Study Of Pathogenicity, Drug Resistance, ERIC-PCR And Distribution Of Aeromonas Veroni Isolated From Megalobrama Amblycephala Pond

Bacterial septicemia disease is a major bacterial disease of Megalobramaamblycephala Yih. This paper we research on Aeromonas veronii which resulted septicemiadisease of Megalobrama amblycephala Yih in Changzhou Wujin. Virulence gene, drugsensitive, ERIC-PCR genotyping and their comparison of different ecological distriutionwere discussed. The aim of this paper is to provide some basic research on the scientificmedicine treatment and the epidemiology of this disease. This paper is divided into thefollowing sections.1Isolation and identification of the Pathogens Aeromonas veroniiA pond of Changzhou Wujin appeared septicemia disease in July2010. In seven daysof tracking sampling, we first used the selective medium of Aeromonas to undertake aninitial screening. Then the strains were identified by the oxidase, catalase and O\F-positive.Then the PCR of Aeromonas-specific primers was detected. Finally, we utilized theornithine decarboxylase and gyrB identify the bacterium into species level. The number ofAeromonas veronii in different niche and the proportion of Aeromonas veronii inAeromonas were calculated.The results showed that: Sequencing of the isolated gyrB has the similar sequencingof the known Aeromonas veronii, the similarity rates was over98%. Ultimately168Aeromonas and56Aeromonas veronii were identified that distributed in the intestinal, gills,surface mucus, sediment, plankton, abdominal cavity, liver, kidneys which indicated thatAeromonas veronii were widely distributed in many niches of the pond. Aeromonas veroniiaccounted for33.4%of Aeromonas which indicated it was an important component inAeromonas. With the improvement of the disease, the number of Aeromonas veronii hasdecreased, but it was still present in the pond.2The detection of virulence gene, drug sensitive test of Aeromonas veronii and the comparison between different ecological niches.This part determined the pathogenicity of Aeromonas veronii by means of theartificial infection. And then the drug sensitive of the isolates to several drugs and the twokinds of virulence gene were tested, analyzed and compared between different ecologicalniches.The results showed that: Aeromonas veronii was pathogenic bacteria. At the standardof109cuf/mL and0.5mL per100g,26of the56bacteria were dead more than40%percentage in48h, which indicated that Aeromonas veronii is one of kind of bacteria thatcould cause fish sepsis. Aeromonas veronii was drug resistance to most of the antibiotics. Itwas drug resistance to ampicillin completely, and the resistance rates to SMZ, noroxin andlomefloxacin reached to94.6%,94.6%and91.1%respectively. It only showed sensitive topolymyxin and gentamycin, and the sensitive rates were64.3%and48.2%respectively.The relevance ratio of alt and hly were26.8%and48.2%respectively. The strains whichshowed the two genes at the same time were over19.6%. The lethallty rates of(alt+、hly+)、(alt+、hly-)、(alt-、hly+)、(alt-、hly-)were41.8%,35%,32.3%,29.2%respectively, whichindicated that lethallty rates might correlate with the two virulence genes. Lethallty rate ofdifferent isolated strains were not obviously different. But the drug resistance rate ofstrains from fish was67.2%, which was significantly higher than the strains from externalenvironment,40%. And the relevance ratio of virulence gene alt、hly from the fish were30.4%and47.8%respectively, which is higher than the external environment(alt10%、hly40%).3The ERIC-PCR genotyping of Aeromonas veronii and the comparison between differentecological niches.In this part genetic types of56Aeromonas veronii were researched by using theERIC-PCR. The amplification of ERIC was clustered by the software of BIO-PROFIL.The phylogenetic tree was set up by using the UPGMA. The various genotypes of strain indifferent niches were compared.The results showed that56Aeromonas veronii was divided into13kind of genotypesat the similarity80%. Type Ⅱ,19, was the maximum of them. Then the number of type Ⅵ,Ⅷ, Ⅴ, were7,6,8,5respectively. The number of type Ⅳ, Ⅶ, Ⅻ, ⅩⅢ were only oneone respectively. Most of the different niches have partial same genotypes. But the type Ⅱwas only appeared on the fish (such as gill, surface mucus and intestinal) and not appeared on the outside environments (such as water, plankton and sediment). In addition, thepathogenic of type Ⅱ was stronger than the average. So we could infer that thepathogenicity of some particular genotypes might have stronger pathogenicity than othergenotypes. In addition, type Ⅳ and Ⅸ were unique genotypes in intestinal, indicatingthat some genotypes might have selective ecological environments.