A quantitative PCR for the detection and quantification of Babesia bovis and B. bigemina

BULING, A.; CRIADO-FORNELIO, A.; ASENZO, G.; BENITEZ, D.; BARBA-CARRETERO, J.C.; MONICA OFELIA JACOBSEN

Veterinary Parasitology

Elsevier

Año: 2007 vol. 147 p. 16 - 25

The haemoparasites Babesia bovis and B. bigemina affect cattle overBabesia bovis and B. bigemina affect cattle over vast areas of the tropics and temperate parts of the world. Microscopicvast areas of the tropics and temperate parts of the world. Microscopic examination of blood smears allows the detection of clinical cases ofexamination of blood smears allows the detection of clinical cases of babesiosis, but this procedure lacks sensitivity when parasitaemia levels arebabesiosis, but this procedure lacks sensitivity when parasitaemia levels are low. In addition, differentiating between similar haemoparasites can be verylow. In addition, differentiating between similar haemoparasites can be very difficult. Molecular diagnostic procedures can, however, overcome thesedifficult. Molecular diagnostic procedures can, however, overcome these problems. This paper reports a quantitative PCR (qPCR) assay involving theproblems. This paper reports a quantitative PCR (qPCR) assay involving the use of SYBR Green. Based on the amplification of a small fragment of theuse of SYBR Green. Based on the amplification of a small fragment of the cytochrome b gene, this method shows both high sensitivity and specificity, andcytochrome b gene, this method shows both high sensitivity and specificity, and allows quantification of parasite DNA. In tests, reproducible quantitative resultsallows quantification of parasite DNA. In tests, reproducible quantitative results were obtained over the range of 0.1 ng to 0.1 fg of parasite DNA. Melting curvewere obtained over the range of 0.1 ng to 0.1 fg of parasite DNA. Melting curve analysis differentiated between B. bovis and B. bigemina. To assess theanalysis differentiated between B. bovis and B. bigemina. To assess the performance of the new qPCR procedure it was used to screen for babesiosis inperformance of the new qPCR procedure it was used to screen for babesiosis in 40 cows and 80 horses. Babesia bigemina was detected in five cows (three of40 cows and 80 horses. Babesia bigemina was detected in five cows (three of these were also found to be positive by standard PCR techniques targeting thethese were also found to be positive by standard PCR techniques targeting the 18S rRNA gene). In addition, B. bovis was detected in one horse and B.18S rRNA gene). In addition, B. bovis was detected in one horse and B. bigemina in two horses using the proposed method, while none was foundbigemina in two horses using the proposed method, while none was found positive by ribosomal standard PCR. The sequences of the B. bigeminapositive by ribosomal standard PCR. The sequences of the B. bigemina cytochrome b and 18S rRNA genes were completely conserved in isolates fromcytochrome b and 18S rRNA genes were completely conserved in isolates from Spain and Argentina, while those of B. bovis showed moderate polymorphism.Spain and Argentina, while those of B. bovis showed moderate polymorphism.