Aeromonasis In Eriocheir Sinensis: Isolation,Identification Of Bacterial Pathogen And Control By Chinese Herbs

Eriocheir sinensis is one of the most economically beneficial freshwater aquaculture products in China.However,with the continuous expansion of breeding density and scale,aeromonasis have caused serious damage to Eriocheir sinensis breeding.Aeromonasis is difficult to prevent and control because of its high incidence,wide variety of pathogenic bacteria,rapid proliferation and strong pathogenicity.Chinese herb is rich in a variety of effective ingredients,which have a good antibacterial effects and enhance the growth performance,non-specific immune function of serum,antioxidant capacity of hepatopancreas and pathogen infection resistance of aquatic animals,so it is widely used in aquaculture.In this paper the pathogenic bacteria of aeromonasis was isolated and identified from Eriocheir sinensis,and the histopathological characterization of hepatopancreas and intestinal tract of naturally infected Eriocheir sinensis was observed.On this basis,the minimal inhibitory concentration（MIC）of 50 kinds of Chinese herb against Aeromonas veronii from Eriocheir sinensis was tested by the tube double dilution method,and the components and proportion of the selected Chinese herb was optimized by the orthogonal t-value method.Besides,the effect of the compound Chinese herb on the non-specific immune function of serum,antioxidant capacity of hepatopancreas and anti-Aeromonas veronii infection of Eriocheir sinensis was evaluated.This paper is expected to provide a technical reference for the prevention and control of Aeromonasis on Eriocheir sinensis breeding.The specific research are as follows:1.The pathogenic strain HXH1 was isolated from hepatopancreas of diseased Eriocheir sinensis and was identified as Aeromonas veronii based on phenotypic characteristics,16S r RNA sequences and gyr B gene sequences analysis.It had an median lethal dose(LD₅₀)of 6.92×10⁴ CFU·m L^-1 for healthy Eriocheir sinensis.Histopathological observation showed that the hepatopancreas cells of diseased Eriocheir sinensis were irregular-arranged,appeared local necrosis with pyknotic nucleus,local epithelial tissues of intestinal mucosa were dissolved and fell off into the intestinal cavity compared with healthy Eriocheir sinensis.2.Aeromonas veronii HXH1 was the instruction strain,HXY1 and HXY3 were the control strains for antibacterial Chinese herb screening.The minimal inhibitory concentration of 50 kinds of Chinese herb against Aeromonas veronii from Eriocheir sinensis was determined by the tube double dilution method,and the results showed that seven kinds of Chinese herb such as Rhizoma Coptidis,Caesalpinia sappan Linn,Radix et rhizoma rhei,Fructus Mume,Spatholobi Caulis,Punica granatum,Moslae Herba,have a good antibacterial effects on Aeromonas veronii with MIC of 7.81-31.25 mg·m L^-1.The orthogonal t-value method were used to optimize the components and proportion of 7 kinds of Chinese herb including Rhizoma Coptidis,Caesalpinia sappan Linn,Radix et rhizoma rhei,Fructus Mume,Spatholobi Caulis,Punica granatum,Moslae Herba,and the results showed that the best group of components were Rhizoma Coptidis,Moslae Herba,Caesalpinia sappan Linn,and Punica granatum with the optimal compatibility ratio 5:4:1:2.The time-kill curve showed that the density of Aeromonas veronii HXH1treated with compound Chinese herb group was reduced by 2.01 log CFU·m L^-1（p<0.05）,2.18 log CFU·m L^-1（p<0.05）,2.77 log CFU·m L^-1（p<0.05）and 2.92 log CFU·m L^-1（p<0.05）compared with that treated with Rhizoma Coptidis,Caesalpinia sappan Linn,Moslae Herba,Punica granatum,which indicated that each component of the compound Chinese herb in this experiment has a synergistic effect.3.The compound Chinese herb were added to the basic feed with 5 g/kg,10 g/kg,15 g/kg feed and fed to Eriocheir sinensis for 45 days,and the basic feed was used as a control.The results show that Acid phosphatase（ACP）,Alkaline phosphatase（AKP）and Lysozyme（LZM）,Superoxide dismutase（SOD）,Catalase（CAT）activity in serum of the test of Eriocheir sinensis than the control were increased by 28.0%to 40.0%,8.9%to34.8%,24.2%to 36.4%,6.3%to 37.5%,10.2%to 41.4%,and Malondialdehyde（MDA）content in serum of the test of Eriocheir sinensis than the control was reduced by 9.9% to 23.1%.And the Acid phosphatase（ACP）,Alkaline phosphatase（AKP）and Lysozyme（LZM）,Superoxide dismutase（SOD）,Catalase（CAT）activity in hepatopancreas of the test of Eriocheir sinensis than the control were increased by 11.7%to 21.3%,3.8%to18.4%,61.6%to 137.9%,8.3%to 30.2%,2.1%to 62.6%,and Malondialdehyde（MDA）content in hepatopancreas of the test of Eriocheir sinensis than the control was reduced by 20.6%to 26.5%.As mentioned above,Acid phosphatase（ACP）,Alkaline phosphatase（AKP）and Lysozyme（LZM）,Superoxide dismutase（SOD）,Catalase（CAT）activity in serum and hepatopancreas of the test of Eriocheir sinensis increased with the increase of compound Chinese herb,and the content of Malondialdehyde decreased with the increase of compound Chinese herb,which indicated that the compound Chinese herb were added to the basic feed with 5 g/kg,10 g/kg,15 g/kg feed can can improve the non-specific immune function and antioxidant function of Eriocheir sinensis.In addition,the addition of compound Chinese herb in the basic feed with 5 g/kg,10 g/kg and 15 g/kg had good protection rates for Eriocheir sinensis against Aeromonas veronii infection,and the relative protection rates were 40.00%,46.67%and 63.33%,respectively.According to the evaluation criteria for the effectiveness of antimicrobial drugs in Clinical Trial Specification for Fishery Drugs,the recommended dosage of compound Chinese herb was 15 g/kg.