Development and Evaluation of a Seminested PCR for Detection and Differentiation ofBabesia gibsoni(Asian Genotype) andB. canisDNA in Canine Blood Samples

Abstract

Canine babesiosis has recently been recognized as an emerging infectious disease of dogs in North America. We sought to develop a seminested PCR to detect and differentiate Babesia gibsoni (Asian genotype), B. canis subsp. vogeli, B. canis subsp. canis, and B. canis subsp. rossi DNA in canine blood samples. An outer primer pair was designed to amplify an ϳ340-bp fragment of the 18S rRNA genes from B. gibsoni (Asian genotype), B. canis subsp. vogeli, B. canis subsp. rossi, and B. canis subsp. canis but not mammalian DNA. Forward primers were designed that would specifically amplify a smaller fragment from each organism in a seminested PCR. The practical limit of detection was 50 organisms/ml of mock-infected EDTA anticoagulated whole blood. The primer pair also amplified an ϳ370-bp fragment of the B. gibsoni (USA/California genotype) 18S rRNA gene from the blood of an experimentally infected dog with a high percentage of parasitemia. Amplicons were not detected when DNA extracted from the blood of a dog that was naturally infected with Theileria annae at a low percentage of parasitemia was amplified. Due to limited sensitivity, this test is not recommended for the routine diagnosis of B. gibsoni (USA/California genotype) or T. annae. The PCR test did not amplify Toxoplasma gondii, Neospora caninum, Leishmania infantum, Cryptosporidium parvum, or canine DNA under any of the conditions tested. The seminested PCR test was able to detect and discriminate B. gibsoni (Asian genotype), B. canis subsp. vogeli, B. canis subsp. canis, and B. canis subsp. rossi DNA in blood samples from infected dogs.

Babesia infection in naturally exposed pet dogs from a north-eastern state (Assam) of India: detection by microscopy and polymerase chain reaction

“…This is in agreement with Matsuu et al (2005) who reported detection limit of light microscopy as approximately 0.001 % parasitaemia compared to PCR capable of detecting 9 parasites per ll of blood similar to 50 organisms/ml of blood reported by Birkenheuer et al (2003).…”

“…So 17.12 % cases remained false negative to Babesia during microscopy. Higher detection of Babesia in PCR compared to microscopy as observed in the present study was also reported by several authors (Birkenheuer et al 2003;Gotsch et al 2009;O'Dwyer et al 2009). The prevalence pattern of this two species recorded in the present study was found similar to those microscopic and serologic findings reported by other workers (Bansal et al 1985;Varshney et al 2003;Senthil Kumar et al 2009).…”

Investigação molecular de Ehrlichia spp. e Anaplasma platys em felinos domésticos: alterações clínicas, hematológicas e bioquímicas

“…Para isto foram utilizados os oligonucleotídeos GAPDH-F (5'-CCT TCA TTG ACC TCA ACT ACA T-3') e GAPDH-R (5'-CCA AAG TTG TCA TGG ATGACC-3') que anelam em uma seqüência especíϐica do gene da enzima gliceraldeído 3-fosfato desidrogenase e resulta em um produto de 400 pb (Birkenheuer et al 2003 Doyle et al (2005). Para a identiϐicação de A. platys foi utilizada a nested polymerase chain reaction (nPCR) foi realizada como descrito por Martin et al (2005), usando oligonucleotídeos 8F ( 5' AGT TTG ATC ATG GCT CAG 3') e 1448R (5' CCA TGG CGT GAC GGG CAG TGT G 3'), que ampliϐica a maior parte do gene 16S rRNA (Dawson et al 1996), na primeira etapa da PCR.…”

Canine bartonellosis: serological and molecular prevalence in Brazil and evidence of co-infection withBartonella henselaeandBartonella vinsoniisubsp.berkhoffii

“…The absence of PCR inhibitors was demonstrated by the amplification of a fragment of the constitutive gene for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein, an enzyme related to the glycolysis pathway that is expressed in all mammal cells [5,7].…”