Research On Detection Of Haemophius Parasuis

Haemophilus parasuis (Hps) is the etiological agent of porcine Glsser's Disease, which can invade and cause severe systemic disease, characterized by fibrinous polyserositis, arthritis and meningitis. Hps is a kind of NAD-dependent, non-motion, Gram-negative, rod-shaped bacterium, which belongs to the family of Pastteurellaceae, the genus Haemophilus. The higher prevalence of Hps in intensive pig farms has become the main reason for the high mortality of young pigs and led to the huge economic losses. Isolation and identification of Hps is a prerequisite for disease prevention. However, no serotyping sera and antigens, the fundamental materials for Haemophilus parasuis research, could be commercially available in China at present. Therefore, the preparation of the above reagents and the establishment of the fast diagnostic method are very important.Firstly, two Hps strains were isolated from suspected specimens collected from Hubei, Hunan, Anhui and Fujian provinces. Phenotypic, biochemical and physiological studies indicated that the bacteria were related to the family Pasteurellaceae . 16S rRNA-based PCR confirmed the isolates and demonstrated that the bacteria are assigned to Hps.Secondly, for preparation of reference sera for serotyping, fifiteen serotypes of Haemophilus parasuis reference strains were cultured, harvested and inactivated prior to being emulsed with adjuvants for raising antibody in rabbits. The specificity,, titer and shelf life of the sera were measured by slide agglutination test and gel diffusion test. The consequences indicated that the sera possessed good specificity and have the retention for 1 month at 4℃ for, 6 months at -20℃and one year in freeze-dried status or -80℃. The reference sera were applied to verify 96 PCR-positive field strains which were proved to be serotype 4, 5 and 14. These results revealed that the prepared serum and relevant assay could be used for serotyping field isolates.Thirdly, for the purpose of rapid diagnosis of Hps infection, a pair of primers was designed to amplify a conserved domain of Hps transferring-binding protein (tbpB) gene and the reaction was optimized. The fragment with size of 288bp could be amplified in Hps serotype 1,3,4,5,6,7,8 ,11,12,13,15 reference strains wheras no amplified products could be detected in 11 serotypes of A. pleuropneumoniae reference strains, Pasteurella, E. coli and Salmonella.. The detection limit of the method was 25 CFU/mL.In conclusion, we have isolated and identified two strains of Hps, fifteen standard positive sera for serotyping were produced and 96 Hps isolates were serotyped. tbpB-based PCR was developed for Hps detection. These methods might be used for further Hps research.