| Bovine anaplasmosis results from infection with A.marginale. A second species, A.centrale, has long been recognised. While A.marginale is the primary pathogen in bovine anaplasmosis, it is not confined to cattle, nor is it the only pathogen of the genus. A. ovis primarily infects sheep, but also goats and some wild animals. There are many methods that detect Anaplasmosis. To date, there has not been a PCR method described that reports amplification of all Anaplasma species and differentiability of A.marginale , A.centrale and A. ovis.An msp4 nested PCR was designed to amplify the major surface protein 4 gene of the rickettisal pathogen: A.marginale, A.eentrale and A.ovis. The nested amplicons in size are 716bp, 431 bp, 310bp, 584bp representing anaplasma species, A. marginale, A. centrale, A.ovis. The nested PCR using the primers of Anaplasma species detect the samples, then differentiate the positive samples using the separate primers. The nested PCR can detect DNAas low as 200 fg. B. bovis, B. bigemina, B. motasi, T. hirci, E. wenyonii, orientalis sp. Nov, T. gondii, T.evansi, cannot be amplified by this method. 1119 samples were detected and of which 9. 4 % were positive, the same as the results of MSP5 nested PCR. It indicates that this method can be use to diagnosis Anaplasmosis. The tests were found that any two of A. marginale, A.ovis and A. centrale can be co-infected.Meanwhile, Primers and TaqMan-MGB Probe were designed from the sequence of major surface protein 4 genes specific to A. marginale. A real-time PCR method was developed for detecting A. marginale, which can detect DNA ofA. marginale as low as 200 f g. A.centrale, A.ovis, B. bovis, B. bigemina, B. motasi T. hirci, E. wenyonii orientalis sp. Nov, T. gondii, T.evansi, cannot be amplified by this method. 180 samples from Jiangsu and Heilongjiang were detected and of which 8.9 % were positive, the same as the results of MSP4 nested PCR. It indicates that this method can be use to diagnosis Anaplasmosis.
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