Detection Of Virulence Genes In Aeromonas Strains And The Influence Of Environmental Factors On Virulence Genes Expression

Aeromonas is an important pathogen which results in aquaculture animals infectingwith disease. The paper carried out the isolation and identification of Aeromonas fromhemorrhagic fish samples in Anhui Province, and detected eight virulence genes inAeromonas. On the basis of preceding study, the influence of environmental factors(temperature, pH, salinity) on virulence gene expression was researched. Throughthis study we hope to provide effective and ecological prevention measures,improve the security of aquatic products and help to promote the sustained andhealthy development of the aquaculture industry.1. Detection of Virulence gene: Vaccinating and cultivating bacteria whichwere isolated from all kinds of diseased fish and extract DNA. According tologged-in nucleotide sequences of Aeromonas in GenBank, we designed andsynthesised eight specific primers, including Aer, Ahy-B, Act, Alt, Epr, Ast, Lip andEla. The result indicated that among58strains of Aeromonas, the genes werepresent in34.5%(Epr),24.1%(Alt),75.9%(Lip),81%(Act),62%(Ela),62%(Ahy-B),34.5%(Ast),41.4%(Aer) of the strains. Corresponding to each type ofvirulence genes, one positive strain was choosed. Listed as follows:①.Aer-47；②.Ela-47；③.Ahy-B-35；④.Act-56；⑤.Lip-17；⑥.Epr-17；⑦.Ast-17；⑧.Alt-50a total of5strains.2. Determination of growth curve: In order to understand the growthcondition of Aeromonas, we detect the growth curve of the5strains which wereselected in the previous chapter. The environmental factors include pH, salinity andtemperature. By measuring OD values and using live bacteria plate countingmethod, the growth condition of the strains was mastered. In pH, Aeromonas grewbest in neutral environment, followed by the acidic environment. It was of worsttolerant to the alkaline environment. In salinity, in addition to No.35strain whichwas most obviously suppressed by elevated salinity, other4strains’ growth wasgradually suppressed with the increasing of salinity. In temperature, lowtemperature’s impact on Aeromonas was more obvious. The bacteria grew more slowly when the temperature was15℃, and when the temperature rose from15℃to35℃, the growth of bacteria also started to speed up.3. Influence of environmental factors on virulence genes expression:Research of environmental factors’ influence on bacterial virulence geneexpression is very important to get a thorough understanding of bacterialpathogenesis. This chapter trys to certain its mechanism in three aspects (pH,salinity and temperature). The growth curve showed that the largest slope wasmainly distributed between11-12hours. We can detect whether the virulence genesexpress or not during this period. First step is to vaccinate and cultivate bacteriaand use the TransZol Up kit to extract RNA，and then conduct RNA reversetranscription and PCR with One-Step RT-PCR SuperMix. Last step is to check theresult through electrophoresis. The results showed that virulence genes expressionquantity is better in the acid environment, low salinity and35℃.