min and 180 min) and relative humidity (70% vs. suspended in water) were tested. After heat treatment in an incubator, the sodium hypochlorite resistance test was used to assess in vitro egg viability at the time of inoculation. Subsequently, the infectivity of the oncospheres was evaluatedby subcutaneous inoculation in mice. Eggs exposed to increasing temperatures were more resistant to heat if suspended in water as compared to eggs exposed on a filter paper at 70% relative humidity. As survival of eggs in water droplets on the vegetables cannot be excluded, further experiments were performed with eggs suspended in water only. Eggs were infectious after heat exposure at 65 °C for up to 120 min, however, no echinococcosis developed after treatment of the eggs at 65 °C for 180 min or at 70, 75 and 80 °C for 7.5, 15 or 30 min

The impact of socio-cultural factors on transmission of Taenia spp. and Echinococcus granulosus in Kosovo

Echinococcus granulosus sensu latu (s.l.) and Taenia hydatigena are common parasites of ruminant intermediate hosts in the Balkans. Transmission is linked mainly to home slaughtering and the feeding of infected organs to dogs. In Kosovo, many old sheep are slaughtered particularly during Eid al-Adha (Feast of Sacrifice). To determine whether this tradition could affect parasite transmission, we compared the probability of 504 dogs to contract taenid infections after deworming during one period before Eid al-Adha and a similar period beginning with this event. Initially, taeniid eggs were detected in 6·2% (CI 4·2-8·6) of the dogs. The prevalence before Eid al-Adha was significantly lower (1·2%, CI 0·4-2·6) as compared with the prevalence after the event (4·3%, CI 2·6-6·3). A comparable trend was apparent at species level for T. hydatigena and E. granulosus. These results indicate that the pronounced increase of taeniid infections, including E. granulosus s.l., after Eid al-Adha is linked to traditional home slaughtering that occurs during this celebration. This particular epidemiological situation provides an opportunity for implementing focussed control activities

Detection of taeniid (Taenia spp., Echinococcus spp.) eggs contaminating vegetables and fruits sold in European markets and the risk for metacestode infections in captive primates

Due to frequent cases of alveolar echinococcosis (AE) in captive primates in Europe, 141 samples of food, which consisting of vegetables and fruits, were investigated for contamination with egg-DNA of taeniids. Each sample consisted of at least 40 heads of lettuce as well as various vegetables and fruits. The samples were purchased at different times of the year: either from September to November (autumn), originating from greenhouses or fields in the Basel region in the North of Switzerland, or in April and May (spring) when fruit and vegetables are sourced from throughout Europe from various wholesalers. Each sample was washed, and the washing water sieved through mesh apertures of 50 μm and 21 μm, respectively. The debris, including taeniid eggs, collected on the 21 μm sieve were investigated by a multiplex PCR-analysis followed by direct sequencing. In 17 (18%) of the 95 samples collected in autumn, taeniid-DNA was detected (Taenia hydatigena in four, Taenia ovis in three, Taenia polyacantha in two and Hydatigera (Taenia) taeniaeformis in five cases). Similarly, in 13 (28%) of the 46 samples collected during spring taeniid-DNA was detected (Echinococcus granulosus s.l. in two, Taenia crassiceps in one, T. hydatigena in two, Taenia multiceps/Taenia serialis in two, Taenia saginata in one and H. taeniaeformis in five cases). Although DNA of Echinococcus multilocularis was not found specifically in this study, the detection of other fox taeniids reveals that vegetables and fruit fed to the primates at the Zoo Basel at different times of the year and from different origin are contaminated with carnivore's faeces and therefore act as a potential source of AE infections

Detection of Echinococcus granulosus and Echinococcus ortleppi in Bhutan

In this pilot study, fecal samples were collected from community dogs around slaughterhouses and from the city of Thimphu (n = 138) as well as from carnivores in the forest area around a farm in Bhutan (n = 28). Samples were analyzed microscopically for the presence of taeniid eggs by the floatation and sieving method. Further molecular analyses of 20 samples of community dogs positive for taeniid eggs confirmed 10 Echinococcus granulosus sensu lato and one Taenia hydatigena case. From 14 environmental fecal samples from the forest area positive for taeniid eggs, one contained E. granulosus s.l., six T. hydatigena and one Taenia taeniaeformis DNA. In the remaining samples considered positive for taeniid eggs, no molecular confirmation could be achieved. Additionally, Echinococcus cysts were collected from locally slaughtered cattle and imported cattle organs. Seven Echinococcus cysts (one fertile) from the local animals and 35 (four fertile) from imported cattle organs were confirmed as E. granulosus (G1–3) by PCR/sequencing. One Echinococcus cyst each from a local animal and from an imported cattle organ (both fertile) were confirmed to be Echinococcus ortleppi (G5). Sterile Echinococcus cysts were also collected from local yaks (n = 10), and all revealed to be E. granulosus (G1–G3). Hospital records of cystic echinococcosis in humans and the presence of Echinococcus spp. in dogs and ungulates indicate the existence of local transmission for both E. ortleppi and E. granulosus in Bhutan

Echinococcus granulosus and other intestinal helminths: current status of prevalence and management in rural dogs of eastern Australia

OBJECTIVES Ascertain the prevalence of intestinal helminths in rural dogs from eastern Australia and Tasmania. Identify farm management practices contributing to the perpetuation and transmission of Echinococcus granulosus. METHODS Helminth infection in dogs was determined microscopically through faecal flotation. Infection with E. granulosus was determined via faecal antigen-capture ELISA and coproPCR. Taeniid eggs were identified using molecular methods. Data on dog management and owner understanding of hydatid disease were collected via questionnaire. RESULTS Faeces were collected from 1425 Australian rural dogs (1119 mainland; 306 Tasmania). Eggs of hookworms were most prevalent, up to 40.2%, followed by whipworms (Trichuris vulpis), up to 21.2%. Roundworms (Toxocara canis and Toxascaris leonine) were least common, up to 6.1%. Taeniid eggs were found in 11 dogs (5 Taenia pisiformis; 2 T. serialis; 4 T. hydatigena); 2 of the T. hydatigena-infected dogs were also E. granulosus coproantigen-positive. Of the 45 dogs found to be E. granulosus coproantigen-positive, 24 were in Tasmania, 16 in NSW, 3 in Victoria and 2 in Queensland. Three Tasmanian coproantigen ELISA-positive dogs were also coproPCR-positive. The most common dog ration was commercial dry food, but half the owners fed raw meat to their dogs and some fed offal of lambs (8.9%) or mutton (7.8%). More than half (69%) of owners weighed their dogs before deworming. Few dewormed their dogs often enough to ensure they remained cestode-free and owners hunting wildlife usually left carcases where they were shot. CONCLUSIONS E. granulosus is still present in Australian rural dogs, including Tasmania, but at low levels. Owner behaviour perpetuates transmission of cestodes

Successful intestinal Echinococcus multilocularis oncosphere invasion and subsequent hepatic metacestode establishment in resistant RccHan™:WIST rats after pharmacological immunosuppression

Susceptibility/resistance to larval Echinococcus multilocularis infection varies greatly depending on host species and strains. Whereas several mice strains and non-human primates are highly susceptible to alveolar echinococcosis, rats and most of humans are considered as more resistant. In this study, we aimed to elucidate factors responsible for host resistance in rats (Experiments A-D). (A) The parasite establishment was not observed in immunocompetent Wistar rats orally inoculated with sodium hypochlorite resistant eggs with/without pig bile, or activated/non-activated oncospheres (NAO). Peritoneal inoculation with NAO or metacestode tissue allowed the parasite establishment in rats. (B) T-cell-deficient athymic nude rats showed complete resistance against the metacestode establishment after oral inoculation with parasite eggs. This finding suggests that T-cell-independent parasite clearance occurred in the animals during early phase of the parasite invasion. Finally, Wistar rats that received pharmacological immunosuppression using either dexamethasone (DMS) alone or methotrexate (MTX) i.p. alone or a combination of these compounds were orally inoculated with the parasite's eggs. As a result (D), successful establishment of metacestode with protoscoleces was observed in all 3 rats treated with DMS (s.c.) alone or in all 6 rats treated with DMS (s.c.) plus MTX but not in 8 rats with MTX alone, suggesting that factors affected by DMS treatment are responsible to regulate the parasite invasion and establishment