The Isolation And Identification Of APP And PCR Detection For APXⅣ Gene

The research isolated a strain of pantomorphic Gram-negative coccobacillus from the lung and amygdale of a pig, which has serious typical I Porcine Contagious pleuropneumonea, by use the plate of ox brain heart infusion (BHI) bouillon and chocolate medium. The cultural characters, bionomics, agar diffusion reaction and biochemistry assessments indicate that it is a serotype I Actinobacillus pleuropneumoniae strain.The pathogenicity experiment indicated that this strain has a strong virulence to the mice. 4 hours after artificial infection, mice appeared weakly, erecting clothing hair and accelerated breathing; 36 hours after artificial infection, mice begin to die. LD₅₀ identify as 7.2 ×10⁸CFU.By dropping nose of young pig about the age of 30 days, the result showed that the strain caused swine to clinical symptom or die. Appearing the symptom of cough, sneeze and hard breath; Main pathological changes showed lung analosis, adhereing to transeptae, arcula cordis adhereing to membrana pleuralis and transeptae, arcula cordis and lung have different level of adherence.A pair of primer was designed according to the sequence of APXIVA gene in GenBank. PCR method for diagnosing APXIVA gene was established by screening the best reaction parameter. The PCR reaction was applied to detect the APXIVA gene, by use the DNA template from standard APP serotype 1, 3 and 7, and from type strains by our laboratory (Salmonella, Staphylococci, streptoc and Pasteurella). The results indicated that APXIVA can be detected in type strain and isolated strain, and a piece of idio-fragment was amplified about 400 bp. The idio-fragment couldn't be detected in negative control, streptoc, Pasteurella, Bacillus coli, staphylococci andSalmonella.DNA was exacted from blood, dung and tissue of swine, which inoculated with isolated strain of APP. APXIVA gene was detected by PCR method using the exacted DNA. 36 hours after artificial infection with actinobacillus pleuropneumoniae, APX IVA gene had been detected from blood by PCR, and the detection rate is 4/6 . 60 hours after artificial infection with actinobacillus pleuropneumoniae, APXIVA gene had been detected from blood of all pigs. APXIVA gene couldn't be detected from dung. The results indicate that the max detection rate of APXIVA gene appeared in lung(6/6) and amygdale(6/6). APXIVA gene could be detected in heart(5/6), heart-blood(5/6), trachea excretion(5/6) and lymphonodi mesenterici(3/6), and none was detected in kidney. There is no report about PCR method for detect APXIVA gene from tissue of swine. This research provided a new method for diagnosing actinobacillus pleuropneumoniae.