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.
Subject(s)
Babesiosis/diagnosis , Dog Diseases/diagnosis , Dog Diseases/parasitology , Animals , Babesia/genetics , DNA, Ribosomal/analysis , Dogs , Molecular Diagnostic Techniques/standards , Polymerase Chain ReactionABSTRACT
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.
A 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.
Subject(s)
Animals , Dogs , Babesiosis/diagnosis , Dog Diseases/diagnosis , Dog Diseases/parasitology , Babesia/genetics , DNA, Ribosomal/analysis , Molecular Diagnostic Techniques/standards , Polymerase Chain ReactionABSTRACT
We hereby propose a novel sensitive, specific, and cost-efficient method to detect Ehrlichia canis and Anaplasma platys DNA from canine whole blood samples by multiplex PCR. Blood samples from hemoparasited dogs attending the Veterinary Hospital at the Universidade Federal Rural da Amazônia-UFRA, Belém, Brazil, were collected in tubes containing EDTA. Amplification of E. canis and A. platys 16S rDNA by nested (n) PCR was successfully achieved by using primers specific to the Anaplasmataceae in the first round of PCR, followed by a second round of PCR using E. canis-specific primers in conjunction with A. platys-specific primers. The amplicons obtained were cloned and sequenced, yielding sequences of 478 and 473 bp (including primers) pertaining to regions of the 16S rDNA of E. canis and A. platys, respectively. The protocol we here propose may help to measure the prevalence of canine monocytic ehrlichiosis (CME) and canine cyclic thrompocytopenia, not only in northern Brazil, where there is no data available, but also elsewhere.
