副猪嗜血杆菌TbpA蛋白对仔猪免疫保护力的研究

发布时间：2018-03-14 19:54

本文选题：副猪嗜血杆菌(HPS)　切入点：转铁结合蛋白A(TbpA)　出处：《安徽农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：副猪嗜血杆菌(Haemophilus parasuis, HPS)是猪上呼吸道的一种常在菌,是猪革拉斯氏病(Glasser's disease)的病原菌。近年来由HPS引起的以猪纤维素性多发性浆膜炎、关节炎、脑膜炎、呼吸困难、高热及高死亡率为特征的传染性疾病呈现逐年上升的趋势。该病已成为影响全球养猪业最为严重的细菌性传染病之一,给养猪业带来了一定的经济损失。在控制副猪嗜血杆菌病方面,通常采用药物防治和疫苗接种等。然而长期大量使用抗菌药物,促使HPS的耐药谱逐渐拓宽,多重耐药现象日益严重；同时HPS血清型众多,各型菌株间的毒力及致病性差异都很大,且相互之间缺乏有效的交叉保护力,致使HPS灭活疫苗的保护效果十分有限。前期的研究结果显示,HPS转铁结合蛋白(TbpA)不仅具有良好的免疫活性,而且对豚鼠具有良好的免疫保护及交叉免疫保护效力。本研究利用重组TbpA蛋白和血清型13型HPS全菌体灭活苗免疫仔猪,通过检测免疫后仔猪的HPS抗体水平和细胞因子(EL-5、IL-10、IFN-γ、TNF-α)水平,观察攻毒后仔猪的采食量、直肠温度、精神状态及死亡率,并进行病理组织学观察,旨在探讨TbpA蛋白对仔猪的免疫保护作用,为HPS新型亚单位疫苗的研究及副猪嗜血杆菌病的防控提供科学依据。首先,选取15头42日龄左右HPS抗体阴性健康大白仔猪,通过Reed-Muench法测定13型HPS的半数致死量。其次,选取25头14日龄左右HPS抗体阴性健康大白仔猪,随机分为5组(编号为A、B、C、D、E),每组5头。A、B两组注射TbpA,剂量分别为2mg、lmg; C组注射13型HPS全菌体灭活苗1mL；D组和E组注射PBS 1mL;佐剂均为弗氏佐剂,接种方法均为颈部肌肉注射。共免疫3次,免疫间隔时间均为14天。一免和二免14天后、三免10天后分别采集仔猪血液,分离血清。应用ELISA方法检测HPS抗体水平和细胞因子(IL-5、 IL-10、IFN-γ、TNF-α)水平。三免10天后用HPS血清型13型菌株以7LD50剂量对仔猪进行腹腔攻毒,测定其免疫保护性,并对每组仔猪的肺脏和脾脏进行病理组织学观察。结果显示,一免后,TbpA免疫组、全菌体灭活苗免疫组HPS抗体水平和细胞因子水平相对于对照组差异不显著(P0.05)；二免后,TbpA免疫组HPS抗体水平和细胞因子水平相对于对照组差异不显著(P0.05),而全菌体灭活苗免疫组HPS抗体水平显著高于对照组(P0.05)；三免后,TbpA免疫组、全菌体灭活苗免疫组的HPS抗体水平和细胞因子水平都显著高于对照组(P0.05),且TbpA免疫组HPS抗体水平显著高于二免抗体水平(P0.05)。三免后第10天以HPS血清型13型菌株7LD50剂量分别对A、B、C、D这4组仔猪腹腔攻毒后,A、C组仔猪存活率为100%,B组仔猪存活率为40%,D组仔猪存活率为0%。A、C、E组仔猪和B组存活的仔猪肺脏和脾脏组织样品的病理学损伤程度与D组和B组死亡的仔猪差异明显。结果表明,HPS的TbpA蛋白对仔猪能够产生免疫保护力,其中2mg免疫剂量、免疫3次,与全菌体灭活苗对同源菌株攻毒的免疫保护力相同,均为100%。
[Abstract]:Haemophilus parasuis (HPS) is a common bacteria in the upper respiratory tract of pigs. It is the pathogen of Glassers disease of swine Graves disease. In recent years, it is caused by HPS caused by cellulosic polyserositis, arthritis, meningitis, dyspnea. Infectious diseases characterized by high fever and high mortality are on the rise year by year. The disease has become one of the most serious bacterial infectious diseases affecting the pig industry in the world. In the control of Haemophilus parasuis disease, drug control and vaccination are usually used. However, a large number of antibiotics are used for a long time, which makes the drug resistance spectrum of HPS widen gradually. The phenomenon of multidrug resistance is becoming more and more serious, at the same time, there are many serotypes of HPS, the virulence and pathogenicity of each strain are very different, and there is a lack of effective cross-protection between each other. Therefore, the protective effect of HPS inactivated vaccine is very limited. Previous studies have shown that TbpA not only has good immunological activity, but also has good immunological activity. In this study, recombinant TbpA protein and serum type 13 HPS inactivated vaccine were used to immunize piglets. The levels of HPS antibody and cytokine EL-5 IL-10 IFN- 纬 TNF- 伪 were detected in piglets after immunization. The feeding intake, rectal temperature, mental state and mortality of piglets after poisoning were observed, and histopathological observation was carried out in order to investigate the immune protective effect of TbpA protein on piglets. To provide scientific basis for the study of new subunit vaccine of HPS and the prevention and control of Haemophilus parasuis disease. Firstly, 15 healthy white piglets with negative HPS antibody for 42 days were selected and the lethal dose of type 13 HPS was determined by Reed-Muench method. A total of 25 healthy white piglets with negative HPS antibody at 14 days old were selected. The mice were randomly divided into five groups (group A, n = 5), group A (n = 5), group A (n = 5), TbpA at a dose of 2 mg / mg, group C, group C, injection of PBS 1 mL of 13 type HPS inactivated vaccine, group D and group E, all adjuvants were Freund's adjuvant and inoculated by intramuscular injection of TbpA. The interval of immunization was 14 days. After 14 days of immunization, the blood of piglets were collected after 10 days of three immunizations. Serum samples were isolated. The levels of HPS antibody and cytokines IL-5, IL-10 IFN- 纬 TNF- 伪 were detected by ELISA method. After 10 days, the piglets were inoculated with HPS serotype 13 strain at the dose of 7LD50 to detect the immune protection. The lung and spleen of each group were observed by histopathology. The levels of HPS antibody and cytokines in the whole bacterial inactivated vaccine group were not significantly different from those in the control group, while the levels of HPS antibody and cytokines in the TbpA immunized group were not significantly different from those in the control group, but the total bacterial inactivation was not significant. The level of HPS antibody in the vaccine immunized group was significantly higher than that in the control group (P 0.05). The levels of HPS antibody and cytokine in the whole bacterial inactivated vaccine immunized group were significantly higher than those in the control group (P0.05), and the HPS antibody level in the TbpA immunized group was significantly higher than that in the second immune antibody level (P0.05). On the 10th day after the third immunization, the dosage of HPS serotype 13 strain 7LD50 was used. The survival rate of four groups of piglets after intraperitoneal attack was 100% and the survival rate of group B was 40%. The survival rate of group D was 0 and that of group B was 0. The pathological damage of lung and spleen tissue samples of group B and group B were higher than that of group D. The results showed that the TbpA protein of HPS could protect the piglets. The dose of 2 mg immunized for 3 times was the same as that of the whole bacterial inactivated vaccine (100%).
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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