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Babesia spp. are tick-borne protozoans that involve birds and mammals in their transmission cycles and cause babesiosis, a severe hemolytic malaria-like disease. Opossums of the genus Didelphis are recognized hosts of tick-borne pathogens. Therefore, exploring tick-borne agents in Didelphis species is important to understand the circulation of pathogens in areas where opossums occur. In this study, we targeted Anaplasmataceae, Babesia, Borrelia and Hepatozoon DNA in ticks, blood and organ samples collected from three hunted Didelphis marsupialis specimens in eastern Guatemala. While the samples were negative for Hepatozoon and bacterial DNA, sequences of Babesia 18S rDNA, cox1 and cytb genes were retrieved from two opossums. Ticks collected on the animals included Amblyomma parvum and an undetermined Ornithodoros sp. The Babesia sp. detected in this study (Babesia sp. THB1-2) clusters phylogenetically within the "Western Babesia group", which includes pathogenic species such as Babesia conradae, Babesia duncani, and Babesia negevi. Our results represent the first record of a Babesia sp. in Guatemala and highlight the importance of D. marsupialis as potential spreaders of ticks and pathogens in Central America.

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Human contact with wild animals in synanthropic habits is often mediated by arthropod vectors such as ticks. This is an important method of spreading infectious agents that pose a risk to human health. Thus, this study aimed to molecularly detect Ehrlichia spp., Anaplasma spp., Borrelia spp., and protozoa of the order Piroplasmida in ticks collected from coatis of Iguaçu National Park (PNI), Paraná, Brazil. This study involved 553 ticks DNA, including Amblyomma spp. larvae, Haemaphysalis juxtakochi nymphs, Amblyomma brasiliense, Amblyomma coelebs, and adults of Amblyomma ovale. The DNA extracted from each sample was subjected to polymerase chain reaction (PCR) targeting the genes 23S rRNA for the Anaplasmataceae family, 16S rRNA for Anaplasma spp., dsb for Ehrlichia spp., flaB, 16S rRNA, hpt, and glpQ for Borrelia spp., and 18S rRNA for Piroplasmid protozoans. DNA from Anaplasma sp. was detected in ticks of the species A. coelebs (4/553); Borrelia sp. DNA was detected in A. coelebs (3/553), A. ovale (1/553), and Amblyomma larvae (1/553); and Theileria sp. was detected in A. coelebs (2/553). All tested samples were negative for Ehrlichia spp. Our study constitutes the newest report in South America of these microorganisms, which remain poorly studied.

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Piroplasmids (order Piroplasmida) are a diverse group of tick-borne protozoa that may cause disease in animals and occasionally in humans. Novel Piroplasmida clades and species have been found in wild animals from Brazil based on the phylogenetic assessment of near-complete 18S rRNA, mitochondrial and heat-shock protein genes. For instance, a putative novel Babesia species has been detected in capybaras and Amblyomma ticks in three Brazilian states. The present work aimed to describe, using phylogenetic assessments based on distinct molecular markers, this novel Babesia species in capybaras and associated ticks (Amblyomma sculptum and Amblyomma dubitatum) sampled in Goiânia city, Goiás state, midwestern Brazil. While the phylogenetic analysis based on both near-complete 18S rRNA and hsp-70 genes positioned the sequences obtained from capybara blood samples into a new clade sister to the Babesia sensu stricto clade, the phylogenetic inference based on the COX-3 amino acid positioned the obtained sequences from capybara blood samples and A. sculptum ticks also into a clade sister to the Theileria sensu stricto clade, highlighting the inappropriateness of this marker inferring evolutionary relationships among piroplasmids. Pairwise distance analysis demonstrated that the divergence rates between the 18S rRNA sequences detected in capybaras and other Piroplasmida already described were very high and ranged from 9.4 to 12.9%. Genotype analysis based on the near-full 18S rRNA sequences of the Piroplasmida detected in capybaras and associated ticks demonstrated the occurrence of high genotype diversity at an intra-species level. In conclusion, phylogenetic analyses based on distinct molecular markers supported the description of Babesia goianiaensis nov. sp. in capybaras and associated Amblyomma ticks. Additionally, a novel phylogenetic clade, apart from the previously described ones, was described in the present study and contributed to untangling the complex evolutionary history of the Piroplasmida.

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The growing proximity of wildlife to large urban niches arouses greater interest in understanding wild reservoirs in the epidemiology of diseases of importance to animal and human health. The aim of the present study was to investigate the presence of piroplasmids in opossums rescued from the metropolitan region of Rio de Janeiro state, Brazil. Blood and bone marrow samples were collected from 15 Didelphis aurita and subjected to DNA extraction and PCR using primers for the 18S rRNA, cox1, cox3, and hsp70 genes of piroplasmids. Clinical and hematological evaluation of the animals was also performed. Five (33.3%) of the 15 opossums tested positive for piroplasms in the nested PCR based on the 18S rRNA, and in two animals, it was possible to observe intra-erythrocytic structures compatible with merozoites. One of the positive animals showed clinical signs of infection such as jaundice, fever, and apathy. Anemia, low level of plasma protein, leukocytosis, and regenerative erythrocyte signs were observed in positive animals. Phylogenetic analysis based on both 18S rRNA and cox-3 genes demonstrated that the piroplasmids detected in D. aurita formed a unique sub-clade, albeit related to piroplasmids previously detected in Didelphis albiventris and associated ticks from Brazil. This study proposes the novel Piroplasmida Clade, namely "South American Marsupialia Group," and reinforces the need for new clinical-epidemiological surveys to understand the dynamics of these infections in didelphids in Brazil.

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The low-land tapir (Tapirus terrestris) is the largest wild terrestrial mammal found in Brazil. Although T. terrestris has been already reported as a host of hemoparasites, the occurrence and genetic identity of Piroplasmida agents in this species is still cloudy. Although it is reported that Theileria equi, an endemic equid-infective agent in Brazil, is occurring in lowland tapirs, these reports are probably misconceived diagnoses since they are solely based on small fragments of 18S rRNA that may not achieve accurate topologies on phylogenetic analyses. The present study aimed to detect and investigate the identity of Theileria spp. in tapirs from Pantanal and Cerrado biomes. Blood-DNA samples from tapirs were screened for a partial (~800 bp) 18S rRNA gene fragment from Piroplasmida and 64 (64/122; 52.46% CI: 43.66-61.11%) presented bands of expected size. Samples were submitted to different protocols for molecular characterization, including near-full length 18S rRNA gene (~1500 bp), and the ema-1 gene from T. equi. Eight sequences were obtained for extended fragments (1182-1473 bp) from the 18S rRNA gene. Moreover, three sequences from partial cox-1 and five from partial hsp70 gene were obtained. None of the samples presented amplifications for the ema-1 gene. Phylogenetic and distance analyses from the 18S rRNA sequences obtained demonstrated a clear separation from tapirs' Theileria spp. and T. equi. Phylogenetic analyses of cox-1 and hsp70 sequences obtained herein also showed a unique clade formed by tapir's Theileria spp. Theileria terrestris sp. nov. is positioned apart from all other Theileria species in 18S rRNA, cox-1, and hps70 phylogenetic analyses. This novel proposed species represents a new Piroplasmida clade, yet to be characterized regarding biological features, vectors involved in the transmission cycles, additional vertebrate hosts, and pathogenicity.

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Feline piroplasmids include the genera Babesia spp., Cytauxzoon spp., and Theileria spp. In Brazil, there are few reports regarding these hemoprotozoans; however, clinicopathological and molecular data are scarce. This study aimed to characterize the clinical relevance of these parasites through hematological, biochemical, and molecular approaches. For this purpose, 166 cats from Brasilia, Federal District, Midwestern Brazil, were screened using a quantitative polymerase chain reaction (qPCR) for piroplasmids based on the LSU4 mitochondrial gene, which resulted in an overall prevalence of 36/166 (21.7%). Twelve of 166 samples (7.2%) were positive for C. felis, while 19/166 (11.4%) were positive for Babesia vogeli. No samples tested positive for Theileria spp. Babesia vogeli and Cytauxzoon spp. LSU4 sequences showed identities of 97-100% and 99.3%, respectively, to US isolates. The hematological and biochemical findings did not differ significantly between the cats that tested positive and negative for piroplasmids. Although the lack of abnormalities in clinical and laboratory parameters does not eliminate the possibility that these cats were sick and recovered, it may suggest that the Brazilian strain of Cytauxzoon spp. is not as pathogenic as that from the USA, despite the high molecular identity with North American isolates.

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Chile is a large country with a marked range of climate conditions that make it an ideal scenario for the study of vector-borne parasites (VBPs); however, knowledge about their distribution is limited to a few confined areas of this country. The presence of Hepatozoon spp., piroplasmids, Leishmania spp. and filarioids was investigated through molecular and serological methods in blood and serum samples of 764 free-ranging rural dogs, 154 Andean foxes (Lycalopex culpaeus), and 91 South American grey foxes (Lycalopex griseus) from six bioclimatic regions across Chile. Hepatozoon spp. DNA was exclusively detected in foxes (43% prevalence), including sequences closely related to Hepatozoon felis (24.1%; only Andean foxes), Hepatozoon americanum (16.2%; only grey foxes), and Hepatozoon canis (1.25%; in one grey fox). Risk factor assessment identified a higher probability of Hepatozoon infection in juvenile foxes. DNA of piroplasmids was detected in 0.7% of dogs (Babesia vogeli) but in no fox, whilst antibodies against Babesia sp. were detected in 24% of the dogs and 25% of the foxes, suggesting a wider circulation of canine piroplasmids than previously believed. A positive association between the presence of antibodies against Babesia and high Rhipicephalus sanguineus sensu lato burden was observed in dogs. Leishmania spp. DNA and antibodies were detected in 0.8% and 4.4% of the dogs, respectively. Acanthocheilonema reconditum was the only blood nematode detected (1.5% of the dogs and no fox). Differences in prevalence among bioregions were observed for some of the VBPs. These results expand our knowledge about the occurrence of vector-borne parasites in Chile, some of which are firstly reported herein. This information will facilitate the diagnosis of vector-borne diseases in domestic dogs and improve the control measures for both domestic and wild canids.

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Canine babesiosis is a disease caused by a parasite of the genus Babesia which destroys red blood cells. Previous studies have shown the presence of Babesia vogeli in rural areas in Costa Rica using molecular techniques. The objective of the present study was to determine the seroprevalence and prevalence of B. vogeli in clinically healthy dogs and their ticks at the national level, both within and outside the Central Valley. Blood samples and ticks from 482 dogs were collected between June 2011 and May 2014, and analyzed by immunofluorescence assay (IFA) and real-time polymerase chain reaction (qPCR); two protocols of endpoint PCR and sequencing were used to confirm qPCR-positive samples. Seroprevalence of canine babesiosis of 5.3% (24/453) was determined at the national level, specifically 2.0% (5/253) within and 9.5% (19/200) outside the Central Valley, respectively. Real-time PCR determined a global prevalence of B. vogeli of 31.3% (125/400): 21.4% (47/220) within the Central Valley and 43.3% (78/180) outside the Central Valley. The endpoint PCR amplified only 10 of the 125 blood samples identified as positive in qPCR. One sample amplified by endpoint PCR was sequenced and identified as B. vogeli. Twelve canines were identified with past infections, seven canines with active infection, and 111 canines with early infection. Two species of ticks were found with B. vogeli: Rhipicephalus sanguineus sensu lato (n = 40) and Amblyomma ovale (n = 1). The prevalence of canine babesiosis at the national level, both within and outside the Central Valley, is reported here for the first time, determining the presence of the piroplasmid throughout the country, with a higher circulation of the agent outside the Central Valley. Only one species, B. vogeli, was detected in the blood of dogs and their ticks. Therefore, veterinarians should consider using qPCR to determine the presence of the parasite in blood donors and before starting treatment of vector-borne disease in dogs.

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Piroplasmida is an order of the phylum Apicomplexa that comprises the Babesia, Cytauxzoon, and Theileria genera. These hemoparasites infect vertebrate blood cells and may cause serious diseases in animals and humans. Even though previous studies have shown that bats are infected by different species of piroplasmids, the occurrence and diversity of these hemoparasites have not been investigated in this group of mammals in Brazil. Therefore, the present work aimed to investigate the occurrence and assess the phylogenetic placement of piroplasmids infecting bats sampled in a peri-urban area from Central-Western Brazil. Seventeen (12.6%) out of 135 animals were positive by nested PCR assay for the detection of Babesia/Theileria targeting the 18S rRNA gene. Eleven sequences of the 17 positive samples could be analyzed and showed an identity of 91.8-100% with Theileria bicornis, Babesia vogeli, a Babesia sp. identified in a small rodent (Thrichomys pachyurus) from the Brazilian Pantanal and a Babesia sp. identified in a dog from Thailand as assessed by nBLAST. A phylogenetic tree was constructed from an alignment of 1399 bp length using analyzed and known piroplasmid 18S rRNA sequences. In this tree, piroplasmid 18S rRNA sequences detected in three specimens of Phyllostomus discolor (Piroplasmid n. sp., P. discolor) were placed as a sister taxon to Theileria sensu stricto (Clade V) and Babesia sensu stricto (Clade VI). An additional phylogenetic tree was generated from a shorter alignment of 524 bp length including analyzed piroplasmid 18S rRNA sequences of bat species Artibeus planirostris and A. lituratus (Piroplasmid sp., Artibeus spp.). The two 18S rRNA sequences detected in Artibeus spp. (Piroplasmid n. sp., Artibeus spp.) were placed within Babesia sensu stricto (Clade VI) into a strongly supported clade (bootstrap: 100) that included Babesia vogeli. The two 18S rRNA sequences of Piroplasmid sp., Artibeus spp. showed a single and a two-nucleotide differences, respectively, with respect to B. vogeli in a 709 pb length alignment. For the first time, the present study shows the occurrence of putative new piroplasmid species in non-hematophagous bats from Brazil.

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C1A cysteine peptidases have been shown to play an important role during apicomplexan invasion and egress of host red blood cells (RBCs) and therefore have been exploited as targets for drug development, in which peptidase specificity is deterministic. Babesia bovis genome is currently available and from the 17 putative cysteine peptidases annotated four belong to the C1A subfamily. In this study, we describe the biochemical characterization of a C1A cysteine peptidase, named here BbCp (B. bovis cysteine peptidase) and evaluate its possible participation in the parasite asexual cycle in host RBCs. The recombinant protein was obtained in bacterial inclusion bodies and after a refolding process, presented typical kinetic features of the cysteine peptidase family, enhanced activity in the presence of a reducing agent, optimum pH between 6.5 and 7.0 and was inhibited by cystatins from R. microplus. Moreover, rBbCp substrate specificity evaluation using a peptide phage display library showed a preference for Val > Leu > Phe. Finally, antibodies anti-rBbCp were able to interfere with B. bovis growth in vitro, which highlights the BbCp as a potential target for drug design.

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Magellanic penguins (Spheniscus magellanicus) are native to Argentina, Chile and the Falkland Islands. Magellanic penguins are highly susceptible to blood parasites such as the mosquito-borne Plasmodium spp., which have been documented causing high morbidity and mortality in zoos and rehabilitation centres. However, to date no blood parasites have been detected in wild Magellanic penguins, and it is not clear whether this is reflective of their true absence or is instead related to an insufficiency in sampling effort or a failure of the diagnostic methods. We examined blood smears of 284 Magellanic penguins from the Argentinean coast and tested their blood samples with nested polymerase chain reaction tests targeting Haemoproteus, Plasmodium, Leucocytozoon and Babesia. No blood parasites were detected. Analysing the sampling effort of previous studies and the climatogeography of the region, we found there is strong basis to conclude that haemosporidians do not infect wild Magellanic penguins on the Argentinean coast. However, at present it is not possible to determine whether such parasites occur on the Chilean coast and at the Falkland Islands. Furthermore, it is troubling that the northward distribution expansion of Magellanic penguins and the poleward distribution shift of vectors may lead to novel opportunities for the transmission of blood parasites.

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Blood parasites are potential threats to the health of penguins and to their conservation and management. Little penguins Eudyptula minor are native to Australia and New Zealand, and are susceptible to piroplasmids (Babesia), hemosporidians (Haemoproteus, Leucocytozoon, Plasmodium) and kinetoplastids (Trypanosoma). We studied a total of 263 wild little penguins at 20 sites along the Australian southeastern coast, in addition to 16 captive-bred little penguins. Babesia sp. was identified in seven wild little penguins, with positive individuals recorded in New South Wales, Victoria and Tasmania. True prevalence was estimated between 3.4% and 4.5%. Only round forms of the parasite were observed, and gene sequencing confirmed the identity of the parasite and demonstrated it is closely related to Babesia poelea from boobies (Sula spp.) and B. uriae from murres (Uria aalge). None of the Babesia-positive penguins presented signs of disease, confirming earlier suggestions that chronic infections by these parasites are not substantially problematic to otherwise healthy little penguins. We searched also for kinetoplastids, and despite targeted sampling of little penguins near the location where Trypanosoma eudyptulae was originally reported, this parasite was not detected.

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Tick-borne infections can result in serious health problems for wild ruminants, and some of these infectious agents can be considered zoonosis. The aim of the present study was the post-mortem detection of hemoparasites in free-living Mazama gouazoubira from Minas Gerais state, Brazil. The deer samples consisted of free-living M. gouazoubira (n = 9) individuals that died after capture. Necropsy examinations of the carcasses were performed to search for macroscopic alterations. Organ samples were collected for subsequent imprint slides, and nested PCR assays were performed to detect hemoparasite species. Imprint slide assays from four deer showed erythrocytes infected with Piroplasmida small trophozoites, and A. marginale corpuscles were observed in erythrocytes from two animals. A. marginale and trophozoite co-infections occurred in two deer. A nested PCR analysis of the organs showed that six of the nine samples were positive for Theileria sp., five were positive for A. phagocytophilum and three were positive for A. marginale, with co-infection occurring in four deer. The results of the present study demonstrate that post-mortem diagnostics using imprint slides and molecular assays are an effective method for detecting hemoparasites in organs.

Patógenos transmitidos por carrapatos podem resultar em sérios problemas de saúde para os ruminantes selvagens, e alguns podem ser zoonoses. O objetivo do presente estudo foi a detecção post mortem de hemoparasitos, em Mazama gouazoubira de vida livre, oriundos de Minas Gerais, através da análise de lâminas de impressão e nested PCR. Foram amostrados nove M. gouazoubira de vida livre, que morreram após a captura. Exames de necropsia foram realizados, e as carcaças foram examinadas para detectar alterações macroscópicas. Amostras dos órgãos foram coletadas para a realização de imprint em lâminas e para nested PCR à procura de hemoparasitos. A análise das lâminas mostrou pequenos trofozoítos de Piriplasmida nos eritrócitos de quatro dos oito animais examinados, e corpúsculos de Anaplasma marginale foram observados nos eritrócitos de dois dos cervídeos. A coinfecção com A. marginale e trofozoítos de piroplasmas ocorreu em dois animais. As análises de nPCR dos órgãos mostraram que seis dos nove animais estavam positivos para Theileria sp., cinco para A. phagocytophilum e três para A. marginale, sendo que a coinfecção ocorreu em quatro cervídeos. Os resultados do presente estudo demonstram que os diagnósticos post-mortem, pelas imprints em lâminas e ensaios moleculares, são métodos eficazes de detecção de hemoparasitos nos principais órgãos parasitados.

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Tick-borne infections can result in serious health problems for wild ruminants, and some of these infectious agents can be considered zoonosis. The aim of the present study was the post-mortem detection of hemoparasites in free-living Mazama gouazoubira from Minas Gerais state, Brazil. The deer samples consisted of free-living M. gouazoubira (n = 9) individuals that died after capture. Necropsy examinations of the carcasses were performed to search for macroscopic alterations. Organ samples were collected for subsequent imprint slides, and nested PCR assays were performed to detect hemoparasite species. Imprint slide assays from four deer showed erythrocytes infected with Piroplasmida small trophozoites, and A. marginale corpuscles were observed in erythrocytes from two animals. A. marginale and trophozoite co-infections occurred in two deer. A nested PCR analysis of the organs showed that six of the nine samples were positive for Theileria sp., five were positive for A. phagocytophilum and three were positive for A. marginale, with co-infection occurring in four deer. The results of the present study demonstrate that post-mortem diagnostics using imprint slides and molecular assays are an effective method for detecting hemoparasites in organs.

Patógenos transmitidos por carrapatos podem resultar em sérios problemas de saúde para os ruminantes selvagens, e alguns podem ser zoonoses. O objetivo do presente estudo foi a detecção post mortem de hemoparasitos, em Mazama gouazoubira de vida livre, oriundos de Minas Gerais, através da análise de lâminas de impressão e nested PCR. Foram amostrados nove M. gouazoubira de vida livre, que morreram após a captura. Exames de necropsia foram realizados, e as carcaças foram examinadas para detectar alterações macroscópicas. Amostras dos órgãos foram coletadas para a realização de imprint em lâminas e para nested PCR à procura de hemoparasitos. A análise das lâminas mostrou pequenos trofozoítos de Piriplasmida nos eritrócitos de quatro dos oito animais examinados, e corpúsculos de Anaplasma marginale foram observados nos eritrócitos de dois dos cervídeos. A coinfecção com A. marginale e trofozoítos de piroplasmas ocorreu em dois animais. As análises de nPCR dos órgãos mostraram que seis dos nove animais estavam positivos para Theileria sp., cinco para A. phagocytophilum e três para A. marginale, sendo que a coinfecção ocorreu em quatro cervídeos. Os resultados do presente estudo demonstram que os diagnósticos post-mortem, pelas imprints em lâminas e ensaios moleculares, são métodos eficazes de detecção de hemoparasitos nos principais órgãos parasitados.

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