Screening Potential Virulent Genes Of Haemophilus Parasuis By SSH And The Establishment Of A Duplex PCR For Identification Of Potential Virulent Strains Of Haemophilus Parasuis

Haemophilus parasuis (HPS) can cause serous fibrinous pleurisy, peritonitis, pericarditis,arthritis and other inflammatory meningoencephalitis in sows Which is called Glasser’sdisease. This worldwide epidemic disease has been a major threat to the pig industry. Inthis study, HPS were isolated and serotyped from these suspected materials collected, inJiangxi Province between December2011to February2013. Representative strains wereselected for screening different genes through suppression subtractive hybridizationexperiment, and then investigated the distribution of these genes, finally, duplex PCR wasestablished for identification and detection in clinic. The specific content as follows.1、Isolation, identification and serotype of Haemophilus parasuis39strains were isolated from these suspected materials collected in Jiangxi Provincebetween December2011to February2013, and these strains were mainly isolated from thelung (51.28%), heart (30.77%), joint fluid (15.38%) and liver (2.56%). All strains wereserotyped by the traditional methods–Kielstein-Rapp-Gabrielson (KRG). The resultsrevealed that serovar5(25.64%), serovar13(20.51%), serovar4(10.26%), serovar12(5.13%) and serovar15(5.13%) were popular in Jiangxi Province.2、Screening of potential virulent genes of Haemophilus parasuisSuppression subtractive hybridization (SSH) was used to screen different genes betweenhigh virulence strain JX1108(serotype13) and non-virulent strain HS197(serotype7),virulent strain HPJX9(serotype12) and non-virulent strain HS81(serotype3).26differentgenes from JX1108, and28different genes from HPJX9were screened. These proteinsencoded by the different genes have high homology with restriction modification system,phage-related products, hypothetical proteins, metabolic proteins, secreted proteins,transport systems, structural protein and recombinase.3、Distribution of potential virulent genes of Haemophilus parasuisThrough the detection of part of different genes in15kinds of serotype reference strainsby PCR, we chosen these possible virulence genes and investigated their distribution in39strains isolated from jiangxi. The results showed that9genes showed virulent preference inthese15genes screened from JX1108, and the frequency of these genes occurred in thesestrains were between23.08-56.41%.11genes showed virulent preference in these17genes screened from HPJX9, and the frequency of these genes occurred in these strainswere between20.51-97.44%.4、The establishment of a duplex PCR for identification of potential virulent strains ofHaemophilus parasuis A duplex PCR was established for detection of16SrRNA and virulence-associated genesVtaA1of HPS, which can identify the potential virulent strains of HPS. It turned out thatfinally optimum concentration of primers of16SrRNA is0.2μmol/L and VtaA1is0.1μmol/L, optimal annealing temperature is61.4℃, and sensitivity of the duplex PCR is10-5ng/mL. Through the detection of39stains, by the duplex PCR, the results showed thatthe amplification of16SrRNA gene was all positive in39Jiangxi strains, and the ratio ofVtaA1gene positive strains was up to97.44%.