DEVELOPMENT OF A TRIPLEX REAL-TIME PCR ASSAY TO DETECT ECHINOCOCCUS SPECIES IN CANID FECAL SAMPLES.

Echinococcosis is a zoonotic disease with great significance to public health, and appropriate detection and control strategies should be adopted to mitigate its impact. Most cases of echinococcosis are believed to be transmitted by the consumption of food and/or water contaminated with canid stool containing Echinococcus spp. eggs. Studies assessing Echinococcus multilocularis, Echinococcus granulosus sensu stricto, and Echinococcus shiquicus coinfection from contaminated water-derived, soil-derived, and food-borne samples are scarce, which may be due to the lack of optimized laboratory detection methods. The present study aimed to develop and evaluate a novel triplex TaqMan-minor groove binder probe for real-time polymerase chain reaction (rtPCR) to simultaneously detect the 3 Echinococcus spp. mentioned above from canid fecal samples in the Qinghai-Tibetan Plateau area (QTPA). The efficiency and linearity of each signal channel in the triplex rtPCR assay were within acceptable limits for the range of concentrations tested. Furthermore, the method was shown to have good repeatability (standard deviation ≤0.32 cycle threshold), and the limit of detection was estimated to be 10 copies plasmid/µl reaction. In summary, the evaluation of the present method shows that the newly developed triplex rtPCR assay is a highly specific, precise, consistent, and stable method that could be used in epidemiological investigations of echinococcosis.

Frequency of parasitic forms in fecal samples of maned wolf in different seasons of the year at the Serra da Canastra National Park, Minas Gerais State, Brazil / [Frequência de formas parasitárias em massas fecais de lobos-guarás em diferentes estações do ano no Parque Nacional da Serra da Canastra, MG]

O Parque Nacional da Serra da Canastra (PNSC), localizado no estado de Minas Gerais, é um importante habitat de inúmeras espécies de animais ameaçados de extinção, como o lobo-guará (Chrysocyon brachyurus). Parasitos de animais selvagens podem representar um problema para os programas de manejo e recuperação de espécies ameaçadas, pois atuam como causa primária ou agravante de inúmeras doenças. Dependendo da época do ano, a suscetibilidade ao parasitismo pode ser maior devido à facilidade de infecção. Com o objetivo de avaliar a frequência de endoparasitos e a sazonalidade dessas parasitoses em diferentes épocas do ano, foram examinadas 103 amostras fecais de lobos-guarás, coletadas no PNSC, durante o período de março de 2017 a agosto de 2019. O número de amostras positivas para pelo menos uma espécie de parasito foi de 47 amostras (45,63%), sendo o outono a estação em que foi encontrada a maior frequência de formas parasitárias, com 60,86% (14/23) de amostras positivas, seguido do inverno, com 52,38% (11/21), verão com 37,5% (15/40), e primavera com 36,84% (7/19). Dentre os parasitos encontrados, Capillaria sp. apresentou a maior frequência, sendo encontrado em 23 amostras (22,33%), seguido de trematódeos, em 15 amostras (14,56%), acantocéfalos, ascarídeos, Trichuris sp. e Ancylostoma sp., em cinco amostras (4,85%), nematoides da superfamília Strongyloidea, Lynxacarus sp., em duas amostras (1,94%), e pentastomídeos em uma amostra (0,97%).(AU)

Rangelia vitalii molecular and histological quantification in tissues comparing crab-eating foxes (Cerdocyon thous) and domestic dogs.

Rangeliosis is a condition transmitted by the tick Amblyomma aureolatum and caused by the protozoan parasite Rangelia vitalii in canids. In domestic dogs, the disease causes a severe hemolytic disease, while in wild canids the piroplasm is often detected without any clinical abnormality. This study aimed to detect and quantify the number of copies of the R. vitalii Hsp70 gene (indirect parasite burden) in several organs of domestic and South American wild canids (Cerdocyon thous and Lycalopex gymnocercus) to elucidate distinct clinical presentations of rangeliosis in these species. A total of seven domestic dogs that died due to rangeliosis and 38 wild foxes were initially included, with all dogs presenting histological and molecular features of rangeliosis, while eight C. thous were positive at the molecular analysis for R. vitalii. Fragments of 22 organs collected from domestic (n = 7) and wild foxes (n = 8) were employed for histological and molecular quantification using real-time polymerase chain reaction aiming at the Hsp70 gene. Histologically, parasitophorous vacuoles were constantly detected in the dogs, while these were detected only in two C. thous. Parasitic burden was significantly higher in the digestive, cardiorespiratory, endocrine, genitourinary, and skeletal-muscle systems of domestic dogs when compared to wild foxes. In the hematopoietic system of wild canids, some organs, such as the lymph nodes and tonsils, presented significantly lower amounts of R. vitalii, while other organs (spleen, bone marrow, and blood) had results similar to those of domestic dogs. Additionally, the central nervous system of both domestic and wild canids presented a similar quantity of R. vitalii. The etiological agent is possibly maintained through an asexual reproductive process (merogony) in both domestic and wild species. Nonetheless, a limited or short-duration schizogony phase occurs in C. thous, which would designate this species as a possible reservoir host for the agent. Dogs, in contrast, would most likely act as accidental hosts, presenting a severe and more pathogenic schizogony phase, resulting in characteristic clinical and pathological rangeliosis.

A shared pathogen: Babesia rossi in domestic dogs, black-backed jackals (Canis mesomelas) and African wild dogs (Lycaon pictus) in South Africa.

In sub-Saharan Africa, babesiosis in domestic dogs is caused primarily by Babesia rossi. Black-backed jackals (Canis mesomelas), which are subclinical carriers of B. rossi, were a likely reservoir host from which infection passed to domestic dogs. The role of other indigenous canids, e.g. African wild dogs (Lycaon pictus), as reservoirs of B. rossi has not been elucidated. The question also arises whether genetic differences have arisen between B. rossi infecting domestic dogs and "ancestral" B. rossi in jackals. In a previous study we found that nearly one-third (27 of 91) of jackals were infected with B. rossi; this was confirmed by 18S rDNA sequence analysis. In this study, the near full-length B. rossi 18S rRNA gene was successfully amplified from 6 domestic dogs and 3 black-backed jackals. The obtained recombinant sequences were identical (100 %) to previously described B. rossi sequences of black-backed jackals in South Africa, and 99 % similar to B. rossi from dogs in South Africa and the Sudan. Although blood specimens from 5 (10 %) of 52 free-ranging African wild dogs (from Kruger National Park, South Africa, reacted with the B. rossi probe on RLB hybridisation, the presence of B. rossi could not be confirmed by amplification and sequencing, nor by multiplex, real-time PCR. Although African wild dogs they can be infected with B. rossi without showing clinical signs, our findings suggest that they are apparently not important reservoir hosts of B. rossi.

Molecular phylogenetic study in Spirocercidae (Nematoda) with description of a new species Spirobakerus sagittalis sp. nov. in wild canid Cerdocyon thous from Brazil.

The nematode family Spirocercidae Chitwood and Wehr, 1932, comprises three subfamilies, Spirocercinae Chitwood and Wehr, 1932; Ascaropsinae Alicata and McIntosh, 1933; and Mastophorinae Quentin, 1970, which occur worldwide. Spirocercids infect canids and can cause severe illness. The crab-eating fox, Cerdocyon thous (Linnaeus, 1766), is a canid that inhabits most of South America, including Brazil and is a host for several parasitic worms, in particular, nematodes. However, few reports or genetic data are available on the spirocercids found in this host. In the present study, we describe a new species of Spirobakerus Chabaud and Bain 1981, from the intestine of two crab-eating foxes from two different biomes in Brazil. Spirobakerus sagittalis sp. nov. presents a) unequal spicules, with a long, thin left spicule with a lanceolated shape at the tip; b) a pair of sessile papillae and a median unpaired papillae located anteriorly of the cloaca, and c) a tuft without spines at the tip of the tail in females. The molecular phylogenetic analysis indicated that S. sagittalis sp. nov. is basal to the other species of the subfamily Ascaropsinae, which was not recovered as monophyletic. Our phylogenies also indicated that Spirocercidae is paraphyletic, given that Mastophorinae did not group with Ascaropsinae and Spirocercinae. We provide the first molecular data on the genus Spirobakerus and expand the molecular database of the spirocercids. However, further studies, including the sequences of other spirocercid taxa, are still needed to infer the relationships within this family more accurately.

Copro-polymerase chain reaction has higher sensitivity compared to centrifugal fecal flotation in the diagnosis of taeniid cestodes, especially Echinococcus spp, in canids.

Prompt and reliable diagnostic tests for taeniid infection in canids are important due to the risk of zoonoses like Echinococcus spp. Current diagnostic methods relying on fecal flotation lack sensitivity and specificity, but this has rarely been quantified due to the challenges in performing adult cestode recovery (the gold standard) in domestic dogs (Canis familiaris). Therefore, we recovered adult Taenia and Echinococcus spp. from intestines, as well as fecal/intestinal material from 484 wild canids trapped for fur in two Canadian provinces (276 foxes - primarily Vulpes vulpes, coyotes - Canis latrans, and wolves - Canis lupus in Québec and 208 coyotes in Saskatchewan). The performances of a newly developed coproPCR for tapeworm DNA detection in dogs, and centrifugal fecal flotation using Sheather's solution, were evaluated against adult cestode recovery. Overall, adult taeniid cestode prevalence (Taenia and/or Echinococcus) was 28 % (95 % CI: 23-33 %) in Québec (62 % (CI: 51-73%) of 74 coyotes, 65 % (CI: 44-82) of 23 wolves, and 11 % (CI: 7-16%) of 179 foxes) and 79 % (CI: 73-84%) of 208 coyotes in Saskatchewan. In Québec, E. canadensis and Taenia spp. were detected in coyotes and wolves, and foxes were only infected with Taenia spp., whereas Saskatchewan coyotes were predominantly infected with E. multilocularis (at significantly higher prevalence, but not intensity, than coyotes in Québec). Compared with centrifugal fecal flotation, the new coproPCR had at least double the sensitivity (58 % vs 23 % in QC coyotes, 57 % vs 23 % in QC wolves, 24 % vs 0% in QC foxes, and 80 % vs 25 % in SK coyotes). Notably, no taeniid eggs were detected on flotations from foxes infected with Taenia spp., and the new coproPCR had highest sensitivity in Saskatchewan coyotes, which were predominantly infected with E. multilocularis. CoproPCR has promising prospects for use in Veterinary clinics and diagnostic laboratories to detect taeniid cestode infections because of its higher sensitivity than faecal flotation methods. This is particularly important for zoonotic Echinococcus spp. where, from a public health perspective, false negatives are a much greater concern than false positives.

Dioctophymosis in a free-ranging maned wolf (Chrysocyon brachyurus): clinical-therapeutic, ultrasonographic and pathological aspects - case report / [Dioctofimose em um lobo guará (Chrysocyon brachyurus) de vida livre: aspectos clínico-terapêuticos, ultrassonográficos e patológicos - relato de caso]

Dioctophymosis is the disease caused by Dioctophyma renale, a large nematode, popularly known as giant kidney worm, and whose definitive hosts are the domestic dog and many wild mammal species. There are some reports of maned wolf parasitism by D. renale, however, in most cases the reports are restricted to the finding of the parasite during necropsy, without clinical assessment. The present report aimed to describe the clinical-therapeutic, ultrasonographic and pathological aspects of D. renale parasitism in a free-ranging maned wolf, emphasizing the treatment with an association of doramectin, praziquantel, pyrantel pamoate, and febantel that resulted in complete elimination of the parasite.(AU)

A dioctofimose é a doença causada pelo Dioctophyma renale, um nemátodo grande, popularmente chamado de verme gigante do rim e que tem como hospedeiro definitivo o cão doméstico e inúmeras espécies de mamíferos silvestres. Existem alguns relatos do parasitismo do lobo-guará por D. renale, contudo, na maioria das vezes, os relatos se restringem apenas ao achado do parasita durante a necropsia, sem a correlação clínica. O presente relato objetiva descrever os aspectos clínico-terapêuticos, ultrassonográficos e patológicos do parasitismo por D. renale em um lobo-guará de vida livre, enfatizando o tratamento com uma associação de doramectina, praziquantel, pamoato de pirantel e febantel, o que resultou na eliminação completa do parasita.(AU)

Pterygodermatites (Multipectines) affinis (Jägerskiöl, 1904) (Nematoda: Rictulariidae), a parasite of Cerdocyon thous (Linnaeus, 1766) (Carnivora: Canidae) from the Brazilian Amazon.

Among the Nematodes of Cerdocyon thous, the genus Pterygodermatites is characteristic for presenting cuticular projections along the body, being mentioned in different hosts in South America, although there are no records of its occurrence in the Amazon Biome. To enable further exploration in terms of their morphology and morphometry, the nematodes collected from cadavers of C. thous were fixed and observed using bright field microscopy and scanning electron microscopy, revealing characteristics compatible with P. (Multipectines) affinis, thus contributing more information about the geographic distribution of the parasite, as well as knowledge of the helminthological fauna of wild mammals in the Brazilian Amazon.

Hemoplasma infection in wild captive carnivores / Infecção por hemoplasmas em carnívoros selvagens cativos

Hemoplasmas are bacteria able to adhere themselves loosely to the plasma membrane of erythrocytes and may parasitize several species of mammals. There are three known species of hemoplasmas that parasitize domestic and wild cats: Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' and 'Candidatus Mycoplasma turicensis'. Dogs are infected by at least two species of hemoplasmas: 'Candidatus Mycoplasma haematoparvum' and Mycoplasma haemocanis. The hemoplasmoses are very important in veterinary clinics, either because of its worldwide distribution and severity of clinical signs, depending on parasite species and host immune competence, or due to its zoonotic potential and capability of infecting endangered species. This study set out to investigate which hemoplasmas species parasitize different captive wild carnivores in order to clarify the epidemiology of hemoplasmoses in wild animals. Furthermore, the research intended to characterize the hematological changes caused by different species of hemotropic mycoplasmas infection in order to establish their clinical importance to wild species and the capacity of these species to become a reservoir of studied agents. Samples of 33 wild felids and 18 wild canids were investigated using polymerase chain reaction (PCR) to detect hemoplasmas DNA and it was observed that the occurrence of infection in these species is 45.5% and 83.3%, respectively. Factors such as age, gender or anaemia are not more frequent in animals positive for the infection. Therefore, it is concluded that infection caused by hemoplasmas in wild carnivores has high prevalence, and either agent pathogenicity is low, or chronic stage is more frequent, resulting in a low rate of diagnosis.(AU)

Hemoplasmas são bactérias capazes de aderir frouxamente à membrana plasmática de eritrócitos e que podem parasitar diversas espécies de mamíferos. São conhecidas três espécies de hemoplasmas que parasitam felídeos domésticos e selvagens: Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' and 'Candidatus Mycoplasma turicensis'. Cães são infectados por ao menos duas espécies de hemoplasmas: Candidatus Mycoplasma haematoparvum' and Mycoplasma haemocanis. As hemoplasmoses são de grande importância na clínica veterinária, tanto pela sua distribuição ubíqua e severidade dos sinais clínicos, a depender da espécie do parasita e imunocompetência do hospedeiro, quanto pelo seu potencial zoonótico e capacidade de infectar espécies ameaçadas. Este estudo visa investigar quais espécies de hemoplasmas parasitam diferentes carnívoros selvagens de cativeiro, a fim de esclarecer a epidemiologia das hemoplasmoses em animais selvagens. Além disso, o trabalho objetivou caracterizar as alterações hematológicas causadas pela infecção por diferentes espécies de micoplasmas hemotrópicos visando estabelecer sua importância clínica para espécies selvagens e a capacidade destas espécies de se tornar reservatórios dos agentes estudados. Amostras de 33 felídeos selvagens e de 18 canídeos selvagens foram investigadas por meio da reação em cadeia da polimerase (RCP) para detectar o DNA dos agentes e foi observado que a ocorrência da infecção por hemoplasmas nestas espécies é de 45,5% e 83,3%, respectivamente. Fatores como idade, sexo ou anemia não são mais frequentes em animais positivos para a infecção. Dessa forma, conclui-se que a infecção causada por hemoplasmas em carnívoros selvagens possui alta prevalência, no entanto ou a patogenicidade dos agentes é baixa ou o estágio crônico da infecção é mais frequente, resultando em uma baixa frequência diagnóstica.(AU)

First confirmed case of Alaria alata mesocercaria in Eurasian lynx (Lynx lynx) hunted in Latvia.

Trematode Alaria spp. have a complex life cycle that includes canids, mustelids, and felids as definitive hosts. However, several mammal species, besides acting as definitive hosts, may also act as paratenic hosts. Alaria spp. life cycles are well studied. Still, there is a lack of knowledge on one host significance as both-the paratenic and definitive host, especially in the case of Eurasian lynx (Lynx lynx). Previous studies have described Eurasian lynx as a definitive host for A. alata although the prevalence has always been low. The aim of the present study was to analyze the occurrence of A. alata in Eurasian lynx in Latvia. The viscera (n = 231) and muscle samples (n = 234) from Eurasian lynx were collected during the hunting seasons from 2004/2005 to 2017/2018. The prevalence of the adult A. alata in Eurasian lynx was 1.7%. However, mesocercariae infection in Eurasian lynx was detected for the first time. These findings suggest that the Eurasian lynx may serve both as paratenic and definitive host for A. alata.

Pterygodermatites (Multipectines) affinis (Jägerskiöl, 1904) (Nematoda: Rictulariidae), a parasite of Cerdocyon thous (Linnaeus, 1766) (Carnivora: Canidae) from the Brazilian Amazon / Pterygodermatites (Multipectines) affinis (Jägerskiöl, 1904) (Nematoda: Rictulariidae), um parasito de Cerdocyon thous (Linnaeus, 1766) (Carnivora: Canidae) da Amazônia brasileira

Abstract Among the Nematodes of Cerdocyon thous, the genus Pterygodermatites is characteristic for presenting cuticular projections along the body, being mentioned in different hosts in South America, although there are no records of its occurrence in the Amazon Biome. To enable further exploration in terms of their morphology and morphometry, the nematodes collected from cadavers of C. thous were fixed and observed using bright field microscopy and scanning electron microscopy, revealing characteristics compatible with P. (Multipectines) affinis, thus contributing more information about the geographic distribution of the parasite, as well as knowledge of the helminthological fauna of wild mammals in the Brazilian Amazon.

Resumo Entre os nematodas de Cerdocyon thous, o gênero Pterygodermatites é característico por apresentar projeções cuticulares ao longo do corpo, sendo citado em diferentes hospedeiros na América do Sul, porém, sem registros de sua ocorrência no Bioma Amazônia. Passível de maior exploração quanto à sua morfologia e morfometria, os nematoides colhidos de cadáveres de C. thous foram fixados e observados em microscopia de campo claro e microscopia eletrônica de varredura, revelando características compatíveis com P. (Multipectines) affinis. Com isso contribuiu com mais informações sobre a distribuição geográfica do parasito, bem como o conhecimento da fauna helmintológica de mamíferos selvagens na Amazônia brasileira.

Hepatozoon sp. gamonts as an accidental finding in synovial liquid from an injured maned wolf (Chrysocyon brachyurus) in southeastern Brazil / Gamontes de Hepatozoon sp. como achado acidental em líquido sinovial de lobo guará (Chrysocyon brachyurus) no sudeste do Brasil

Abstract A free-living, adult male maned wolf (Chrysocyon brachyurus) was referred to the Governador "Laudo Natel" - FCAV/Unesp veterinary hospital after being found with skin lesions and a fracture on the right pelvic limb, which had to be amputated due to compromised integrity. Around 20 days later, bilateral accentuated swollen on humerus-radius-ulna articulation was observed. The synovial liquid was drained and sent to the laboratory for synovial cytology with Rosenfeld staining that revealed predominantly degenerated neutrophils with karyolytic chromatin associated with intracellular inclusions suggestive of Hepatozoon sp. gametocytes. Blood and synovial liquid samples were submitted to molecular analysis, aiming to amplify the Hepatozoon spp. 18S rRNA gene fragment. Despite the positioning of the found Hepatozoon sequence together with Hepatozoon canis previously detected in domestic carnivores, the BLAST analysis showed only 98% identity with H. canis. To the best of the authors' knowledge, this is the first time a Hepatozoon was detected in the synovial liquid by clinical pathology and molecular analyses.

Resumo Um lobo guará (Chrysocyon brachyurus) adulto, macho, de vida livre foi encaminhado para atendimento no hospital veterinário Governador "Laudo Natel" - FCAV/Unesp após ser encontrado com lesões de pele e fratura em membro pélvico direito, sendo amputado devido a comprometimento da integridade do membro. Aproximadamente 20 dias após a chegada ao hospital, foi notado acentuado aumento de volume bilateral em região de articulação úmero-rádio-ulnar. O líquido sinovial foi drenado e enviado para análise citológica com coloração de Rosenfeld, revelando a presença de neutrófilos degenerados com cromatina cariolítica associados a inclusões intracelulares sugestivas de gametócitos de Hepatozoon sp. Amostras de sangue e líquido sinovial foram submetidas a análises moleculares visando amplificar um fragmento do gene 18S rRNA de Hepatozoon spp. Apesar da sequência de Hepatozoon detectada se posicionar filogeneticamente no mesmo clado que H. canis previamente detectado em carnívoros domésticos, o resultado da análise do BLAST mostrou somente 98% de identidade com H. canis. De acordo com o conhecimento dos autores, esta é a primeira vez que Hepatozoon foi detectado no líquido sinovial por meio de patologia clínica e análises moleculares.

Secretome of the carcinogenic helminth Spirocerca lupi reveals specific parasite proteins associated with its different life stages.

Spirocerca lupi is a parasitic and carcinogenic nematode of canids distributed in tropical and subtropical regions around the world. The excretion-secretion proteins (PES) of S. lupi have been suggested to play a role in the pathogenesis of its infection. We aimed to identify the PES of different stages of S. lupi and search for proteins that would be useful for diagnostic, therapeutic and vaccination purposes as well as understand their functions. A nano-UPLC mass spectrometry de novo analysis was performed on proteins collected from cultures of S. lupi L3 larvae, L4 females, adult females and adult males from naturally infected hosts. A total of 211 proteins were identified in all cultures. Accordingly, 117, 130, 99 and 116 proteins were detected in L3 larva, L4 females, adult females and adult males, respectively, with a strong correlation in the biological replicates (Pearson coefficients > 0.73). Fourty-four proteins were detected in all developmental stages, 64 were stage-specific and 49 were exclusively identified in L4 females. Cell compartment enrichment analysis revealed that proteins common to all stages were cytoplasmatic (p < 9.x10-6), whereas L4 unique proteins were in collagen trimers, and macromolecular complexes (p < 0.00001). Functional enrichment analysis of proteins showed significant enrichment in lipid metabolism in L3-unique proteins (p<0.00005), in mannose metabolism and protein de-glycosylation for L4-unique proteins (p < 0.00004), and in phosphorus metabolism in proteins shared by all stages (p <  2.1 x10-9). Interestingly, annexin 6, associated with cancer in humans, was detected in all life stages, but in a larger abundance in L4 females and adults. These findings indicate that S. lupi establishes complex interactions with its hosts by an arsenal of proteins expressed in different patterns in each life stage which influence the pathogenesis and oncogenesis of S. lupi and may be used as potential targets for diagnostic assays, drug targets or vaccine candidates.

Harmful Parasitoses on the Russian Southern Far East under Climatic and Demographic Changes.

Progressive expansion of the most hazardous human parasitoses caused by trematodes, cestodes and nematodes has been found on the south of the Russian Far East. Decelerating expansion of the trematode Clonorchis sinensis, an agent of clonorchiasis towards the southern Primorye Territory from the Amur River basin, that began 10-15 years ago, was revealed. A prognosis was made on the activation of the natural foci of clonorchiasis and paragonimiasis. Circulation possibilities are discussed of the highly pathogenic trematode Pagonimus heterotremusar in the South Asian regional ecosystems. Our experiments showed that the freshwater gastropods of the Parajuga genus from the Amur River basin and those of Stenothyra genus from Primorsky Territory were resistant to this trematode infection. Nevertheless, this does not exclude the possibility of this parasite penetration into the Far East region via infection of the local gastropods of other genera.

Hepatozoon sp. gamonts as an accidental finding in synovial liquid from an injured maned wolf (Chrysocyon brachyurus) in southeastern Brazil.

A free-living, adult male maned wolf (Chrysocyon brachyurus) was referred to the Governador "Laudo Natel" - FCAV/Unesp veterinary hospital after being found with skin lesions and a fracture on the right pelvic limb, which had to be amputated due to compromised integrity. Around 20 days later, bilateral accentuated swollen on humerus-radius-ulna articulation was observed. The synovial liquid was drained and sent to the laboratory for synovial cytology with Rosenfeld staining that revealed predominantly degenerated neutrophils with karyolytic chromatin associated with intracellular inclusions suggestive of Hepatozoon sp. gametocytes. Blood and synovial liquid samples were submitted to molecular analysis, aiming to amplify the Hepatozoon spp. 18S rRNA gene fragment. Despite the positioning of the found Hepatozoon sequence together with Hepatozoon canis previously detected in domestic carnivores, the BLAST analysis showed only 98% identity with H. canis. To the best of the authors' knowledge, this is the first time a Hepatozoon was detected in the synovial liquid by clinical pathology and molecular analyses.

Evidence for camels (Camelus bactrianus) as the main intermediate host of Echinococcus granulosus sensu lato G6/G7 in Mongolia.

Cystic echinococcosis (CE), the parasitic disease caused by the larval stage of Echinococcus granulosus sensu lato (s.l.), is a global public health problem. In Mongolia, despite wide distribution of human CE, not enough information is available on the prevalence and molecular characterization of CE in livestock and its zoonotic linkage with human cases. We investigated the distribution of human CE cases and livestock population using statistical models to get insight into the zoonotic linkage. The incidence of human CE cases increased by a factor of 1.71 for one interquartile range increment in the density of the camel population. No significant association was observed with other livestock species. The samples collected from 96 camels and 15 goats in an endemic region showed a CE prevalence of 19.7% and 6.7%, respectively. All livestock CE were E. granulosus s.l. G6/G7 species of the E. granulosus s.l. complex. The genetic diversity was investigated using the haplotype network based on full cox1 gene analysis of the samples collected from livestock CE and nucleotide sequences previously reported from human CE and wild canids infection in Mongolia. Four haplotypes were identified within the livestock samples, two of which had not been previously reported. A common haplotype was identified among humans, camels, goats, and a wolf, all of which were within the same geographical area. A mixed infection of E. granulosus s.l. G6/G7 with different haplotypes in the intermediate host was identified. To the best of our knowledge, this is the most comprehensive description of the current epidemiological situation of CE in Mongolia with substantial evidence that camels might be the main intermediate host of E. granulosus s.l. G6/G7 in Mongolia. Moreover, our result presents the first report in the country to provide insight into the prevalence of E. granulosus s.l. G6/G7 in livestock.

Serological evidence of Leishmania infection by employing ELISA and rapid tests in captive felids and canids in Brazil.

Visceral leishmaniasis (VL) is a zoonosis and the dog is considered the most important urban reservoir. Cases in cats have been reported, but little is known about Leishmania infection and disease in wild felids and canids kept in captivity in endemic areas. Thus, the serological pattern of wild felids and canids kept in captivity at the Belo Horizonte Zoological Garden was investigated using two primary antigens for conventional ELISA: k39 and rKDDR, as well as three serological rapid kits: Dual Path Platform (DPP®) immunochromatographic test, rKDDR Immunochromatographic assay and ELISA SNAP Leishmania IDEXX®. A total of 21 serum samples, 13 of wild felids and 8 wild canids of varying age and sex were evaluated. The results obtained in the tests were analyzed by agreement using Kappa coefficient, and between ELISA antigens all the analysis performed had showed significant agreement among both of them, as well between the three immunochromatographic tests. The results demonstrated that there is serological evidence of wild animals seropositive for Leishmania antibodies at the Belo Horizonte Zoological Garden, and that all the antigens and rapid tests used can be employed in serological screening for VL in wild felids and canids.

Neospora caninum DNA in feces of crab-eating fox (Cerdocyon thous - Linnaeus, 1776) from northeastern Brazil.

Neospora caninum is an obligate intracellular tissue cyst-forming coccidian parasite and it was first described in dogs. Despite the relevance of wild canids in the transmission chain of N. caninum, there are few studies in Brazil. The aim of the present study was to detect N. caninum DNA in feces of free-range and captive crab-eating fox (Cerdocyon thous) from different states of northeastern Brazil. Fecal samples of eighteen crab-eating foxes (fifteen individually and three pools) were collected in sterile containers and were kept cool at -20 °C until further processing. All fecal samples were subjected to DNA extraction. A nested PCR targeting the ITS-1 gene was performed for N. caninum. All the positive bands were extracted from the gel and purified. Forward and reverse strands were sequenced and the nucleotide sequences obtained were compared with N. caninum sequences deposited in Genbank. The alignment was edited and phylogenetic reconstruction was based on the ITS1 gene sequences. Thirteen stool samples were PCR-positive for N. caninum DNA. Nine out of thirteen positive samples showed similarity between 99%-100% for N. caninum in relation to the sequence U25044.1 stored at GenBank. The crab-eating fox could have an important role in the sylvatic cycle of Neospora caninum in Brazil. Experimental infections studies involving these wild canids may confirm if the crab-eating foxes are definitive hosts of N. caninum.

Detection of Rickettsia spp. and Bartonella spp. in Ctenocephalides felis fleas from free-ranging crab-eating foxes (Cerdocyon thous).

Fleas are insects with a worldwide distribution that have been implicated in the transmission of several pathogens. The present study aimed to investigate the presence of Rickettsia spp. (Rickettsiales: Rickettsiaceae) and Bartonella spp. (Rhizobiales: Bartonellaceae) in fleas from free-ranging crab-eating foxes Cerdocyon thous (Linnaeus, 1766) (Carnivora: Canidae) from Rio Grande do Sul, southern Brazil. Fleas were collected manually from animals and used for the molecular detection of Rickettsia spp. and Bartonella spp. Twenty-nine C. thous were sampled in six municipalities. Four foxes were parasitized by 10 fleas, all of which were identified as Ctenocephalides felis (Bouché, 1935) (Siphonaptera: Pulicidae). DNA from Rickettsia felis Bouyer et al., 2001 and Rickettsia asembonensis Maina et al., 2016 were found in three and eight fleas, respectively. In four fleas, DNA of Bartonella sp. was identified. Phylogenetic analysis grouped Bartonella sp. together with other genotypes previously reported in C. felis worldwide. The scenario described in the present study highlights a Neotropical canid parasitized by the invasive cosmopolitan cat flea, which in turn, is carrying potentially invasive vector-borne microorganisms. These findings suggest that C. felis is adapted to wild hosts in wilderness areas in southern Brazil, hypothetically exposing the Neotropical fauna to unknown ecological and health disturbances.

Variation in the prevalence and abundance of acanthocephalans in brown-nosed coatis Nasua nasua and crab-eating foxes Cerdocyon thous in the Brazilian Pantanal.

Host infection by parasites is influenced by an array of factors, including host and environmental features. We investigated the relationship between host sex, body size and age, as well as seasonality on infection patterns by acanthocephalan in coatis (Procyonidae: Nasua nasua) and in crab-eating foxes (Canidae: Cerdocyon thous ) from the Brazilian Pantanal wetlands. Between 2006 and 2009, we collected faecal samples from these hosts and analyzed for the presence of acanthocephalan eggs. Prevalence, abundance and intensity of eggs of acanthocephalans were calculated. Egg abundance was analyzed using generalized linear models (GLM) with a negative binomial distribution and models were compared by Akaike criteria to verify the effect of biotic and abiotic factors. Prevalence of acanthocephalans was higher in the wet season in both host species but did not differ between host sexes; however, adult crab-eating foxes showed higher prevalence of acanthocephalan eggs than juveniles. In contrast, prevalence of acanthocephalan eggs found in coatis was higher in coati juveniles than in adults. Host age, season and maximum temperature were the top predictors of abundance of acanthocephalan eggs in crab-eating foxes whereas season and host sex were predictors of egg abundance in coatis. The importance of seasonality for abundance of acanthocephalan was clear for both host species. The influence of host-related attributes, however, varied by host species, with host gender and host age being important factors associated with prevalence and parasite loads.