The Construction Of B739₁343 Mutant Strain In Riemerella Anatipestifer CH-1 And Evaluation Of Protective Effect

Riemerella anatipestifer?R.anatipestifer?is the etiological agent of acute septicemia and infectious polyserositis of 1?8 weeks of ducks,chickens,goose,and other avian species.R.anatipestifer infection can give rise to high contagiosity and mortality between farm ducks and result in major economic losses in the poultry industry.Among clinically isolated strains,R.anatipestifer serotypes 1,2 and 10 are the most prevalent with high-level virulence in China.However,little is known about pathogenicity of this pathogen based on nutritional metabolism associated with iron acquisition although several virulence have been reported.Iron is essential element for all most organisms,and the ability of the pathogen to obtain iron from the host is a key determinant of virulence.Conversely,host innately limit the availability of free iron aganist invading pathogens termed nutritional immunity.In order to overcome host iron-withholding defenses,therefore most bacterial pathogens must have evolved highly sophisiticated systems to acquire iron for successfully sustaining infection.In the study,we first identified the function of B739₁343 gene correlated to iron uptake in R.anatipestifer,then evaluated if the mutant strain RA-CH-1?B739₁343 is able to be developed as a potential candidate of live attenuated vaccine.The main results as follows:1.The construction of RA-CH-1 B739₁343 mutant strain and the complementation strainGenome analysis showed that B739₁343 gene could be involved in iron uptake,and it is highly conserved among different R.anatipestifer serotypes.To identify the role of B739₁343 gene,we construct the mutant strain and the complementation strain.The first,we applified the upstream,downstream of B739₁343 gene and SpcR cassette,then the three fragments were ligated using overlap PCR.The result fragments were cloned into the plasmid pEX18GM,resulted in pEX18GM::B739₁343-USD.The second,the pEX18GM::B739₁343-USD were further introduced into CaCl2-competent E.coli strain S17-1 cells.The plasmid pEX1 8GM::B739₁343-USD was transferred to the recipient strain R.anatipestifer CH-1 through conjugation.The transconjugants were screened with blood agar plates supplemented with Kan and Spc.The gene deletion mutant strains were identified by mutiple-PCR.In addition,the B739₁343 gene of R.anatipestifer CH-1 was cloned into the shuttle plasmid pLMF03,then the resulting plasmid pLMF03::B739₁343 was introduced into RA-CH-1?B739₁343 mutant strain by conjugation to construct RA-CH-1?B739₁343 complementation strain.2.The functional identification of B739₁343 gene in RA-CH-1Growth curve in TSB liquid medium showed that B739₁343 disruption did not damage in the growth of R.anatipestifer CH-1.However,the addition of iron chelator Dip impaired the growth of RA-CH-1?B739₁343 more seriously than that of wild type.Complementation strain restored growth to WT growth levels.These data suggested that the B739₁343 plays a pivotal role in R.anatipestifer CH-1 iron acquisition under iron-starvation conditions.To further strengthen this suggestion,the bacteria were also tested for growth on the TSA plate with or without Dip.There was no any effect on the growth on TSA plate.However,it was observed that R.anatipestifer CH-1?B739₁343 pLMF03 was not able to grow on iron-limited TSA plate,and the complementation strain RA-CH-1AB739₁343 pLMF03::B739₁343 restored the growth.These results indicated that the B739₁343 gene of R.anatipestifer CH-1 was involved in ferric iron utilization.The transcription level of the B739₁343 in iron rich medium and iron depleted medium were measured by qRT-PCR,the results showed that the transcription of B739₁343 was not regulated by iron.3.The virulence evaluation of RA-CH-1?B739₁343 mutant strainThe B739₁343 was involved in iron utilization of RA-CH-1,it was hypothesized that the B739₁343 was also involved in the the virulence of R.anatipestifer strain CH-1.Firstly,LD50 were evaluated,the vilurence of the mutant strain CH-1?B739₁343 was attenuated by more than 104-fold,compared to its wild-type strain CH-1.Then,in a duck co-infection model,the mutant strain RA-CH-1?B739₁343 was out-competed by the wild-type strain CH-1 in the blood,livers and brains of infected ducks,indicating that expression of B739₁343 contributed to colonize in these tissues.It was indicated that the B739₁343 gene plays an important role in virulence of R.anatipestifer CH-1.4.Evaluation of RA-CH-1?B739₁343 mutant strain as attenuated vaccineSince the mutant strain RA-CH-1?B739₁343 was significantly attenuated in pathogenicity,the potential use of RA-CH-1?B739₁343 as a live attenuated vaccine was evaluated.Firstly,the results of evaluation of vaccine safety showed that mutant strain had no effect on the healthy of ducks.Next,we checked the serum antibody titers by ELISA,we found that RA-CH-1?B739₁343 vaccine enhanced specific humoral immune response in ducks.Finally,immunization with the mutant strain RA-CH-1?B739 1343 protected 83.33%ducks against challenge with the high dose wild-type stain RA-CH-1.It was suggested that the mutant strain RA-CH-1?B739-1343 could be used as a potential candidate of live attenuated vaccine.