The Epidemiological Investigation Of Duck Circovirus

Duck circovirus (DuCV) is listed as a tentative member of the Genus circovirus by the most recent 8th ICTV Report. Ducks infected by DuCV exhibit disordered feather, retarded growth, reduced body weight.In the present study, we conducted the epidemiological investigation from etiology and serology two aspects.1. Epidemic investigation of duck circovirus in spontaneous diseased duck flocks in ChinaThe tissues samples of 343 spontaneous diseased ducks from 36 duck flocks were collected in Shandong, Jiangsu, Sichuan, Fujian and Guangdong of China, and the DNA was extracted to be detected for duck circovirus (DuCV) by hybridization and PCR. The results shown that all the 5 provinces existed DuCV, and the individual positive ratio of DuCV is 81.63%(280/343) and the flock positive ratio of DuCV is 94.44 % (34/36).Besides, the coinfection of DuCV and REV was investigated,the positive ratio is 40.12%(67/167).The results indicate that the infection of DuCV was existing in wide range in duck flocks of China. The DuCV-positive ducks had a higher rate of infection by REV, comparing with DuCV-negative ones.2. Prokaryotic Expression and Antiserum Preparation of Replication Protein of Duck circovirusRep gene was amplified from genomic DNA of Duck circovirus (DuCV) FJ0601 strain by polymerase chain reaction(PCR)and then cloned into pGEX-6P-1 according to the right open reading frame(ORF).The recombinant plasmid was transformed into E.coli BL21 strain for expression with the induction of IPTG. After SDS-PAGE, the expressed specific band was excised from the gel and injected into four-week-old mice 3 times, then the antiserum was collected from the mice and used for IFA with the positive froze histological section of spleen, thymus and bursa that had been tested by PCR. The focuses of infection composed of positive staining cells were found in the three immune organs infected by DuCV, which shows that the fusion protein of Rep in vitro expressed has some epitopes of natural Rep.3. Development of Indirect ELISA for Detection of Antibodies to DuCV and preliminary applicationThe partial Cap gene of DuCV was amplified by PCR method and cloned in prokaryotic vector pET-32a(+)vector to construct the recombinant plasmid. After the induction of IPTG,the purified recombinant protein can be antigen to found an indirect ELISA method to detect the antibody of DuCV.The result showed that the Cap gene of DuCV can expressed steadily and efficiently in E. coli. Western blot analysis proved the renaturation protein has good immunoreactivity against DuCV positive antibody. The indirect ELISA method for the detection of DuCV specific antibody in duck serum was established ,after the optional working circumstances for the ELISA assay ( antigen concentration:4μg/ml; optimal serum dilution:1:50; positive Cut-off value:0.298) with chessboard titration. The results showed that the individual positive ratio is 15.61% in healthy duck flocks; 13.37% (individual) , 88.89% (colonial) in duck flocks; 22.33% (individual), 75% (colonial) in breeder duck flocks. The method was assessed for reproducibility and specificity. From the assay of 1614 serum sample,we learn that the method can be applied in serum diagnosis of DuCV infections.