| Sturgeon is one of the important species of freshwater aquaculture in China,and occupies an important position in China’s freshwater aquaculture industry.At present,the sturgeon farming industry shows a rapid growth trend,but as the sturgeon farming intensification and scale continue to increase,bacterial diseases continue to occur.Aeromonas hydrophila is a member of the genus Aeromonas in the family Vibrioceae,widely living in freshwater,silt,soil and other environments,often causing fish and sturgeon and two sacrificial animals to die of disease.At present,antibiotics and chemical drugs are commonly used in the prevention and treatment of bacterial diseases.However,if they are used unreasonably or irregularly for a long time,they will cause bacterial resistance and lead to serious environmental bacterial pollution.Vaccine prevention has the advantages of good effect and low pollution.It is widely used in sturgeon aquaculture.In this study,based on the early isolation of Aeromonas hydrophila from sturgeon,by comparing the inactivation conditions including formaldehyde concentration(0.05%,0.1%,0.2%,0.3% and 0.4%),inactivation temperature(4 ℃,28 ℃,30 ℃,and 37 ℃)and inactivation time(4h,8h,12 h,and 24h)to prepare inactivated aquamonas hydrophila vaccine for sturgeon.Then select 140 sturgeon,then divide into control group,vaccine group,adjuvant group and vaccine + adjuvant group,each group of 35 tails,with sterile PBS solution,inactivated vaccine,astragalus polysaccharide and inactivated vaccine + astragalus Polysaccharides are injected.At 1d,7d,14 d,21d and 28 d after injection,blood samples of tail vein of sturgeon were randomly collected,5 tails / group,and routine biochemical indexes of sturgeon blood were measured by ELISA(aspartate aminotransferase AST,alanine aminotransferase ALT,alkaline Phosphatase AKP,etc.)and immune-related indicators(superoxide dismutase SOD,lysozyme LYZ,immune antibody Ig G,acid phosphatase(ACP)and complement C3).At the 28 th day after the immunization,the newly prepared aeromonas hydrophila solution(2.5 × 108 cfu/m L)was used for injection challenge,and the immune protection rate of each experimental group was determined.On the 14 th day,the sturgeon was culled.Five fish in each group were taken,and the liver,spleen and intestinal tissues of the sturgeon were taken for pathological section to observe the lesions.The main experimental results are as follows:(1)Optimal inactivation conditions for Aeromonas hydrophila: when the bacterial solution is placed at 4℃,the final concentration of formaldehyde is 0.05% and 0.1%,and the bacteria can survive after 24 hours of inactivation;at 28℃,the final concentration of formaldehyde 0.05%,inactivation can detect bacteria survival after 24 h inactivation,final formaldehyde concentration is 0.1%,bacteria survival can be detected after inactivation 4h;at 30℃,final formaldehyde concentration is 0.05%,bacteria survival can be detected in inactivation 24h;When the temperature is 37℃,the final concentration of formaldehyde is 0.05%,and the survival of bacteria can be detected after 4 hours of inactivation,and the survival of bacteria is not detected in other inactivation conditions.The optimal inactivation conditions are obtained by comparison: 0.2% formaldehyde solution,28 ℃ inactivation temperature 8h inactivation time.(2)Test results of routine biochemical indexes of experimental sturgeon blood: On the 7th day,the T-BIC content of the control group was the highest at 14.56 ± 6.05umol/L,and the content of the vaccine + adjuvant group was 11.46±5.85umol/L was the lowest,the difference between the two groups Significant(P <0.05),the CREA content of the control group was the lowest at 85.06 ± 22.36umol/L,which was significantly different from the vaccine group(P <0.05),and the TP value of the control group was significantly different from the data of the vaccine group and adjuvant group(P <0.05),The control group had the highest TG content,the adjuvant group had the lowest content,and the difference between the two groups was significant;on the 28 th day,the vaccine group had the highest ALP content of 7.88 ± 3.61u/L,which was significantly different from the control group(P <0.05),and the control group ALB content The lowest content was 34.00±6.15g/L,and the highest was 41.88 ± 4.24g/L in the adjuvant group.The difference between the two groups was significant(P <0.05);the rest showed no significant difference(P> 0.05).(3)Test results of blood sturgeon related indexes of experimental sturgeon: After the 7th day of the experiment,the SOD activity of the vaccine group and the vaccine + adjuvant group were higher than the control group,the vaccine + adjuvant group was the highest,the vaccine group was the second,and the vaccine + adjuvant was the 28 th day.The peak value o f the adjuvant group was 168.45±5.38u/m L,and the peak value of the vaccine group was 151.36 ± 8.37 u / m L at 21 d,which was significantly different from the control group(P <0.05);after the start of the experiment,the adjuvant group had the highest LYZ activity at 21 d The peak value was 9.22 ± 0.57 U / L,which was significantly different from the control group(P <0.05);the Ig G content was highest in the vaccine groups on the 1d,7d,21 d,and 28 d,and the peak value on the 21 d was 12.60 ± 0.47ng/m L,which was significantly different from the control group(P<0.05);after the first day of the experiment,the ACP activity of the vaccine group was the highest,and the peak value was 10.51 ± 0.64IU/L on the 21 st day,which was significantly different from the control group(P <0.05);It was 153.96 ± 6.96 ug / m L,and the vaccine + adjuvant group was 141.02 ± 8.54ug/m L,the control group was higher than the vaccine + adjuvant group,and the difference was significant(P <0.05),the rest was not significant(P> 0.05).(4)Test result of the immune protection rate of experimental sturgeon: After challenge to the experimental sturgeon,the control group began to die on the 3rd day.During the continuous observation for 14 d,all the sturgeons in the control group died,and 2 vaccine groups died,there were 4 deaths in the adjuvant group.The calculated immune protection rates of the control group,vaccine group,adjuvant group and vaccine + adjuvant group were 0%,80%,60% and 100%,respectively.(5)Observation of lesions of experimental sturgeon after challenge: on the 14 th day after challenge(test 42d)in the control group,hepatic cords in the liver tissue were disordered and hepatic cell vacuoles were degenerated;small necrosis of the spleen;intestinal tissue There were pathological changes such as inflammatory cells and cell necrosis;the intestinal,liver and spleen tissues in the adjuvant group showed slight pathological changes;the vaccine group and vaccine + adjuvant group showed no obvious pathological changes.In summary,this study successfully prepared an inactivated aquamonas hydrophila vaccine for sturgeon.After immunization injection,it can effectively improve the routine biochemical indicators and immune-related indicators of sturgeon.The immune protection rate is more than 80%,and it has no obvious damage to the liver,spleen and intestinal tissues of sturgeon.The results of these studies provide a theoretical reference for the prevention and treatment of sturgeon hydrophila. |
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