Immunological Evaluation On The Expression System Of Infectious Pancreatic Necrosis Virus VP2-VP3in Trout Intestinal Lactobacillus

Infectious pancreatic necrosis virus (IPNV) is a pathogen that causes infectious pancreatic necrosis disease in salmon fish (IPN). The disease is highly contagious and contacts, IPN virus mainly against juvenile salmonid species. The mortality rate as high as90%or more, the surviving fish carriers of the virus lifelong, is a potential source of infection. The main symptom of this disease is solid organs such as liver and pancreas hemorrhage, necrosis. The disease epidemic widely with a high range of incidence and mortality, brought huge economic losses each year to fish farming around the world, becoming the first class in fish port quarantine quarantine.In this study, isolated lactobacilli with Lactobacillus selective medium MRS from rainbow intestine. After doing Gram staining, select Gram-positive, no exercise, no spores, rod-shaped bacteria conducted catalase, oxidase activity and nitrate reduction experiments, strains obtained was purified by plate scribing. All strains isolated test sugar fermentation reaction. Identification results are classified by "Bergermine bacteria Classification Manual".To further confirming, Lactobacillus16s rRNA primers were designed specifically.The sequencing results showed the sequence of isolates were highly consistent with Lactobacillus plantarum. Although many scholars have reported isolated Lactobacillus, but isolated Lactobacillus from the intestine of rainbow trout is the first time.Tolerance to the environmental stress plays an important role when the microorganisms are used in industry or are applied as probiotics. The five selected strains were all displayed a high survival in all of the conditions of pH3.0,8%NaCl,0.5%bile,7gl-1trypsin, and10g1-lpepsin (Fig.1), whereas all of the strains can’t live in pH2.0. On the basis of temperature tests, all of the5strains can live almost normally after disposed to60℃for10min, but none colonel formed after dealt with80or100℃.Antimicrobial activities of the bacterial isolates were tested for growth inhibition of9pathogenic microorganisms. In this investigation, we found that all of the five selected strains with the capacity to suppress growth of both Gram-positive (Staphylococcus aureus, Listeria, Streptococcus dysgalactiae, Streptococcus iniae) and Gram-negative (Colibacillus, Pasteurella, Salmonella typhimurium, Edwardsiella tarda, Aeromonas hydrophila) pathogens.The use of probiotics has been increasingly viewed as an alternative to antibiotic treatment in reducing the incidence of disease in aquaculture. In this report, antibiogram tests against24 antibiotics were performed using Sensi-Disc (6mm) to determine the antibiotic susceptibility of the selected strains. It is noteworthy that the strains isolated in our current study were resistant to vancomycin and highly sensitive to erythrocin, This is consistent with many previous studiesThe colonization potential of the selected strains in rainbow trout intestines L1026, L1212and Lactobacillus KLDS1.0344as control was conducted by using5(and6)-carboxyfluorescein diacetate, succinimidyl ester (cFDA-SE), intragastrically administered rainbow trout juveniles. Three rainbow trout of each group were killed on days1,2,4,6,10and15, the intestines were divided into3parts and the presence of cFDA-SE-labeled L.plantarum was monitored by a Epics Elite flow sorter. The rate of wash-out of L1026, L1129-2, and KLDS1.0344from the intestinal mucosal surface was expressed from the residual percentage of lactobacilli labeled with cFDA-SE extract from the mucosal surface versus the time after orogastric intubation. Around all the strains, the number of the residual lactobacilli performed decline from1to4day. Then, the parent of the isolated strain L1026displayed increase on6day, decline on10day and another increase on15day; the number of the isolated strain L1212rise continuously from6to15day after orogastric intubation; however, the control group fed with KLDS1.0344displayed decrease persistently.In this study the constructed plasmid pPG2-VP2-VP3was transformed to Lactobacillus plantarum which were isolated from the intestine of rainbow trout. The recombinant strains were induced by1%lactose. By Western-blotting, apparent target band was observed on NC membrane (approximately66KD). Showed that the protein was expressed.In this study, we used a mixture of bacterial suspension and alginate coated particles bait. Studied the survival number of cell when with or without sodium alginate. The results showed that adding sodium alginate group showed a higher colony formation than the other group. In the research of add1%,2.5%,5%sodium alginate and drying in20℃,32℃and37℃, results showed add2.5%sodium alginate and drying at20℃display the maximum number of colony formation.Rainbow trout were divided into7groups, fed with bait coating with pPG-2-VP2-VP3/L1212, besides, host bacteria L1212, PBS and pattern strain pPG612-VP2+3/L393as control. We detected the special IgM level in the rainbow trout’s serum by indirect ELISA method at days0,31,50and66after immunizations. The results showed that the groups fed with recombinant bacteria serum antibodies appeared at31d after immunization. At the same time set up a continuous and interval immune group. Results showed the antibody levels of the interval group were slightly higher than the continuous, the gap between the two groups was not significant (p>0.05); In addition, after the booster immunization, antibody levels increased gradually, reaching the highest at66d, differences were significant (p<0.01) compared with the control group, and discrepancy predominance than the basic immunization group (p<0.05). Juvenile rainbow trout were challenged with IPNV (sp strain), and each group for continuous monitoring. The results showed that: the IPNV VP2-VP3recombinant subunit oral vaccine prepared using the method of coating particles bait play a protective role in the process of IPNV infection to some extent.These results confirm that this study IPNV VP2-VP3recombinant subunit oral vaccine prepared using the method of coating particles bait play a protective role in the process of IPNV infection. All theses work established a good foundation for further study on the new and effective recombinant oral fish vaccine.