Establishment Of Detection Methods By Bordetella Avium OMP With Labelled Antibody Technique And Detection Of Biological Characteristics Of Related Toxins

Bordetalla avium disease was a high-contacted infectious disease.In our country,it was discovered by Rui-liang Zhu in 1990.It could arouse that chicken-embryo death;low incubation;chicking acuting death and chicken opHthalmia so on.The infection rate of this disease was 10%～50%,it could bring up illness-chicken grew up lowly, the low redound upon of feedingstuff,and great economy loss.Recent years, the immunity of outer-membrane-protein of bacteria in cell-wall was paid attention to by science workers.the test testified that OMP with well immunity could stimulate not only humoral immunity and cell immunity. The study indicates that the labelled antibody technique used Bordetella avium OMP is rapid, reliable and sensitive and it has the potential for use as an fast diagnosis for Bordetella avium.The pathogenesis of Bordetella avium is related to its virulence factors. Presumably, these factors may play an important role in pathology and immunity. In order to solve problems mentioned above, Pure endotoxin,raw dermonecrotic toxin and fibrillae hemagglitinins of Bordetella avium were prepared and chicken embryo tracheal ring model was established for detecting biological characteristics of Bordetella avium.It preliminarity revealed relationship between Bordetella avium toxins and its pathogenicity.The research divides two parts:Part One: Preparation of High Titer Immuno Serum and Establishment of detection methods by Bordetella avium OMP with labelled antibody techniqueIn this study, out membrane proteins of B.avium strains P5 and P8 were prepared by improved Wooldridge method. We could determine contents of Bordetella avium OMP by Bradford, do SDS-PAGE electropHoresis of Bordetella avium OMP and prepare OMP immunity to immunity rabbits for preparation of high titer immuno serum. And then detections of indirect ELISA and indirect immunofluorescence were established by optimization and screening of conditions. The result showed that: the contents of Bordetella avium OMP was 320 ug/mL. The protein molecular weight of P5 and P8 strains is respectively 58,47,41,36,24KD, 58,47,41,38,21,16KD. The detection technique of labelled antibody was simple, rapid and strong specificity. It can be used to study on the epidemiology and serology of bordetella avium. This method laid a foundation of fast diagnosis for bordetella avium.Part Two: Extraction and detection of biological characteristics of Bordetella avium related toxinsPure endotoxin,raw dermonecrotic toxin and fibrillae hemagglitinins of Bordetella avium were prepared to used for animal test of rabbits,Balb/c mice,chicks,SPF chicken embryos,guinea pigs. Pathogenicity and biological characteristics of Bordetella avium were researched and explored by animal test. At the same time, chicken embryo tracheal ring model was established to verify the existence of tracheal cytotoxin and its pathogenic effects on tracheal cilia epithelium. The results showed that: endotoxin of Bordetella avium can effectively lead to acute septicemia lesions of animals. DNT caused little white rats,chicks death and muscle tissue damaged. FHA only agglutinated erythrocytes of guinea pigs, it had no agglutination to erythrocytes of rabbit and chicken. When Bordetella avium P5,P8 were diluted 10-8.5,10-8.3 times , abscission and injury of 50% of chicken embryo tracheal rings cilia could be caused by inoculating 0.1mL Bordetella avium.This result preliminarily verified the existence of tracheal cytotoxin and revealed pathogenic mechanism of Bordetella avium on tracheal cilia.The biological characteristics of these toxins played an important role for studing Bordetella avium vaccines.