Isolation And Identification Of Pathogenic Bacteria And In Vitro Bacteriostasis Chinese Herb Compound Selection For Canker Disease Of Schizothorax Grahami

From 2012 to the present,through epidemiological investigations,it was found that a number of Schizothorax grahami aquacultured in Bijie,Jiangkou and other places,Guizhou Province have successively developed a disease characterized by skin rupture and muscle ulceration before and after the dorsal fin.According to the characteristics of the disease,it is called"canker disease".This study isolated and purified the pathogenic bacteria from body surface ulcers,liver and kidneys by bacterial isolation method under aseptic condition.The pathogen was identified based on morphological observation,biochemical characteristics together with sequence analysis of 16S r DNA,dna J and cpn60.The pathogenicity of pathogenic bacteria were determined by artificial infection and the drug susceptibility was detected by Kirby-Bauer disk diffusion method.What is moe,31 common Chinese herbal aquatic extracts were prepared by decoction method,the diameter of the bacteriostatic circle of each flavored Chinese herbal medicine was determined by oxford cup method,and the minimum inhibitory concentration（MIC）and minimum sterilization concentration（MBC）were determined by double dilution method.Finally,6 kinds of Chinese herbal medicines with good antibacterial activity in vitro were selected,and the partial bacteriostatic concentration index（FIC）test of 15 groups of binary prescriptions was carried out by the checkerboard method,and the 25 groups of compounds were matched according to the orthogonal design table,and the susceptibility test was carried out.The results showed that the bacterial colonies with uniform morphology and size were isolated and purified from the diseased S.graham and BL61C was randomly selected as experimental bacteria.The results of morphological observation,identification of physiological and biochemical characteristics and sequence analysis of housekeeping genes showed that the pathogenic bacteria isolated was Aeromonas veronii.Artificial infected fish by intraperitoneal BL61C injection showed same symptoms as that of natural ones and the identical pathogenic bacteria were isolated from the tissue again,which proved that A.veronii BL61C was the pathogen causing epidermis ulcer of S.grahami,with the LC₅₀was 5.09×10⁷CFU/m L.Drug sensitivity test showed that the isolated bacterium was highly sensitive to eight kinds of antibiotics including Spectinomycin,Azithromycin,Levofloxacin,Chloramphenicol,Florfenicol,Ceftazidime,Claforan and Cefotaxime,while resistant to 20 drugs including Claricid,Cystatin and Tetracycline.Among the 31 kinds of Chinese herbal medicines,6 kinds of Chinese herbal medicines such as Galla Chinensis,Radix Sanguisorbae,Fructus Chebulae,Lignum Sappan,Radix Scutellariae and Rhizoma Coptidis had the best antibacterial effect.No antagonism occurred in 15 groups of combination prescriptions.The combination of Chinese Gallnut and Radix Sanguisorbae,Fructus Chebulae and Radix Scutellariae had the best effect,and the FIC were 0.31 and 0.50,respectively,showing a synergistic effect.Compound F16（G.Chinensis:F.Chebulae:L.Sappan:R.Scutellariae:R.Coptidis:R.Sanguisorbae=4:4:2:5:3:1）with the best anti-bacterial effect,the diameter was 23.2 mm,and MIC and MBC were 0.98mg/m L and 1.95mg/m L,respectively;it could be used as the preferred bactericidal combination of A.vernoii from S.grahami.The results of this study determined that the pathogen of canker disease in S.grahami in Bijie,Guizhou Province was A.vernoii,and 6 kinds of Chinese herbal medicines and their compounds were preliminarily screened with good inhibitory effect on the isolated bacteria BL61C in vitro.The above results provide scientific basis and reference for the prevention and treatment of canker disease in Kunming split-bellied fish,and provide a theoretical basis for the use of Chinese herbal medicine to inhibit bacteria and fishery.