Pathogenicity Of Two Pathogenic Bacteria And Their Protective Effect Of Inactivated Vaccines On Crucian Carp

With the development of aquaculture,bacterial diseases are on the increase,and the current prevention and control of bacterial diseases is mainly based on the use of antibacterial drugs to inhibit pathogenic bacteria,which has caused problems such as environmental pollution and increased bacterial drug resistance.As an efficient and environmentally friendly biological product,vaccines are an important means of preventing and controlling various diseases caused by pathogenic microorganisms.Two strains of pathogenic bacteria were isolated by our team from the water and diseased fish in the Ren River,Chongqing.This experiment is intended to investigate the isolation and identification of the two strains of bacteria,the pathogenicity test on crucian carp and the immunological effect and immunoprotective effect of inactivated vaccine on crucian carp:1.two strains of pathogenic bacteria,named JS-1 and JW-4,were isolated from the water column and from gill and skin lesions of diseased fish.morphological,physiological,biochemical and molecular identification of 16S rDNA showed that the bacteria JS-1 was Staphylococcus sciuri and JW-4 was Aeromonas veronii.Phylogenetic analysis showed that JS-1 clustered with S.sciuri and JW-4 clustered with A.veronii.The results of the drug sensitivity tests showed that JS-1 was resistant to benzocillin,ceftazidime and furazolidone,while JW-4 was resistant to penicillin,benzocillin,ampicillin,carbenicillin,furazolidone and medetomycin,and sensitive or moderately sensitive to other drug sensitive papers.The results showed that the two strains of bacteria were highly resistant to the drug.2.After the acute toxicity test to determine the 96 h LD₅₀of the two strains of bacteria,four test groups were set up for each strain of bacteria based on the LD₅₀of the two strains（JS-1,5×10⁶cell/ml;JW-4,2×10⁶cell/ml）,and three parallel groups were set up for each test group,bacteria were mixed into the feed and fed to the carp.The concentrations were control group（CG,0 cell/g feed）,low concentration group（LG,10⁴cell/g feed）,medium concentration group（MG,10⁵cell/g feed）and high concentration group（HG,10⁶cell/g feed）for a 28-day trial period.lysozyme（LZM）,glutamic oxalacetic transaminase（GOT）and glutamic pyruvic transaminase（GPT）activities were measured in serum and liver of all concentration groups of JS-1 and JW-4.Histopathological observation of liver,kidney and spleen in CG and MG groups and determination of TNF-αand IFN-γgene expression in liver and spleen were performed.The results revealed that the GOT and GPT activities in serum and liver of LG,MG and HG groups showed an increase followed by a decrease and LZM activity showed an increase after treatment with both strains compared to the control group.In the test groups with different concentrations of JS-1,serum GOT and GPT activities were significantly higher in the HG group than in the CG group at day 1（p<0.05）,and serum LZM and liver GOT activities were significantly higher in the MG group than in the CG group;the activities of serum LZM and liver GOT in LG group were significantly higher than those in CG group on day 7（p<0.05）;at day 14,serum LZM activity was significantly higher in the HG group than in the CG group（p<0.05）and liver GOT activity was significantly lower in the MG and HG groups than in the CG group;at day 21,liver GOT activity was significantly lower in the MG group than in the LG group;at day 28,serum GPT was significantly lower in the LG group than in the MG group（p<0.05）and serum LZM activity was significantly higher in the LG group than in the CG group（p<0.05）.In the test groups with different concentrations of JW-4,the GOT activity in the liver was significantly higher in the LG group than in the CG group at day 1（p<0.05）;at day 7,serum GPT was significantly higher in the HG group than in the LG group（p<0.05）,liver GOT activity was significantly higher in the HG group than in the other three groups（p<0.05）,and liver GPT activity was significantly higher in the MG group than in the CG group（p<0.05）;at day 14,serum LZM activity was significantly higher in the LG group than in the CG group（p<0.05）,and liver GOT and GPT activities were significantly lower in the LG and MG groups than in the CG group（p<0.05）;at day 21,serum GPT activity was significantly lower in the LG group than in the CG group（p<0.05）,serum LZM activity was significantly higher in the HG group than in the CG group（p<0.05）,liver GOT and GPT activities were significantly lower in the HG group than in the CG group（p<0.05）;at day 28,serum GOT and GPT activities were significantly higher in the CG group than in the other three groups（p<0.05）,serum LZM activity was significantly higher in the LG and HG groups than in the CG group,and liver GOT activity was significantly lower in the HG group than in the CG group（p<0.05）.Histopathological observations showed varying degrees of lesions in the liver,spleen and kidneys of the carp.After treatment with JS-1,the liver in the MG group had irregular nuclei and vacuolated cells;the spleen was severely congested and locally necrotic;and in the kidney,the renal capsule gap was enlarged and the renal tubercle was swollen.After treatment with JW-4,there was localized necrosis in the liver with fragmented and lysed nuclei;irregular arrangement of lymphocytes and other cells in the parenchyma of the spleen with distorted nuclei;The cells of the tubules and glomeruli in the kidney are indistinguishable from each other and the nuclei are variable in shape.The trend for both TNF-αand IFN-γgene expression increased and then decreased and peaked at 14 days,with a significant increase in both the MG group of JS-1 and JW-4 compared to the CG group at day 14（p<0.05）.The results showed that JS-1 and JW-4 were highly pathogenic to crucian carp,causing varying degrees of damage to crucian carp organs and stimulating the expression of non-specific immune genes.3.Three test groups,JS-1,JW-4 and CG,were set up according to the 96h LD₅₀of the two bacterial strains,with three parallel groups each.10⁷cell/ml of inactivated vaccine made by adding F（?）rster’s complete adjuvant was injected into each of the JS-1 and JW-4 groups,and an equal amount of saline was injected into the CG.To determine LZM,alkaline phosphatase（AKP）,GOT,GPT,superoxide dismutase（SOD）and catalase（CAT）activities in serum and liver,α-amylase and lipase activities in the intestine,and to determine specific IgM levels and total IgM levels in serum.After 28 days of immunisation with inactivated vaccine,100μl of live bacteria at 10 times the concentration of inactivated vaccine was injected and the rate of immune protection was measured.Crucian carp after immunization with JS-1inactivated vaccine,liver CAT activity was significantly lower than that of the CG group at day 1（p<0.05）;at day 7,serum GOT activity,GPT activity and liver GPT activity were significantly lower than in the CG group（p<0.05）,serum CAT activity was significantly higher（p<0.05）than in the CG group;serum GOT,serum SOD,liver AKP and liver GPT activities were significantly lower than in the CG group（p<0.05）and serum CAT activities were significantly higher（p<0.05）than in the CG group at day 14;at day 21,serum GOT and liver AKP activities were significantly lower than those in the CG group（p<0.05）and intestinal lipase activities were significantly higher than those in the CG group（p<0.05）;at day 28,serum GOT,liver GPT and liver AKP levels were significantly lower than those in the CG group（p<0.05）,serum LZM,intestinal amylase and intestinal lipase activities were significantly higher than those in the CG group（p<0.05）.After JW-4 inactivated vaccination,at day 1,serum CAT activity was significantly higher than in the CG group（p<0.05）;at day7,serum LZM and CAT activities were significantly higher than in the CG group（p<0.05）,serum AKP,GOT and GPT activities were significantly lower than in the CG group（p<0.05）;at day 14,serum AKP and GPT activities were significantly lower than in the CG group（p<0.05）;at day 21,serum CAT activity and intestinal amylase activity were significantly higher than in the CG group（p<0.05）and liver GPT activity was significantly lower than in the CG group（p<0.05）;at day 28,LZM activity in serum and liver was significantly higher than in the CG group（p<0.05）,and serum AKP,liver GOT and liver GPT activities were significantly lower than in the CG group（p<0.05）.There was no significant difference in liver SOD activity between all groups（p>0.05）.Both specific IgM and total IgM levels were significantly increased after injection of JS-1 and JW-4 inactivated vaccines compared to the CG group（p<0.05）.Immunoprotection rates within 10 days of performing the regression infection test were 83.3%in the JS-1 group and 75%in the JW-4 group,a significant difference compared to the CG group（p<0.05）.The results showed that JS-1 and JW-4inactivated vaccines were non-toxic to crucian carp and also stimulated the immune response of crucian carp and caused specific antibodies to be produced.The above results showed that the feeding of diets mixed with S.sciuri JS-1 and A.veronii JW-4 was highly toxic to crucian carp and caused significant pathological reactions in the organs of crucian carp.The inactivated vaccines of both strains of bacteria were not harmful to the crucian carp organism and would promote the immune response and digestibility of the carp to some extent.The combination of IgM content and immune protection rate indicated that JS-1 and JW-4 could promote the production of antibodies in the organism and could achieve the effect of protecting crucian carp after infection with the bacteria.Combined with the pathogenicity of JS-1 and JW-4 and the effectiveness of inactivated vaccines,this could provide a theoretical basis for disease control in aquaculture.