Canine piroplasmids: Molecular detection and laboratory characterization in dogs from Brasilia, Brazil, with the first molecular evidence of dog exposure to a novel opossum-associated Babesia sp.

Canine piroplasmid infections can be caused by Babesia spp., Theileria spp. and Rangelia vitalii. In Brazil, canine babesiosis caused by Babesia vogeli is endemic and reported throughout the country. On the other hand, Rangeliosis caused by R. vitalii has only been described so far in the South and Southeast regions. Despite that, studies analyzing the laboratory and molecular characterization of these hemoprotozoa are still scarce. To investigate the occurrence, the laboratory features, the molecular characterization, and the diversity of piroplasmids from Midwestern Brazil, a survey was performed using blood samples obtained from 276 domestic dogs from Brasília, Federal District, Midwestern Brazil. A broad-range quantitative PCR (qPCR) targeting the mitochondrial large subunit ribosomal DNA (LSU4) was used to detect piroplasmid DNA. The overall molecular occurrence of piroplasmids was 11.2% (31/276), with 9.7% (27/276) of the sequences identified as Babesia vogeli (98-100% identity to B. vogeli isolate from the USA). Based on a partial 18S rRNA sequence pairwise alignment (-250 bp), 1.4% (4/276) of the sequences showed only 76.8% identity with B. vogeli but 100% identity with opossum-associated Babesia sp. (MW290046-53). These findings suggest the exposure of dogs from Brazil to a recently described Babesia sp. isolated from white-eared opossum. None of the analyzed dogs was positive for Theileria spp. or R. vitalii. Subsequently, all positive sequences were submitted to three additional PCR assays based on the 18S rRNA, cox-1, and cytb genes, aiming at performing a haplotype network analysis. Haplotype network using cox-1 sequences showed the presence of six different haplotypes of B. vogeli; one of them was shared with isolates from Brazil, the USA, and India. When including animals co-infected with other vector-borne diseases, piroplasmid-positive dogs had 2.3 times higher chance of having thrombocytopenia than the negative ones. The molecular results demonstrated that the compared Babesia vogeli sequences showed a low variability as well as evidence of exposure to a putative novel opossum-associated Babesia sp. in dogs from Midwestern Brazil.

Piroplasmid infection is not associated with clinicopathological and laboratory abnormalities in cats from Midwestern Brazil.

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.

A new piroplasmid species infecting dogs: morphological and molecular characterization and pathogeny of Babesia negevi n. sp.

INTRODUCTION: Babesiosis is a protozoan tick-borne infection associated with anemia and life-threatening disease in humans, domestic and wildlife animals. Dogs are infected by at least six well-characterized Babesia spp. that cause clinical disease. Infection with a piroplasmid species was detected by light microscopy of stained blood smears from five sick dogs from Israel and prompted an investigation on the parasite's identity. METHODS: Genetic characterization of the piroplasmid was performed by PCR amplification of the 18S rRNA and the cytochrome c oxidase subunit 1 (cox1) genes, DNA sequencing and phylogenetic analysis. Four of the dogs were co-infected with Borrelia persica (Dschunkowsky, 1913), a relapsing fever spirochete transmitted by the argasid tick Ornithodoros tholozani Laboulbène & Mégnin. Co-infection of dogs with B. persica raised the possibility of transmission by O. tholozani and therefore, a piroplasmid PCR survey of ticks from this species was performed. RESULTS: The infected dogs presented with fever (4/5), anemia, thrombocytopenia (4/5) and icterus (3/5). Comparison of the 18S rRNA and cox1 piroplasmid gene sequences revealed 99-100% identity between sequences amplified from different dogs and ticks. Phylogenetic trees demonstrated a previously undescribed species of Babesia belonging to the western group of Babesia (sensu lato) and closely related to the human pathogen Babesia duncani Conrad, Kjemtrup, Carreno, Thomford, Wainwright, Eberhard, Quick, Telfrom & Herwalt, 2006 while more moderately related to Babesia conradae Kjemtrup, Wainwright, Miller, Penzhorn & Carreno, 2006 which infects dogs. The piroplasm forms detected included tetrads (Maltese cross), merozoite and trophozoite stages whose average size was larger than stages of other canine Babesia spp. belonging to the Babesia (s.l.) and B. gibsoni Patton, 1910, and smaller than other canine Babesia (sensu stricto) spp. Of 212 O. tholozani ticks surveyed, 11 (5.2%) harbored DNA of the new species of Babesia. CONCLUSIONS: Babesia negevi n. sp. is described based on morphological and genetic characterization and phylogenetic analyses. The species is named after the Negev desert of southern Israel, where the first infected dog originated from. Despite co-infection in four dogs, the fifth dog had fatal disease attesting that B. negevi n. sp. infection requires clinical attention. Incriminating O. tholozani or another tick species as the vector of Babesia negevi n. sp., would require additional studies.