Epidemiological Investigation,prevention And Control Of Aeromonas Hydrophila Infectionin Megalobrama Amblycephala In Jiangsu Province

Aeromonas hydrophila(AH)belongs to Aeromonas of Vibrionaceae.It exists in sewers,septic ponds,sediments,rivers,lakes,and transportation vehicles.It is a widespread commensal bacteria.Pathogenic strains can infect most aquatic and terrestrial animals,especially fish.Megalobrama amblycephala is commonly known as Wuchang,and its largest breeding area in Jiangsu Province,mainly located in Yixing and Wujin.In 2015,200 million tons were produced.Aeromonas hydrophila is the main cause of bacterial sepsis outbreak in megalobrama amblycephala.At the peak of incidence from June to October each year,the detection rate of Aeromonas hydrophila was over 90%,the incidence rate was 60-70%,and the mortality rate varied from 15%to 25%.In order to control the pathogen of Aeromonas hydrophila and reduce the morbidity and mortality of megalobrama amblycephala,antibiotics and antimicrobials are often used in over doses for a long time in clinic.In order to develop healthily,the most fundamental food safety problem must be solved.In view of this,this study is conducted an epidemiological investigation of Aeromonas hydrophila in the main producing areas of megalobrama amblycephala in Jiangsu Province,and found effective drugs to inhibit this pathogen through pathogenicity,drug susceptibility test and drug resistance gene screening.At the same time,the effect of traditional Chinese medicine(TCM)prevention and control was evaluated to provide reference for prevention and control measures in clinic.The specific research results are reported as follows:1.Epidemiological investigation of Aeromonas hydrophila in megalobrama amblycephala in Jiangsu ProvinceFrom Apr.2018 to Oct.2018,a field survey was conducted on megalobrama amblycephala aquaculture farms suspected of having bacterial sepsis in 6 urban and county areas,such as Yixing,Wujin,Liyang,Jintan,Baoying and Sihong,in the main producing areas of megalobrama amblycephala in Jiangsu Province,and 149 samples of megalobrama amblycephala were collected.A total of 101 strains of Aeromonas hydrophila were identified from 149 samples by biochemical reactions combined with 16S rRNA PCR.This indicates that Aeromonas hydrophila infection exists in the main producing areas of megalobrama amblycephala in Jiangsu Province.The infection status of Aeromonas hydrophila in each body-weight of fish was confirmed.Aeromonas hydrophila infections exist in almost every fish with a body-weight more than 0.1 kg.The peak period of the outbreak was June,July,and August.The outbreak water temperature ranged from 26℃ to 35℃,with 30℃ to 34℃ as the most prone water temperature.The rate of onset is rapid.The incidence can reach 90%,and the mortality rate is more than 10%.The use of sensitive antibiotics can rapidly suppress outbreaks,but long-term overdose of antibiotic use makes clinical resistance very common.After treatment,the fish pond will have repeated outbreaks or even two or three outbreaks.2.Pathogenicity of Aeromonas hydrophila in megalobrama amblycephala in Jiangsu ProvinceThe virulence gene carriage of 101 strains of Aeromonas hydrophila in Jiangsu province was detected.Virulence genes aerA,hlyA,altA,ahpA and astA were detected by PCR.97 strains were detected with aerA,and the detection rate was 96.04%.93 strains were detected with ahpA,and the detection rate was 92.08%.89 strains were detected by hlyA,and the detection rate was 88.12%.83 strains were detected with altA,and the detection rate was 82.18%.78 astA strains were detected,with a detection rate of 77.23%.Among 101 strains of Aeromonas hydrophila,the difference in the number of virulence genes was found.The highest proportion of virulence genes was 66.34%,with all five virulence genes.Strains with three virulence genes accounted for 14.85%,while those with two virulence genes accounted for the least 7.92%.According to the results of virulence gene detection,with different virulence gene combinations were selected for animal challenge experiment to evaluate the pathogenicity of the strains.The LD50 of WJ2011BJ41 was 7.9×104 CFU/mL,indicating a virulent strain.The strains with five virulence genes,aerA+hlyA+altA+ahpA+astA+were virulent strains(LD50<1×106 CFU/mL).The strains with four virulence genes,aerA+hlyA+altA+ahpA+astA-were virulent strains(LD50<1×106 CFU/mL).One thirds of the strains with aerA+hlyA-altA-ahpA+astA+three virulence genes were virulent strains(LD50<1×106 CFU/mL).all of the strains with two virulence genes,aerA+hlyA-altA-ahp+astA-,were attenuated strains(LD50>1×106 CFU/mL).3.Drug resistance and in vitro bacteriostasis of Aeromonas hydrophila in megalobrama amblycephala in Jiangsu ProvinceThe resistance rates of 101 strains of Aeromonas hydrophila to 10 common drugs and Yugeili detected by K-B disk method showed that 101 strains of Aeromonas hydrophila showed different resistance to tested antibiotics.The highest resistance rate for penicillin G was 98.02%.The lowest resistance rate for florfenicol was 11.88%.The resistance rate for enrofloxacin was 80.20%.The resistance rate for Yugeili(the main component is Scutellaria fragrans)was 26.6%.At the same time,101 strains of Aeromonas hydrophila showed different levels of drug resistance pattern.100%of Aeromonas hydrophila isolates were resistant to two or more drugs.27.72%of Aeromonas hydrophila isolates were the most resistant to the five drugs,P+AMX+ENR+SMZ+LEV.Four quinolone resistance genes of 101 Aeromonas hydrophila isolates were amplified by PCR.The main genotypes are qnrB,qepA,qnrS,qnrB/qnrS.Among them,the detection rate of qnrB resistance gene was the highest,which was 14.85%.The genotype detection rates of qnrB/qepA and qepA/qnrS were the lowest,which were 1.98%.The minimum inhibitory concentration(MIC)of Yugeili against Aeromonas hydrophila strain WJ2011BJ41 was detected to be 122.4 μg/mL by multiple dilution method.For the growth curve of WJ2011BJ41,both low concentration(1 MIC)and high concentration(4 MIC)of Yugeili could inhibit the growth of the bacteria after adding equal amount of bacterial suspension.Yugeili with concentration of 2 MIC and 4 MIC could completely kill Aeromonas within 10 h,and the bactericidal effect was significantly different among the treatment groups(P<0.05).The effect of Yugeili on the hemolysis of WJ2011BJ41 was measured by 5%sheep Yang red blood cells.The hemolysis rate was 79%when the concentration of Yugeili was 64 μg/mL,70.8%when the concentration was 128 μg/mL,and 43.9%when the concentration was 256 μg/mL.The inhibition of hemolysis was significant(P<0.05).The relative conductivity of Yugeili to WJ2011BJ41 was measured by a conductometer to reflect cell membrane permeability.At 0 hours,the relative conductivity of Yugeili and control groups at different concentrations was equal.After 1 hour post-treatment,the relative conductivity showed a significant change.The order of relative conductivity from large to small was:4 MIC>2 MIC>1 MIC>control group.It indicated that Yugeili could impair the cell permeability of Aeromonas hydrophila,and the effect was concentration-dependent with the concentration of Yugeili.The experiment showed that the concentration of Yugeili at 102.4 μg/mL could significantly weaken the swimming ability of WJ2011BJ41(P<0.05).4.Prevention and control of Yugeili on Aeromonas hydrophila infection in megalobrama amblycephalaYugeili prevention group(2.5 g/kg、5 g/kg、10 g/kg),Yugeili treatment group(5 g/kg,10 g/kg,20 g/kg),florfenicol prevention group(0.5 g/kg),florfenicol treatment group(1 g/kg),and negative and positive control groups were set up.The model was made by intraperitoneal injection,and each fish was injected with 0.2 mL of WJ2011BJ41 bacterial solution with a concentration of 1.0×105 CFU/mL.The mortality of fish was observed and the cumulative mortality was calculated at 0,6,12,24,48,72,96,120,144 and 168 h after challenge.Sampling analysis was performed before(0h)and 24,72,168,336 h after challenge.(1)The cumulative mortality rate was analyzed.The cumulative mortality of Yugeili prevention group(5 g/kg),Yugeili treatment group(20 g/kg)and florfenicol treatment group(1 g/kg)after 36 h of challenge was significantly lower than that of the positive control group(P<0.05),indicating that Yugeili has a significant role in the prevention and treatment of bacterial sepsis caused by Aeromonas hydrophila.(2)Effect of Yugeili on blood physiological and biochemical indexes of megalobrama amblycephala.In this experiment,Yugeili prevention group(2.5 g/kg,5 g/kg)and Yugeili treatment group(10 g/kg,20 g/kg)could significantly increase the TP content in blood of megalobrama amblycephala.The GPT and GOT levels in the Yugeili prevention group(5 g/kg,10 g/kg)and Yugeili treatment group(10 g/kg,20 g/kg)were significantly lower than those in the positive control group.The activity of AKP in serum of Yugeili prevention group(2.5 g/kg,5 g/kg)and Yugeili treatment group(5 g/kg,10 g/kg,20 g/kg)was significantly higher than that of positive control group.The activity of LSZ in blood of megalobrama amblycephala was significantly increased in Yugeili prevention group(2.5g/kg,5 g/kg)and Yugeili treatment group(10 g/kg,20 g/kg).Yugeili prevention group(5 g/kg),Yugeili treatment group(10 g/kg,20 g/kg)can significantly increase the blood C3,C4 content of megalobrama amblycephala.Yugeili prevention group(5 g/kg)and Yugeili treatment group(10 g/kg,20 g/kg)could significantly increase the number of leukocytes in blood and respiratory burst activity of megalobrama amblycephala.SOD activity of megalobrama amblycephala was increased to some extent in Yugeili prevention group(5 g/kg)and Yugeili treatment experimental group(20g/kg).The concentration of MDA was significantly reduced in the Yugeili prevention group(2.5 g/kg、5 g/kg、10 g/kg)and the Yugeili treatment experimental group(5 g/kg,10 g/kg,20 g/kg),thus reducing the lipid peroxidation injury of megalobrama amblycephala.(3)Effect of Yugeili on liver tissue structure of megalobrama amblycephala.After 7 days of treatment,the shape of hepatocytes was normal in Yugeili prevention group(5 g/kg,10 g/kg)and Yugeili treatment group(5 g/kg,10 g/kg,20 g/kg),and no pathological changes such as hepatocyte degeneration and necrosis were observed.(4)Effects of yugeili on the total number of Aeromonas hydrophila in tissues and blood of Megalobrama amblycephala.After challenge,the number of Aeromonas hydrophila in liver,kidney and blood of Megalobrama amblycephala in yugeili treatment group(5 g/kg,10 g/kg,20 g/kg)was significantly lower than that in positive control group(P<0.05).(5)Effect of Yugeili on prevention and control of bacterial sepsis of megalobrama amblycephala in fish ponds:the incidence of bacterial sepsis using Yugeili(10 g/kg)was 30.00%,and that using Yugeili(5 g/kg)was 25.00%.Compared with the incidence 45.00%of the control group,the incidence decreased by 33.33%and 44.44%,respectively.