Rapid Detection Of Pathogen Bacteria And Development Of Killed Vaccines For Aquaculture Animals

Vibrio anguillarum and Streptococcus iniae, the causative agent of vibriosis and streptococcus disease, are evastating fish pathogen prevailing in worldwide fishery industries and accounting for severe economic losses. The rapid on-site detection and effective vaccination for these pathogens are in urgent need. In this study, a modified dot blotting method was developed for the on-site determination of V. anguillarum. We also developed vaccines against V. anguillarum and S. iniae by screening different adjuvants based on formailn whole-cell killed pathogenic bacteria as antigens. This work will provide useful imformation for the sanitary control of V. anguillarum and S. iniae.In this study, a mouse monoclonal antibody (MAb) against V. anguillarum MVM425 was generated using hybridoma technology and its isotype was characterized. Based on MAb 6B3-C5 labeled with CdSe/ZnS QDs, a modified dot blotting mothod was developed for the on-site determination of V. anguillarum. It was found that the method had no cross-reactivity with other bacteria than V. anguillarum in aquaculture. The detection limit for V. anguillarum was 1×103 CFU/ml in cultured bacterial suspension samples, which was 100-fold higher sensitivity than that by reported colloidal gold immunochromatographic test strip.V. anguillarum vaccines were prepared by using formalin killed whole-cell bacteria as antigen. By mixed with oligosaccharide H3 as a nonspecific immunologic stimulant, the challenged RPS at 28 days after immunization reached 100%, while the number dropped down to 75.3% when challenged at 60 days after immunization. On the other hand, the immune group which used MONTANIDETM ISA 763A as oil adjuvant had a continuous immune protection, when challenged with V. anguillarum at 60 days after immunization, the RPS still maintained 85.2%, showed a great application prospect.S. iniae HT701 was also isolated and identified from diseased tilapia. The biochemistry characteristics, antibiotics susceptibilities and virulence to model fish of this isolate were analyzed extensively. By mixed with S. iniae HT701 ferrichrome transporter protein FtsB which expressed and purified in this study as immune stimulus and Mobil Marcol 52 as oil adjuvant, formailn killed whole-cell vaccine against S. iniae HT701 was prepared and tiapia was immuned though intraperitoneal injection. The serum antibody level and transcriptional level were also analyzed. Results showed that FtsB possessed strong immunogenicity, while Mobil Marcol 52 oil adjuvant could extend the immuno protection phase and reduce immune mortality compared to other oil adjuvant.The RPS at 28 days and 60 days after immunization reached 72% and 65%,respectively, showed great long-term protection against S. iniae.