Development And Preliminary Application Of Enzyme-Linked Immunosorbent Assay For Porcine Contagious Pleuropneumonia

Actinobacillus pleuropneumoniae ( APP ) is the etiological agent of porcine contagious pleuropneumonia, a severe respiratory contagious disease effecting swine industry worldwide. The clinical signs vary from acute to chronic, and asymptomatic carriers also occur frequently. Acute pleuropneumonia is characterized by necrotic and hemorrhagic lung lesions with fibrinous pleuritis and high mortality. Chronically infected pigs have less obvious clinical signs but show reduced growth rates. The surviving pigs can remain as carriers in the herd and cause a new outbreak when immunity is weakened.Fifteen serotypes of APP have been recognized so far. There is no or weak cross reaction in different serotypes, which brings many difficulties to diagnosis and prevention of the disease. The conventional serological tests, such as Complement Fixation Test (CFT), Indirect Hemagglntination Test (IHA ), only test the antibodies induced by the antigens in the bacterial surface and diagnose Pleuropneumonia caused by homologous serotypes. The same problem exists in prevention by vaccine inoculation. The killed vaccine of APP can induce certain protective immunity, only to homologous serovar, not to heterologous serovar. The goal of the study is to develop a sensitive, species-specific diagnostic method able to detect all serotypes.The expression plasmid pET-ApxII were tansformed into E.coli BL21(DE3).Clones of higher expression level were selected, and grown in the presence of IPTG at 37 C to induce expression. The conditions of expression were optimized and the protein was purified form inclusion bodies, denatured and renatured. Using the renatured protein as antigen an indirect enzyme-linked immunosorbent assay(ELISA) was developed to detect antibodies to Actinobacillus pleuropneumoniae. The comparison between this method and IHA showed that the ELISA had good speciality, sensitivity and reliability for clinic serum samples. The average positive rate of Porcine Contagious pleuropneumonia is above 63% using the Apx II -ELISA method to detect 2503 clinic serum samples.The 2726 bp gene encoding subunit A of transferring- binding protein ( TbpA ) of Actinobacillus pleuropneumoniae serotype 2 was amplified by PCR and cloned into plamid pET-28b. The positive clones were confirmed by enzyme digestion and sequencing analysis, and transformed to E.coli BL21 cells. Protein expression form the clones was analyzed by SDS-PAGE and Western blot. After purification, the protein was applied as antigen to develop indirect ELISA. The comparison between IHA and theTbp-ELISA was performed by simultaneously detecting 117 clinical samples. The experimant demonstrated the diagnostic potential of the transferring-binding protein (Tbp) for actino bacillus pleuropneumoniae.