Optimization of a molecular method for the diagnosis of canine babesiosis

Abstract

Babesiosis is a hemolytic disease caused by protozoans of the genus Babesia (Apicomplexa). This disease occurs worldwide and is transmitted by ticks to a variety of mammals, including humans. The objective of the present study was to optimize a molecular approach for the detection of a fragment of 18S rDNA of Babesia canis, Babesia vogeli, Babesia rossi or Babesia gibsoni based on a single semi-nested Polymerase Chain Reaction (PCR), and compare the efficiency of this approach with that of a simple PCR protocol. To this end, 100 blood samples collected from dogs with suspected hemoparasite infections were analyzed. A comparison of the results of simple PCR and semi-nested PCR indicated a highly significant difference (p value = 0.0000). While only five (5%) of the samples tested positive using the simple protocol, 22 (22%) were positive using the snPCR technique. The results of this study reinforce the findings of previous studies, which have demonstrated the greater sensitivity of tests based on nested or semi-nested PCR. Therefore, to avoid false-negative results due to low levels of parasitemia, we suggest the preferential use of this protocol in epidemiological studies of canine babesiosis, particularly those that require reliable estimates of the prevalence of infection.Keywords: Babesia, molecular diagnosis, semi-nested PCR, eastern Amazonia. ResumoA babesiose é uma doença hemolítica de ocorrência mundial, causada por protozoários do gênero Babesia (Apicomplexa), que são transmitidos por carrapatos a diversos mamíferos, incluindo o homem. O objetivo deste estudo foi otimizar um método molecular para a detecção de fragmento do 18S rDNA de Babesia canis, Babesia vogeli, Babesia rossi ou Babesia gibsoni com base em uma única semi-nested (snPCR), comparando sua eficiência com um protocolo de PCR simples. Para isso, 100 amostras de sangue de cães com suspeita de hemoparasitoses foram analisadas e, enquanto o protocolo de PCR simples indicou somente 5% (5/100) de amostras positivas, o protocolo de snPCR, com 22% (22/100) de amostras positivas, apresentou maior sensibilidade (p valor = 0,0000). Este resultado está de acordo com outros estudos que mostram a maior sensibilidade de detecção dos testes baseado em nested ou snPCR. Assim, como uma forma de prevenir resultados falso-negativos devido à baixa parasitemia, sugere-se que este protocolo seja preferencialmente usado nos estudos epidemiológicos de babesiose canina, em especial naqueles que tratam da sua prevalência.

Molecular characterization of Babesia vogeli in dogs from Belém, northern Brazil

“…Despite the fact that several studies have been conducted on Babesia spp., those by Spolidorio et al (2013) and Moraes et al (2014) appear to be the only published reports of the occurrence of the genus in northern Brazil. Spolidorio et al (2013) conducted a serosurvey and an indirect immunofluorescence assay with the antigens B. vogeli, Ehrlichia canis, and six Rickettsia species, while Moraes et al (2014) used the same molecular method as used in the present study.…”

“…Spolidorio et al (2013) conducted a serosurvey and an indirect immunofluorescence assay with the antigens B. vogeli, Ehrlichia canis, and six Rickettsia species, while Moraes et al (2014) used the same molecular method as used in the present study. Whereas Spolidorio et al (2013) detected anti-B.…”

“…The molecular detection of Babesia spp was performed in a semi-nested PCR (snPCR), as described by Moraes et al (2014). Briefly, a primer set targeting a partial sequence (394 bp) of the 18S rDNA gene was used in an initial reaction.…”

“…In Brazil, canine babesiosis is typically caused by B. vogeli, which is known to occur in the south (Trapp et al, 2006;Jojima et al, 2008;Vieira et al, 2013), southeast (Dell-Porto et al, 1990, 1993Spiewak, 1992;O'Dwyer, 2000;Costa-Junior et al, 2009;Lemos et al, 2012), midwest (Souza et al, 2004;Duarte et al, 2011;Spolidorio et al, 2011), north-east (Carlos et al, 2006;Lima et al, 2006;Ramos et al, 2010;Silva et al, 2012), and north (Spolidorio et al, 2013;Moraes et al, 2014). In addition, there have been some reports of B. gibsoni from the south of the country (Trapp et al, 2006;Jojima et al, 2008).…”

“…Regarding canine piroplasms occurring in northern Brazil, their molecular characterization is limited to that conducted by Moraes et al (2014), who reported a short fragment (394 bp) of the 18S rDNA gene from B. vogeli in one dog from the Belém metropolitan area, Pará State. Molecular methods have been used for the description of genotypically novel pathogens by their small subunit rRNA (ssrRNA) gene sequences, which have been applied for the comparison of geographically different strains.…”