Establishment And Application Of SCOTS Technique In Screening Funcational Genes Of Haemophilus Parasuis

Haemophilus parasuis, a non-motile, small, pleomorphic rod-shaped bacillus in the family Pasteurellaceae, is a common commensal bacterium of the upper respiratory tract of conventional pigs. However, H. parasuis can also invade and cause severe systemic disease (Gl(a|¨)sser's disease) characterized by fibrinous polyserositis, polyarthritis and meningitis, which has received more attention in the past decade due to the increasing economic losses in the pig industry worldwide, especially in the complicated disease of porcine high heat disease in China in last year (2007). Until now, there are 15 serotypes have been described and about 20% strains are nontypable. serotype 4, 5, 13 are most prevalent. The main virulence factors are CPS, LPS, OMP, TBP, neuraminidase. New virulence fsctors will be determined based on the development of new technology.1. Estabilishment of selective capture of transcribed sequences (SCOTS) techniqueSelective capture of transcribed sequences (SCOTS) was originally described by Graham and Clark-Curtiss in 1999 for the identification of genes expressed by Mycobacterium tuberculosis upon growth in macrophages. The technique was subsequently used for the capturing the bacterial genes expressed in tissues of infected animals.In this study, we aimed to employ SCOTS to identify genes differentially expressed by Haemophilus parasuis in necrotic lung tissue at the end of the acute stage of infection (7 days postinfection). Thirty days healthy piglets were infected with Haemophilus parasuis 0165 strain and we extracted the RNA of lung and bacteria cultured under normal growth conditions. cDNA samples obtained from the necrotic lung in infectious pigs took six rounds of SCOTS before dot blot hybridization. Then we established a reverse dot blot(RDB) method for screening the differentially-expressed genes of Haemophilus parasuis in vivo and in vitro from the selective capture of transcribed sequences (SCOTS) by optimizing the hybridization conditions including the kinds and concentration of target DNA, probe concentration, and UV irradiation time. The results showed that the optimal conditions of Reverse dot blot (RDB) included that the concentration of probe was 50 nanogram per milliliter, the energy of ultraviolet light was 0.15 joule per square centimeter, the kind of target DNA was PCR product, the products of PCR was 1 microgram.2. The application of SCOTS for screening the genes response to heat stress in Haemophilus parasuisThe aim of the study reported here was identification of differential expression of H. parasuis genes in response to heat stress by SCOTS. Forty potential virulence factors were identified. Results from this study demonstrate the suitability of SCOTS for the elucidation of gene expression in H. parasuis and may contribute to a better understanding of the pathogenesis of H., parasuis infections.Until now, compared too many Gram-negative bacteria there is little information available on the mechanisms of pathogenicity or gene expression of H. parasuis in vivo. With the development of new technologies, new virulence factors will been identified. In this study, we installed and optimized two technologies SCOTS and RDB to screen the differential expression genes of H. parasuis. The up-regulated genes responsed to heat stress were obtained by these methods, and the next step of research will study the gene to supply the biological evidences for prevention and cure the Glasser's disease caused by H. parasuis.