Detection and molecular characterization of piroplasms species from naturally infected dogs in southeast Brazil

Abstract

Rangelia vitalii is a protozoon described from dogs in the south and southeast regions of Brazil. It is phylogenetically related to Babesia spp. that infects dogs, but data on this enigmatic parasite is still limited. The aim of this work was to detect piroplasm species in dogs in the state of Rio de Janeiro, Brazil, by 18S rRNA gene-based PCR assay, restriction fragment length polymorphism (RFLP) and sequence analyses. Of 103 dogs examined, seven (6.8%) were positive for Babesia spp. by PCR. The amplified products were digested by restriction enzymes to differentiate the Babesia species, and one sample was identified as Babesia vogeli. The pattern observed for the other six amplification products did not match with pattern described for large Babesia infecting dogs. Sequencing analysis confirmed these six samples as R. vitalii, with high homologies (99-100%) with a sequence from south Brazil. This study confirms the presence of Babesia vogeli and Rangelia vitalii circulate in domestic dogs in Teresópolis, Rio de Janeiro, Brazil.Keywords: Babesia vogeli, Rangelia vitalii, piroplasms, dogs, PCR. ResumoRangelia vitalii é um protozoário que infecta cães e foi descrito nas regiões Sul e Sudeste do Brasil. R. vitalii é filogeneticamente próxima à Babesia spp., mas dados deste misterioso parasito ainda são escassos. O objetivo deste trabalho foi detectar a presença de piroplasmas em cães naturalmente infectados no estado do Rio de Janeiro, através da amplificação do gene 18S rRNA pela PCR, clivagem com enzimas de restrição (RFLP) e caracterização genética através do sequenciamento. De 103 cães, sete (6,8%) foram positivos para Babesia spp. pela PCR. Os produtos amplificados foram digeridos por enzimas de restrição para a diferenciação das espécies de Babesia e uma amostra foi identificada como Babesia vogeli. O padrão de amplificação observado nas outras seis amostras não correspondeu ao padrão descrito para babesias que infectam cães. O sequenciamento das seis amostras confirmou ser uma espécie geneticamente idêntica a R. vitalii apresentando grande homologia (99-100%) com a sequência do sul do Brasil. Este estudo confirma a presença de Babesia vogeli e Rangelia vitalii infectando cães em Teresópolis, Rio de Janeiro, Brasil.

Clinical and hematological evaluation of Rangelia vitalii-naturally infected dogs in southeastern Brazil

“…Rangelia vitalii and Babesia vogeli are morphologically indistinguishable, and species differentiation is done by PCR and genetic sequencing (LEMOS et al, 2012).…”

“…Forward primer Bab 143-167 (5´-CCG TGC TAA TTG TAG GGC TAA TAC A-3´) and reverse primer Bab 858-834 (5´-CCT CTG ACA GTT AAA TAC GAA TGC C-3´) were used in the PCR reaction, which amplified fragments of approximately 700 bp from the gene that encodes the 18S region of piroplasm RNA (SOARES et al, 2011;LEMOS et al, 2012).…”

“…The primers used in this study amplify other canine piroplasms and identification of R. vitalii was only possible by genetic sequencing (SOARES et al, 2011;LEMOS et al, 2012). DNA sequencing was performed on the 36 samples that were found to be positive in PCR assays.…”

“…Differentiation of these species is done by molecular diagnostics by PCR and genetic sequencing. Genes that amplify piroplasms by partial sequences of the 18S rRNA and the heat shock protein 70 (hsp70) genes (SOARES et al, 2011;LEMOS et al, 2012) have been used for parasite DNA detection.…”

Rangeliosis: histopathological analysis, hematology and molecular detection of canine Rangelia vitalii in Rio Grande do Sul, Brazil

“…There is a need for a different method of diagnosis because of the similarity of this disease with canine babesiosis (MORITA et al, 1995;LEMOS et al, 2012). Hence, the objective of this research was to characterize cases of canine rangeliosis identified in the Setor de Patologia Veterinária at Universidade Federal do Rio Grande do Sul (SPV-UFRGS) based on epidemiology, hematology and gross and histopathology in addition to the molecular detection and sequencing of genetic material.…”