| Actinobacillus(A.) pleuropneumoniae is a highly infectious porcine respiratory tract pathogen. The disease caused by this bacterium is encountered worldwide and frequently causes severe losses in infected herds.The first observations of this disease was made by Pattison et al.(1957), now occurs in most pig-keeping countries. Infected animals develop disease symptoms ranging from acute fibrinous pneumonia and pleuritis with high mortality to chronic lung lesions resulting in reduced growth rates. Pigs that survive an infection develop a protective immune response but can still be carriers of the pathogen. The rapid onset and severity of disease means that losses may occur before antibiotic therapy has time to become effective. Attempts to immunize against A. pleuropneumoniae disease have been hampered by the occurrence of two biotypes with at least 15 serotypes and the lack of reliable cross-protection between serotypes upon immunization with conventional formaldehyde inactivated bacterin vaccines. The construction of isogenic mutants, which cannot persist in the host over an extended period, may solve that problem by inducing the development of a cross-protective immune response. To date, commercial vaccines are based on whole inactivated bacteria or subunits, which include capsular polysacharide, lipopolysaccharide, and outer membrane proteins of various combinations of serotypes 1, 2, 5, 7 and 9 of A. pleuropneumoniae. Results obtained with these vaccines have offered, at least, homologous protection against the serotypes which were used to prepare the vaccine material.In contrast, natural and experimental infection can induce protection against any heterologous serotype. Pigs surviving a natural infection with A. pleuropneumoniae develop immunity to the organism and are protected from clinical symptoms in further infections with homologous and heterologous serotypes. However, the antigens that produce this solid protective immunity have not been clearly identified, and this crossprotection has not yet been achieved with vaccines produced commercially.Thus, live attenuated vaccines might be a viable approach to solve this problem. The methods described previously for the construction of mutant strains of A. pleuropneumoniae have some disadvantage. The study includes:1 bio-characteristics studies of APPAPP strain GD serotype 1 was isolated, the experiments of culture character, satellitism, CAMP test, sugar ferment test and urease test was accord of APP biology feature. Liquid culture medium additive were tested and the culture medium in which Actinobacillus pleuropneumoniae grew well was found out. Growth curve of APP serotype1 in TSB was determined by culture turbidity measurements, the stationary phase was begin at 24th hour. During the 1st and 24th 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. The pathogenicity experiment indicated that this strain had a strong virulence to mice. LD50 identify as 1.14×10~9 CFU.2 Initial establishment of PCR method for diagnosing apxⅣA geneA pair of primer was designed according to the sequence of apxⅣA gene in GenBank. PCR method for diagnosing apxⅣA gene was established by screening the best reaction parameter. The PCR reaction was applied to detect the apxⅣA gene, by use the DNA template from standard APP serotype 1 and 7, and from type strains by our laboratory(Salmonella, streptoc and Haemophilus parasuis). The results indicated that apxⅣA can be detected in type strain and isolated strain, and a piece of idio-fragment was amplified about 400 bp. The idio-fragment couldn't be detected in negative control, Salmonella, streptoc and Haemophilus parasuis.3 Construction of the apxⅡC gene deleted vector pBSCA(-) by OE-PCRThe apxⅡgene consist of the apxⅡC gene encoding posttranslational activator, and apxⅡA gene encoding the structural toxin. When the apxⅡC gene was inactivated, virulence of the strain was decreasing obviously, but the expression of Apx1TA still occurs in this strain. In our work, the complete DNA encoding ApxⅡprotein of APP strain GD serotype 1 was amplified by polymerise chain reaction(PCR). The DNA fragment was further cloned and the complete sequences were determined. Compared with the apxⅡCA gene of APP 1,2, 3 and 7 serotypes strain, it was found that there was over 99% as identity.In this study, The apxⅡC gene was knocked out by OE-PCR. The transfer vector pBSCA(-) was constructed by cloned the truncated apxⅡCA gene into plasmid pBS-T.4 Construction of the recombinant plasmid transfer vector PBSCA(-)OSA for the Mutants of APP In this study, the 157bp omlA-P gene from APP, the 1421bp sacB gene from Bacillus subtilis and the 1251 amp~r gene were amplified by PCR. The transfer vector pBS-OSA was constructed by cloned the three DNA fragments including apxⅡC gene into plasmid pBS-T. Then the recombinant plasmid transfer vector PBSCA(-)OSA was construct by ligated the-omlA-sacB-amp~r-cassette into the vector pBSCA(-). |
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