The Research Of Genetic Toxoid Bacterin Against Porcine Contagious Pleuropneumonia

Porcine contagious pleuropneumonia (PCP) caused by Actinobacillus pleuropneumoniae (APP) is a severe, contagious pulmonary disease of pigs that cause important economic losses in industrialized pig production worldwide. This respiratory disease can be either characterized by acute, extensive, and respiratory hemorrhagic or chronic, localized lesion associated with pleuritis. The first observation of this disease was made by Pattison et al. (1957), and currently this disease occurs in most pig-keeping countries. Immunization is the major method to prevent and control the disease. The commercial vaccines to prevent PCP in China is inactivated bacterin vaccine. The bacterin vaccine can only reduce mortality, not morbidity and chronic infection against same serotype, little cross-protection against heterologous challenge. Therefore, the development of safer and more effective vaccine against porcine pleuropneumonia has been required to prevent the disease and diminish the economic losses. The study to the virulence factor of the APP show that the exotoxins (Apx toxins) secreted by APP played an important role in the pathogenesis of porcine pleuropneumonia and were important protective antigens equally, the vaccine with exotoxin toxoid improved protection markedly. In order to improve bacterin effectiveness, a new genetic toxoid bacterin was explored.1. The growth curve determination of APP serotype 7Three different media which were usually used to culture APP were compared and Tryptic Soy Broth (TSB) was selected to culture APP in our laboratory. Growth cycle of APP serotype 7 in TSB was determined by culture turbidity measurements and the number of viable cells, the seventh and tenth hour was stationary phase, and mean generation time was about 30min. During the first and fourth hour of incubation, culture turbidity was proportional to the number of viable cells and the bacteria of this phase could be used as challenge inocula.2. Cloning and expression of structural gene of Apx IAccording to the structural characteristic of apx I, the prokaryotic expression plasmids pET- I CA and pET- I A were constructed, which were used to express Apx I . After induction by IPTG, a high expression of fusion protein was obtained. SDS-PAGE analysis and Western blot showed that the fusion protein was 110 kDa and the protein was specific to antisera against APP. Apx I expressed by pET- I CA possessed haemolytic characteristic.3. Immunocompetence study of the expressed protein of Apx IThe expressed protein was purified and refolded, then emulsified with Freund's incomplete adjuvant in equal volumes to get vaccine, then BALB/c mice were immunized. Native Apx I secreted by APP serotype 10 and un-refolding expressed Apx I were used to immunize mice at the same time. Immunized mice were challenged with lOXLDso of serotype 10. Results showed both refolding and un-refolding inclusion body possessed good immunocompetence and could substitute native Apx as supplements of the bacterin.4. Protective Efficacy of the Genetic toxoid Bacterin against APP in MiceThe recombinant protein of Apx I , Apx II and ApxIII was purified and mixed with APP serovar 7, which is prevalent in China, then emulsified with Freund's incomplete adjuvant in equal volumes to get subunit bacterin. Groups of BALB/c mice were immunized and challenged intraperitoneally with 5 X LDso of serovar 1 and serovar 7. Antibodies were detected with ELISA. After second immunization, antibody level increased obviously and the immune protection rate against serovar 1 and serovar 7 reached 83.3%(10/12) and 91.7% (11/12) respectively. These preliminary results showed the toxoid bacterin had good protection against homologous and heterologous APP, which built a good foundation for the further research of high efficacy vaccine against porcine pleuropneumonia.5. Protective efficacy of the genetic toxoid bacterin against APP in pigletsThe efficacy of a new genetic toxoid bacterin containing recombinant Apx toxins and serovar 7 of APP was determined. Weaned piglets w...