Development Of The Triple Inactivated Oil Emulsion Vaccine Of Duck Tembusu Virus?Duck Escherichia Coli And Riemerella Anatipestifer

Duck Tembusu virus(DTMUV)? Escherichia coli(E.coli) and Riemerella anatipestifer(RA) are in the duck industry in our country in recent years is widely popular pathogens, they with all sorts of bacteria in clinic often take the form of mixed infection, supplies a series of economic caused serious losses. Cloth Sue virus disease in order to prevent the duck, colibacillosis and duck plague, this is the first study to use laboratory preserved duck temple Sue GDNS2010.1 virus strains and a duck were identified for O1 serotype strains sources identified and a strain e. coli serotype of duck plague creamer's coli was developed as an antigen temple Sue virus, duck duck source escherichia coli and duck plague creamer's coli sanlian inactivated vaccines. The concrete research content is as follows:1 The identification and analysis of Duck Tembusu GDNS2010.1 virus strain Escherichia coli(E.coli) and Riemerella anatipestifer(RA) Duck Tembusu GDNS2010.1 virus strain was cultured stably through the Muscovy duck embryos passage. The virus titer of allantoic fluid was 10-2.68 ELD50/0.2m L measured in accordance with the Reed-Muench method. Duck Escherichia coli was identified as Escherichia coli O1 strain through Mac Conkey agar plate culture ? Gram staining ?Biochemical tests?16sr RNA sequencing and Serotype test. Duck Escherichia coli grown in TSB 10 h, measured concentration of1.1×109CFU/m L, with OD600 value of 0.115. Riemerella anatipestifer(RA) was identified as RA-2 strain through TSA serum plate culture Gram staining?Biochemical tests?16sr RNA sequencing and Serotype test. The Minimal lethal dose of E.coli O1 strain and RA-2 strain were 0.2m L 1×107 CFU/m L?0.5m L ×107 FCU/m L, respectively.2 Development of the triple inactivated oil emulsion vaccine of Duck Tembusu virus ?Duck Escherichia coli and Riemerella anatipestifer Duck Tembusu virus ? Duck Escherichia coli and Riemerella anatipestifer were inactivated by 0.2% formaldehyde for 24 h in 37 ? thermostat, and then made into inactivated vaccine aqueous phase by 1:1: 1 mixed. The aqueous phase mixed with oil phase in the ratio of 2:3 emulsion into bivalent inactivated oil emulsion vaccine. After the physical properties and sterility tests, Vaccine safety tests and immunity protection test were carried out. The results confirmed that the vaccine was safe and reliable to test animals and there was a protective effect to duck egg production. The immunization program was explored in this study, there were three immune groups, the first group were only immunized at 10 days; the second group were immunized at 10 days, and immunized again at 21 days later; the third group were immunized at 10 days, and immunized again at 21 days later, and immunized at 100 days for the third time. ELISA and micro agglutination test were respectively detected for antibodies level of Tembusu virus and E.coli?RA. ELISA results showed that the first and second program from various periods of serum were negative. Tembusu virus antibody can be detected with ELISA in the third immunized after 21 days. Neutralizing antibodies showed Sheldrake which had second-immunization produced the peak antibody with titer of 1:70.79 at 21 days after the second immunization.The third-immunization produced the highest antibody titer of 1:77.62 at 14 days after the third immunization. And then the Neutralizing antibodies titer would decline. Until the third immunized after 60 days Tembusu virus antibody can be detected. Micro-agglutination test show Sheldrake which had immunized 30 days produced the highest average antibody titer of 6.76 log2. The second group of second-immunization produced the highest average antibody with titer of 8.33 log2, After then Sheldrake can maintain a higher level of E.coli antibodies. Sheldrake can produced the peek average antibody titer of 9.00 log2 at second-immunization 30 days later. Sheldrake can produced peek average antibody titer of 9.00 log2 at third-immunization 21 days later, and then Sheldrake can maintain a higher level of RA antibodies. The attacking result showed that the Sheldrake which had third-immunization could have 100% protection facing E.coli and RA attacked. The Sheldrake which had third-immunization egg production rate dropped from 80% to 60% after Duck Tembusu virus attacking, and then the egg production could back to the level before the attacking a week later.