發(fā)布時間：2018-09-08 08:59

【摘要】：牛支原體(Mycoplasma bovis,M.bovis)是感染牛的一種重要的致病性病原體,主要引起牛的肺炎、乳腺炎和關(guān)節(jié)炎等多種疾病。美國于1961年首次從患乳腺炎的牛乳中分離得到該病原。國內(nèi)于2008年首次從患肺炎的犢牛肺臟中分離得到,此后在國內(nèi)逐步蔓延傳播,造成了巨大的經(jīng)濟損失,為研發(fā)防控牛支原體病的有效疫苗,在綜合前期分析研究的基礎(chǔ)上,本文對牛支原體生長滴度測定、抗原菌懸液濃縮、滅活疫苗制備以及犢牛免疫效果評價等疫苗研發(fā)關(guān)鍵技術(shù)進行了試驗研究,主要研究內(nèi)容及結(jié)果如下:1 TTC應(yīng)用于牛支原體活菌計數(shù)研究及牛支原體擴大培養(yǎng)效果測定為探討氯化三苯基四氮唑(TTC)應(yīng)用于M.bovis菌落計數(shù)的可行性及實驗條件,通過平板計數(shù)法進行了TTC濃度篩選;采用傾注培養(yǎng)法對M.bovis擴大培養(yǎng)效果進行了菌落形成單位(Colony forming unit,cfu)計數(shù),通過顏色變化單位(Colour change unit,ccu)法進行了濃度檢測,并測定了630 nm處OD值及培養(yǎng)物p H的變化。結(jié)果:培養(yǎng)基中TTC質(zhì)量濃度在0.3~0.6 g/L時菌落正常生長且顯色效果明顯,TTC質(zhì)量濃度較低時菌落顯色效果不明顯,較高時則會抑制M.bovis的生長,TTC作用最佳質(zhì)量濃度為0.4 g/L;CFU法測得菌液濃度為1.8~2.42×108 CFU/m L,CCU法測得牛支原體菌株在96 h時的生長滴度為5.0×108 CCU/m L,兩種方法檢測結(jié)果基本一致;而不同時段牛支原體培養(yǎng)物OD值變化范圍在0.024~0.213,在對應(yīng)時間點OD值與上述兩種方法未表現(xiàn)出一致性,OD值測定僅可做為培養(yǎng)效果檢測時的輔助方法;培養(yǎng)物p H變化與OD值測定卻表現(xiàn)出一定的相關(guān)性。結(jié)果表明:TTC可應(yīng)用于M.bovis活菌計數(shù),菌落呈醒目紅色且特征明顯,可有效、準確地對M.bovis進行活菌計數(shù);CFU法與CCU法均可應(yīng)用于牛支原體擴大培養(yǎng)效果測定。2牛支原體生長曲線測定及不同培養(yǎng)基生長效果比較通過顏色變化單位法(Colour change unit,ccu)對牛支原體新疆分離株在牛支原體專用液體擴大培養(yǎng)基中不同培養(yǎng)階段的生長滴度進行了測定并繪制了生長曲線,同時采用活菌計數(shù)法對牛支原體菌株在牛支原體專用液體擴大培養(yǎng)基、改良Thiaucourt’s培養(yǎng)基、牛心湯培養(yǎng)基、葡萄糖血清肉湯培養(yǎng)基中的培養(yǎng)效果進行了比較。結(jié)果表明:活化培養(yǎng)后的牛支原體菌株在牛支原體專用液體擴大培養(yǎng)基中生長良好,該培養(yǎng)基相比其他三種培養(yǎng)基具有一定的經(jīng)濟優(yōu)勢,可作為制備疫苗時牛支原體抗原獲取的最優(yōu)培養(yǎng)基。3牛支原體滅活疫苗的研制采用透析法、高速離心法及超濾濃縮法對M.bovis培養(yǎng)物進行了濃縮試驗對比研究,測定不同處理方法濃縮物蛋白含量,并利用CFU與CCU法測定了超濾濃縮后濃縮液濃度;應(yīng)用ISA 206雙相佐劑制備M.bovis滅活疫苗,先后進行了疫苗無菌檢測、物理性狀檢測、穩(wěn)定性與保存期試驗、動物安全性試驗,結(jié)果顯示超濾濃縮法處理得到的濃縮物蛋白濃度最高,CFU法與CCU法檢測結(jié)果基本一致;所制備疫苗物理性狀良好,4℃可保存180 d、37℃可存放3 d,受試動物接種后未見異常反應(yīng),注射部位均正常,未見化膿及紅腫現(xiàn)象。結(jié)果表明:超濾濃縮法適用于M.bovis培養(yǎng)物的濃縮,可用于疫苗生產(chǎn)時抗原的富集;疫苗安全可靠,為M.bovis滅活疫苗的規(guī)�；a(chǎn)提供了一定的方法與技術(shù)支持。4牛支原體滅活疫苗免疫小鼠最小免疫劑量及抗體消長規(guī)律初步研究應(yīng)用牛支原體滅活疫苗對小鼠進行最小免疫劑量及抗體消長規(guī)律初步研究。將制備的疫苗分別以0.1、0.2、0.3、0.4 m L/只的不同劑量,經(jīng)腿部肌肉途徑免疫小鼠,結(jié)果顯示最小免疫劑量為0.2 m L/只;以0.2 m L/只劑量免疫小鼠,免疫3周后,抗體滴度達到最高峰,隨后抗體滴度開始下降,但仍保持相對穩(wěn)定,5周后抗體滴度表現(xiàn)出下降趨勢。5牛支原體滅活疫苗免疫犢牛的免疫保護性評價隨機選取10-15日齡犢牛20頭,免疫組10頭于10-15日齡首免,間隔10天進行二免,肌肉注射3 m L/每頭,未免疫組10頭不注射疫苗,注射等劑量生理鹽水。測定兩組犢牛二免后14 d抗體水平并在二免后21 d隨機選取相同日齡免疫與未免疫犢牛各3頭進行攻毒保護試驗,每頭犢牛經(jīng)鼻腔黏膜接種(滴注)2 m L、氣管注射3 m L牛支原體培養(yǎng)物(1.8×109 CFU/m L),記錄犢牛體溫,觀察臨床癥狀,檢測血清抗體水平。攻毒后28 d剖檢觀察肺臟臨床解剖學(xué)及病理組織學(xué)變化,記錄犢牛肺臟病變指數(shù)并采集病變肺臟組織進行牛支原體分離培養(yǎng)。結(jié)果:免疫組犢牛在二免后14 d血清牛支原體抗體達到0.404(OD450);3頭攻毒犢牛均保持良好的精神狀態(tài)且體溫變化不明顯,臨床癥狀綜合評分為3,肺臟病變指數(shù)為2,肺臟解剖學(xué)及病理組織學(xué)觀察無異常且肺臟中未分離回收到牛支原體。未免疫組犢牛同期血清牛支原體抗體為0.142(OD450);3頭攻毒犢牛均先后出現(xiàn)體溫升高、輕度咳喘、膿性鼻液,明顯消瘦等癥狀,臨床癥狀綜合評分為11,肺臟病變指數(shù)為17,肺臟尖葉、心葉及部分膈葉均表現(xiàn)明顯肝變,兩頭犢牛表現(xiàn)胸膜與肺臟黏連,一頭犢牛整個尖葉廣泛分布黃白色蠶豆狀大小壞死性結(jié)節(jié),與自然感染病例相同,病理組織學(xué)觀察顯示肺泡腔和支氣管中浸潤有大量中性粒細胞,支氣管管壁充血、水腫,肺臟部分壞死灶呈均質(zhì)紅染,為典型的化膿性及壞死性肺炎變化,3頭未免疫組犢牛病變肺組織中均分離回收到M.bovis。
[Abstract]:Mycoplasma bovis (M. bovis) is an important pathogen of bovine pneumonia, mastitis, arthritis and other diseases. The pathogen was first isolated from the milk of cows suffering from mastitis in 1961 in the United States. It was first isolated from the lungs of calves suffering from pneumonia in China in 2008 and has been in the country since then. In order to develop an effective vaccine to prevent and control Mycoplasma bovis disease, on the basis of comprehensive preliminary analysis, the key technologies of vaccine development, such as determination of Mycoplasma bovis growth titer, concentration of antigen suspension, preparation of inactivated vaccine and evaluation of calf immune effect, were studied. The main research contents and results are as follows: 1. TTC application in Mycoplasma bovis viable count study and Mycoplasma bovis enlarged culture effect determination to explore the Triphenyltetrazolium chloride (TTC) application in M. bovis colony count feasibility and experimental conditions, through plate counting method for TTC concentration screening; The colony forming unit (cfu) was counted, and the concentration of the colony forming unit (ccu) was measured. The OD value and the change of the culture P H were measured at 630 nm. Results: The colony grew normally and showed obvious colour effect when the concentration of TTC in the medium was 0.3-0.6 g/L. The best mass concentration of TTC was 0.4 g/L; the concentration of CFU was 1.8~2.42 *108 CFU/m L, and the growth titer of Mycoplasma bovis was 5.0 *108 CCU/m L at 96 h by CCU method. The results of the two methods were basically the same; but the best mass concentration of TTC was 0.4 g/L. The OD values of the cultures ranged from 0.024 to 0.213. The OD values were not consistent with the above two methods at the corresponding time points. The determination of OD values could only be used as a supplementary method for the detection of culture effect, but the changes of culture P H showed a certain correlation with the determination of OD values. CFU method and CCU method can be used to determine the effect of Mycoplasma bovis in the enlarged culture. 2 The growth curve of Mycoplasma bovis and the growth effect of different media were compared. Colour change unit (ccu) was used to detect Mycoplasma bovis in Xinjiang isolates of Mycoplasma bovis. The growth titers of Mycoplasma bovis strains in different culture stages were measured and their growth curves were plotted. Meanwhile, the effect of Mycoplasma bovis strains in liquid enlarged culture medium for Mycoplasma bovis, improved Thiaucourt's culture medium, bovine heart soup culture medium and glucose serum broth culture medium were studied by counting live bacteria. The results showed that the strains of Mycoplasma bovis grew well in the special liquid expanded medium for Mycoplasma bovis. Compared with the other three media, this medium had certain economic advantages and could be used as the optimal medium for obtaining Mycoplasma bovis antigen in the preparation of vaccine. 3 The development of inactivated Mycoplasma bovis vaccine was made by dialysis. Methods: The concentration of M. bovis culture was studied by high-speed centrifugation and ultrafiltration. The protein content of M. bovis culture concentrate was determined by different treatment methods, and the concentration of concentrated solution after ultrafiltration was determined by CFU and CCU methods. The results showed that the protein concentration of the concentrate was the highest by ultrafiltration and concentration, and the results of CFU and CCU were basically the same. The prepared vaccine had good physical properties, and could be stored at 4 ~C for 180 days, 37~C for 3 days. No abnormal reaction was observed after inoculation, and the injection site was normal. The results showed that the ultrafiltration method was suitable for the concentration of M. bovis culture and could be used for antigen enrichment during vaccine production; the vaccine was safe and reliable, which provided a certain method and technical support for the large-scale production of M. bovis inactivated vaccine. 4 Minimum immune dose and antibody elimination of mice immunized with bovine mycoplasma inactivated vaccine A preliminary study on the length rule of mice immunized with inactivated Mycoplasma bovis vaccine was carried out. The mice were immunized with the vaccine at different doses of 0.1,0.2,0.3,0.4 m L per mouse through the leg muscle route. The results showed that the minimum immune dose was 0.2 m L/mouse, and 0.2 m L/mouse was immunized with the vaccine at different doses. After 3 weeks, the antibody titer reached its peak, then began to decline, but remained relatively stable. After 5 weeks, the antibody titer showed a downward trend. 5 Immunoprotective evaluation of calves immunized with inactivated Mycoplasma bovis vaccine was randomly selected from 10 to 15 days of age. 10 calves in immunization group were immunized for the first time from 10 to 15 days of age. The levels of antibodies were measured 14 days after the second immunization and randomly selected 3 calves of the same age immunized and 21 days after the second immunization. Each calf was inoculated (dripped) 2 ml through nasal mucosa and 3 ml Mycoplasma bovis by tracheal injection. The body temperature of calves was recorded, clinical symptoms were observed and serum antibody levels were detected. The clinical anatomy and histopathological changes of lungs were observed 28 days after treatment. The pathological index of lungs of calves was recorded and Mycoplasma bovis was isolated and cultured by collecting pathological lung tissues. Mycoplasma antibody was 0.404 (OD450); all the three calves maintained good mental state and had no significant changes in body temperature. The comprehensive score of clinical symptoms was 3, the index of lung lesion was 2, the pulmonary anatomy and histopathology were normal, and the antibody of Mycoplasma bovis was not isolated and recovered from the lungs. The clinical symptoms were 11, pulmonary lesion index was 17, pulmonary apical lobe, heart lobe and part of diaphragm lobe showed obvious liver degeneration, pleura and lung adhesion in two calves, and yellow and white in the whole apical lobe of one calf. Histopathological observation showed that there were a large number of neutrophils infiltrated in alveolar cavity and bronchus, the wall of bronchial tube was congested, edema, and some necrotic lesions of lung were homogeneous red stained, which was a typical change of suppurative and necrotizing pneumonia. The pathological changes in the lung tissues of the three unimmunized calves were all the same. Separation and recovery to M.bovis.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.4

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