Embryos Pathogen Detection And Bordetella Avium Outer Membrane Protein (OMPA) Expression And Immunogenicity

Bordetalla avium has been studied rather intimately now, which could transmission by horizontal and both, so during subculture in poultry, the disease continued to enlarge, cause more damage to poultry. 764 dead chick embryos were collected to investigate the major bacterial and viral factors that caused the death of chick embryos in Shandong. The major virus were detected by nucleic acid probes technology and ALV-p27 antigen detection kit, and the major bacteria were detected by traditional methods and the 23S rRNA gene sequence analysis. The multiple infection rates of bacteria and virus that statistics by this study. The multiple infection rates of Bordetella avium and the major virus occupyed a larger proportion, which were 23.56%.There was no specific vaccine, currently. To assess the evolution of Bordetella avium by 23S rRNA genes, the homologies between Bordetella avium strains LL and XT were 99.2%~99.7%. The nucleotide sequence homologies between LL and AM167904 were 77.1%, and amino acid sequence homologies were 83.2%. and the purified protein were used to immune 4 weeks old female BALB/c mice, which had provided a theoretical basis for the further study of Bordetella avium membrane protein subunit vaccine.Part One:The Multiple Infection of Embryonal Diseases in Large-scale Broiler Breeder Farms and Establishment of multiplex PCR detection for pathogens of chicken embryosIn order to investigate the major bacterial and viral factors that caused the death of chick embryos in Shandong of China, 17 to 19-day-old dead chick embryos were collected from three large-scale broiler breeder farms in Shandong. The methods of morphological characteristics, biochemistry identification, serological diagnosis and the 23S rRNA gene sequence analysis were used to identify different bacteria. Bordetella avium (23.56%), Escherichia coli (46.47%), Salmonella (56.28%), Pseudomonas aeruginosa (45.18%) and Staphylococcus (15.71%) were found to be the major bacterial factors to embryonic death. ALV (13.00%), REV (20.00%) and CIAV (23.00%) were detected by nucleic acid probes technology and ALV-p27 antigen detection kit. The multiple infection rates of bacteria and virus that statistics by this study have provided a theoretical basis for better prevention and treatment of embryoncal disease.Based on the gene sequences of OMPA in Bordetella avium, invA in Salmonella, phoA in Escherichia coli and toxR in Pseudomonas aeruginosa, four pairs of specific primers were designed and synthesized to amplify the special DNA sequences by multiplex PCR. The sensitive and specific multiplex PCR essay had provided an effective basis for rapid detection of pathogens.Part Two:Evolution analysis of 23SrRNA genes of Bordetella aviumAssess the evolution of Bordetella avium by 23S rRNA genes. The homologies between eight Bordetella avium strains were 99.2%~99.7%, were 92.4% ~92.5% with one strain in GenBank, and were 98.5% ~ 99.2% with rabbit Bordetella bronchiseptica strain and porcine Bordetella bronchiseptica strain. The Bordetella avium strains and Bordetella bronchiseptica strains isolates in domestic have a certain degree of genetic difference with foreign countries strains. 23S rRNA sequence analyses may be a reliable and rapid way for identifycation of Bordetella avium in diagnose of Bordetella avium.Part Three:Cloning and Expression of Outer Membrane Protein A (OMPA) from Bordetella aviumIn order to study the immunogenicity of OMPA protein of Bordetella avium LL, the fragment of OMPA was cloned onto the appropriate pGEX 4T-3 expression vector and transformed into Escherichia coli BL21 (DE3) competent cells, and the purified protein were used to immune 4 weeks old female BALB/c mice. Nucleotide sequence analysis of the OMPA protein gene of LL indicated that it encodes 199 amino acids which result in a protein with a molecular mass of 21,3178 Da. The sequence was submitted to NCBI, and the accession number was HM545298. There were different from the nucleotide sequenc of AM167904 which encodes 194 amino acids. The nucleotide sequence homologies between HM545298 and AM167904 were 77.1%, and amino acid sequence homologies were 83.2%. The antibody titer produced by the OMPA protein of HM545298 was 5.4, which could not play a role of immune protection, it was determined that a single dose of OMPA protein vaccine failed to elicit serum antibodies against the OMPA protein, which had provided a theoretical basis for the further study of Bordetella avium membrane protein subunit vaccine.