Pathogenicity Analysis Of Aeromonas Veronii From Lateolabrax Maculatus And Effect Evaluation Of Inactivated Vaccine And Chinese Herbal Medicine

Aeromonas veronii is a major pathogen in the culture of Lateolabrax maculatus,and it can cause bacterial septicemia of L.maculatus,mainly manifested by abdominal congestion,hemorrhage,pathological changes of spleen,kidney,liver,and other organs,bringing certain environmental and economic harm.At present,fish vaccine and Chinese herbal medicine are two important ways of prevention and control bacterial diseases.This research on A.veronii bacterial septicemia caused by the pathogenic bacterium,and through the preparation of the monovalent/bivalent inactivated A.veronii vaccine and aqueous extract of Chinese herbal medicine to explore fish with vaccines and drugs in A.veronii of bacterium antibacterial effect,aimed for bacterium A.veronii prevention and control to provide data and theoretical support.（1）This study selected A.veronii to L.maculatus toxicity screening,bacteria selected high virulence strain-18BJ181 as the pathogenic bacterium of A.veronii explore strains.Physiological and biochemical experiments found that A.veronii 18BJ181 bacterium of O/129 resistance,β-Galactosidase,and other 16 kinds of physiological and biochemical reaction is positive.It grows well between p H 7 and 9,and is resistant to amoxicillin,madinomycin,penicillin,and sulfamethoxazole.It carries five virulence genes,including aerolysin（aer）,cytotoxic enterotoxin（act）,glycerophospholipid-cholesterol acyltransferase（gca T）,type IV pilus（tap A）and flagellin（fla）.Artificial infection with A.veronii-18BJ181 caused acute death of L.maculatus within 24 h,accompanied by abdominal hemorrhage,anal swelling,hemorrhage and congestion of kidney and liver,and swelling of the spleen.Histopathology showed that A.veronii 18BJ181 caused hemorrhage of myocardial hemorrhage,myofibrillar edema and rupture,hepatocellular steatosis,congestion and hemorrhage in the liver,kidney with marked necrosis and desquamation of the renal tubular epithelial and the hemorrhage in the interstitial tissue,numerous lymphocytes and macrophages aggregated around ellipsoids,aggregation of inflammatory cells,and hemozoin accumulation in the spleen.Bacterial load test results showed that the bacterial loads of A.veronii in the spleen and liver reached the peak at 8h after infection,and the bacterial load in blood and kidney reached the peak at 12 h and24 h after infection,respectively.The Median lethal dose(LD₅₀)under intraperitoneal injection and intramuscular injection were 8.5×10⁵cfu·g^-1 and 4.2×10⁵cfu·g^-1,respectively.（2）The acute death of L.maculatus caused by A.veronii-18BJ181 was analyzed.The L.maculatus began to die 1 h after infection with 18BJ181,and the time of death was concentrated within 12 h.The results showed that the 8 h group caused severe abdominal fluid and hemorrhage,blackened spleen,hemorrhage,degeneration,and necrosis of hematopoietic tissue and lymphoid tissue.In the 24 h group,the bleeding was abated,the spleen was dark red and the hemosiderin accumulation was observed.Transcriptomics showed that 2665 DEGs were up-regulated and 2242 DEGs were down-regulated in the8 h group after the infection of A.veronii-18BJ181.In the 24 h group,1102 DEGs were up-regulated and 984 DEGs were down-regulated.By GO and KEGG enrichment analysis,the Chemokine signaling pathway and NOD-like receptor signaling pathway were found to have the highest enrichment number of differentially expressed genes related to immune pathways.（3）The monovalent formaldehyde inactivated vaccines of 18BJ181 and 18QW235virulent strains of A.veronii were prepared.The monovalent formaldehyde inactivated vaccines with the final concentration of 0.2%for 48 h were safe to a certain extent.The antibody agglutination titer between homotypes in serum was as high as 1:480,while the antibody agglutination effect between heterotypic was poor,with the highest value of 1:192.The changes of acid phosphatase（ACP）,alkaline phosphatase（AKP）,lysozyme（LZM）,superoxide dismutase（SOD）,and malondialdehyde（MDA）in serum were different at different time,and the ability of respiratory burst was significantly enhanced（P<0.05）.After 28 days of immunization,the relative protection survival of the monovalent inactivated vaccine against A.veronii was 92.3%and 53.6%,respectively,and the relative protection survival between the heterotypic was poor.It was inferred that A.veronii 18BJ181 and 18QW235 were two different serotypes.The bivalent inactivated vaccine of A.veronii with different volume ratios was constructed,and the bivalent inactivated vaccine showed good safety after 24 h inactivation at the final concentration of 0.5%.The immune response could be produced 10 days after immunization,and the immune duration could be up to 56 days.The bivalent inactivated vaccine of A.veronii with different volume ratios had different immune protective effects on different strains at different times.The levels of ACP,AKP,LZM and SOD in mucus increased significantly on 21 d and after immunization（P<0.05）.After 49 days of immunization,serum Ig M content was consistent with that of the control group,and serum lactate dehydrogenase（LDH）content was significantly decreased（P<0.05）.In order to combine with the batch production steps of the vaccine and optimize the preparation process,the bivalent inactivated vaccine of A.veronii（containing extracellular products）was prepared in this study.After immunization for 10 days,it was found that the immune protective effect of the bivalent inactivated vaccine without extracellular products was better than that of the bivalent inactivated vaccine without extracellular products,with the highest relative protection of 96%.The serum Ig M content and the serum high-density lipoprotein（HDL）were significantly decreased（P<0.05）,the serum LDH content was significantly increased（P<0.05）.The storage life of A.veronii inactivated vaccine could be stored at 4℃for 4 months,and it had a good shelf life.（4）Eighty-two single and compound Chinese herbal medicines with low cost were screened in vitro and water extracts were prepared.The results of the drug sensitivity test showed that 36 Chinese herbal medicines had inhibitory effects on A.veronii.Among them,Prunus mume,Caesalpinia sappan,Galla chinensis,Syzygium aromaticum,and Artemisia argyi had a strong inhibitory effect on A.veronii in vitro.The antibacterial zone diameter ranged from 15 to 25 mm,MIC<125 mg·m L^-1,and MBC<500 mg·m L^-1.In vitro antibacterial activity of the compound,Chinese herbal medicine was found to be good against A.veronii,with a diameter of>19 mm,MIC was 15.6 mg·m L^-1,and MBC was31.25～62.5 mg·m L^-1.Combined antibacterial index analysis showed that they did not affect.P.mume,C.sappan,and Lonicera japonica were selected to evaluate the safety of Chinese herbal medicine against L.maculatus in vivo and the effect of Chinese herbal medicine on A.veronii.The results showed that the water extract of C.sappan and L.japonica had good safety to L.maculatus,but the antibacterial effect in vivo was low.In this study,the pathogenicity of A.veronii to L.maculatus was analyzed from multiple perspectives including artificial infection,pathology and transcriptomics,and the monovalent inactivated vaccine,bivalent inactivated vaccine and Chinese herbal medicine water extract of A.veronii were prepared respectively to evaluate their prevention and control effects on A.veronii to L.maculatus,building a foundation for the research on breeding diseases of L.maculatus.