Studies On The Relationships With Phages And Acute Hepatopancreatic Necrosis Disease(AHPND)

With the rapid development of aquaculture industry, the aquaculture animal diseases appear constantly, causing great economic losses. In recent years, a new shrimp disease, acute hepatopancreatic necrosis disease(AHPND), occurred and become a troubled disease in shrimp farming industry. So far, we still do not understand the pathogenic mechanism of this disease. In 2013, Tran reported that they had separated the pathogen of AHPND. The pathogen was a kind of Vibrio parahemolyticus, as a result, the cause of AHPND had confirmed. In the process of exploring the pathogenic microorganisms causing AHPND, a phage that may associated with AHPND had caused attention to Flegel. However, they did not give us any subsequent related reports.In this article, we used five isolates of V.parahemolyticus, the five strains were all separated and identified by workers in our laboratory. We used the primers AP2 which was announced by Flegel and Lo. AP2 can isolate the strains that related to AHPND. After PCR detection, we confirmed that the 5 isolates we used in this article were all AHPND related strains.In this experiment, we used four different concentration of mytomycin C to test the sensitivity of the 5 isolates of V.parahemolyticus. After the sensitivity test, we made sure that the appropriate concentrate of mytomycin C that used to induce the V.parahemolyticus was 0.5μg/ml. After the inducing of mytomycin C(0.5μg/ml), we selected 2 isolates of V.parahemolyticus(No. 20130629002S01 and 20130726001S01) in which might have lysogenic phages. We made this conclusion mainly based on the growth curve of the 5 isolates of V.parahemolyticus, the trend of the curve showed that a losogenic phage changing into virulent.After super centrifuging of the lysogenic phage, we observed the phage particles by transmission electron microscopy. As a conclusion, the 2 isolates of V.parahemolyticus we mentioned above truly had lysogenic phage. Then we further viewed the two phage and they belong to different types of phages. Phage 1 may belong to myovirus while phage 2 belong to getah virus. The results of this article may remind that the phages had no relationship with AHPND.The artemia nauplii was used to conduct the pathogenicity of the 5 isolates of V.parahemolyticus. The result showed that the two isolates of V.parahemolyticus which had lysogenic phages had some degree of virulence. However, the most virulent isolates of V.parahemolyticus did not have lysogenic phages. Hence, we consider that the virulence of artemia nauplii had some relevance with the AHPND related plasmids while it had nothing to do with the lysogenic phages. As a result, the existence of phage can not add any virulence to the isolates of V.parahemolyticus. The Master of our laboratory,Wang Hailiang had separated the suspicious virulence protein gene vpp19 and constructd a specific test method to identify the AHPND.In addition, Sirikharin had separated the suspicious bacterial toxin and constructed a PCR detection method to AHPND. As a result, in combination with the other results of this article, we had made a conclusion that some of the isolates of V.parahemolyticus may have lysogenic phages, however, the lysogenic phages had nothing to do with the virulence of the isolates of V.parahemolyticus. So far, the reasons causing difference still do not clear.In this article, we tested the host range of the two lysogenic phages vp1 and vp2, the results showed that vp1 could infect one of the strains of V.parahemolyticus, phage vp2 could not infect any of the strains we used in this experiment.In this article, we used electric transformation method to transfer the sequence coding protein VPP19 to the SM10 competence cells. The positive strains were preserved. In this test, we found out that the positive strains with VPP19 protein sequence could be developed in the 2216 E culture solution. As a result, the positive strains with VPP19 sequence could be used to the artificial infection experiment in shimp. We used the arab sugar to induce the VPP19 protein in order to indentify where the protein existed in supernatant or precipitation. We found out that the protein VPP19 may exist both in the supernatant and the precipitation. The result lay a foundation in the further analysis of this protein.