Purification Of ApxⅠ,ApxⅡ,ApxⅢ Toxins From Porcine Actinobacillus Pleuropneumoniae And Pathogenicity And Immunogenicity In Mice

The contagious porcine pleuropneumonia is a respiratory tract infection in pigs caused by Actinobacillus pleuropneumoniae (APP). It can infect every stage of the pigs. The clinical symptoms of infected pigs breathe hardly. The pathological changes are that the lung has different degree of hyperaemia, haemorrhage and fibroid pneumonia. The incidence and mortality of the disease are usually above 20%. The occurance tendence of porcine contagious pleuropneumoniae in our country is rising now, and it has brought serious econimic damages to modern pig industry.The virulence factors of APP include Capsular Polysaccharide, lipopolysaccharide, outer membrane proteins, transferrin binding proteins and Haemolysins, etal. Hemolysin is the most important pathogenic factor, while research has shown that this hemolytic toxin is also an important factor in immunogenicity. In this experiment the toxins ApxI, ApxⅡ, ApxⅢwere purified. The pathogenicity and immunoprotection of the three toxin was also studied to provide theoretical basis for the development of effective multi-component subunit vaccine and for the effective prevention and control of porcine contagious pleuropneumonia. This study was also significant to protect and promote the healthy development of modern pig industry.The following two parts were included in the studies:Part I: Extraction and Purification of Actinobacillus pleuropneumoniae hemolysin. The Actinobacillus pleuropneumonia serotype 10,7,3 strains were cultured separately according to the characteristics that serotype 10 only secrets ApxI, serotype 7 only secrets ApxⅡand serotype 3 only secrets ApxⅢ. And the toxin ApxI, ApxⅡ, ApxⅢwere extracted and purified. The Actinobacillus pleuropneumoniae serotype 10,7,3 strains isolated from Shandong province had been cultivated in the LB medium(containing 0.02%NAD) , and optimum secretion conditions of toxin ApxI, ApxⅡ, ApxⅢwere optimized.The Actinobacillus pleuropneumoniae serotype 10, 7, 3 strains were cultured under optimal secretion conditions. The culture medium was centrifuged at 5000 r/min for 20 minutes at 4℃, and the supernatant fluid was taken. And ammonium sulfate fractionation, dialysis, concentration, and Sephadex G-200 gel filtration were used to purify APP toxin ApxI, ApxⅡ, ApxⅢfrom the supernatant fluid which culutured the Actinobacillus pleuropneumoniae serotype 10, 7, 3 strains. After purified, the toxin ApxI, ApxⅡ, ApxⅢwas analyzed by SDS-PAGE and Western blot. The results showed that the optimum secreted time of toxin ApxI, ApxⅡ, ApxⅢwas separately 12h, 12h and 24h and the optimum degree of saturation of ammonium sulfate was 60%.The result of SDS-PAGE of the purified toxins indicated that the molecular size was separately 105 kDa, 103kDa, 120kDa. In Western-blot, specific bands of the toxin ApxⅠ, ApxⅡ, ApxⅢappeared in the corresponding molecular size, indicating that the three toxins have good immunoreactivity.Part II: Study of the pathogenicity and immunoprotection on miceThe pathogenic and the immunoprotection of three kinds of purified toxin were studied separately. In order to verify the toxicity of ApxI, ApxⅡ, ApxⅢ, the half lethal dose of the purified toxin was determined in the pathogenic experiment, and four groups were done at the same time, control group, ApxI group, ApxⅡgroup and ApxⅢgroup. The mice of testing groups were injected with the dose of 400μg each, after 1d, 7d, 14d, they were killed by orbital bleeding to carry out blood test and blood biochemical indicators, changes in body weight were recorded in the whole range. The results of the half lethal dose of ApxI, ApxⅡ, ApxⅢwere 80.9 mg / kg, 108.6 mg / kg, 128.8 mg / kg respectively; ApxI affected the growth, blood and blood biochemical parameters of mice most, followed by ApxⅡ, ApxⅢ. In the immune protection experiments, the best immunization dose of ApxI, ApxⅡ, ApxⅢwas measured respectively, and the results were 40μg, 50μg, 80μg. According to the best immunization dose, four kinds of subunit vaccine (ApxⅠ+ ApxⅡ, ApxⅡ+ ApxⅢ, ApxⅠ+ ApxⅢ, ApxⅠ+ ApxⅡ+ ApxⅢ) were prepared, which were used to the immunization of mice at 30 days and 40 days. Serotype 1,3,5,7,10 of APP were used to attacks the mice when they were 60 days old. The average protection rates of serotype 1, 3, 5, 7, 10 were 87.5%, 80%, 84.1%, 97.5%, which show that the subunit vaccine contains 3 kinds of hemolysin was superior to the remaining three contain two kinds of hemolysin. The whole research shows that toxins ApxI, ApxⅡ, ApxⅢhave some immunoprotection function, which provides the basis theory for the development of new types of efficient pig pleuropneumonia vaccines.