| Summary: | 碩士 === 國立中興大學 === 獸醫病理生物學研究所 === 99 === Actinobacillus pleuropneumoniae (APP) is an important causative agent of porcine pleuropneumonia. APP causes acute, hemorrhagic, fibrinous and necrotic pleuropneu- monia in pigs and results in great economic loss in the swine industry. APP exotoxins (Apx) are important virulence factors. There are four types of Apx toxins (ApxI~IV). Among these, ApxIV is APP-specific and produces only in vivo by all serotypes. The aims of this study were to produce and characterize the recombinant ApxIV (rApxIV) proteins, and to develop a rApxIV-based ELISA for detection of APP infection in pigs.
The rApxIV-1, -2, -3 and -4 were expressed in E. coli. Rabbits were immunized with purified rApxIV-1, -2, -3 or -4 and antisera collected for characterization. The results of Western blot analysis showed that rabbit anti-rApxIV-1 or -2 serum had no cross-reactivity toward native ApxI, II and III, and rApxIV-1 did not react with sera specific to rApxI, II and III. These results indicated that rApxIV-1 had the highest specificity which was then used as antigen in the development of an ELISA.
To test whether rApxIV-ELISA was able to detect APP-infected pigs, 7 weeks old pigs were intratracheally challenged with different doses of APP serotype 1 and sera were collected weekly until 6 weeks post challenge. Antibodies to ApxIV in sera were evaluated using ELISA and Western blot analysis. The results of ELISA showed that the antibody titer increased at 1 week after challenge in both high- and low-dose challenged pigs, and the magnitude of antibody elevation seemed to correlated to the challenge dose. Western blot results did not show apparent trend of antibody increase after challenge. ELISA seemed to have higher sensitivity than Western blot analysis. Further, 48 sera of SPF piglets were tested with rApxIV-ELISA to determine the baseline of antibody titer. In addition, sera of 15 pigs in a commercial farm were collected every 3 to 4 weeks from 7 till 29 weeks of age, and analyzed with rApxIV-ELISA. It was found that the sera of pigs at 8 weeks of age had the lowest antibody titers, which increased gradually and remaintained high till 29 weeks of age. It indicated that the farm may have subclinical APP infection.
To evaluate the rApxIV-ELISA on detection of infection of different APP serotypes, ICR mice were intraperitoneally challenged with APP serotype 1 or 2 and anti-ApxIV antibody monitored. It was found that the antibody titer increased at 1 week after challenge, and maintaind until 4 weeks after challenge, but antibody titers of APP serotype 2 challenged mice remained low throughout the experiment.
In conclusion, both rApxIV-1 protein and anti rApxIV-1 antisera have highest specificity. rApxIV-1 based ELISA detects APP serotype 1 infection in pigs and mice. The application of rApxIV-ELISA on detection of different APP serotypes infection requires further investigations.
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