Outcomes of Trypanosoma cruzi and Trypanosoma evansi infections on health of Southern coati (Nasua nasua), crab-eating fox (Cerdocyon thous), and ocelot (Leopardus pardalis) in the Brazilian Pantanal.

The occurrence of Trypanosoma spp. in wild carnivore populations has been intensively investigated during the last decades. However, the impact of these parasites on the health of free-living infected animals has been largely neglected. The Pantanal biome is the world's largest seasonal wetland, harboring a great diversity of species and habitats. This includes 174 species of mammals, of which 20 belong to the order Carnivora. The present study aimed to investigate the effect of Trypanosoma evansi and Trypanosoma cruzi infections and coinfections on the health of the most abundant carnivores in the Pantanal: coati (Nasua nasua), crab-eating fox (Cerdocyon thous), and ocelot (Leopardus pardalis). We captured 39 coatis, 48 crab-eating foxes, and 19 ocelots. Diagnostic tests showed T. cruzi infection in 7 crab-eating foxes and 5 coatis. Additionally, 7 crab-eating foxes, 10 coatis, and 12 ocelots were positive for T. evansi. We observed coinfections in 9 crab-eating foxes, 8 coatis, and 2 ocelots. This is the first report of T. evansi and T. cruzi infection on the health of free-living ocelots and crab-eating foxes. We showed that single T. evansi or T. cruzi infection, as well as coinfection, caused some degree of anemia in all animals, as well as an indirect negative effect on body condition in coatis and crab-eating foxes via anemia indicators and immune investment, respectively. Furthermore, the vigorous immune investment observed in sampled coatis, crab-eating foxes and ocelots infected by T. evansi, T. cruzi and coinfected can be highly harmful to their health. Overall, our results indicate that single and combined infection with T. evansi and T. cruzi represent a severe risk to the health of wild carnivores in the Pantanal region.

Unraveling the key to the resistance of canids to prion diseases.

One of the characteristics of prions is their ability to infect some species but not others and prion resistant species have been of special interest because of their potential in deciphering the determinants for susceptibility. Previously, we developed different in vitro and in vivo models to assess the susceptibility of species that were erroneously considered resistant to prion infection, such as members of the Leporidae and Equidae families. Here we undertake in vitro and in vivo approaches to understand the unresolved low prion susceptibility of canids. Studies based on the amino acid sequence of the canine prion protein (PrP), together with a structural analysis in silico, identified unique key amino acids whose characteristics could orchestrate its high resistance to prion disease. Cell- and brain-based PMCA studies were performed highlighting the relevance of the D163 amino acid in proneness to protein misfolding. This was also investigated by the generation of a novel transgenic mouse model carrying this substitution and these mice showed complete resistance to disease despite intracerebral challenge with three different mouse prion strains (RML, 22L and 301C) known to cause disease in wild-type mice. These findings suggest that dog D163 amino acid is primarily, if not totally, responsible for the prion resistance of canids.

Teste de ELISA indireto para diagnóstico sorológico de leishmaniose visceral em canídeos silvestres / Indirect ELISA for the serological diagnosis of visceral leishmaniasis in wild canids

Na América do Sul, alguns canídeos silvestres são considerados reservatórios naturais da Leishmania chagasi. A resposta imunológica desses animais à Leishmania é pouco conhecida, havendo a necessidade de métodos diagnósticos adequados para esse fim. No presente estudo, é descrita a padronização do ensaio imunoenzimático indireto (ELISA) para o diagnóstico sorológico de leishmaniose visceral em canídeos silvestres brasileiros. Foram estudadas amostras de soro e plasma de 12 canídeos cativos: sete lobos-guará (Chrysocyon brachyurus), três raposinhas (Lycalopex vetulus) e dois cachorros-do-mato (Cerdocyon thous). As amostras de um C. brachyurus e uma L. vetulus, cativos em área endêmica para LV, que apresentavam doença clínica e positividade em testes de Imunofluorescência Indireta e Reação em Cadeia de Polimerase, foram utilizadas como controles positivos. Foram comparados os conjugados anti-IgG de cão e proteína A, ambos ligados a peroxidase, cujos testes detectaram quatro (04/12) e três (03/12) C. brachyurus soropositivos para anticorpos anti-Leishmania sp., respectivamente. As médias das densidades ópticas (DOs) das amostras negativas foram nitidamente mais baixas do que as médias das DOs dos positivos tanto no ELISA com anti-IgG de cão (4,8 vezes) como com proteína A (15,5 vezes). Os soros de três C. brachyurus positivos no ELISA indireto foram avaliados por Western blotting e identificaram 22 bandas, sendo imunodominantes as de peso molecular de 19, 22, 24, 45 e 66 kDa. Os testes ELISA com a proteína A e o conjugado anti-IgG de cão apresentaram respectivamente concordância excelente (Kappa = 1; p<0,001) e moderada (Kappa = 0,8; p<0,0015), com o Western blotting. Ambos foram, portanto, considerados adequados a avaliações de triagem de animais cuja resposta humoral de anticorpos indica contato com o parasito, úteis para subsidiar estudos para adequação de metodologias específicas para os canídeos silvestres.

In South America, some wild canids are considered natural reservoirs of Leishmania chagasi. The immunological response of wild canids to Leishmania is not well understood, and the development of diagnostic methods is necessary for such purpose. In the present study, the standardization of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of visceral leishmaniasis (VL) in Brazilian species of wild canids is described. Serum and plasma samples from 12 captive wild canids were studied: seven from maned wolves (Chrysocyon brachyurus), three from hoary foxes (Lycalopex vetulus), and two from crab-eating foxes (Cerdocyon thous). Samples from C. brachyurus and L. vetulus, both captive in an endemic area for VL, presenting clinical disease and positivity in Indirect Immunofluorescence Reaction and Polymerase Chain Reaction tests were used as positive controls. The antibody anti-dog IgG and Protein A, both conjugated with horseradish peroxidase, were compared in indirect ELISA tests which detected four (04/12) and three (03/12) seropositive C. brachyurus for anti-Leishmania antibodies, respectively. The ELISA tests were able to clearly distinguish negative from positive samples, as the mean optical density (OD) of the negative samples was 4.8 and 15.5 times lower than those of the positive ones either using anti-dog IgG and Protein A, respectively. Samples from three ELISA - positive C. brachyurus were analyzed by Western blotting and identified immunodominant bands of 19, 22, 24, 45 and 66 kDa, among 22 protein bands detected. The ELISAs with protein A and anti-dog IgG showed respectively excellent (Kappa = 1.0; p<0.001) and moderate (Kappa = 0.8; p<0.0015) agreement with the Western blotting assay. The ELISA tests showed to be adequate for screening studies to identify antibody responses, thus indicating contact with Leishmania infection by wild canids.