Construction And Charaterization Of Actinobacillus Pleuropneumoniae Quintet Gene Deletion Mutant Vaccine Strain

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, severe and often fatal respiratory disease of swine that occurs worldwide.Currently, vaccination is still the main measures for prevention and control of the disease. Therefore, there is a great prospects to obtain one safe, efficient, good cross-protective vaccine for Actinobacillus pleuropneumoniae. Animals are induced immune response when infected attenuated vaccine strain. So, attenuated vaccine can provide well cross-protection for heterologous serotype strain infection.1. Construction and characterization of arcA deleted mutant strain based on strain CH04A precisely defined quintet gene deletion mutant YWJ05(opxⅠC/apxⅡC/apxlV-orfl/cpxAR/arcA) of Actinobacillus pleuropneumoniae was constructed based on quartet gene detetion mutant CH04(apxⅠC/apxⅡC/apxⅣ-orf1/cpxAR) by transconjugation and counterselection to knock out arc A gene. When compare to the CH04, the tendency of the growth of the YWJ05is cutted down. Furthermore, in order to gain more information about the characterization of two mutant strains,the adherence and invasion assay were preformed by using PK-15in vitro, and the ability of antiphagocytic were tested in pulmonary alveolar macrophages assaay. In additionand, an PMN-mediated killing assay was implaied to analysis the difference between two mutant strains. Compared with CH04. all the results demonstrated that the YWJ05mutant strain had a decrease in the ability of reject killing2、Vir μ Lence in miceThe levels of virulence of two mutant strains were compared in an experimental infection in mice. BALB/c mice were immunized by intraperitoneal injection with200u L of either the mutant strains, and statistics the quantity of surviving mice of each group was recorde7days post-challenge, The data indicated that the virulence of the YWJ05was similar with the CH04.3. Protection in miceMice were immunized individually mutants, and PBS was used as negative control. In order to know about the ability of mutant lymphocytes proliferation, the MTT assay was carried out by the spleen form the seventh day after the secondly immunization. Compared with the CH04group, the YWJ05group have a significant decrease in the ratio of OD490nm, indicating that the YWJ05provoke more higher level of the immune response. Incubated the WT with the serum obtained from each immune group, and incubated with whole blood of the healthy mouse,we found that the killing rate of the YWJ05group have a significantly increase than that of the CH04group, it demonstrated that the YWJ05strain can induce more higher expression of antibodies. Serum samples harvested at day14and28following immune were detected by ApxIVA ELISA, the results suggested the YWJ05strain can elicit a higher level of ApxIVA antibodies against ApxIVA, which coincided with the result of Mouse whole-blood killing bacteria assay.Typing the antibodies of serum samples which harvest at day7following the second immune.the result demonstratd the level of IgGl had a significantly increase than that of the IgG2a in two mutant strains group, it indicated that the mutant strains induces a bias towards a Th2-type immune response. Which coincide with the result of the level of cytokine in the culture supernatants of splenocytes isolated from immunized mice. A significant increase was observed in the expression of IL-6、TNF-α, the level of IL-6was more higher than TNF-a in each mutant strain group, It indicated that the two mutant strains provocated the Humoral Immunity in the mouse.All immunized mice were challenged intraperitoneal injection with a lethal dose of3×107CFU(30LD50) at the28th day, the two mutant strains exhibited a survival rate of100%following challenge with the wild strain, even through there was an descent in the virulence after knock out the arcA gene, the rate of the protection still very high, in order to further confirmed this result, we made paraffin section of the lung gained at the7th day after the challenge, the result demonstrated that the YWJ05group had a slight lesion.