Isolation, Identification And Development An Elisa Assay For Serum Antibodies Detection Of Brachyspira Hyodysenteriae

Swine Dysentery (SD) caused by the anaerobic intestinal Spirochaete Brachyspira hyodysenteriae is a contagious mucohaemorrhagic diarrhoeal and economically important disease in many piggeries through the word. The definitive diagnosis of SD primarily relies on the isolation and identification of the spirochaete, nevertheless culture and PCR testing is time consuming, relatively costly, and may not identify pigs that intermittently excrete low numbers of spirochaetes. Therefore, establishment a rapid and accurate diagnostic method of B.hyo detection from pig-farms is urgent and necessary.According to the previous studies and conditions of the lab, a rapid laboratory method about isolation and identification of B.hyo was successfully established. Detected the conservative NADH oxidase (nox) gene of B.hyo by using PCR out of clinical samples firstly; then,cultured the samples demonstrated by PCR in an anaerobic bag with the differential medium BAM-SR (Blood Agarose Medium with Spectinomycin and Rifampicin) and stained the isolates with the modified Warthin-Starry method; diagnosed with16S rRNA gene sequence analysis finally. In this study,13samples were detected positive and one strain named SHB1was isolated and cultured from254pigs’large intestinal mucosa and fecal samples.An outer membrane lipoprotein Bhlp30.7with specification and immunogenicity was obtained after the expression and purification from the E.coli prokaryotic express system. The Bhlp30.7expression and purification conditions were optimized. This study established an indirect ELISA with the reference sera and the recombinant Bhlp30.7as an ELISA antigen and optimized the conditions of the indirect ELISA. The expression of the30.7KDa histidine-tagged fusion protein Bhlp30.7was induced by0.1M IPTG for6h. The optimal binding buffer containing100mM imidazole was selected in purification of the target protein. The titration experiment results indicated that the best parameters were2μg/mL coated-antigen and200-fold serum dilution.This study was to express a His-tagged fusion protein His6-Bhlp30.7(Histidine-tagged Brachyspira hyodysenteriae lipoprotein30.7kD) as the ELISA antigen for detecting SD by using609sera collected from28herds of the surrounding areas of Shanghai. Compared with the history of SD incidence in all herds and.PCR results, a cut-off value was set for the assay. A cut-off value4.28(P<0.05) and the suspected positive critical point4.49were obtained.46positive sera,38suspected sera and525negative sera were detected from609serum samples, accounted for7.56%,6.24%and86.2%respectively of the total. The positive rate of sows was14.1%accounted of its population. The piglets’ was0.75%and the finishing-pigs’ was1.2%. The sows population have the highest infection risk of B.hyo from the above experimental data analysis. The indirect ELISA for the detection of porcine serum antibodies can be used as an infection risk assessment method, which provides a scientific basis for prevention and control of SD.