Isolation, Identification And Biological Characterization Of Riemerella Anatipestifer In Parts Of Anhui Province

Riemerella anatipestifer(RA) infection is a acute or chronic contagious disease of domestic young ducks,turkeys,and various other domestic and wild birds.It accounts for significant economic losses due to high morbidity and mortality,known as is a major disease confronting the duck industry through the world.To date,21 serotypes have been reported,little cross reaction was found between different serotypes.In addition,resistant strains appear with the application of antibiotics The iterative antibiotics treatment is responsible for the occurrence of resistant strains in infected ducks.So,it is becoming more and more urgent to diagnose the early RA infection,predict the potential RA serotype change,and seek for effective vaccine in order to control and prevent the RA infectious disease.In the present study,a pair of primer was designed and synthesized based on 16S rRNA gene sequence from RA strain.PCR was then performed with these primers in the assay of lesion samples from the suspended infection.It demonstrated the PCR amplification is RA specific with 20pg of the minimum DNA amount for detection.The optimal experiment showed that the brain tissue is the preferential tissue in the assay. Notably,the PCR developed in this study is highly specific,much sensitive,and practically useful in monitoring the RA infectious disease.Out of 108 tissue samples of infected ducks collected in 6 different regions including Chuzhou,Liuan,Anqing,Hefei,Chaohu and Bengbu in Anhui Province,26 of freshly isolated strains were gotten with 24.07%of the positive percentage.Among them,strains isolated from Chuzhou accounts for 57.69%(15/26).Our results showed that the RA infection is commonly distributed in Anhui area,and Chuzhou is one of regions it happens seriously.20 strains were further identified as serotype 1 which stands for the predominant serotype with 76.92%.The other 6 isolated strains have not been yet identified as certain type.These results demonstrated that there are at least two serotypes being prevalent in Anhui province.Considering the previous reporter that serotype 1 was the unique type in 2000,we speculate that RA infection appears in a trend of diversity.We next determined the nucleotide sequences of the 16S rRNA and cam genes in the isolates,and compared them with the known data in GenBank.Our results showed that the sequences of the novel strains in our study for the 16S rRNA are completely identical with the previously described sequences.We come to the conclusion that 16S rRNA gene has no obvious variation according to the geographic distribution,serotypes,as well as within the same type but different strains.Likewise,cam gene doesn't show any significant variation among its different serotypes,or within the same serotype but different strains.Specifically, cam gene has no variation among differently virulent strain.In other works,cam gene could tell the discrepancy of virulence between strains.The virulences of eight isolated strains were determined by animal regression test. Our data show that CZ9 and AQ2 fall into avirulent strains,CZ1,AQ1 and LA4 were mild strains;CZ2,CZ12 and HF1 were virulent strains.Ducks immunized with CZ9 could resist the challenging of CZ12 and HF1 with a survival percentage of 60%and 80%,respectively. CZ9 strain features with stronger immunogenicity than AQ2.Therefore,CZ9 can be chosen as candidate for attenuated vaccine.With it for vaccination,the optimal dose for the induction of the effective protection to recipient duck was determined to be 8.0×108CFU/ 0.1mL.All these preliminary data supplied a solid foundation for further investigation of RA and the relative infectious disease.