The Virulence Of The Aeromonas Hydrophila

Aeromonas hydrophila is the most common pathogenic bacteria in the freshwater, which often cause of large outbreak on aquatic animals , Usually, It belongs to conditional pathogenic bacteria which is only moderate virulence. It must be achieved high survival rate and no pollution ,Meanwhile,It must maintain the stability of its properties, such as morphological characteristics, cultivating characteristics, physiological and biochemical characteristics, genetic characteristics of chemical composition and cell for the veracity and precisenes of the research.Therefore,it is so important about the storage of the strains.However,at present,it is hard to control the characteristics of the strains can not mutate,To a great extent,it was caused by the excessive subculture or incongruous preservation method. It is necessary to adjustment some factors to guarantee the bacterial strains to decline the variation to the minimum. In this study, To investigate the Virulence Factors-Pathogenicity Relationships of A.hydrophila, five virulence factors were detected by PCR amplification. At the same time, In order to analysis the relationship between the weaken virulence during subculture and storage and the distribution of the virulence genes.The LD50 was quantified by acute toxic test on Carassais auratus gibebio and the virulence gene were determined during subculture and storage, For the temperature and culture medium factots which are able to cause virulence of the strains weaken ,it had been studied their effect on distribution of the virulence genes to discuss the mechanism of the virulence weakened.21 strains which were identified as A.hydrophila were selected the experimental subject. To investigate the Virulence Factors-Pathogenicity Relationships of A.hydrophila, five virulence factors were detected by PCR amplification, namely the aerolysin (aerA), hemolysin (hlyA), extracelluar protease (ahpA), enterotoxin (altA), Anti metalloproteinases(ast). The LD50 was quantified by acute toxic test on Carassais auratus gibebio; Antimicrobial susceptibility test was used the Kirby-Bauer method. Except that the pathogenicity of Hong12 of which the LD50 is 109 cfu is weak.The other eight strains are all strong relatively, of which the LD50 are all around 106 cfu/ml, Presence of aerA, ahpA, hlyA, altA and ast virulence genes was identified among 77.8%, 88.9%, 100%, 100%, 88.9% of tested strains. By comparing the results of the detection of virulence genes and the results of acute toxicity test of the A.hydrophila on Carassais auratus gibebio ,It was found that the association of aerA and ahpA should be thus be considered as a possible virulence marker , highly related to the pathogenicity ; and ast may be related to the pathogenicity ; however , neither hlyA nor altA is related to pathogenicity . The 21 strains bacteria were all identified as A.hydrophila by means of biochemical and molecular identification.They were found 100% insensitive to Penicillin, Carbenicillin,Yancomycin; modertate sensitive to Syntactic, Sulfonamide, Cefotaxime, Rifampicin; all highly sensitive to Furantoin,Chloromycetin, Acheomycin, Ofloxacin.To study the stability of virulence genes of A.hydrophila strains during subculture, 10 isolated A.hydrophila strains were subcultured to 20 times on LB agar., Then we observed the change of LD50 and distribution of five virulence factors (aerA, hlyA, ahpA, altA, ast) of A.hydrophila , the virulence genes were amplified respectively by PCR from template DNA of the 10 strains and their corresponding 20 times subcultured strains , The results showed that the LD50 of Carassais auratus gibebio weakened even losed resulting from subculture, The distribution of virulence genes is no significant differences among original strains, the tenth and the twentieth times.except that ahpA gene losed extremely resulting subculture, the genotype of virulence genes was instable relatively.In the meantime , To study the stability of virulence genes of A.hydrophila strains during storage, 4 isolated A.hydrophila strains were subcultured on LB agar. Then we observed the change of LD50 and distribution of five virulence factors (aerA, hlyA, ahpA, altA, ast) of A.hydrophila , the virulence genes were amplified respectively by PCR from template DNA of the 4 strains and their corresponding 3 days,1 week, 2 weeks, 4 weeks, 8 weeks, 6 weeks and 32 weeks strains, The results showed that in preservation period, single virulence gene changes with different characteristics, that doesn't have a consistent changes in law; namely after period of preservation,virulence genes would have different degree to negative , the existence of virulence genes aerA, ahpA, ast is relevant with the virulence.Furthermore, Temperature and medium which may affect the virulence were selected to study the influence on the distribution of virulence genes. the three level of 25℃,4℃,-20℃were choosed and three mediums of nutrient broth(NB)brain heart infusion broth(BHIB), LB broth were choosed to storage the strains for 1 month,At the same time,the virulence genes were amplified respectively by PCR from template DNA of the strains . The experimental results show that in 4℃storage conditions, the genotype is stable highest, and in 25℃conditions, aerA gene is prone to lost, In -20℃condition, the altA, hlyA which have little relation with virulence is prone to lost, so,it do not affect virulence, and the brain heart infusion broth(BHIB) medium doesn't fit as the medium of A.hydrophila for storage.In short, this paper on the virulence characteristics of 21 strains of A.hydrophila provides further theoretical data for the thorough research of the character of pathogenic bacteria.