Identification And Pathogenicity Study Of Aeromonas Veronii From Procambarus Clarkii And Screening Of Antibacterial Chinese Herbal

With the development of Procambarus clarkii(crayfish)aquaculture,the disease becomes more and more serious.In May 2016,a lot of death cases of Procambarus clarkii were reported in a crayfish farm at Qianjiang City,Hubei Province.The P.clarkii were found to be weekness of limbs and insensitive in response of stimulation.Numerous dead P.clarkii were found around the pond and on the top of waterweeds.In order to clarify the aetiology of the disease,we isolated and identified a strain of Aeromonas veronii from diseased P.clarkii.The A.veronii virulence gene distribution and genotyping status were studied by virulence gene detection and multi-locus sequence analysis.The histopathology of the diseased P.clarkii was further observed by paraffin section technique.Moreover,we test 30 Chinese herbals for their antibacterial activities against A.veronii.This study provided a fundamental knowledge for the prevention and control of crayfish bacteria dieases.The results were as follows:1.We isolated a strain of Gram-negative bacteria(QD160502)from the hepatopancreas of diseased P.clarkii.It was identified as A.veronii based on morphological observation,biochemical identification and physiology characteristics.The 16 S r DNA and gyr B sequence(KX585901 and MN189983)of QD160502 showed high homology with in Gen Bank.Artificial infection experiment proved QD160502 is the pathogen of the disease..The antimicrobial sensitivity test showed that QD160502 was more sensitive to enrofloxacin,norfloxacin,tetracycline,doxycycline etc.,and resistant to penicillin,streptomycin,kanamycin etc.The detection of virulence genes suggested that there are three virulence genes including aerolysin(aer),cytotoxic enterotoxin(act)and flagellinfla(fla).The results of multi-locus sequence typing showed that QD160502 was clustered with two A.veronii strains from Italy.2.The histological results are as follows: After challenging for 12 h,some transferred vacuoles containing granular materials were found in hepatopancreas blasenzenllen cells;after challenging for 24 h,inflammatory cell infiltration were observed in hepatopancreas,the intestinal connective tissue were atrophied and vacuole-like dilations were appeared in the myocardial tissue;after challenging for 48 h,mass of hepatopancreas restzellen cells and blasenzenllen cells were disintegrated,intestinal mussel flods was decreased and vacuole-like dilations was increased in the myocardial tissue;after challenging for 72 h,hepatopancreas and intestinal epithelial cells were impaired with necrosis and hyaline blood cells aggregation were appeared in myocardial tissue.3.The antibacterial efficacy of 30 kinds of Chinese herbals against A.veronii were detected in vitro by using the agar diffusion method.The minimal inhibitory concentration(MIC)and minimal bactericidal concentration(MBC)were investigated by double dilution method.The results showed that A.veronii were highly sensitive to Caesalpinia sappan,Rhus chinensis,Sanguisorba officinali and Prunus mume,of which the MIC and MBC were below 10 mg/m L.C.sappan had the best antibacterial effect on the A.veronii and the MIC and MBC of C.sappan were both 0.98 mg/m L.The MIC/MBC values of Melia azedarach,Bretschneidera sinensis,Punica granatum and Senecio scandens were both bwtween 15.63 and 31.25 mg/m L.The MIC/MBC value of Radix paeoniae rubra、Myrica rubra、Pterocarya stenoptera and other 5 herbals were both bwtween 125 and 250 mg/m L.Areca catechu,Lithospermum erythrorhizon,Macleaya cordata and other 11 herbals had only a little or no antibacterial efficacy against A.veronii.