Establishment Of An Indirect ELISA For Detection Of Antibodies Against Haemophilus Parasuis And Screening Of In Vivo Induced Antigen

Haemophilus parasuis (HPS) has been paid widely attention as the pathogen of Gl?sser's disease which characterized by polyserositis, arthritis, dyspnea, high fever and high mortality rate. Gl?sser's disease has recently emerged as one of the typical bacterial diseases in swine industry in the worldwide. Although significant studies on HPS diagnosis, epidemiology and control have been recently been reported, the factors involved in the increased prevalence of HPS infections are unclear. More important areas, such as potential virulence factors, revised diagnosis, etc, need further study.In this study, an indirect ELISA assay based on the supernatant of sonicated HPS serovar 5 cells was established. Chessboard titration test showed that the optimal concentration of coating antigen wass80μg / mL, and incubated at 37℃for 2 h then overnight at 4℃. The optimal dilution of serum and IgG-HRP was 1︰320 and 1︰15000, respectively. The criteria evaluation was as follows: the serum was positive if S/N≥2.5, negative if S/N<2.5. Reproducibility and specificity tests confirmed that the developed assay had good stability and specificity. Compared with indirect haemagglutination (IHA), the indirect ELISA was more sensitive. So this assay can be used as a tool for diagnosis, quarantine of HPS.IVIAT was employed to identify protein gene expressed by HPS during pig infection in this study. Convalescent sera from pigs infected with HPS reference strain of serotype 5 were pooled, absorbed against in vitro-grown HPS serotype 5 and E.coli BL21(DE3)and used to probe a genomic expression library in E. coli BL21(DE3)constructed from HPS reference strain of serotype 5. 10 positive clones were identified after 2 round screening, and 12 ORFs were then found by sequencing positive clones. The proteins encoded by these ORFs were as follows: six proteins involved in metabolism and biosynthesis including 5-methyltetrahydropteroyltriglutamate -homocysteine S-methyltransferase(metE), DNA-binding protein, hypothetical protein excinuclease ABC subunit B, nitrate reductase, putative deoxyribonucleotide triphosphate pyrophosphatase and DNA polymerase III subunit beta; four transportproteins including transferring,putative Mg²⁺ and Co²⁺ transporter CorB, VtaA5,amidophosphoribosyltransferase;one regulatory protein of regulator for maltose metabolism; one signal transduction associated protein of putative extracellular serine protease. Analysis of these protein genes by RT-PCR demonstrated that metE and VtaA5 are expressed during pig infection, but not during in vitro growth. The results suggested that metE and VtaA5 may play important roles in HPS infection and be useful candidates for diagnosis and vanccine development.