Separation And Identification Of Pasteurella Multocida Serotype D From Swine Atrophic Rhinitis, Preparation And Characterization Of Monoclonal Antibody Against Pasteurella Multocida Serotype D

Porcine atrophic rhinitis(PAR) is one of the important disease of respiratory organ in pigs. The disease can lead to severe economic losses to the swine industy. Toxigenic Pasteurella multocida (DNT~+Tm) serotype D are pathogens of this disease. With the development of intensivism in China,its endangers swine industry increasingly. The key of the depurating PAR is to establish rapid diagnosis for it.In this study 80 strains of Pasteurella mutocida(Pm) from 226 swabs of weaned piglet, collected from the five scale pig farms in Guangxi province, were separated using the bacterial separation and culture method, and then classified using biochemistry testing. And 9 of 80 are toxigenic Pasteurella mutocida pathogen respectively, which further conformed by mouse lethality assay,guinea pig skin necrosis experiment and PCR,and 8 of 9 are DNT~+Pm serotype D,the other one is DNT~+Tm serotype A. The results of microbial sensitivity test showed that the 80 strains were insensitive to Streptomycin, Amoxycillin, Penicillin, Clindamycin, and were sensitivity to Amikacin, Ampicillin,Canamycin,Tobramycin,Gentamicin,Ciprofloxacin,Vibramycin,Sulfa methoxazoci.Then six hybridoma cell line designated as 1B11, IH7, 5A5, 5B4, 6C10 and 6C11, secreting monoclonal antibodies (MAbs) against DNT~+Tm serotype D were produced by fusing mouse myeloma cells(SP2/0) with spleen cells from Balb/c Immunized with DNT~+Tm serotype D and subsequently three-time limited dilution method. The titres of ascetic fluids of the 1B11,1H7 and 5A5 werel:25600,l:6400 and 1:3200 respectively when measured by indirect ELISA. The result of specific reaction showed that the 1B11, 1H7 and 5A5 recognized with the DNT~+Tm serotype D, while the e coli, salmonella typhimurium, streptococci, aureas staphylococcus showed negative reactions;the 6C11 showed cross reactions with WGX3 and e coli; and the 5B4 showed cross reactions with WGX1, WGX2, WGX3, WGX5 and salmonella typhimurium. Antigen binding epitopes analysis showed the 1H7and 1B11 reacted with the disparate antigen determinant. They will have potentiality application in ELISA detection against DNT+Pm serotype D.