| Rabbit Pasteurellosis caused by Pasteurella multocida is one of the most significant bacterial diseases of rabbits and causes considerable economic losses in large production units throughout the world. The disease is characterised by various clinical symptoms, including respiratory distress, genital infections, abscesses, otitis, and septicaemia, but infection by P. multocida can also appear without manifesting any clinical signs. Pathogenic serotypes of the disease are complex and the immune effect between different serotypes is not satisfactory, and popular serotypes are different in different areas, which brings greater difficulty in prevention and treatment work of Rabbit Pasteurellosis. In this study, in order to investigate the rabbit pasteurellosis epidemiology in western Henan and provide a scientific measures for effective prevention and treatment, the auther had done some research. The contents and results are as followes:1. Isolation and Identification of Rabbit Pasteurella MultocidaIn order to find out the rabbit pasteurellosis epidemiology in western Henan and to provide a scientific measures for effective prevention and treatment, the rabbit suspected Pasteurella multocida infection from western Henan were anatomy-checked and the Pasteurella multocida were isolated from the year2003to2005.18strains were identified and were numbered as P1, P2……P18, which the P1, P2……P11from the meat rabbit, P12, P13……P18from the Rexyabbit. According to the fluorescence that they showed on serum agar plate, The18isolates were classfied into three categories:Fo-fluorescence (10strains); Fg fluorescence(5strains) and medium type(3strains). Susceptibility testing showed more than half of strains were resistant to common antibiotics norfloxacin, gentamicin, furoxone; and only a few strains were resistantto doxycycline and ciprofloxacin; and mostly of the18strains were moderately sensitive or highly sensitive to adriamycin, erythromycin, kanamycin, tetracycline, penicillin and ampicillin. The lethal dose on newborn rabbits of ten strains (P1, P2, P5, P6, P8, P10, P11, P13, P15and P16) was below20cfu. They had stronger pathogenicity and accounted for55.6%of all18isolated strains. The lethal dose of five strains (P1、P2、P8、P13、P16) among them was below10cfu. It showed they were velogenic strains. The virulence of different fluorescent type has different toxicity, Fo-type and Fg-type had stronger toxicity to newborn rabbits and mice than the medium.2. The Immunological Characteristics Study of Rabbit Pasteurella multocida IsolatesIn order to find out the rabbit Pasteurella multocida serotype prevalence in western Henan, the18isolates were typed on the serological identification. The IHA test was used to identify the capsule antigen, the results showed that18isolates were all belonged to group A capsule antigen. Agar-proliferation was used to identify the heat-resistant bacterial antigen, the results showed that18isolates were grouped two main types:14isolates were type1(77.8%) and4isolates were type3(22.2%). The P1、P2、P5、P6、P8、P10、P11、P13、 P15、P16isolates with stronger toxicity were cultivated in improved Martin broth and were treated with formaldehyde to be made inactivated vaccine. Vaccination was carried out in mice to test immunogenicity and the mice were attacked with homologous strain, standard strain C51-2and standard strain C51-3on the21st day after vaccination, respectively, and the protection rate reached60%were9,7and4isolates, respectively. The above-mentioned10isolates inactivated vaccine were selected to inoculate newborn rabbits to test immunogenicity on rabbits, and the rabbits were attacked with homologous strains, standard strain C51-2and standard strain C51-3on the14th day after vaccination, respectively, and the protection rate reached60%were7,4and3isolates, respectively.The interactive immune test was done using the P1(serotypeA:1) and P2(serotypeA:3) strains which had better immunogenicity on rabbits, the results showed that vaccine prepared with type1bacteria P1had a certain interaction immune to type3bacteria, but the vaccine prepared with type3bacteria P3had weaker interaction immune to type1bacteria.3. Development of the Rabbit Pasteurella Multocida Bivalence propolis inactived vaccineFor the effective control of rabbit pasteurellosis in western Henan, protecting the healthy development of rabbit production industry, the Pasteurella multocida isolates of P1(serotype A:1) and P2(serotype A:3) were used as vaccine strains and were cultivated in improved Martin broth for culture of antigen. The inactivated antigen were produced into pasteurella bivalence inactivated propolis vaccine assisted by the propolis as immune-enhancer which has broad-spectrum biological activity. The results of mice immunization showed that the bivalence inactivated propolis vaccine was safe and reliable and had no obvious interference, strong immunity produced14days after immunization, and the recent protection rates for pasteurellosis was almost100%. Succeeded in using mice replacement of rabbit to test bivalence vaccine efficacy, and this method can reduce reduce test cost, and more sensitive, specific, objective and simple. Using challenge method for pasteurellosis, modified micro-aggregation test for antibody titer, to check the immunity period and storage time of this vaccine. The result showed that the immunity period is more than6months,storage time is12months at4℃～8℃;6months at15℃～30℃.4. Establishment and application of PCR method for detecting rabbit pasteurella multocidaIn order to establish a quick detection method of the rabbit pasteurellosis, a pair of specific primers was designed according to the highly conservative region of the outer membrane protein (OmpH) gene sequence of13Pasteurella multocida in Genbank. By comparing the DNA extraction methods and optimizing the PCR system and procedure, a PCR based assay for detecting rabbit pasteurella multoceda was developed. The size of PCR amplified fragment was526bp. A specific fragment of526bp could be amplified with either the rabbit and poultry standard strains of C51-2and C48-1or the18laboratory separation strains from rabbits. But the amplification results were negative with the rabbit E. coli, Streptococcus, B.bronchiseptica and Rabbit Staphylococcus aureus. The results of sensitivity detection showed that the strip could be seen clearer when the template DNA was only lpg. It suggested that the sensitivity of the PCR method is better. The PCR reaction can be completed successfully and get accurate diagnosis as long as the pathogenic nucleic acid exists in the samples.40samples from from Luoyang and Sanmenxia rabbits were examined by the developed PCR assay and72.5%were positive. Comparison of the assay with the conventional bacterioscopy revealed that the sensitivity of the PCR assay was2.08times higher than that of the bacterioscopy. The results showed that the PCR method is extremely value in clinical diagnosis. |
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