| The channidae fish is rich in nutrients and easy to raise,it is an important freshwater fish species in China.Hybrid snakehead was obtained by interspecific hybridization of Channa maculata♀and C.argus♂,which showed excellent economic characters and high disease resistances.With high density culture and rapid expanding production,problems of its diseases become more and more serious.Aeromonas schubertii is an emerging pathogen causing the disease with the symptom of white spots in internal organs in recent years.Because of its acute onset,high mortality and strong host specificity,it has caused great harm to snakehead culture.At present,the prevention and treatment of the bacterial disease is mainly focused on the use of antibiotics,and the resulting environmental pollution and bacterial resistance problems have great potential risk for the aquaculture industry and the entire ecological environment.As a highly efficient and environmentally friendly biological product,vaccine is an important tool in preventing and controlling diseases caused by various pathogens.As a fish pathogen,A.schubertii has not yet been reported on the vaccine research both in China and other countries.This study summarizes the results of the classification and identification of A.schubertii,the preparation of inactivated vaccine,the protective effect of vaccines and the changes of related immune indicators after vaccination.The results of this study are helpful to further research on the pathogenesis of A.schubertii and the prevention and control methods against it in aquaculture.Taking hybrid snakehead and A.schubertii as experimental subjects,using physiological and biochemical characteristics test,modified plate method detection,micro-double dilution method,PCR amplification and sequencing analysis,artificial injection infection,characteristics and pathogenicity of the 4 strains of A.schubertii(WL-4,ZL-1,ZL-13,D075)isolated from hybrid snakehead have been analyzed and compared.The results showed that the physiological and biochemical characteristics of these 4 A.schubertii were basically the same and the similarity of the 16S rRNA and gyrB genes of them were 100%and 99.57%respectively.Four strains are clustered into one group though cluster analysis.Different stains of A.schubertii(WL-4,ZL-1,ZL-13,D075)have hemolytic,protease activity and lipase activity,and carry virulence genes such as hlyA,ela,lip,ahal,aer;Four strains isolated from hybrid snakehead showed different pathogenicity.The mortality rate of hybrid snakehead by artificial injection of WL-4,ZL-1,ZL-13 and D075 at 1.2×105 CFU/mL solution was30%,40%,70%and 80%respectively.The antibiotic sensitivity and antibiotic resistance gene are also different among them.Two strains of pathogenic strains D075 and WL-4 were screened,and the inactivated-bacteria vaccine was prepared by inactivation with 0.2%formaldehyde.After injection of the immunized experimental fish,the samples were collected periodically to analyze the peripheral blood cell parameters between the experimental group and the control group,non-specific immune index,serum antibody titer and anti-infective immune protection rate.The results show that the inactivated vaccine prepared by D075 and WL-4 strain can cause non-specific and specific immune response in hybrid snakehead;Compared with the control group,the number of peripheral blood leukocytes,hemoglobin content,serum lysozyme,NO content,AKP,POD and other activities of WL-4 immune hybrid snakehead increased more significantly within 0-4 weeks;The immune protection effect of D075 and WL-4vaccine reached the peak in the 4th-12th week,the highest immune protection rate could reach 40%and 56%respectively,and showed a downward trend after 12 weeks.There was cross immune protection between WL-4 and D075.At 12 weeks,the cross-protection rates were 50%(WL-4 immunization,D075 challenge)and 44%(D075 immunization,WL-4 challenge).The vaccine was prepared using WL-4 strain.The effects of immune dose and number of immunizations on the immunoprotective effect of the inactivated vaccine of A.schubertii were compared.The results showed that when different concentrations of vaccine immunize hybrid snakehead,the immune effect of 10~9 CFU/mL was the best.After 2 weeks of immunization,the relative immune protection rate was 39%,the relative immune protection rate increased gradually in 2-4 weeks,the relative immune protection rate fluctuated in 8-12 weeks,and the immune protection rate reached 78%at 12 weeks,at 28 weeks,the relative immune protection rate of the vaccine decreased to 0;the antibody titer level of the immunized fish serum was correlated with the relative immune protection rate,and the immune protection rate was 78%when the antibody titer was 1:512;After 4 weeks of immunization at 10~9CFU/mL,the remaining half of the hybird snakehead was immunized twice at the same dose.After 8 weeks of secondary immunization,the immune protection rate was12%,which was significantly lower than the 10~9 CFU/mL primary immune protection at 12 weeks.Improper selection of secondary immunization time and immune dose will have a negative impact on the immunoprotective effect of A.schubertii vaccine.The physiological and biochemical characteristics of these 4 A.schubertii were basically the same,but there are still some differences on pathogenicity and antibiotic sensitivity among the four strains of A.schubertii isolated from hybrid snakehead.The non-specific and specific immune responses of hybrid snakehead caused by whole-bacteria inactivated vaccines derived from different strains showed different performances,but cross-immunization protection exists;proper immune dose and number of immunizations are closely related to the immunoprotective effect of whole-bacteria inactivated vaccines.Further study should be conducted on strain selection,optimal immunization dose and secondary immunization time of A.schubertii. |
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