The Analysis Of Antigenicity Of Isolated Riemerella Anatipestifer And Establishing Of Latex Agglutination Test

Riemerella anatipestifer disease, also called duck infectious serositis, is a contagious disease of many types of poultry, such as domestic ducks, turkeys, geese and other many kinds of birds, caused by Riemerella anatipestifer(RA). Infected domestic ducks show the main clinical symptoms which were depression, ataxia, opisthotonos, dysentery. The disease is accompanied with pathological changes, such as fibropericarditis, perihepatitis, salpingitis, meningitis, conjunctivitis, arthritis, septicemia and so on. RA causes high morbidity and mortality with various serotypes, but the lack of cross-protection between different serotypes causes difficulties to the prevention and control of the disease, RA causes important economic losses to the duck industry.Four bacteria were isolated from affected ducks in duck farms from Heilongjiang, Shandong, Guangdong province, respectively,and identified as Riemerella anatipestifer,designated HLGl, SD0901, SD0902and GD0901, according its morphology, serologic test and biochemical characteristics. The four isolates were high sensitive to enrofloxacin, ofloxacin and cephalosporin V, but resistive to kanamycin and gentamycin. The cloning and sequencing of ompA genes of the isolates showed that ompA gene was1164bp in length, which shared89%to100%similarity with other RA reference strains, Furthermore, SPF ducks were subcutaneously injected in cervical part at a dosage of5X108CFU of isolate HLGl and showed the clinical signals of RA infection and pathological changes in heart, liver, spleen, lung, brain and kidney.The Riean0791, Riean1110and Riean1852genes of HLGl strain of Riemerella anatipestifer were amplified using3pairs of primers and cloned into pMD18-T vector. Sequencing showed that lengths of ORFs encoding Riean0791, Riean1100and Riean1852were1404bp,1437bp and1374bp, respectively. Riean0791, Riean1110and Riean1852were cloned into pPRO-EX-Htb to construct the recombinant plasmids pHTb-Riean0791, pHTb-Riean1110and pHTb-Riean1852. The recombinant plasmids were transformed into the E.coli BL21(DE3) plysS and induced by IPTG. SDS-PAGE showed that the rRiean0791, rRiean1110and rRiean1852were about55ku,56ku and54ku and in the form of inclusion body. Western blot analysis was indicated that rRiean0791, rRiean1110and rRiean1852were recognized by the positive serum of RA. SPF ducks were immunized with rRiean0791, rRiean1110and rRiean1852and produced high level of antibody titers by ELISA and immune protective rate were5/6,0/6and2/6, respectively. Western blot analysis showed that native Riean0791, Riean1110and Riean1852protein of HLGl strain was mainly in membrane protein of bacteria by positive duck serum of immunized by rRiean0791, rRiean1110and rRiean1852.A latex agglutination test based on rRiean0791was constructed to detect specific antibodies of RA. The optimization conditions were that the concentration of carboxylated latex was1%, the quantity of rRiean0791was200μg and the time was6hour. LAT had good reaction with RA positive serum apparently, and showed no reaction with PBS, BBS, negative duck serum and other positive serum. The LAT had better specificity, sensitivity and repetitiveness. The agreement rate between RA latex agglutination test and indirect ELISA was91.04%. In this study, isolation and identification of RA from ducks and construction of latex agglutination test provides the method to diagnosis and monitoring SPF ducks and a rapid diagnostic method to prevent and control RA in field.