Isolation And Identification Of Pathogenic Bacteria And In Vitro Bacteriostasis Chinese Herb Compound Selection For Hemorrhagic Disease Of Ictalurus Punctatus

Ictalurus punctatus is one of the most important economic fish in China,as the scale of culture expands,there were many trouble appeared which caused by bacterial disease.Disease caused by bacterial were the most widely distributed and harmful disease of Ictalurus punctatus,which can often cause bleeding or congestion on the surface,viscera,intestine,cloaca and other bleeding ulceration.So far,the bacteria that have been found to cause the death of Stenotrophomonas maltophilia.Aeromonas hydrophila、Aeromonas media、Aeromonas veronii、Aeromonas sobria,et al.In Oct 2018,in Suiyang,Guizhou,Ictalurus punctatus suffered from mass-mortalities caused by hemorrhagic disease,In order to explore the cause of this disease,experiments were carried out:isolated the pathogenic bacteria from sick Ictalurus punctatus,and then pathogenic bacteria were identification by morphological observation、physiological and biochemical characterization、molecular identification with 16S rDNA and gyrB genes、artificially infected and its antibiotic sensitivity、10 virulence genes and 45 antibiotic sensitivity tests were carried out.On those basis,the diameter of inhibition circle、the minimum inhibitory concentration(MIC)and the minimum bactericidal concentration(MBC)of 41 kinds of Chinese herbs and their compound to pathogenic bacteria in Ictalurus punctatus were determined respectivelyThe experimental results showed that cloacal aperture of diseased Ictalurus punctatus were red,swollen and convex,with the intestines being hyperemia and intussusception,the pathogenic bacteria was identified as Aeromonas hydrophila by morphological observation、physiological and biochemical characterization、molecular identification with 16S rDNA and gyrB genesThe artificially infected experimental results showed that artificially infected Ictalurus punctatus showed similar symptoms to that of the natural,The LD50 of pathogenic bacteria to Ictalurus punsctatus which body length are 15～20 cm was 4.75×106 cfu/mL;carrying 7 virulence genes of aerA、hly、aha、ahp、ast、laf、OMPs and no three virulence genes of act、alt、epa.Sensitivity of S5-X1 to 45 common antibiotics was detected,and results showed that the strain was highly sensitive to 13 drugs,including azithromycin,furantoin,doxycycline,and moderately sensitive to 3 antibiotics,including polymyxin B,ceftazidime and cefotaxime,resistant to 29 antibiotics such as amoxicillin,tetracycline and teicoplaninIn the Chinese herbal test section,The results showed that 14 of 41 Chin ese herbs had bacteriostasis circle,27 of 41 Chinese herbs had no bacteriostas is circle,the lowest of MIC was Coptischinensis Franch which the concentratio n is 7.81 mg/mL,comprehensive diameter of inhibition circle、MIC and MB C results,comprehensive screening of 6 Chinese herbs with better bacteriostati c effect:Punica granatum L、Terminalia chebularetz、Violaphilippica、Caesalpi niasappanL、Coptischinensis Franch·Mumefructus.Drug combination results sho wed the percentage that there was no antagonism between the 6 herbs was 933%,the percentage that positive effect was 80%.The results of 25 groups acco rding to preparation tables of mixed chinese herbal medicine showed that M-10 compound group which scale is Punicagranatum L:Terminalia chebularetz:Viola philippica:CaesalpiniasappanL:Coptischinensis Franch:Mumefructu=2:5:1:2:3:4 h ad the best comprehensive bacteriostatic effectThe results of this study indicate that the pathogenic bacteria of hemorrhagic disease in Guizhou is Aeromonas hydrophila.The results provide a theoretical basis and reference for the effective prevention of hemorrhagic disease and provide a theoretical basis for the further development of compound Chinese herbal medicine to suppress bacteria and clinical application.