| The giant freshwater prawn Macrobrachium rosenbergii is an important freshwater shrimp in China due to its rich nutrition and short culture cycle.However,with the expansion of farming scale and the deterioration of the water ecology,epidemic infectious diseases of M.rosenbergii have become a significant problem,limiting high-quality development.In recent years,outbreaks of mass mortalities of M.rosenbergii occurred in different farms of Jiangsu province,China.The epidemiological investigation showed that Aeromonas veronii was the major pathogen which caused repeated infections in M.rosenbergii.In 2020,outbreaks of mass mortalities occurred at a M rosenbergii farm in Jiangdu county of Yangzhou City,Jiangsu Province.The diseased prawns showed red body,red hepatopancreas,reduced vitality,decreased appetite,and reduced sensitivity to external factors.In order to identify the pathogens causing diseases in M.rosenbergii,the potentially pathogenic bacteria were isolated and identified,the virulence characteristics of the isolated bacteria and its induced immune response in M.rosenbergii were explored.Further,the Chinese herbals formulas against the isolated bacteria were also selected,and its effects on non-specific immune response and disease resistance of M.rosenbergii evaluated.The results of this study will provide a theoretical basis for controlling the disease in M.rosenbergii aquaculture.The main research results are as follows1.The dominant strain JDM1-1 was isolated the hepatopancreas of diseased M.rosenbergii,and identified by morphological characteristics,physiological and biochemical characteristics,sequence alignment of 16S rRNA gene and phylogenetic analysis.The pathogenicity of the starin JDM1-1 was determined by histopathological observation,artificial infection experiments and virulence factors and virulence related genes detection.Besides,the drug sensitivity of the pathogenic bacteria was evaluated by Kirby-Bauer method.The results showed that the pathogen causing the mass mortalities M.rosenbergii was Aeromonas veronii,and the LD50 of the representative strain JDM1-1 to M.rosenbergii was 1.4×105 CFU/mL.Histopathological analysis revealed that this bacterial infection caused considerable histological lesions of M.rosenbergii,including increasing the hepatic tubule lumen and the gap between the hepatic tubules,disappearing of the brush border.The results of virulence related gene detection showed the strain JDM1-1 carried outer membrane protein A(ompA),lipase(lip),adhesion factor(tapA),escherichia coli alpha-hemolysin(hlyA),filaggrin A(flgA),filaggrin M(flgM),filaggrin N(flgN)and aerolysin(aer),and virulence factors detection showed that the strain JDM1-1 had the activities of gelatinase,lipase,lecithase,protease and hemolysin.In addition,the results of antimicrobial susceptibility analysis showed that the strain JDM1-1 was sensitive to 18 antibiotics,including ciprofloxacin,erythromycin and aminotransm,etc.It was moderately sensitive to 10 drugs such as clarithromycin and tetracycline,etc.,and resistant to 9 drugs,including piperacillin and vancomycin,etc.2.In order to investigate the immune response of M.rosenbergii against A.veronii,the expression levels of six selected immune-related genes(ALF1,ALF2,ALF3,C-lectin,Crustin and Lysozyme)were monitored at different point of time in hepatopancreas and intestine of M.rosenbergii infected with A.veronii by quantitative real-time PCR(qRT-PCR).Results showed that the expression levels of the these immune-related genes were up-regulated from 6 to 48 h after A.veronii infection.The expression level of Crustin reached a peak at 6 hpi,the expression level of C-lectin and Lysozyme peaked at 12 hpi,and the expression level of ALF1,ALF2 and ALF3 peaked at 24 hpi in the hepatopancreas;the expression levels of ALF1,ALF2,ALF3,C-lectin,Crustin and Lysozyme reached a peak at 24 hpi in the intestine.The results revealed that these immune-related genes participated in the immune response and can be considered as potential immune indexes for monitoring health status of M.rosenbergii.3.In order to screen the Chinese herbals and compound formulas against A.veronii isolated from M rosenbergii,the inhibitory effects of 42 kinds of water extractions from Chinese herbals were determined by agar diffusion and dilution method.The results showed that Sanguisorba officinalis and Chebula Retz had the best antibacterial effects against A.veronii,followed by Paeoniae Radix Alba and DarkPlum Fruit.Meanwhile,the two kinds of single Chinese herbals with better antibacterial effect were selected,and the fraction inhibitory concentration index(FICI)indicated that the combination of S.officinalis and C.Retz was characterized by an additive effect.Further,Paeoniae Radix Alba was selected as an immune enhancer,these three Chinese herbals were prepared for compound Chinese herbal in a 1:1:1 ratio,the minimum inhibitory concentration(MIC)and the minimum bactericidal concentration(MBC)of the compound Chinese herbal against A.veronii were determined.The results showed the MIC of this compound Chinese herbal against A.veroni was 7.813 mg/mL,and the MBC was 15.625 mg/mL.In addition,the effects on non-specific immune response and disease resistance of M.rosenbergii were evaluated by immersion administration with the compound Chinese herbal(7.8 mg/mL).The results showed the activities of SOD,AKP and ACP were significantly higher than those in control groups,and the expression levels of ALF1,HSP70,Lysozyme,proPO,SOD and TLR1 genes were significantly up-regulated in compound Chinese herbal treated group.After challenged with A.veronii(1.4×105 CFU/mL),the survial rate of M.rosenbergii was significantly higher in compound Chinese herbal treated group.The findings provide a theoretical basis for control diseases caused by A.veronii in M.rosenbergii by application of Chinese herbals. |
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