No abstract

Hepatozoon parasites comprise intracellular apicomplexan parasites transmitted to vertebrate animals by ingestion of arthropods definitive hosts. The present work aimed to investigate the occurrence of Hepatozoon spp. in wild animals, domestic dogs and their respective ectoparasites, in southern Pantanal region, central-western Brazil, by molecular techniques. Between August 2013 and March 2015, 31 coatis (Nasua nasua), 78 crab-eating foxes (Cerdocyon thous), seven ocelots (Leopardus pardalis), 42 dogs (Canis lupus familiaris), 110 wild rodents (77 Thichomys fosteri, 25 Oecomys mamorae, and 8 Clyomys laticeps), 30 marsupials (14 Thylamys macrurus, 11 Gracilinanus agilis, 4 Monodelphis domestica and 1 Didelphis albiventris), and 1582 ticks and 80 fleas collected from the sampled animals were investigated. DNA samples were submitted to PCR assays for Hepatozoon spp. targeting 18S rRNA gene. Purified amplicons were directly sequenced and submitted to phylogenetic analysis. A high prevalence of Hepatozoon among carnivores (C. thous [91.02%], dogs [45.23%], N. nasua [41.9%] and L. pardalis [71.4%]) was found. However, ticks and fleas were negative to Hepatozoon PCR assays. By phylogenetic analysis based on 18S rRNA sequences, Hepatozoon sequences amplified from crab-eating foxes, dogs, coatis and ocelots clustered with sequences of H. canis, H. americanum and H. felis. The closely related positioning of Hepatozoon sequences amplified from wild rodents and T. macrurus marsupial to Hepatozoon from reptiles and amphibians suggest a possible transmission of those Hepatozoon species between hosts by ectoparasites or by predation. Hepatozoon haplotypes found circulating in wild rodents seem to present a higher degree of polymorphism when compared to those found in other groups of animals. Although rodents seem not to participate as source of Hepatozoon infection to wild carnivores and domestic dogs, they may play an important role in the transmission of Hepatozoon to reptiles and amphibians in Pantanal biome.

Hemotrophic mycoplasmas (hemoplasmas), Bartonella sp., Hepatozoon sp. and Cytauxzoon felis are prominent pathogens that circulate between cats and invertebrate hosts. The present study aimed to detect the presence of DNA from hemoplasmas, Bartonella sp., Hepatozoon sp. and Cytauxzoon felis, and then confirm it by means of sequencing, in blood samples from cats in Cuiabá, MT, Brazil. From February 2009 to February 2011, blood samples with added EDTA were collected from 163 cats that were being housed in four different animal shelters in the city of Cuiabá, state of Mato Grosso, Brazil and from 15 cats that were admitted to the veterinary hospital of the Federal University of Mato Grosso (UFMT). Out of the 178 cats sampled, 15 (8.4%) were positive for hemoplasmas: four (2.2%) for Mycoplasma haemofelis, 12 (6.7%) for 'Candidatus M. haemominutum' and one (0.5%) for 'Candidatus M. turicensis'. One cat (0.5%), a patient that was attended at the veterinary hospital, was coinfected with M. haemofelis, 'Candidatus M. haemominutum' and 'Candidatus M. turicensis', based on sequencing confirmation. Four cats were positive for Bartonella spp.: three (1.7%) for B. henselae and one (0.5%) for B. clarridgeiae. None of the animals showed Cytauxzoon sp. or Hepatozoon sp. DNA in their blood samples. This study showed that cats housed in animal shelters in the city of Cuiabá, state of Mato Grosso, are exposed to hemoplasmas and Bartonella species.

Hemotropic mycoplasmas are known to cause anemia in several mammalian species. The present work aimed to investigate the occurrence of Mycoplasma spp. in wild mammals, domestic dogs and their respective ectoparasites, in southern Pantanal region, central-western Brazil. Between August 2013 and March 2015, 31 Nasua nasua, 78 Cerdocyon thous, seven Leopardus pardalis, 42 dogs, 110 wild rodents, and 30 marsupials were trapped and ectoparasites (ticks and fleas) found parasitizing the animals were collected. Mammals and ectoparasites DNA samples were submitted to conventional PCR assays for Mycoplasma spp. targeting 16S rRNA and RnaseP genes. Twenty-four N. nasua, three C. thous, two domestic dogs, one L. pardalis and one wild rodent were positive for 16S rRNA PCR protocols. Fourteen N. nasua samples were also positive in RnaseP PCR. No marsupial or arthropod showed positivity for Mycoplasma spp. The phylogenetic analyses based on 16S rRNA gene showed that all sequences obtained from dogs, two sequences obtained from C. thous and ten sequences obtained from N. nasua showed to be closely related to Mycoplasma haemocanis/Mycoplasma haemofelis species. Genotypes closely related to 'Candidatus Mycoplasma haemominutum' and Mycoplasma haemomuris were detected in the L. pardalis and in the wild rodent, respectively. Probably a novel Mycoplasma genotype, closely related to a sequence obtained from a Brazilian capybara was detected in 14 N. nasua, based on a concatenated phylogenetic analysis of 16S rRNA and RnaseP genes. The present study revealed that wild animals in southern Pantanal region, Brazil, are exposed to different species of hemoplasmas.

The present work provides details of a novel qPCR assay to diagnose infections caused by Bartonella species.

Tick-borne pathogens affect a wide range of vertebrate hosts. To identify tick-borne pathogens among dogs from Campo Grande, MS, Brazil testing seropositive for Leishmania infantum (syn. L. chagasi), a serological and molecular study was conducted to detect Ehrlichia canis, Anaplasma platys and Babesia vogeli in 60 serum and spleen samples. A confirmatory diagnosis of L. infantum based on serological and molecular assays was also performed, as was sequence alignment and phylogenetic analysis to assess the identity of the parasite species infecting these animals. IgG antibodies to Ehrlichia spp., B. vogeli and L. infantum were found, respectively, in 39 (65%), 49 (81.6%) and 60 (100%) of the sampled dogs. Twenty-seven (45%), fifty-four (90%), fifty-three (88.3%), two (3.3%) and one (1.6%) dog were positive, respectively, for E. canis, Leishmania spp., Leishmania donovani complex, Babesia sp. and Anaplasma sp. in PCR assays. After sequencing, the amplicons showed 99% of identity with E. canis, B. vogeli, A. platys and Leishmania chagasi isolates. The findings of this study indicate that L. infantum-seropositive dogs from Campo Grande are exposed to multiple tick-borne pathogens, which should therefore be included in the differential diagnosis of dogs with clinical suspicion of leishmaniasis.Keywords: Ehrlichia canis, Babesia vogeli, Anaplasma platys, Leishmania infantum, dogs, co-infection. ResumoPatógenos transmitidos por carrapatos atingem uma variedade de hospedeiros vertebrados. Para identificar os agentes patogênicos transmitidos por carrapatos entre cães soropositivos para Leishmania infantum no município Campo Grande-MS, foi realizado um estudo sorológico e molecular para a detecção de Ehrlichia canis, Anaplasma platys e Babesia vogeli em 60 amostras de soro e baço, respectivamente. Adicionalmente, foi realizado o diagnóstico confirmatório de L. infantum por meio de técnicas sorológicas e moleculares. Também foi realizado o alinhamento e análise filogenética das sequências para indicar a identidade das espécies de parasitas que infectam esses animais. Anticorpos IgG anti-Ehrlichia spp., anti-B. vogeli e anti-L. infantum foram detectados em 39 (65%), 49 (81,6%) e 60 (100%) dos cães amostrados, respectivamente. Vinte e sete (45%), cinquenta e quatro (90%), cinquenta e três (88,3%), dois (3,3%) e um (1,6%) cães mostraram-se positivos na PCR para E. canis, Leishmania spp., Leishmania donovani complex, Babesia sp. e Anaplasma sp., respectivamente. Após o seqüenciamento, os amplicons mostraram 99% de similaridade com isolados de E. canis, B. vogeli e A. platys e Leishmania chagasi. Os resultados deste estudo indicaram que os cães soropositivos para L. infantum de Campo Grande, MS, são expostos a vários agentes transmitidos por carrapatos, e, portanto, devem ser incluídos no diagnóstico diferencial em cães com suspeita clínica de leishmaniose.

Piroplasmoses are one of the most prevalent arthropod-borne diseases of animals. The present work aimed to investigate the occurrence of piroplasmid in wild mammals, domestic dogs and ectoparasites in southern Pantanal region, central-western Brazil. For that purpose, blood or tissue samples from 31 Nasua nasua, 78 Cerdocyon thous, 7 Leopardus pardalis, 42 dogs, 110 wild rodents, and 30 marsupials, and 1582 ticks were submitted to PCR assays for piroplasmid targeting 18SrRNA and hps70 genes. Seven dogs, one C. thous, five L. pardalis, three N. nasua, six wild rodents, eight Amblyomma parvum, two Amblyomma sculptum and one Amblyomma ovale were positive for piroplasmid-PCR assays. Genotypes closely related to Babesia vogeli were detected in six dogs and five wild rodents. While genotypes closely related to Babesia caballi were detected in one C. thous, one dog, one A. ovale and one A. sculptum, genotypes closely related to Babesia bigemina and Babesia bovis were detected in four A. parvum ticks. Four sequences obtained from A. parvum, three coatis and one wild rodent were closely related to Theileria equi. Cytauxzoon spp. was detected in four ocelots. The present study revealed that wild and domestic animals in Brazilian southern Pantanal are exposed to different piroplasmid species.

New genotypes of Anaplasmataceae agents have been detected in wild carnivores, birds and deer in Brazil. The present work aimed to investigate the presence of Ehrlichia and Anaplasma species in rodents sampled in Brazil. Additionally, a newly designed quantitative 5' nuclease real-time multiplex PCR for Ehrlichia and Anaplasma spp. detection based on groEL gene amplification was designed, showing high specificity and sensitivity (10 groEL fragment copy/μL). Between 2000 and 2011, different rodent species [n=60] were trapped in 5 Brazilian biomes. Among 458 rodent spleen samples, 0.4% (2/458) and 2.4% (11/458) were positive for Ehrlichia and Anaplasma spp., respectively. Of 458 samples, 2.0% (9/458) and 1.1% (5/458) were positive for Anaplasma sp. and Ehrlichia sp., respectively, using conventional 16S rRNA PCR assays. Maximum Likelihood phylogenetic analyse based on a small region of 16S rRNA genes positioned the Anaplasma genotypes in rodents near Anaplasma phagocytophilum or Anaplasma marginale and Anaplasma odocoilei isolates. Ehrlichia genotypes were closely related to E. canis. There was a low occurrence of Anaplasma and Ehrlichia in wild and synanthropic rodents in Brazil, suggesting the circulation of new genotypes of these agents in rodents in the studied areas.