Identification,Pathogenicity And Immunological Control Of Pathogens Of Grass Carp Hemorrhagic Disease

Septicemia(blood poisoning)is the most common epidemic in the aquaculture industry in China.With the development of intensification model and severe deterioration of water environment,the outbreak of hemorrhagic disease caused by Aeromonas infection is extremely serious.The symptoms are extremely serious.The disease has a short onset time and a high mortality rate,which brings huge economic losses and severely hinders the healthy development of the aquaculture industry.This study intends to deduce the pathogenesis of the disease through the isolation,identification and pathogenicity of pathogens.On this basis,probiotics are added to fish feed for application in the production of grass carp,and the probiotics’fermented feed is tested for its ability to resist disease after bacterial infection to explore probiotics as an immune stimulant against bleeding disorders.The main findings are as follows:The diseased fish was obtained from in Xinxiang.The skin,liver,spleen,and intestine tissues of the diseased grass carp were obtained.After screening and rescreening,four strains of opportunistic pathogens were isolated from diseased fish,after which the molecular identification of 16SrDNA was performed.The results confirmed the four strains as Aeromonas veronii,Shewanella sp.,Bacillus cereus,and Aeromonas salmonicida.Four kinds of strains were used to conduct infection experiments on grass carp.Experimental has revealed that Aeromonas veronii was the primary strain.Multiple virulence-related factors were discovered,including aerolysin(Aer),cytotoxic enterotoxin(Act),and the outer membrane protein(Omp AII),which was detected only in Aeromonas veronii.The determination of LC50 in grass carp was3.6×10~4 CFU/mL over 96h,whereas the safe concentration was 9.11×10~3 CFU/mL.The grass carp were challenged with 1.82×10~5,2.81×10~6,1.82×10~8 CFU/mL of the Aeromonas veronii strain for 48 h.Following 2.81×10~6 CFU/mL incubation and then were treated for 24 h,48 h,72 h and 96 h.Whereupon the expression of IL-8 and TNF-αwas up-regulated in the kidney,liver,spleen,and heart using qRT-PCR.The correlation between inflammation-associated genes and intestinal inflammation of grass carp was analyzed.The results showed that grass carp were infected by pathogenic bacteria.The mRNA level of IL-8 and TNF-αtranscriptional increased obviously.The grass carp was infected with concentrations of 1.82×10~5,2.81×10~6,1.125×10~7,and 1.82×10~8CFU/mL.SDH,lysozyme,PK,ALT and AKP were detected in serum and liver tissues at different times.The results showed that the activity of ALT,SDH,lysozyme and AKP enzymes in serum increased significantly after infection with pathogenic bacteria,and the enzymes of ALT,SDH,lysozyme and AKP in the high concentration group was significantly higher than that in the control group(p<0.05).The PK enzyme activity in the high concentration group compared with the control at 96 hours after the infection was significantly reduced(p<0.05).The activity of PK,ALT and AKP enzymes in liver tissue showed a significant increase with increasing infection concentration(p<0.05).Probiotics(Lactobacillus bulgaricus,Bacillus subtilis,and Clostridium butyricum)with a concentration of 1.82×10~5 CFU/mL were sprayed onto common feed at a rate of 3%and fed grass carp infected with pathogens.The results showed that the expression of IL-8 in the liver,kidney and intestine tissues of the grass carp infected with probiotic fermentative feed was significantly higher than that fed to the basal diet(p<0.05);At the same time,the expression levels of TNF-αin liver,skin,spleen,and intestinal tissues were significantly higher than those fed basal diet(p<0.05).Compared with the control group,AKP and ALT enzyme activities were significantly higher at 16 days(p<0.05);PK,SDH,and lysozyme enzyme activity did not significantly increase with feeding time(p>0.05).The mortality rate of grass carp fed with probiotic fermentation material was significantly lower than that of the fed basic material group,that is,probiotic fermentation material could significantly increase the survival rate of grass carp after infection with pathogenic bacteria.The results showed that the probiotic fermented feed can improve the fish resistance,enhance the immunity,and reduce the incidence of infection by pathogens.This result provides a good reference for the probiotics to improve the role of aquaculture environment.Innate immune stimulants are a promising field because they are biodegradable and biocompatible,while being safe to the environment and human health.Therefore,the probiotic fermentation feed provides a theoretical basis for the control of diseases in aquaculture and even the development of vaccines,and brings new ways to improve the aquaculture environment.