Detection and molecular characterization of a canine piroplasm from Brazil

Abstract

In the beginning of the 20th century, a new canine disease was reported in Brazil under the name "nambiuvú", whose etiological agent was called Rangelia vitalii, a distinct piroplasm that was shown to parasitize not only erythrocytes, but also leucocytes and endothelial cells. In this new century, more publications on R. vitalii were reported from Brazil, including an extensive study on its ultrastructural analysis, in addition to clinical, pathological, and epidemiological data on nambiuvú. However, a molecular analysis of R. vitalii has not been performed to date. In the present study, we performed molecular phylogenetic analyses of R. vitalii based on fragments of the genes 18S rRNA and the heat shock protein 70 (hsp70), amplified by PCR performed on blood samples derived from five clinical cases of dogs presumably infected with R. vitalii in southern Brazil. In addition, we examined Giemsa-stained thin blood smears from these same dogs. DNA sequences (604-bp) of the 18S rRNA gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (95%) with Babesia sp. China-BQ1. DNA sequences (1056-bp) of the hsp70 gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (87%) with Babesia bigemina. Phylogenetic analyses inferred from either of the two genes resulted in the newly genotype being placed in the Babesia spp. sensu stricto clade with very high bootstrap support (95-100%) in three analyses (Neighbor-Joining, Maximum parsimony, and Maximum likelihood). Giemsa-stained thin blood smears from the dogs were shown to contain piroplasm organisms within erythrocytes, monocytes and neutrophils (individual forms), and schizont-like forms within neutrophils, in accordance with literature reports of R. vitalii. Based on these results, we conclude that R. vitalii, the etiological agent of "nambiuvú" in southern Brazil, is a valid species of piroplasm. Further studies are required to evaluate the validity of the genus Rangelia.

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

“…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.…”

Clinical, hematological, parasitological and pathoanatomical profiles of Rangelia vitalii infection in a dog: a case report

“…O ciclo de vida deste protozoário consiste no estágio de desenvolvimento eritrocítico e na forma exoeritrocitária, ocorrendo no citoplasma de neutrófilos, monócitos e células endoteliais (FHIGERA et al, 2010;SOARES et al, 2011;DA SILVA et al, 2011). Na fase aguda da doença são encontrados principalmente no sangue circulante e encontrados, também, em corte histológico de fígado, baço, pulmão, rim e medula óssea (LORETTI; BARROS, 2005).…”

“…Rangelia vitalii foi descrito pela primeira vez por Pestana (1910) e reavaliado por Carine e Maciel (1914), porém recentemente foi validado por técnicas moleculares (SOARES et al, 2011).…”