Journal
VETERINARY PARASITOLOGY
Volume 187, Issue 1-2, Pages 17-22Publisher
ELSEVIER
DOI: 10.1016/j.vetpar.2012.01.008
Keywords
B. bigemina; PCR; AMA-1; Mongolia; Cattle
Categories
Funding
- program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN)
- Japanese Ministry of Education, Science, Sports, Culture, and Technology
- Japan Society for Promotion of Science (JSPS), Japan
- Grants-in-Aid for Scientific Research [22380154, 23405041, 10F00420] Funding Source: KAKEN
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We developed a new nested PCR (nPCR) assay based on the Babesia bigemina apical membrane antigen-1 (AMA-1) gene sequence for parasite-specific detection. The primers were designed to amplify 738-bp and 211-bp fragments of the AMA-1 gene by primary and nested PCRs, respectively. The assay was proven to be specific for the B. bigemina, whereas the previously established Spel-AvaI nPCR assay amplified not only the target fragment of B. bigemina but also a homologous one from Babesia ovata. The AMA-1 nPCR assay was also evaluated using field DNA samples extracted from 266 bovine blood samples collected from Mongolia in 2010. In a comparative evaluation, 90(33.8%) and 25(9.4%) of the blood samples showed positive reactions for B. bigemina by the Spel-AvaI nPCR and AMA-1 nPCR assays, respectively. The sequencing analysis of the nPCR products confirmed that the AMA-1 nPCR method had specifically detected the target B. bigemina DNA. However, 4 different kinds of sequences were determined among the Spel-AvaI nPCR amplicons. Two of them were derived from B. bigemina and B. ovata, while the origins of the others were unknown. In the current study, the presence of B. bigemina was clearly demonstrated among Mongolian cattle populations by the current nPCR assay for the first time. Furthermore, our findings also indicate that the AMA-1 nPCR assay may be a useful diagnostic tool for the specific detection of B. bigemina.(C) 2012 Elsevier B.V. All rights reserved.
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