兔波氏杆菌病是由波氏杆菌(Bordetella Bronchiseptica, Bb)引起的一种兔多发性呼吸道疾病，该病在兔群中感染率高，病程长久，较难治愈，已成为影响养兔业发展的重要原因。本试验通过对波氏杆菌毒力菌株的筛选，攻毒模型的建立，不同剂量毒力菌株全菌灭活抗原免疫兔后的抗体消长测定，灭活抗原对兔的免疫保护力测定，对波氏杆菌的免疫原性进行探讨；同时本研究应用建立的波氏杆菌TaqMan荧光定量PCR检测方法测定人工滴鼻感染兔体内各脏器中波氏杆菌含量，对波氏杆菌在感染兔体内的感染分布进行初步探讨，为波氏杆菌病的防控提供理论基础。 1. 波氏杆菌人工感染兔模型的建立 为建立波氏杆菌人工感染兔的动物模型，本实验室用不同兔场分离鉴定的6株波氏杆菌进行ICR小鼠腹腔攻毒实验，采用寇氏法分别计算6株波氏杆菌对小鼠的半数致死量，筛选出毒力较强菌株FX-1。用FX-1菌株通过静脉、胸腔、腹腔、滴鼻、皮下5种不同途径对兔人工感染，对不同攻毒途径下感染兔的临床症状及病理变化观察比较，筛选出静脉攻毒作为对兔的最终攻毒途径。通过静脉攻毒途径测定FX-1菌株对兔的半数致死量，菌株FX-1对40日龄兔的半数致死量为6.61×109CFU。波氏杆菌人工感染兔模型的建立为后续波氏杆菌免疫原性和致病性的研究奠定了基础。 2. 波氏杆菌免疫原性研究 为探讨波氏杆菌的免疫原性，本试验测定了FX-1菌株生长曲线，制备波氏杆菌灭活抗原，用不同剂量FX-1灭活抗原对兔进行免疫，应用间接ELISA方法检测比较不同免疫剂量下兔的抗体消长规律，测定波氏杆菌免疫保护率。试验结果表明：FX-1菌株对数生长区为4-16h，该阶段细菌浊度与活菌数线性关系为lgCFU=1.3716 OD600+6.6516，R2=0.9954。免疫1mL FX-1灭活抗原剂量组在免疫后21天内抗体持续上升，21天至105天内抗体滴度维持在213左右，105天后抗体效价出现下降趋势，133天时降至211.14；免疫2mL组，免疫后21天内抗体滴度持续上升，21天至133天内抗体滴度都维持在213左右且无下降趋势。免疫组与对照组在免疫后第21天用2倍LD50剂量进行静脉攻毒，免疫组保护率为87.50%（21/24），对照组死亡率为95.83%（23/24），为波氏杆菌疫苗的研发奠定了基础。 3. 波氏杆菌兔体内感染分布 利用波氏杆菌毒力因子CyaA为目的基因设计特异性引物和探针，以阳性克隆质粒作为定量检测标准品建立标准曲线，以提取的波氏杆菌基因组DNA为模板进行特异性、灵敏性和重复性试验，建立TaqMan荧光定量波氏杆菌检测方法。对兔进行波氏杆菌滴鼻攻毒感染，于攻毒感染后第6天和第15天剖杀采集样本（心、肝、脾、肺、肾、气管），运用建立的TaqMan荧光定量PCR检测方法测定各脏器中的波氏杆菌含量，探索波氏杆菌在感染兔体内各脏器中的分布。运用全菌包被ELISA方法检测感染兔血清中抗波氏杆菌IgG滴度。本试验建立了具有较好特异性、敏感性和重复性的TaqMan荧光定量波氏杆菌检测方法。兔在感染后第6天和第15天波氏杆菌菌含量测定表明，波氏杆菌主要感染分布于气管和肺，在感染后波氏杆菌含量显著高于其它4个脏器（P<0.01）。其余4个脏器（心、肝、脾、肾）中细菌含量差异不显著（P>0.05）。感染兔在感染后血清中抗波氏杆菌IgG滴度逐渐上升。本试验为波氏杆菌病的防治提供了理论基础。

The rabbit Bordetella bronchiseptica disease is a rabbit multiple respiratory diseases caused by Bordetella Bronchiseptica (Bordetella Bronchiseptica, Bb). It has become one of the most important factors that influence the development of the rabbit industry induced high infection rate, long duration carrying bacteria and difficulty treatment. The immunogenicity of Bb was studied in present study including the screening of virulent strains, establishing the challenge model, detecting the immune response and protection efficiency of whole cell inactivated antigen by using indirect ELISA method. Colonization regularities of Bb in chanllenged rabbits were also studied through TaqMan quantitative PCR methods after Bb intranasally challenge. This study provided a theoretical basis for future prevention against Bb infection in rabbits. 1. The establishment of rabbit model artificially infected with the Bordetella Bronchiseptica In this study, six strains of Bb isolated and identified from different rabbit farms were used to intraperitoneal challenge experiments in ICR mice to establish the rabbits artificially infected animal model. Karber method was used to calculate the median lethal dose of Bb chanllenge in mice. Strains of FX-1 had been screened as the virulent strain. Five challenge ways such as vein injection, thoracic injection, abdominal cavity injection, nose dropping and subcutaneous injection were tested for selecting the best challenge way in future experiment of rabbits artificially infected with Bb. The results showed that intravenous inoculating was a best way to challenge rabbits by observing the clinical symptoms and pathological changes as a result of chanllenge experiment. The median lethal dose of strain FX-1 to 40-day-old rabbits was 6.61×109CFU. The establishment of rabbits artificially infected animal model laid the foundation for the subsequent Bb immunogenicity and pathogenicity study. 2. The study of Bordetella Bronchiseptica immunogenicity In present experiments, growth curve of strain FX-1 were determined. Rabbits were immunized with different doses of inactivated Bb antigen. Antibody regularities of different immunization doses were detected using indirect ELISA method as well as the pretection percentage. Logarithmic growth area of FX-1 strain was 4-16h, the bacterial turbidity of the stage and the linear relationship of the number of viable cells for lgCFU = 1.3716OD600+6.6516, R2 =0.9954. After 1mL FX-1 immunization, immunization antibody continued to rise from 21 days to 105 days with the titers remained at the level of 213.Then the antibodies decreased from 105 days until to 133 days with the titer of 211.14. After 2mL FX-1 immunization, antibody titers continued to rise within the first 21 days. From 21 days to 133 days the antibody titer maintained about 213 without having any downward trend. The groups (1mL) were intravenous challenged on 21st day after the immunization. The protection rate of immunized group on 21st day was 87.50%（21/24）, the death rate of the control group was 95.83%(23/24). This study laid the foundation for the Bb vaccine research and development. 3. The distribution regularities of Bordetella in infected rabbits The regularities distribution of Bb in infected rabbits after intranasal challenge were studied in this experiment. Specific primers and probes was designed according to the gene CyaA-the virulence factor of Bb, extracted Bb genomic DNA were used as template, a specificity, sensitivity and reproducibility TaqMan fluorescent quantification method was established to explore the distribution of Bb in rabbits infected body organs. Rabbits were intranasally challenged with Bb，Different organ samples (heart, liver, spleen, lung, kidney, trachea) were collected and detected 6 day and 15 day post-challenged by using the established Bb TaqMan fluorescent quantitative detection method . The results of determination bacterial content in various organs in infected rabbits with intranasally challenged at each time point showed that bacterial infection mainly located in the trachea and lungs with significantly higher than other four organs (P<0.01). The difference of bacterial content in other 4 organs（heart,liver,spleen,kindy）was not significant（P>0.05) . The anti-Bb IgG drop degrees of infected rabbits rose gradually. Pathogenic mechanism was studied in the present experiment providing a theoretical basis for the prevention and treatment against Bb.