| Micropterus salmoides is a very important freshwater aquaculture species in China.In 2021,the total output of M.salmoides has exceeded 700,000 tons.However,with the continuous expansion of the scale of M.salmoides breeding,its disease problems are becoming more and more serious.Bacterial diseases have always threatened the healthy breeding of M.salmoides and caused huge losses to the breeding industry of M.salmoides.In this study,an epidemiological investigation was conducted on different M.salmoides farms in Chizhou,Anhui,Wuhan,Hubei,and Foshan,Guangdong.A total of 39 strains of pathogenic bacteria were isolated from the diseased M.salmoides.After16S r DNA sequencing,it was found that 23 strains of pathogenic bacteria were highly homologous to Aeromonas veronii.Then the physiological and biochemical identification,drug resistance evaluation and multilocus sequence typing of the 23isolates were carried out.The results showed that the physiological and biochemical characteristics of the 23 isolates were highly consistent with the standard strains of A.veronii.There were 13 sequence types(STs)in 23 strains,of which 12 STs were newly submitted.The results of drug resistance evaluation showed that all the isolated strains were sensitive to cefuroxime ofβ-lactams and aztreonam of monoamides,and resistant to vancomycin of glycopeptides,penicillin ofβ-lactams and clindamycin of lincosamides.The resistance rate of enrofloxacin to quinolones approved in aquaculture was 86.96%,followed by sulfamethoxazole of sulfonamides and doxycycline of tetracyclines,with resistance rates of 78.26%and 60.87%,respectively.The resistance rate to florfenicol of chloramphenicol was relatively low,only 20.69%.The pathogenicity of 13representative strains of STs was evaluated and the isolate HZAU2283 with strong pathogenicity was screened.After that,the regression infection experiment of the strain was carried out.It was found that the symptoms of the disease after infection with A.veronii HZAU2283 were the same as those of the disease after infection with A.veronii in the natural state.The main pathological changes were congestion and redness of the gill cover,jaw and fin base,redness and swelling of the anus.After dissecting the diseased fish,it was found that the liver,spleen and kidney were swollen and congested.To further study the strain,the whole genome of the isolate HZAU2283 was sequenced,and then the whole genome data were compared and annotated with multiple databases.It was found that the types of virulence factors of the isolate HZAU2283 were mainly adhesion factors,secretion systems and motility invasion adhesion factors.To explore the prevention and control methods of A.veronii from M.salmoides,in this study,two virulent strains of HZAU2283(ST2131)and HZAU2227(ST2213)were isolated and prepared into bivalent inactivated vaccines,and to evaluate the immune protection effect of the five-gene deletion strain of Aeromonas hydrophila constructed in the previous study.They were immunized by intraperitoneal injection or immersion,respectively.After 28 days of secondary booster immunization,high concentration(1.0×10~8CFU/m L)and low concentration(1.0×10~7CFU/m L)of A.veronii HZAU2283 were used to scratch and soak the M.salmoides in different immune groups.We found that the relative protection rates of bivalent inactivated vaccine injection and immersion immunization groups were 88.89%and 44.44%,respectively.The relative immune protection rates of A.hydrophila attenuated vaccine injection and immersion immunization were 44.44%and 60.00%,respectively.The detection of serum agglutinating antibody titer showed that the bivalent inactivated vaccine and attenuated vaccine could effectively activate the acquired immune response of M.salmoides regardless of intraperitoneal injection or immersion immunization.The agglutinating antibody titer induced by intraperitoneal injection of A.veronii bivalent inactivated vaccine was the highest. |
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