Establishment Of Real-time Fluorescence PCR Methods For Diagnosing Bovine Babesiosis And Its Applications

Bovine babesiosis is an economically important tick-borne disease of cattle in tropical and subtropicalregions of the world. The disease is mainly caused by two bovine intraerythrocytic protozoan parasites,Babesia bovis and Babesia bigemina. The clinical signs induced by these parasites are similar, ascharacterized by fever, anemia, andicterus in the infected cattle. It is great significant to establish anaccurate, sensitive, rapid pathogen detection method for early diagnosis, treatment and epidemiologicalinvestigations of bovine Babesiosis.(Ⅰ)To detect the B.bovis, a real-time PCR method was established with a pair of primers and aTaqMan probe designed according to the conserved sequence of18S rRNA genes in GenBank. The resultsshowed that: the limit detection of real-time PCR was about1.31×101copies/μL for the target gene whichwas1,000times higher sensitivity than that of conventional PCR, and no cross-reaction with otherpiroplasmasida in cattle. The reproducibility tests in inter-assay and intra-assay indicated that thecoefficients of variation were less than3%. A total of23clinical samples were tested by the real-time PCRcomparing with conventional PCR, and positive rates were52.17%(12/23) and30.43%(7/23),respectively.(Ⅱ)A pair of primers and a TaqMan probe was designed according to the conserved sequence of the18S rRNA gene of B. bigemina, and a TaqMan real-time PCR method was established for detection of B.bigemina. In result, the sensitivity of the method was1.1×101copies/μL which was1000times highersensitivity than that of conventional PCR. And the reproducibility tests in inter-assay and intra-assayindicated that the coefficients of variation were0.21%-1.14%and0.31%-1.50%, respectively, which wereless than2%. Moreover, the method was also highly specificity and had no cross-reaction with other bovinepiroplasms. A total of30clinical samples were tested by the established real-time PCR and theconventional PCR, respectively, the positive rates by the real-time PCR was30%, but16.7%positive bythe conventional PCR.(Ⅲ) The established real-time PCR were applied to bovine babesiosis detection for the suspected sampleswhich collected from various parts of Xinjiang yak (cattle). The B.bovis positive rate was7.12%andB.bigemina positive rate was6.10%; The highest positive rate of B.bovis and B.bigemina were at the Kashi,13.33%,and11.67%, respectively; Calf infection rate of the B.bovis and B.bigemina were higher than adultcattle, the positive rate were9.52%and8.73%, respectively; The Babesia positive rate of Yak were higherthan other varieties. Comparing real-time PCR, ELISA and direct microscopic examination, real-time PCRwas the most specific and accurate motheds for diagnosing Babesiosis.The real-time PCR method for detection of B.bovis and B. bigemina was sensitive, specific and rapiddetection technology which can be used for early diagnosis and daily monitoring. It will provid a strongguarantee for the tick-borne bovine piroplasmosis control and epidemiological investigation.