Molecular diagnosis of diphyllobothriasis in Spain, most presumably acquired via imported fish, or sojourn abroad

Human diphyllobothriasis is sporadically detected in Spain. Diphyllobothrium latum and Diplogonoporus balaenopterae have been identified. In the study, four cases of presumably imported diphyllobothriasis in Spanish patients were appraised. Molecular diagnosis allowed us to identify 'exotic' fish tapeworms such as Diplogonoporus balaenopterae in one patient and Diphyllobothrium pacificum in the others.

Genetic identification of an oxyurid from a captive, black-handed spider monkey—implications for treatment and control

Parasites are of major clinical significance in captive primates in zoos, particularly those with direct life cycles. Oxyurid nematodes can be a persistent problem, as infection intensity and environmental contamination with infective eggs are usually high. Observations at the Basel Zoo in Switzerland have revealed that particularly black-handed spider monkeys (Ateles geoffroyi) exhibit continuous oxyurid nematode infection(s), despite regular deworming with anthelmintics. In the present study, using a molecular approach, we were able to identify the nematode (Trypanoxyuris atelis) causing this ongoing problem, and we are now evaluating a practical treatment and control regimen to tackle this parasite problem.

Treatment of echinococcosis: albendazole and mebendazole - what else?

The search for novel therapeutic options to cure alveolar echinococcosis (AE), due to the metacestode of Echinococcus multilocularis, is ongoing, and these developments could also have a profound impact on the treatment of cystic echinococcosis (CE), caused by the closely related Echinococcus granulosus s.l. Several options are being explored. A viable strategy for the identification of novel chemotherapeutically valuable compounds includes whole-organism drug screening, employing large-scale in vitro metacestode cultures and, upon identification of promising compounds, verification of drug efficacy in small laboratory animals. Clearly, the current focus is targeted towards broad-spectrum anti-parasitic or anti-cancer drugs and compound classes that are already marketed, or that are in development for other applications. The availability of comprehensive Echinococcus genome information and gene expression data, as well as significant progress on the molecular level, has now opened the door for a more targeted drug discovery approach, which allows exploitation of defined pathways and enzymes that are essential for the parasite. In addition, current in vitro and in vivo models that are used to assess drug efficacy should be optimized and complemented by methods that give more detailed information on the host-parasite interactions that occur during drug treatments. The key to success is to identify, target and exploit those parasite molecules that orchestrate activities essential to parasite survival.

Immunoblotting for the serodiagnosis of alveolar echinococcosis in alive and dead Eurasian beavers (Castor fiber)

A novel species-specific anti-beaver-IgG-alkaline-phosphatase conjugate was synthesized for the development of a new serological test for echinococcosis in beavers. Two different ELISAs conventionally used for human Echinococcus multilocularis serology (Em18-ELISA and Em2-ELISA) yielded diagnostic sensitivities of 0% and 46%, respectively. In contrast, the subsequently developed immunoblotting assay gave an 85% diagnostic sensitivity (11 out of 13 beavers with alveolar echinococcosis were immunoblotting-positive, i.e. showed reactivity with a specific 21 Mr band), and maximal specificity. In conclusion, this immunoblotting assay should be the method of choice for use in serological studies on E. multilocularis in Eurasian beavers, and the test proved suitable to investigate both animals alive and post-mortem.

Comparative assessment of ELISAs using recombinant saposin-like protein 2 and recombinant cathepsin L-1 from Fasciola hepatica for the serodiagnosis of human Fasciolosis.

Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2) and cathepsin L-1 (recCL1), were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG) conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory-secretory products (FhES) from adult stage liver flukes was assessed by receiver operator characteristic (ROC) analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n=20), patients with other parasitic infections (n=87) and patients with malignancies (n=121). The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy) employing the threshold (cut-off) to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls.

Echinococcus P29 antigen: molecular characterization and implication on post-surgery follow-up of CE patients infected with different species of the Echinococcus granulosus complex.

The protein P29 is a potential serological marker for post-treatment monitoring of cystic echinococcosis (CE) especially in young patients. We now have demonstrated that P29 is encoded in the Echinococcus genus by a single gene consisting of 7 exons spanning 1.2 kb of DNA. Variability of the p29 gene at inter- and intra-species level was assessed with 50 cDNA and 280 genomic DNA clones isolated from different E. granulosus s.l. isolates (E. granulosus sensu stricto (G1), E. equinus (G4), E. ortleppi (G5), E. canadensis (G6), E. canadensis (G7) and E. canadensis (G10)) as well as four E. multilocularis isolates. Scarce interspecies polymorphism at the p29 locus was observed and affected predominantly E. granulosus s.s. (G1), where we identified two alleles (A1 and A2) coding for identical P29 proteins and yielding in three genotypes (A1/A1, A2/A2 and A1/A2). Genotypic frequencies expected under Hardy-Weinberg equilibrium revealed a high rate of heterozygosity (47%) that strongly supports the hypothesis that E. granulosus s.s. (G1) is predominantly outbreeding. Comparative sequence analyses of the complete p29 gene showed that phylogenetic relationships within the genus Echinococcus were in agreement with those of previous nuclear gene studies. At the protein level, the deduced P29 amino acid (AA) sequences exhibited a high level of conservation, ranging from 97.9% AA sequence identity among the whole E. granulosus s.l. group to 99.58% identity among E. multilocularis isolates. We showed that P29 proteins of these two species differ by three AA substitutions without implication for antigenicity. In Western-blot analyses, serum antibodies from a human CE patient infected with E. canadensis (G6) strongly reacted with recombinant P29 from E. granulosus s.s. (G1) (recEg(G1)P29). In the same line, human anti-Eg(G1)P29 antibodies bound to recEcnd(G6)P29. Thus, minor AA sequence variations appear not to impair the prognostic serological use of P29.

Laboratory diagnosis of ruminant abortion in Europe.

Abortion in ruminants is a major cause of economic loss worldwide, and the management and control of outbreaks is important in limiting their spread, and in preventing zoonotic infections. Given that rapid and accurate laboratory diagnosis is central to controlling abortion outbreaks, the submission of tissue samples to laboratories offering the most appropriate tests is essential. Direct antigen and/or DNA detection methods are the currently preferred methods of reaching an aetiological diagnosis, and ideally these results are confirmed by the demonstration of corresponding macroscopic and/or histopathological lesions in the fetus and/or the placenta. However, the costs of laboratory examinations may be considerable and, even under optimal conditions, the percentage of aetiological diagnoses reached can be relatively low. This review focuses on the most commonly occurring and important abortifacient pathogens of ruminant species in Europe highlighting their epizootic and zoonotic potential. The performance characteristics of the various diagnostic methods used, including their specific advantages and limitations, are discussed.

Alveolar hydatid disease (Echinococcus multilocularis) in the liver of a Canadian dog in British Columbia, a newly endemic region.

An adult dog that lived in central British Columbia was examined because of a history of lethargy and vomiting. Histology, immunohistochemistry, and polymerase chain reaction (PCR) examination of a hepatic mass confirmed the presence of an alveolar hydatid cyst, the first description of Echinococcus multilocularis in British Columbia. We provide recommendations for case management and remind practitioners in endemic areas of western Canada that dogs can serve as definitive and, rarely, intermediate hosts for E. multilocularis.

First cases of besnoitiosis in cattle in Switzerland

Bovine besnoitiosis has been diagnosed in neighboring countries but not in Switzerland so far. This disease occurs endemically in France and focal outbreaks have been reported in Germany and Italy. To determine if Besnoitia besnoiti is introduced into Switzerland through the import of breeding cattle from France, a systematic serological survey was performed. A total of 412 breeding cattle (from 114 farms) imported from France into Switzerland between 2005 and 2011, were serologically examined for antibodies against B. besnoiti using a commercial ELISA kit (PrioCHECK© Besnoitia Ab 2.0, Prionics AG, Zurich, Switzerland). Sixty-four (15.5 %) animals reacted positive in ELISA. The serologic diagnosis was confirmed by an indirect immunfluorescence test (IFAT) and a Western blot (WB) in only 2 Limousin cows imported from France on a farm in Eastern Switzerland. Subsequently, this whole herd (n = 16) was examined clinically and serologically and 2 additional Limousin cows imported from Germany also reacted positive in the three serological tests. One of these cows presented B. besnoiti tissue cysts in the scleral conjunctiva and typical skin lesions in the head region. The infection was further confirmed cytologically, histopathologically and by PCR. It can be concluded that the parasite is most likely being introduced into Switzerland through the import of infected animals.