本文選題：肺炎克雷伯氏菌 + 分離鑒定　； 參考：《山東農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：肺炎克雷伯氏菌是一種條件致病菌,過去人們認(rèn)為其對畜禽的危害不大,故一直未引起人們足夠的重視。但是近年來關(guān)于肺炎克雷伯氏菌引起水貂等毛皮動物疾病的報道卻在迅速增加,加之各種抗菌藥物的廣泛使用,導(dǎo)致該菌的耐藥性越來越強,且多重耐藥菌株大量出現(xiàn),肺炎克雷伯氏菌病在水貂等毛皮動物養(yǎng)殖業(yè)中的危害越來越嚴(yán)重,因此隨著環(huán)境的改變,肺炎克雷伯氏菌作為人畜共患傳染病的病原,應(yīng)引起大家的廣泛重視。由于目前市場上尚沒有有效的肺炎克雷伯氏菌疫苗用于該病的防治,致使該病得不到合理有效的防治,嚴(yán)重制約著水貂等毛皮動物養(yǎng)殖業(yè)的發(fā)展。因此本研究擬以槐花多糖、弗氏佐劑等為免疫佐劑,制備了不同佐劑的肺炎克雷伯氏菌滅活疫苗,為肺炎克雷伯氏菌的免疫防控提供新的技術(shù)支撐及理論依據(jù)。1.山東部分地區(qū)水貂肺炎克雷伯氏菌的分離鑒定為確診近兩年造成水貂死亡的主要細(xì)菌性病原,本研究在流行病學(xué)調(diào)查的基礎(chǔ)上,對新鮮病死水貂進(jìn)行臨床癥狀觀察、病理學(xué)檢查,同時取病變組織肝、肺、氣管為材料分離純化細(xì)菌,對所分離的細(xì)菌進(jìn)行形態(tài)特征、理化特性檢查、藥敏試驗和16S rRNA基因的分子鑒定。結(jié)果顯示,從剖檢病死水貂的肺、肝、氣管組織等均檢出同種細(xì)菌,生化特性相似,分離菌株對頭孢曲松鈉和阿米卡星敏感,綜合分析,該分離菌株為克雷伯氏菌屬的肺炎克雷伯氏菌。2.槐花多糖對肺炎克雷伯氏菌滅活疫苗免疫增強效果的研究依照本實驗室改進(jìn)的水提醇沉法提取槐花多糖,并將甲醛滅活的肺炎克雷伯氏菌與槐花多糖或弗氏不完全佐劑等體積配比制備疫苗。將160只清潔級昆明小白鼠(雌性)隨機分為4組,于3、7、12天時,每組每只小白鼠分別注射等量的槐花多糖佐劑肺炎克雷伯氏菌滅活疫苗、弗氏佐劑肺炎克雷伯氏菌滅活疫苗、全菌體滅活疫苗和PBS。在首次免疫后的第7、14、21、28、35、42、49天每組隨機選取3只小白鼠采血檢測其血清抗體效價、白細(xì)胞介素-2(IL-2)含量、白細(xì)胞介素-4(IL-4)含量,外周血CD4+和CD8+T淋巴細(xì)胞數(shù)量水平。同時,在首次免疫后的14天每組隨機選取20只進(jìn)行攻毒保護(hù)試驗,連續(xù)7天記錄小白鼠生存狀態(tài)并統(tǒng)計免疫保護(hù)率。結(jié)果顯示,肺炎克雷伯氏菌滅活疫苗能夠刺激機體產(chǎn)生特異性抗體、提高細(xì)胞因子分泌量和外周血CD4+和CD8+T淋巴細(xì)胞數(shù),并為小白鼠提供50%的免疫保護(hù)。另外,槐花多糖和弗氏佐劑能夠顯著提高小白鼠體液免疫和細(xì)胞免疫水平且槐花多糖效果更優(yōu)(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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.92

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