| In recent years,with high density hybrid snakehead(Channa maculate♀×C.argus♂)culture systerm promotion,water quality of aquaculture in poor,the frequent occurrence of disease,restricts to sustainable and healthy development.Visceral sarcoidosis caused by Aeromonas schubertii is one of the common diseases of hybrid snakehead.A.schubertii is one of the important pathogens in aquaculture,caused prevalent diseases in freshwater,marine,soil,intestines of vertebrate and is also a threaten to people’s health and life.It caused serious economic losses to aquaculture industry.Currently,disinfectants,antibiotics and other chemicals are still main way to prevent and treat bacterial disease caused by A.schubertii,but there are many problems happen such as bacterial resistance,drug residues in aquaculture food.Immunization is the most economical and effective way to prevent fish diseases.However,no vaccine against A.schubertii has been reported.Therefore,the development of a vaccine against A.schubertii is of great significance to the prevention of the disease.However,inactivated vaccines of A.schubertii,which mainly respond to humoral immunity,are ineffective,and attenuated vaccines not only induce humoral immunity,but also produce strong cellular immune response.Therefore,the development of attenuated vaccine of A.schubertii is more advantageous in the prevention of hybrid snakehead sarcoidosis.In this study,A.schubertii strains were isolated and identified,and attenuated strains were screened as candidate strains of attenuated vaccine through fish test.The safety and effectiveness of the attenuated strain for vaccine preparation were evaluated.At the same time,the expression changes of related immune factors of hybrid snakehead after immunization were studied,which laid a foundation for the development of attenuated vaccine of hybrid snakehead A.schubertii.The main results are as follows:1.Isolation and identification of A.schubertii,screening of attenuated strains and analysis of its characteristicsIn this study,the physiology,biochemistry characteristicsand16S r RNA gene sequence of four A.schubertii strains isolated from the diseased culture fish were analyzed and the pathogenicity were tested and compared.In comparison with the type strain A.schubertii,four strains were almost same as the type strain in major physiological and biological characteristics.Moreover,16S r RNA gene of four strains were 100%similar to other three A.schubertii strains.The challenge tests were carried out by artificially infecting healthy hybrid snakehead with the pure culture of the isolated strain.The test results showed that the pathogenicity of OM170701 was weak,and the mortality of experimental group is very low.The results of growth characteristics showed that the OM170701 strain’s optimum growth temperature was28℃,optimal salinity was 0.5 and optimum p H was 7.2.Establishment and application of tissue distribution,growth and decline rules and detection methods of vaccine candidatesTo investigate the tissue distribution of A.schubertii in hybrid snakehead,a sensitive protocol for the detection of A.shubertii was established.The Taq Man real-time PCR was developed with a pair of primers and Taq Man probe targeting the conserved region of A.schubertii rpo D gene.The specificity,sensitivity and repeatability of the method were verified,The correlation efficiency for standard curves of C_Tvalue and plasmid copy number was 1.000 and the amplification efficiency was 94.705%.The assay was highly specific for amplification from A.schubertii,and had no detection of other 14 pathogenic bacteria including Aeromanas hydrophila,Aeromonas veronii,Aeromonas sobria and Nocardia seriolae.The detection limits of recombinant plasmid and pure culture bacteria were 4.76 copies/μL and 15 CFU/m L,respectively.The tissue distribution and growth of OM170701 in hybrid snakehead were studied by this method.The results showed that OM170701was distributed in intestine,liver,muscle,gill,spleen and kidney.It stayed in the liver for the longest time(6 days)and in the gill for the shortest time(3 days).And it’s not detected in the brain the brain of hybrid snakehead.3.Evaluation of immune potency of attenuated vaccine against A.schubertiiIn order to explore the immune efficacy of Live-attenuated vaccine(LAV)for hybrid snakehead.In this study,we constructed Live-attenuated vaccine and Formalin killed cell.The use of intraperitoneal injection and immersion immune hybrid snakehead,respectively.To investigate the he expression levels of IL-8、IL-1β,complement C3 and Ig M of hybrid snakehead,the method of fluorescence quantitative RT-PCR were established.The candidate strain of attenuated vaccine could induce expression level of IL-8,IL-1β,complement C3 genes up-regulated in the liver of hybrid snakehead,and the relative expression was the highest at 72h after immunization,which was 4.95 times,5.57 times,3.94 times of the control group,respectively.The relative expression of Ig M in the immunized group was significantly higher than that in the control group(P<0.05),and the relative expression level of Ig M in the immunized group was 17.26 times of control group on the 28th day.The relative protective survival of LAV by intraperitoneal injection was 65.0%,significant higher than FKC’s 30.1%;and the relative protective survival of LAV by immersion administration was 26.1%,little higher than FKC’s17.4%LAV groups were significant higher than control group(P<0.05).Based on the above,LAV in intraperitoneal injection way can cause stronger immune response than FKC in intraperitoneal injectionw ay.Live-attenuated vaccine are good candidate vaccine in preventing A.shubertii disease. |
