Isolation And Identification Of Actinobacillus Pleuropneumoniae And Development Of NqrA-ELISA

This study started with biological characteristics of bacteria, the bacteria was isolated from the lung of suspected symptom swine and identificated by Gram staining microscopic examination, Cultural character evaluation, Biochemical character evaluation, PCR, Serological type evaluation etc.. This bacteria is Gram-negative and coccobacillary form by Gram staining microscopic examination. It didn’t grow in the plain agar or MacConkey culture, but grew well in TSA (contained serum and NAD), its growing demands serum and NAD. The results of the bacteria’s biochemical reactions with urea, sucrose, xylose, maltose, raffinose, mannite, ONPG, nitrate reduction and glucose were positive, and CAMP test was positive. The result of PCR reaction was positive. The bacteria could react with Actinobcillus Pleuropneumoniae (APP)1standard positive serum but not with other types serum. In sum, the isolated bacteria is APP serotype1。Through the drug sensitivity test, the bacterial strain displayed drug resistance to Polymyxin B, Lincomycin, Vancocin and Aminoglycoside, and it had medium sensitivity to cephalo-type antibiotics which are often used in clinic and showed the sensitivity to Penicillins and Quinolones. The growth curve of bacteria indicated that the bacteria’s growth was on obvious logarithmic phase during2h-22h after inoculation, and became decreased after24h. The mouse pathogenicity test revealed the bacteria could cause the death during24h after inoculation.Referring to the APP NqrA gene on NCBI and the restriction site of this gene, a pair of primers was designed to amplify NqrA gene by PCR, which used the DNA of the isolated bacterial strain as template。The PCR products had been inserted in pMD19-T Simple Vector to sequencing identification. The sequence, which was correct by sequencing identification, was connected with pET32a Vector after enzymatic digestion to make up the recombinant plasmid pET32-NqrA. Introduced it to Colon bacillus.and induced the prokaryotic expression of Colon bacillus. By means of SDS-PAGE and Western Blot, determined expression of NqrA in Colon bacillus and was antigenicity.Collected and purified the expressed protein as the antigen to create the indirect ELISA. Optimize the conditions of ELIS A through the experiment, the result revealed the optimal antigen coated concentration was5.2μg/mL by this method, the serum dilution was1:160and the optimal dilution of ELISA secondary antibody IgG was1:10000. The tests of sensitivity, specificity and repeatability show that the created ELISA method has high sensitivity and its reactions were negative with Colon bacillus, Salmonella, Pasteurella, Swine fever, Porcine pseudorabies and PRRS positive serum, and positive with partial Haemophilus parasuis positive serum. The coefficient of variation for intra-repeatability test was4.20%-9.51%and the coefficient of variation for inter-repeatability test was6.06%11.79%. Applied the created ELISA method and indirect hemagglutination test (IHA), ApxIV-ELISA kit to detect120copies of clinical serum, the relevance ratio of positive one is46.6%by ELISA, the relevance ratio of positive one is40%by IHA, the relevance ratio is53.3%by ApxIV-ELISA kit. The above results reveal that the created ELISA method can provide a better reference to clinical detection。However, it has cross reaction with the part of Haemophilus parasuis positive serum, which requires further optimization of ELISA method and criterion.