Preliminary Study On Immunogenicity And Immunoproteomics Of Extracellular Products Of Aeromonas Veronii TH0426

Aeromonas veronii is a kind of Human-animal-fish zoonotic pathogen,which is widely existed in the natural environment,and causes massive fish death and other aquatic animal disease.Some countries have classified A.veronii as quarantine items.The studies on A.veronii are gradually conducted at many countries.Extracellular products(ECPs)are products that are secreted by the pathogen in the process of growth and reproduction.It has good immunogenicity and can cause the host to produce immune response,so it can considered to be an effective protective antigen.At present,there is no study on the immunological effect of Aeromonas veronii extracellular products.So,in this study,Aeromonas veronii TH0426 strain was used as experimental material to prepare its extracellular products,and analyze the immunoprotective effect of extracellular products on koi and the immunogen proteomics of the extracellular product to identify the immunogenic protein.In order to study the component and function of extracellular products(ECPs)from Aeromonas veronii TH0426.In this test,we study ECP's pathogenicity,enzyme activity and immunogenicity,which was prepared by ultrafiltration.The results showed that the ECPs included protease,lipase,amylase and heamlyase.It was proved to be lethal to koi,and the LD50 does was 391.6 ?g protein/per fish.We used 240 carp koi to study the effect of extracellular products on immune protection,which were randomly assigned to four groups,and each group had 60 koi.The group one were intraperitoneally injected with ECPs protein with Freund's adjuvant;the group two were intraperitoneally injected with ECPs and inactivated A.veronii TH0426 strain combined with freund's adjuvant(FKC+ECPs);the group three were intraperitoneally injected with inactivated A.veronii TH0426 strain combined with freund's adjuvant(FKC);the group four were intraperitoneally injected with sterile PBS as the control group.Carp koi were immunized after one week in laboratory conditions.At the third week we continued the second immunity and observed the changes of immune parameters.We collected the blood of 5 koi randomly from each group every week and the koi were humanely sacrificed by anesthesia euthanasia.We detected the IgM,IL-1?,TNF-?,and LZM in blood.At the 4th week after first immunity,20 koi from each group were intraperitoneally challenged with 0.2mL(2×106cfu/mL,10×LD50)A.veronii strain TH0426,then we observed the RPS of each group and evaluated the immune protective effects.The results showed that immunize ECPs could protective koi against A.veronii,and the immune effect is best,the RPS was 75%.Furthermore,the RPS of FKC+ECPs and FKC were 50% and 70% respectively.The immune parameters also showed that immunize ECPs can induce the immune response of koi and can form a higher protective effect.The extracellular products were analyzed by immunoglobulin 2-DE and 2-DE gel immunoblot analysis to identified the immunogenic proteins,and the PCR method was used to detect the distribution of three immunogenic proteins in different isolates of Aeromonas veronii.The results showed that the extracellular products contained 11 kinds of immunogenic proteins,including some known immunogenic proteins,such as Peptidase S8,Long-chain fatty acid outer membrane transporter,Membrane protein,Aerolysin-like toxin,Major outer membrane protein OmpAI,Maltoporin and so on.Three genes of interesting proteins,Membrane protein,Long-chain fatty acid outer membrane transporter and Peptidase S8,were detected among 100 strains of Aeromonas veronii by PCR amplification,the distribution rates were 97%(97/100),89%(89/100)and 77%(77/100)respectively.