The Functional Identification Of B739₁208 In Riemerella Anatipestifer CH-1 And The Virulence Evaluation Of The Gene Mutant Strain

Riemerella anatipestifer is a kind of gram negative bacteria, which cause seriously economic losses to the duck industryworldwilde. The bacteria is able to infect one-eight weeks of ducks, goose, chicken and other poultry, which is characterized by fibrinous pericarditis, perihepatitis. meningitis. This disease leads to a large number of ducklings death or slow growth. Iron is an essential nutrient element for almost all living organisms, including bacteria. It is an important cofactor for many proteins and enzymes, which is involved in the process of electron transfer, nucleic acid synthesis and redox reactions. In order to infect successfully, pathogen have evolved multiple mechanisms. Since iron acquisition systems of bacteria are related to the virulence, we constructed a mutant strain, and evaluated if it is able to as a potential attenuated vaccine.The main results as follows:1. The construction of RA-CH-1 B739 1208 mutant strainSequence analysis showed that B739₁208 was related to iron uptake. The homologues of B739₁208 are conserved in all RA strains. In order to identify the functions of the gene, we need to construct the mutant strain. Firstly, we designed specific primers and applified the upstream, downstream and anti-Spc-fragment of B739₁208 gene. Then these three sections were connected together by enzyme digestion method, and the connecting section was inserted into the suicide vector pEX18Gm. Secondly, the recombinant suicide plasmid was transformed into the E.coli S17-1. and the mutation in the chromosomal genome of RA was generated by transferring the suicide vector from E.coli S17-1 into the RA-CH-1 strain. Then, we screened positive clones by kanamycin and spectinomycin. Finally, the positive clones were identified by PCR. Growth curve showed that the mutant strain was slower in TSB medium than the wild type.2. The functional identification of B739₁208 in RA-CH-1In iron limited condition, the mutant strain RA-CH-1 AB739₁208 grew more slowly than wild type. It suggested that B7391208 was involved in iron uptake. In contrast, the mutant strain had more ability to uptake hemin than wild type. One possible explanation was that hemin uptake-relating genes were upregulated in the mutant strain, instead of wild type. Real-time PCR result showed that the transcription of B739₁208 had not significant change in iron limited condition, which suggested that the gene B739₁208 was not regulated by iron.3. The virulence evaluation of the RA-CH-1 B7391208 gene mutant strainWe used 109 CFU RA-CH-1 wild type and RA-CH-1 AB739₁208 to challenge 3-days ducklings respectively. Compared with the number of death in each group, it showed that the fatality rate of the mutant strain was reduced by 50%. Then, the result of LD50 of each group has been measured and showed that it increased more of decuple in the mutant group. It meant the virulence of mutant strain has declined by more than tenfold. Meanwhile, the result of the competition of wild type and mutant strain also has been tested and showed that the former had been weak significantly in vivo. Finally, the result of the number of bacteria in every organ after challenged 3-days ducklings by wild type and mutant strain has been calculated, and showed the number of the latter was much less than the former. In summary, these results shown that the mutant strain was attenuated significantly.