Characterization And Study Of Immunoprotection Against Mice Of Three Apx Toxins Of Actinobacillus Pleuropneumoniae

Actinobacillus (A.)pleuropneumoniae is a gram-negative rod that causes porcine pleuropneumonia, a highly infectious, often fatal disease of pigs leading to severe economic losses worldwide.Based on the characteristic of Actinobacillus pleuropneumoniae serotype 10 strain only secretes ApxⅠ, serotype 7 strain only secretes ApxⅡand serotype 3 strain secretes ApxⅢ. We cultivated serotype 7, serotype 10 and serotype 3 strains respectively as the routine method. The highly purified ApxⅠ, ApxⅡand ApxⅢwere gained with collecting supernatant of bacterium, method of salting out and gel filtration chromatography. In the basis of the above results, this experiment preliminary studied the biologic activity, physiological nature as well as immunogenicity of the three toxins. ApxⅠ, ApxⅡand ApxⅢwhich were emulsified with Freund's complete adiuvant as antigen to immunize Mus musculus albus for three times, every immune is two weeks interval. After every immune, antibodies were detected with method of ELISA. Groups of Mus musculus albus were challenged intraperitoneally with fatal dose of serotypel0, serotype7 and serotype 3 after two weeks when the third immunization was finished.Through method of polyacrylamide gel electrophoresis(SDS-PAGE), we found that the molecular weight of ApxⅠ, ApxⅡand ApxⅢwere 105kD, 103kD and 120kD respectively. We studied their biologic activity and physiological nature and the results showed the three toxins still had the hemolysis after heated at 60℃for 30min and 100℃for 10min respectively; ApxⅠ, ApxⅡand ApxⅢhad bioactivity with processing of 5％。formaldehyde for an hour and 1mg/ml protease K, but the bioactivity loss obviously; ApxⅠ, ApxⅡand ApxⅢhad the most biological activity among pH6～8. Experimental results indicated that ApxⅠhad the strong hematolysis and cytotoxicity, ApxⅡhas the moderately hematolysis, and the weak cytotoxicity, ApxⅢonly has the strong cytotoxieity, but non-hematolysis.Cross neutralization test and agar diffusion reaction were done with the purified three toxin proteins and immune serum of Actinobacillus pleuropneumoniae serotype 1～10. The result indicated that there were cross reaction among serotypel, 5 and 10; serotype3, 6 and 8; serotype 1, 4, 5 and 7 respectively. In the agar diffusion reaction where ApxⅠwas antigen, sediment line appeared between sample bores of serotype 1, 5 and 7 and antigen bores respectively; In the agar diffusion reaction where ApxⅡwas antigen, sediment line appeared between sample bores of serotype 1, 4, 5 and9 and antigen bores respectively; In the agar diffusion reaction where ApxⅢwas antigen, sediment line appeared between sample bores of serotype 4, 6 and 8 and antigen bores respectively; The above results indicated that APP had many antigen binding sites, and could generate Cross-testing between some serotype antisera and antigen easily. Groups of Mus musculus albus were immuned by the three toxins proteins, and they were challenged intraperitoneally with homologization strains after the third immune. These preliminary results showed the ApxⅠ, ApxⅡand ApxⅢhad good protection against homologous APP, whose immune protection rates were 80％(8/10), 70％(7/10) and 80％(8/10) respectively, it builted a good foundation for the further research of high efficacy vaccine against Actinobacillus pleuropneumoniae.