Isolation, Identification Of Bordetella Bronchiseptica From Swine And Biological Characteristic Research Of Inactivated Vaccine Strain

Bordetella bronchiseptica (Bb) is an important pathogenic bacterium and results in acute and chronic respiration system disease. Bb is found to infect numerous mammals, such as swine, canine, rabbit, guinea, pig ferret, koala bear, et ah Human beings are also infected by Bb, especially children and immunocompromised individuals. Furthermore, it can cooperate with other pathogenic germs. The interaction increases incidence and severity of respiratory system disease.The pathogencity of B. bronchiseptica is associated with colony phase. Phase I has heat-labile capsule, short flagella and dermonecrotic toxin (DNT). Compared with phase II and phase III, it is the highest virulent strain. As a main pathogenic factor, DNT, which is encoded by dnt gene, plays a key role in pathogenesis. Although the toxin is a exotoxin, it remains in the cytoplasmic compartment of bacterial cells and releases after bacterium lysis.This research focused on Bb and DNT. The results were summarized as follows:1. A PCR was used to detect B. bronchiseptica, combined with microscope observation and biochemical reaction, 31 strains of Bb phase I were isolated from clinic samples. Among those, 30 strains were isolated from lung and 1 strain was isolated from nasal swabs. Every isolate was studied on antibiotics sensitivity, hemagglutination test and dermonecrosis assay in the infant mouse.2. Based on the nucleotide sequences of dnt gene published in GenBank, three pairs of primers were designed to partly amplify the dnt gene of Bb. The subsections were ligated to holotoxin dnt gene by digestion. The complete gene was cloned into pET-28a, and the constructed recombinant plasmid pET-dnt was sequenced. The sequence was 99.6% homolous to other three published dnt sequences in GenBank. Then the plasmid was transformed into E.coli BL21. A 140 kDa recombinant holotoxin, named rDNT, was expressed. Dermonecrosis assay indicated that the protein had biological activity.3. Tryptic Soy Broth with serum (TSB+S) was selected to culture Bb in liquid medium. Growth curve of Bb in TSB+S was determined by culture turbidity measurements and the number of viable cells. The hours from eighth to thirty-second were logarithmic phase, and latter hours were stationary phase. During the logarithmic phase, culture turbidity was proportional to the number of viable cells.4. ZK1217,JS3928,QH0814 and HH0809 were used to compare the pathogencity by mice . The results showed Bb had spleen atrophy toxicity. HH0809 from nasal swabs had the highest pathogenicity, the LD50 was 4.4×10~5 CFU/mL. It could be used as...