Identification Of The Serotype And Investigation Of The Main Virulence Factors Of Vibrio Parahaemolyticus Strains From The Coastal Seashell In Ningbo

Vibrio parahaemolyticus is a Gram negative,halophilic,rod-shaped bacterium that distributed worldwide in the estuarine environment,especially in fishes,shellfish and seafood products.It has been considered as one of the most important seafood-borne pathogens vibrio species primarily involved in causing gastrointestinal illness in the countries and regions with long coastlines.V.parahaemolyticus is considered to be the causative agent in 50%to 70%of all cases of diarrhea associated with the consumption of fishery products in the summer months.Humans caused by this organism are almost exclusive with consumption of raw or inadequately cooked food,or any food cross-contaminated,by handling raw seafood in the same environment or by rinsing with contaminated seawarter.V.parahaemolyticus has been listed one of the pathogenic vibrios endangering net-cage cultured large yellow croaker(Pseudosciaena crocea) in coastal areas of China.Techniques of quick detection and isolation for pathogenic microorganisms have been concentrated on recent years.Polymerase chain reaction(PCR) is an effective detection technology,which can be used to quickly detect pathogenic microorganisms. Therefore,to improve a reliable,rapid and convenient methods for identification and typing of V.parahaemolyticus isolates from seafoods and processing plants are crucial to identify the principal contamination points along the processing lines and establish the critical control points thereafter.The PCR method was developed for rapid,specific and sensitive identificationg of V.parahaemolyticus from seafoods using primers targeting the toxR gene specific for the bacterium.A total of 71 V.parahaemolyticus strains were isolated,and all of them amplified the 340-bp toxR gene fragment.It could differentiate between V.parahaemolyticus and other vibrio spp respectively.The thermostable direct hemolysin(TDH) and TDH-related hemolysin(TRH),thermolabile hemolysin(TLH) are the main virulence factors of V.parahaemolyticus.To investigate the distribution of the virulence of V.parahaemolyticus among different strains,we isolated V.parahaemolyticus from seashell and oysters obtained from the area of Ningbo in China,determined the serogroups,phenotypically and genotypically characterized TDH and TRH,and investigated the presence of the toxR gene.The TRH gene was not identified in any of the strains except BJI.1997;none strains were Kanagawa phenomenon(KP) positive and amplified the 269-bp TDH gene fragment.The TLH gene was identified in all of the strains.Objective to understand the serotype distribution and resistance to antibiotics of the V.parahaemolyticus isolated from seashell products.Using the method of the standard technique GBPT 478917-2003 for serotype tested.The minimum inhabitory concentration method was used to test the antibiotic susceptibility,including Ampicillin,Kanamycin, Streptomycin Sulfate,Tetracyyline,Neomycin Sulfate,Gentamycin Sulfate, Spectinomycin,Oxytetracycline Tablets,Rifampin was studied.The results showed that 20 strains of V.parahaemolyticus mostly consisted of two serum groups:O1 50%(10/20), O5 35%(7/20),autoagglutination 15%(3/20).Susceptibility tests showed that serological type O1:KⅢof V.parahaemolyticus is susceptible to Kanamycin,all strains are resitant with AMP,T-tested showed that there was no significant difference sensitivity between NEO and STR tested strains(P＞0.05).After 96h fish acute toxicity test,the results showed that the toxicity of the different serological type of V.parahaemolyticus for Carassius auratus gibelio var.Songpu(Bloch) has distinctness diversity.Formalin killing cells of V.parahaemolyticus were prepared with four strains:NB-001,NB-002,NB-003, NB-004,which showed different serological type:NB-001(O1:KⅢ),NB-002(O1:KⅥ), NB-003(O1:KⅦ),NB-004(05:KⅣ).The differernce of the antigenicity among the bacterins prepared with these four strains was investigated by monitoring of agglutinating antibody titer.High agglutinating antibody titer was detected against immunized antigen, but low cross-agglutinating antibody titer was found.Challenge test with live pathogenic bacteria resulted in high survival percent against the corresponding one but low percent to the other two strains.It was proved that the antigenicity showed difference among the bacterins prepared with these four strains.The results showed that the phagocytic activity and lysozyme activity in the serum of immunized group were significantly higher than the control group(t-test,p＜0.05).