Isolation And Identification Of Cetobacterium Somerae From Micropterus Salmoides And Effects Against Nocardia Seriolae Infection

The largemouth bass(Micropterus salmoides),one of the most important economical freshwater fish has been widely cultivated in China in recent years.However,bacterial diseases have greatly hindered the development of largemouth bass farming industry.Using antibiotics is the main approach to control aquaculture bacterial diseases,but the abuse of antibiotics not only leads to the drug resistance of pathogens but also causes food safety issues.Under the circumstance of restricting and substituting antibiotics in aquaculture,it is extremely urgent to develop an environment-friendly and effective method to of controlling bacterial diseases.Currently,probiotics have been widely used in aquaculture to improve the growth performance,immune response and disease resistance.Furthermore,the indigenous probiotics have better colonization capacity and security than the exogenous probiotics.Therefore,the development of new indigenous probiotics is important for the sustainable development of aquaculture.Cetobacterium somerae is the predominant bacteria in the gut of freshwater,and it’s abundance is closely related to the fish health.In present study,we isolated a Cetobacterium somerae strain named C.somerae LJ from the gut of largemouth bass and analyzed it’s physiological biochemistry characteristic and genome.Then in vivo experiment was conducted to evaluate the effect of C.somerae on intestinal health,immune parameters and resistance against Nocardia seriolae of largemouth bass.The main results were shown as follows:1.We isolated a C.somerae LJ from the gut of largemouth bass using anaerobic culture approach and analyzed it’s physiological biochemistry characteristic and genome.The results showed that LJ produce acid during growth and possessed resisting against oxygen and 2%bile salt.There are gene clusters associated with the synthesis of short chain fatty acid and vitamin B12 in the genome of LJ.Multiple genes related to stress resistance were also found in the genome.In addition,the results of safety assessment based on the whole genome sequences showed no typical virulence factor and less antibiotic genes,indicating the high security of LJ.2.We added C.somerae LJ into the basil diet of largemouth bass at low dose(10~6CFU/g)and high dose(10~8CFU/g)and then fed the fish for 8 weeks.The results showed that both LD and HD groups didn’t improve the growth performance compared to control group.However,the HD group increased the expression of genes related to gut barrier(ZO-1 and Claudin-1)remarkably and reduced the intestinal redox potential(P<0.05).Meanwhile,the HD group enhanced the serum enzyme activities related to nonspecific immunity(ACP,AKP,LZM and SOD)and reduce the expression of TNF-α,IL8,IL-1βand IL15 that are related to pro-inflammation(P<0.05).Furthermore,after challenged by N.seriolae,the survival rate of largemouth bass in HD group(57.5%)was significantly increased compared to the control(37.5%)and LD groups(42.5%)(P<0.05).3.The diet containing DSS can induced gut injury on M.salmoides after feeding for 7days.The symptoms included the intestinal structure damage,the significant expression reduction of ZO-1 and Claudin-1,the lower enzyme activities of SOD and CAT,as well as the remarkable elevation of the intestinal inflammation level(P<0.05).The intestinal damage of largemouth bass induced by DSS contributed to the N.seriolae infection through the gut and leaded to higher morality.While the diet containing C.somerae LJ could repair the intestinal damage to some extent as well as recover the normal intestinal environment and barrier function.Consequently,the HD group inhibited the infection of N.seriolae through gut and reduce the morality of M.salmoides significantly(P<0.05).Taken together,C.somerae LJ have resistance against oxygen and bile salt.The in vivo experiment showed that C.somerae LJ plays an essential role in maintaining stable intestinal environment and intact barrier function.Moreover,LJ can enhance the resistance against N.seriolae,which can be further developed and utilized as a new indigenous probiotic in aquaculture.