Isolation, Identification Of Bordetella Bronchiseptica From Rabbits And Some Biological Characteristics Research Of These Strains

Bordetella bronchiseptica is an aetiological agent of potential infection and acute or chronic inflammation in respiratory tract of mammals. As for rabbits, it is characterized by rhinitis, sneezing, dyspnea and seromucous secretion flowing from nasal cavity. One group includes Bordetella bronchiseptica, B.pertussis and B.parapertussis, and other four species including B.avium belong to another group. They are members of gram-negative aerobic rods and cocci section of uncertain affiliation, Bordetella genus. With the development of rabbitry in our country, B.bronchiseptica infection and co-infections with Pasteurella multocida and Staphylococcus et.al are increased notably. It has been an important disease which caused significant mortality and morbidity in rabbits and led to vast economic losses in rabbit industry in China. In this study, B.bronchiseptica strains was isolated from rabbits and identified by microbiological assay; an experimental infection model of rabbit challenged by B.bronchiseptica isolates was established. In addition, the immune characteristics and adhesion properties between different phases of B.bronchiseptica were researched.1. Isolation, identification of B.bronchiseptica from rabbits: During the period from October 2005 to December 2006, B.bronchiseptica was isolated from nasal cavity of rabbits suffering from rhinitis in four experimental rabbit fields around Nanjing and a commericial field in Zibo Shandong province, and identified by microbiological assay. The prevalence of B. bronchiseptica infection in rabbits had been investigated and some biological characterictics of B. bronchiseptica had been studied. 56 B.bronchiseptica strains were isolated from 194 clinical samples. The isolation rate was from 17.87 % to 39.39 % and 28.86% on average. The results showed that B.bronchiseptica was an important pathogen which could cause rhinitis of rabbits and the rate of isolation was relatively high. It also revealed some seasonal relativities. These isolates had special characteristics of cultivation, morphology, biochemistry and antibiotics sensitivity. 2. The establishment of experimental infection model of rabbit challenged by B. bronchiseptica: The difference of virulence to ICR mice and the pathogenicity to weaned rabbits among 19 B.bronchiseptica isolates were researched. The pathogenicity to weaned rabbits challenged by B.bronchiseptica isolate BJL0504 (phaseâ… ) with different inoculating ways were evaluated and one isolate strain would be selected from three high pathogenic strains as a candidate vaccine strain. The results showed that 11 isolates were characterized as very high virulent, another 3 isolates were high virulent, still another 3 isolates were low virulent and 2 isolates were avirulent to ICR mice. B.bronchiseptica isolate BJL0504 showed very high virulent to ICR mice and rabbits, and BALM0503 was avirulent to ICR mice but it revealed high pathogenicity to rabbits. Other isolates showed variant pathogenicity to ICR mice and rabbits. The mortality of rabbits infected by intravenous injection and pulmonary inoculation was higher and the clinical symptoms and lesions were more severe than that of rabbits inoculated by nasal drip. B.bronchiseptica isolate BJL0504 remained high pathogenicity to rabbits after freeze-dry preserved for 15 months and the pathogenicity of the other two strains did not change, so BJL0504 isolate could be a candidate strain for vaccine.3. The protective immunity study on different phases of B.bronchiseptica in ICR mice: In order to study the immune responses and protection of different phases of B.bronchiseptica, the characterization of cultivation and pathogenicity between two different phases were researched, then the time of protective immunity presence and protective efficacy were evaluated in ICR mice inoculated with B.bronchiseptica phaseâ… and phaseâ…¢inactivated vaccine. The results of the dynamic test were positive in B.bronchiseptica F₁～F₆ and negative in B.bronchiseptica F₇～F₁₂, and other biochemical reaction remained the same. According to the hemolytic characteristic, B.bronchiseptica F₁ and B.bronchiseptica F₉ were grouped to phaseâ… and phaseâ…¢, respectively. The results of protective efficacy revealed that they could produce efficient protection in ICR mice challenged by different phases of B.bronchiseptica.4. Study on the adhesion characteristic of B.bronchiseptica to HEP-2 cell line: HEP-2 cell line was used as a model in vitro to compare the adhesion characteristic of B.bronchiseptica phaseâ… with phaseâ…¢and carry out adhesion dynamic test of B.bronchiseptica phase I. The results showed that B.bronchiseptica isolate BJL0504 phaseâ… could adhere to HEP-2 cell line in vitro, while phaseâ…¢could not. It was showed that pathogenicity was relative to their phase. The result of the dynamic test showed that the amounts of phase I of B.bronchiseptica adhered to HEP-2 cells increased while the time extended and reached the highest point after 165 min.To summarize, B.bronchiseptica was isolated and identified successfully and some important biological characteristics were studied. The B.bronchiseptica isolates with high pathogenicity were preserved successfully. Typical clinical cases were copied by challenging with B.bronchiseptica by different methods and the pathologic changes were studied. Different phases of B.bronchiseptica with high pathogenicity were used to study the immune potency and the adhesion to HEP-2 cell line. All of these observation has great significance for diagnose and prevention of the disease and the design of new vaccine.