| Haemophilus parasuis(HPs)is the pathogen of Glasser's disease,which can causes fibrinous polyserositis,arthritis and meningoencephalitis.The serotype is widely regarded as one of important virulent symbol in HPs.IscR is transcriptional regulation factor with special structure which can bind specific fraction of DNA to regulate gene expression.In this study,the prevalence of serovar in Zhejiang province was investigated,meanwhile,the relation between IscR and genes associated with capsular polysaccharide(CPS)was explored.Multiplex PCR was used to identify the serotype of HPs strains isolated from 113 farms during 2010-2015 in Zhejiang.Meanwhile,the presence of six potential virulence-associated genes(B14,D45,nhaC,fhuA,hhdA,hhdB)in these strains were detected,and then the correlation of the six genes with serotypes was analyzed.The results showed that serovar 5/12,4,13 and 14 were the major prevalent serotypes in Zhejiang and no isolate of serovar 3,9,11 and 15 was detected;the primary serotype prevalence during 2010-2015 did not change significantly;co-infection of two serotypes were detected in several farms.The results of potential virulent genes detection showed that the presence ratio of D45 gene in virulent strains(82.4-87.5%)was higher than that in the avirulent strains;the proportion of virulent strains(55%)harboring fhuA gene was lower than that of moderate virulent and avirulent strains(70.6-90%);the testing rate of the remaining four genes(B14,nhaC,hhdA,hhdB)was high in all strains,the results indicated that there was no clear correlation between the six potential virulence-assoiciated genes and serotypes.The expression of funA,wbgY,capD,wza,and funK genes in the capsule locus of a wild strain(290A1-WT)and the IscR mutant(290A1-?IscR)were determined by RT-PCR,and capsule of both strains was observed by transmission electron microscope when the two strains were cultured in TSB and interacted with the serum of swine.The results showed that the expression of wza was significantly higher in 290A1-?IscR than that in 290A1-WT(P<0.05)when the strains were cultured in TSB;capD expression was significantly higher in 290A1-?IscR than that in 290A1-WT(P<0.05)when OD600 was 0.2;the expression of other genes tested were relatively stable.The expression of capD,wza and funK were significantly higher in 290A1-?IscR than that in 290A1-WT at all tested time points during interaction with swine serum;funA expression was significantly lower after 10 min interaction with swine serum,while wbgY expression was significantly higher after 20 min interaction with swine serum in 290A1-?IscR than that in 290A1-WT.The results of transmission electron microscope showed that there was no capsule in both strains when they were cultured in TSB,and the capsule of 290A1-WT got 100 nm after interacting with serum,but it had no changes in 290A1-?IscR.This study provided new information on the expression and regulation of capsular polysaccharide biosynthesis-related genes in HPs.Electrophoretic Mobility Shift Assay(EMSA)was used to detect whether IscR can bind the promoters of capD and wza(p-capD and p-wza).Preparing polyclonal antibody of IscR to observe whether it can express in vitro by western blot.It indicated that IscR did not bind p-capD and p-wza directly,and using western blot can not detected the expression of IscR in vitro.In conclusion,clearly pointing out the prevalent serotype provided some reference of prevention and control of HPs in Zhejiang province,and exploration of influence on capsular polysaccharide biosynthesis-related genes with IscR provided new information on the expression and regulation of capsular polysaccharide in HPs. |
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