槐花多糖对水貂肺炎克雷伯氏菌灭活疫苗的免疫增强效果的研究

发布时间：2018-04-15 21:23

本文选题：肺炎克雷伯氏菌 + 分离鉴定　；参考：《山东农业大学》2015年硕士论文

【摘要】：肺炎克雷伯氏菌是一种条件致病菌,过去人们认为其对畜禽的危害不大,故一直未引起人们足够的重视。但是近年来关于肺炎克雷伯氏菌引起水貂等毛皮动物疾病的报道却在迅速增加,加之各种抗菌药物的广泛使用,导致该菌的耐药性越来越强,且多重耐药菌株大量出现,肺炎克雷伯氏菌病在水貂等毛皮动物养殖业中的危害越来越严重,因此随着环境的改变,肺炎克雷伯氏菌作为人畜共患传染病的病原,应引起大家的广泛重视。由于目前市场上尚没有有效的肺炎克雷伯氏菌疫苗用于该病的防治,致使该病得不到合理有效的防治,严重制约着水貂等毛皮动物养殖业的发展。因此本研究拟以槐花多糖、弗氏佐剂等为免疫佐剂,制备了不同佐剂的肺炎克雷伯氏菌灭活疫苗,为肺炎克雷伯氏菌的免疫防控提供新的技术支撑及理论依据。1.山东部分地区水貂肺炎克雷伯氏菌的分离鉴定为确诊近两年造成水貂死亡的主要细菌性病原,本研究在流行病学调查的基础上,对新鲜病死水貂进行临床症状观察、病理学检查,同时取病变组织肝、肺、气管为材料分离纯化细菌,对所分离的细菌进行形态特征、理化特性检查、药敏试验和16S rRNA基因的分子鉴定。结果显示,从剖检病死水貂的肺、肝、气管组织等均检出同种细菌,生化特性相似,分离菌株对头孢曲松钠和阿米卡星敏感,综合分析,该分离菌株为克雷伯氏菌属的肺炎克雷伯氏菌。2.槐花多糖对肺炎克雷伯氏菌灭活疫苗免疫增强效果的研究依照本实验室改进的水提醇沉法提取槐花多糖,并将甲醛灭活的肺炎克雷伯氏菌与槐花多糖或弗氏不完全佐剂等体积配比制备疫苗。将160只清洁级昆明小白鼠(雌性)随机分为4组,于3、7、12天时,每组每只小白鼠分别注射等量的槐花多糖佐剂肺炎克雷伯氏菌灭活疫苗、弗氏佐剂肺炎克雷伯氏菌灭活疫苗、全菌体灭活疫苗和PBS。在首次免疫后的第7、14、21、28、35、42、49天每组随机选取3只小白鼠采血检测其血清抗体效价、白细胞介素-2(IL-2)含量、白细胞介素-4(IL-4)含量,外周血CD4+和CD8+T淋巴细胞数量水平。同时,在首次免疫后的14天每组随机选取20只进行攻毒保护试验,连续7天记录小白鼠生存状态并统计免疫保护率。结果显示,肺炎克雷伯氏菌灭活疫苗能够刺激机体产生特异性抗体、提高细胞因子分泌量和外周血CD4+和CD8+T淋巴细胞数,并为小白鼠提供50%的免疫保护。另外,槐花多糖和弗氏佐剂能够显著提高小白鼠体液免疫和细胞免疫水平且槐花多糖效果更优(P0.05)。
[Abstract]:Klebsiella pneumoniae (Klebsiella pneumoniae) is a kind of conditional pathogen.However, in recent years, the reports that Klebsiella pneumoniae cause fur animal diseases such as mink have been increasing rapidly, and the extensive use of various antimicrobial agents has led to the increasing drug resistance of Klebsiella pneumoniae and the emergence of a large number of multidrug resistant strains.Klebsiella pneumoniae is becoming more and more serious in fur animal breeding such as mink. With the change of environment, Klebsiella pneumoniae, as the pathogen of zoonotic infectious disease, should be paid more and more attention.Because there is no effective vaccine against Klebsiella pneumoniae on the market at present, there is no reasonable and effective prevention and cure of the disease, which seriously restricts the development of fur animal breeding such as mink.Therefore, the inactivated vaccine of Klebsiella pneumoniae with different adjuvants was prepared with Sophora japonica polysaccharide and Freund's adjuvant as immune adjuvants, which provided new technical support and theoretical basis for the prevention and control of Klebsiella pneumoniae.The isolation and identification of Klebsiella pneumoniae from mink in some areas of Shandong Province were confirmed as the main bacterial pathogens that caused the death of mink in recent two years. Based on the epidemiological investigation, the clinical symptoms of fresh diseased dead mink were observed.Pathological examination, liver, lung and trachea were taken as materials to isolate and purify bacteria. Morphological, physical and chemical characteristics of the bacteria were examined, drug sensitivity test and molecular identification of 16s rRNA gene were carried out.The results showed that the same bacteria were found in lung, liver and trachea of dead mink by dissection, and the biochemical characteristics were similar. The isolated strains were sensitive to ceftriaxone sodium and amikacin.The isolated strain is Klebsiella pneumoniae.Study on the effect of Sophora japonica Polysaccharide on immune Enhancement of Klebsiella pneumoniae inactivated Vaccine; extraction of Sophora japonica polysaccharides from Sophora japonica by water extraction and alcohol precipitation improved in our laboratory.The vaccine was prepared by mixing formaldehyde inactivated Klebsiella pneumoniae with Sophora japonica polysaccharide or Freund's incomplete adjuvant.160 Kunming mice (females) of clean grade were randomly divided into 4 groups. Each group was injected with an inactivated vaccine of Klebsiella pneumoniae, an adjuvant of Sophora japonica polysaccharides, and an inactivated vaccine of Klebsiella pneumoniae with Freund's adjuvant.Whole bacterial inactivated vaccine and PBSs.The serum antibody titer, interleukin-2 (IL-2) level, interleukin-4 (IL-4) content, CD4 and CD8 T lymphocytes in peripheral blood were measured in 3 mice in each group on day 7, 14, 21, 28, 35, 42, 49 days after the first immunization. The serum antibody titers, interleukin-2 (IL-2), interleukin-4 (IL-4) levels and the levels of CD4 and CD8 T lymphocytes in peripheral blood were determined.At the same time, 20 mice in each group were randomly selected 14 days after the first immunization. The survival status of the mice was recorded for 7 days and the immune protection rate was counted.The results showed that the inactivated Klebsiella pneumoniae vaccine could stimulate the production of specific antibodies, increase the secretion of cytokines and the number of CD4 and CD8 T lymphocytes in peripheral blood, and provide 50% immune protection for mice.In addition, Flos Sophorae polysaccharide and Freund's adjuvant could significantly improve the humoral and cellular immunity of mice, and the effect of Sophora japonica polysaccharides was better than that of P0.05.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.92

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