Molecular epidemiology and diagnostics for Echinococcus multilocularis in canid definitive and intermediate hosts

Echinococcus multilocularis shed by canid definitive hosts causes alveolar echinococcosis (AE) in rodent intermediate hosts as well as human and dog aberrant hosts. AE is debilitating in humans and dogs, resulting in serious health challenges and expenses for both veterinary and public health. This thesis addresses needs to improve molecular and serological diagnostics for E. multilocularis in wild canids and dogs. I adapted and evaluated an in-house copro-qPCR-MCA which has comparable diagnostic sensitivity and specificity with the ‘gold standard’ adult cestode recovery, and superior diagnostic sensitivity (92% vs 39%) when compared to conventional fecal flotation techniques. I demonstrated E. multilocularis prevalence of 72% in coyotes in Saskatchewan, a highly endemic region, and using the copro-qPCR-MCA, 17% in coyotes from a newly endemic and highly populated area of British Colombia, and 10% in foxes from islands in the western Canadian Arctic. I also demonstrated parasite stage specificity of the Em95 antigen for serological diagnosis of canine AE in coyotes with intestinal infections, suggesting that serology for the Em95 antigen is likely to be an excellent tool for detecting cases of AE in dogs in North America, where this disease is increasingly described. My thesis also described 27 cases of canine AE from western Canada, highlighting important clinical, epidemiological, and economic information for veterinary practitioners and dog owners and indicating that dogs with AE may serve as indicators of parasite range expansion and risk to humans. Finally, molecular epidemiology revealed that the haplotypes present in intestines of wild canids and AE cases in dogs and humans in prairie regions of western Canada are highly pathogenic and zoonotic European strains, compared to the N2 strain previously described in central North America. In the western Canadian Arctic, my thesis reports, for the first time, that the established haplotype of E. multilocularis is N1 North American Arctic strain, and not Asian strains reported from the west coast of Alaska, nor the N2 or European strains established in populated regions of southern Canada. Therefore, the Canadian Arctic remains vulnerable to introduction of Asian and European strains with higher zoonotic potential. This thesis provides useful diagnostic tools for large scale prevalence studies of E. multilocularis in canids, as well as data on the molecular epidemiology and prevalence which can guide the formulation of control and prevention policies

Intestinal infection with Echinococcus multilocularis in a dog.

OBJECTIVE To raise veterinary awareness of a newly recognized parasitic threat to canine and human health, highlight the increasing availability of molecular parasitological diagnostics and the need to implement best practices of cestocidal use in high-risk dogs. ANIMAL A young Boxer dog with vomiting and bloody diarrhea, suspected diagnosis of inflammatory bowel disease. CLINICAL PRESENTATION, PROGRESSION, AND PROCEDURES Bloodwork revealed inflammation, dehydration, and protein loss, addressed with supportive therapy. Fecal culture revealed only Escherichia coli. On centrifugal flotation, tapeworm eggs (which could be Taenia or Echinococcus spp) and, unusually, adult cestodes of Echinococcus were observed. The referring veterinarian was contacted to initiate immediate treatment with a cestocide due to zoonotic potential. Diagnosis was confirmed with a coproPCR which has higher sensitivity for Echinococcus spp than fecal flotation alone. DNA was identical to an introduced European strain of E multilocularis currently emerging in dogs, people, and wildlife. Since dogs can also self-infect and develop hepatic alveolar echinococcosis (severe and often fatal), this was ruled out using serology and abdominal ultrasound. TREATMENT AND OUTCOME Following cestocidal treatment, fecal flotation and coproPCR were negative for eggs and DNA of E multilocularis; however, coccidia were detected and diarrhea resolved following treatment with sulfa-based antibiotics. CLINICAL RELEVANCE This dog was serendipitously diagnosed with E multilocularis, acquired through ingestion of a rodent intermediate host likely infected from foxes and coyotes. Therefore, as a dog at high risk of reexposure from eating rodents, regular (ideally monthly) treatment with a labeled cestocide is indicated going forward

Molecular dynamics and combined docking studies for the identification of Zaire ebola virus inhibitors

<p>Ebola virus (EBOV) is a lethal human pathogen with a risk of global spread of its zoonotic infections, and <i>Ebolavirus Zaire</i> specifically has the highest fatality rate amongst other species. There is a need for continuous effort towards having therapies, as a single licensed treatment to neutralize the EBOV is yet to come into reality. This present study virtually screened the MCULE database containing almost 36 million compounds against the structure of a Zaire Ebola viral protein (VP) 35 and a consensus scoring of both MCULE and CLCDDW docking programs remarked five compounds as potential hits. These compounds, with binding energies ranging from –7.9 to –8.9 kcal/mol, were assessed for predictions of their physicochemical and bioactivity properties, as well as absorption, distribution, metabolism, excretion, and toxicity (ADMET) criteria. The results of the 50 ns molecular dynamics simulations showed the presence of dynamic stability between ligand and protein complexes, and the structures remained significantly unchanged at the ligand-binding site throughout the simulation period. Both docking analysis and molecular dynamics simulation studies suggested strong binding affinity towards the receptor cavity and these selected compounds as potential inhibitors against the Zaire Ebola VP 35. With respect to inhibition constant values, bioavailability radar and other physicochemical properties, compound A (MCULE-1018045960-0-1) appeared to be the most promising hit compound. However, the ligand efficiency and ligand efficiency scale need improvement during optimization, and also validation via in vitro and in vivo studies are necessary to finally make a lead compound in treating Ebola virus diseases.</p

Canine Alveolar Echinococcosis: An Emerging and Costly Introduced Problem in North America

Alveolar echinococcosis (AE), caused by the metacestode of Echinococcus multilocularis, is emerging in both dogs and people in North America. Here, we review 27 cases of canine AE opportunistically reported since the index case was described in 2009 in Western Canada. We describe clinical presentation, diagnosis, treatment, outcome, and source of canine infection, based on genetics of the parasite isolated from some canine cases. Diagnosis of AE was by histopathology and/or PCR on DNA extracted from metacestodes. The median age of dogs at diagnosis was 4 years (range 1–12), which is low compared to neoplasia, the most common differential diagnosis. There was no sex predilection and different breeds were involved, but there were a disproportionate number of boxers and beagles relative to their representation in the general canine population. The most common potential risk factors included contact with wildlife and visits to off leash areas. Abdominal distension was the most common clinical sign at presentation, and medical imaging generally revealed an abdominal mass. On histopathology, protoscoleces were observed in 7 out of 14 dogs. In 7 cases, DNA sequences were most similar to European (versus North American) haplotypes, identical to those recently reported in coyotes as definitive hosts in North America, and different between eastern and western North America, implying multiple introduction events. Dogs that were not treated (n = 6) had 16% survival in the first 100 days in comparison with 82% survival of treated dogs (n = 11). Direct costs to the owner of treating canine AE ranged from 1,317 to 12,655 CAD depending on the situation at the onset of treatment. This study provides important clinical, epidemiological, and economic information for veterinary practitioners and regulators for importation of dogs, and for public health, as dogs with AE may serve as indicators of parasite range expansion and risk to humans.Peer Reviewe