| Riemerella anatipestifer infection,also known as infectious serositis,is a contagious disease of domestic ducks,geese,turkeys,and various other domestic and wild birds.Infected ducks will cause slow growth,weight loss,reduced feed conversion,meat quality decline,and high mortality and other symptoms,in the breeding and treatment of diseases such as duck industry to our country brought huge economic losses.There are at least 21 serotypes of Riemerella anatipestifer that have been identified,and full genome sequence of nine strains have been released in GenBank.There are more thantwo thousand genes in Riemerella anatipestifer,but only a few genes are known.In China,the main methods to control Riemerella anatipestifer infection include vaccination and antimicrobial drugs.In previous study,our results showed that most of R.anatipestifer strains were resistant to kanamycin.In this study,we want to find out the gene(s)involved in kanamycin resistance using random transposon mutagenesis.Test one:Two pairs of primers were designed according to the sequence of dnaB gene and 16 S rRNA of Riemerella anatipestifer CH3 strain.The optimized reaction temperature was 60 ?.The results showed that two specific bands(364bp and 627 bp respectively)were amplified from all the detected R.anatipestifer strains with different serotypes by the duplex PCR,while no products was detected from other bacterial species,such as avian pathogenic Escherichia coli,avian Pasteurellamultocida,Salmonella enteritidis and Flavobacterium johnsoniae.The sensitivity tests showed that the established duplex PCR could detect17.33ng/mL of Hxb2 bacterial genomic DNA,or 2.9×102CFU/mL of HXb2 bacteria.In addition,R.anatipestifer from the liver and brain tissues of infected ducks were detected bythe duplex PCR,the results showed that,using DNAs from liver and brain tissues,the coincidence of duplex PCR with bacterial isolation was 100%(9/9)and 77.8%(7/9)respectively.It suggested that the duplex PCR could be used todetected R.anatipestiferfrom liver samples.In conclusion,the duplex PCR we established could not only identify isolated R.anatipestifer strains rapidly and effectively,but also detect them from clinical tissue samples.Test two:Sensitivity to kanamycin in Riemerella anatipestifer field strains.17 suspected R.anatipestifer isolates from Guizhou University were selected to identify their species,determine the serotypies and do drug sensitivity test.The results showed that the strains belonged to R.anatipestifer serotype 1,2,5 and 15 respectively,and all these strains were resistant to Kanamycin.In addition,the commonly used R.anatipestifer strains in our laboratory,such as CH3,HXb2,Yb2 and WJ4,were also resistant to Kanamycin.Test three :The plasmid pEP4351 was transformed into Escherichia coli BW19851 to construct BW19851(pEP4351)as a donor strain,and the plasmid pEP4351 was introduced into Yb2 by using bi-parental mating method..The minimum inhibitory concentration(MIC)of erythromycin and polymyxin for RA and BW19851 was determined by the broth dilution method.Since erythromycin resistance gene on Tn4351 and strain Yb2 was resistant to polymyxin,the results of MIC showed that TSA agar containing 0.3?g/mL erythromycin plus125?g/mL polymyxin was suitable to screen positive tansconjugants.The mutant strains were further screened by kanamycin resistance test,and the mutant strains with reduced kanamycin resistance were tested.The minimum inhibitory concentration of the mutant was tested to determine the mutant whether the mutant has lost resistance to kanamycin or is significantly less resistant.The mutant strain DK-2,which was significantly attenuated by kanamycin,was selected and the insertion site of transposon was amplified by genome-step method.The insertion site of the transposon was determined by genomic walking method in the L27 subunit of ribosomal 50 S.For constructing the complemented strain for DK-2,the 50 S L27 gene ORF of wild strain was amplified from wild type Yb2 by PCR.The 50 S L27 ORF was ligated into Escherichia coli-Riemerella anatipestifer pRES to generate the complemented plasmid pRES-50S-L27.Then the complemented plasmid was introduced into the mutant DK-2 by mating,and the complemented strain cDK-2 was obtained by erythromycin and polymyxin resistance screening and PCR identification.The growth curves and susceptibility testing of wild type Yb2,DK-2 and cDK-2 were determined.The results showed that there is no significantlydifference on the growth curves of wild type Yb2,DK-2 and cDK-2,and the MIC of DK-2was about 8 fold lower than that of Yb2 and DK-2.The kanamycin susceptibility tablets of the complemented strain cDK-2 and the wild type Yb2 had a diameter of 0 and were tolerant to kana,while the kanamycin susceptibility of the mutant DK-2 was 10 mm in diameter.In addition,drug sensitivity tests showed that the sensitivity of mutant DK-2 to Neomycin and rifampicin was increased compared with that of wild-type Yb2 and cDK-2.In conclusion,in this study,our results showed that 50 S L27 was involved in kanamycin resistance in R.anatipestifer Yb2 by constructing random tansposon mutagenesis library.This study lays the foundation for further study on the function of 50 S ribosomes and 50 S L27 subunits of Riemerella anatipestifer. |
PDF Full Text Request