Serosurvey Of Canine Brucellosis And Developing A Rapid Diagnosis Of Brucella By Real-time PCR

Canine brucellosis is a zoonosis caused by Brucella spp., which is easily spreading in kennel. The continuous separation of Brucella canis suggests that the epidemic trend of canine brucellosis has increased in recent years in China. With the number of pet dogs is increasing, the prevention and control of canine brucellosis should also get more attention. This research investigated the prevalence of canine brucellosis in Chengdu and the surrounding areas, and initially established a rapid detection method for canine brucellosis detection by Real-time PCR.1. Serosurvey of canine brucellosis:the serum samples are from Chengdu Boai animal hospital, Jintang kennel and stray dogs,1010 in all. Smooth and rough of Rose Bengal plate agglutination test were used in screening test of all collected samples. RBPT positive serum samples were further reexamined using smooth and rough of Standard tube Agglutination Test. The results showed that seropositive samples of smooth and rough RBPT were 14 and 19, respectively; 33 in all. The seroprevalence of smooth and rough RBPT were 1.39% and 1.88%, respectively. The seroprevalence of RBPT was 3.27%. The RBPT seropositive samples were reexamined using standard tube agglutination test. The results showed that seropositive samples of smooth and rough of SAT were 1 and 12, respectively; 13 in all. The seroprevalence of smooth and rough SAT were 0.10% and 1.19%; the seroprevalence of SAT was 1.29%. This study showed that stray dogs have a highest seroprevalence, followed by the dog, then the Boai animal hospital in which the seroprevalence of canine brucellosis is the lowest.2. Establishing a rapid detection method for canine brucellosis via Real-time PCR: A pair of specific primers was designed directed to bcsp31, a shared gene of the Brucella. The bcsp31 gene fragment was cloned into pGM-T vector. Recombinant pGM-T-bcsp31 was used for the preparation of Real-time PCR standard curve. Primer specific test results showed that genome DNA of 8 kinds of standard strains of Brucella was amplified the same fragment, while control strains were none amplified, indicating that the method has good specificity. From the results of sensitivity analysis of this Real-time PCR, we can see that the ordinary PCR can only minimally amplify 10-2 magnitude concentration of DNA, while the established quantitative PCR method can minimally amplify 10"4 magnitude concentration of DNA, of which the sensitivity is 100 times higher than ordinary PCR.33 RBPT positive serum samples were detected by this Real-time PCR method, and 10 of them were positive amplified. Real-time PCR test was carried out on 100 randomly selected RBPT negative samples,6 samples showed positive amplification. The Real-time PCR established in this study could detect Brucella spp. in seronegative samples. But for seropositive samples, there were differences between Real-time PCR test and SAT results. Two methods can be used in detecting canine brucellosis. There would be greater results when combining both two methods.