Isolation And Identification Of Pathogen Bacterial From Farmed Pseudosciaena Crocea And Development Of Antibacterial Compounds

This present studies focused on the identification of the pathogenic bacteria of Pseudosciaena crocea and their correlative antibacterial drug prevention.In terms of pathogen bacteria identification,bacteria were isolated mainly from livers,kidney and skin lesion of disease Pseudosciaena crocea and their identification was created by the results of physiological,biochemical,biolog automatic identification system and molecular characters.At the same time,the virulence of pathogenic bacteria was ascertained according to the result of the artificial infection.In the development of antibacterial compounds studies,the antimicrobial activities of antibiotics and Chinese traditional medicine to 5 virulent strains in vitro were compared by the disc,agar diffusion and microcalorimetry method.Moreover,the influence of different Chinese traditional medicine and different concentrations of prescription on Oreochromis niloticus growth performance,nonspecific immune indexes and disease-resistant ability was investigated to provide the basis for the application of antibacterial compounds.The main results of this study are as follows:1.A total of 44 strains of bacteria were isolated from three batches of disease Pseudosciaena ecroca.Of which,38 strains according to primary biochemical identification were classified as eleven genus,including twenty-four strains of Vibrio,four Aeromonas strains,each two strains of Yersinia,Acinetobacter and so on.Partial of differ genus or species strains were selected for further identification by the method of biolog automatic system and 16S rRNA sequences analysis,the results indicated eleven out of fourteen strains were accurately identified as Vibrio damsela?2?,Vibrio parahaemolyticus?2?,Vibrio hollisae?1?,Vibrio fluvialis?1?,Vibrio alginolyticus?1?,Aeromonas schubertii?1?,Aeromonas veronii?1?,Morganella morganii?1?,Pseudomonas pseudoalcaligenes?1?,while other three strains could not be identified to species but with the same genus relationship.2.Based on the artificial infection test of 11 strains which separated from Pseudosciaena ecroca,strong virulence strains were selected to determinate the median lethal dose(LD₅₀)in Oreochromis niloticus.The results showed five out of eleven strains?Aeromonas veronii,Vibrio fluvialis,Vibrio alginolyticus,Vibrio vulnificus,Pseudomonas pseudoalcaligenes?were deadly to fish,their LD₅₀0 were 1.08×10³ CFU/g,8.52×10² CFU/g,3.06×10² CFU/g,4.92×10³ CFU/g,3.13×10⁵ CFU/g respectively.Therefore,the disease model of Oreochromis niloticus was constructed by artificial infection of 5 pathogen strains.3.Moreover,the antibiotic and Chinese traditional medicine were screened against these five virulence strains in vitro,the results indicated the proportion of strains resistant to twenty-four antibiotics were 29.2%,54.2%,20.8%,20.8%,41.7%respectively.However,ten out of twenty herbs showed better antibacterial effect against pathogenic bacteria in vitro with the average MIC range from 0.11 to 7.20 mg/mL.Among them,the inhibiting effect of Chinese herbs A,H,I were further confirmed by the method of microcalorimetry,which show the same value of MIC as results based on agar dilution method.4.Base on the disease model of Oreochromis niloticus,the influence of adding 5%feed rate of four Chinese traditional medicines?Chinese herbs A,H,I,M?on growth,nonspecific immunity and disease resistance to A.veronii in Oreochromis niloticus was assayed.The results showed that Oreochromis niloticus supplemented herbs had a significant increase in growth property.Among them,Chinese herb M showed the best effect with its weight gain rate?WGR?and specific growth ratio?SGR?increased by 21.25%,15.34%?P<0.05?respectively,and feed conversion ratio?FCR?reduced by 19.5%?P<0.05?compared to the control group.The serum and liver lysozyme?LZM?,superoxide dismutase?SOD?,catalase?CAT?,acid phosphatase?ACP?and alkaline activities?AKP?of experimental group improved in a certain degree and the challenge test also indicated the mortality rate significantly decreased?P<0.05?.Based on the result above,the optimal effect for improving nonspecific immunity and antibacterial ability were added Chinese herbs H,I,M for 21 d and Chinese herb A for 28 d.5.On the basis of four Chinese traditional medicines additives research,One prescription contains four graded level?0,2.5%,5%,10%?,were respectively added into the basal diet for the Oreochromis niloticus and fed to fish for four weeks.The growth property,nonspecific immune response was investigated each seven day.Then,the fish was infected by five pathogenic bacteria and mortalities were recorded.The results showed WGR and SGR of fish fed with control diet were significantly lower than the group with prescription?P<0.05?.Especially the 5%prescription,which showed the best effect with its WGR and SGR increased by 15.71%,11.88%?P<0.05?respectively,and FCR reduced by 19.5%?P<0.05?.The serum LZM,SOD and AKP activities of prescription groups showed a gradual upward trend during four weeks.While the activities value of CAT and ACP presented a drop trend after the first upward.There were no significant differences of ACP activities among groups fed with different doses of both herbs.Toxicity test showed that 2.5%prescription had wide spectrum antibacterial ability.Its immune protective rate of A.veronii,V.fluvialis,V.alginolyticus,V.vulnificus,P.pseudoalcaligenes was75%,100%,100%,100%,66.7%respectively.It is concluded that the optimal dose of prescription was 2.5%-5%.In summary,five pathogenic bacteria from Pseudosciaena ecroca were detected,and the drug sensitive test showed partial of strains had generated drug-resistant while 10 kinds of Chinese traditional medicine had better antibacterial effect against pathogenic bacteria in vitro.In addition,the influence of four herbs and different concentrations of prescription on Oreochromis niloticus wide spectrum antibacterial ability was investigated.This research achievement hopefully to provide reference value to the diagnosis and drug control of pathogenic bacteria in cultivated Pseudosciaena ecroca.