本文選題：牛支原體 + 分離培養(yǎng)��； 參考：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：牛支原體從發(fā)現(xiàn)到現(xiàn)在一直沒有引起人們足夠的重視,也沒有列入重大疫病的名單,但是牛支原體在牛養(yǎng)殖業(yè)引起的損失一直都非常巨大,許多的報道已經(jīng)說明牛支原體病呈世界性分布,牛支原體的致病力非常強(qiáng),主要導(dǎo)致牛乳房炎、肺炎,這也是人們了解和發(fā)現(xiàn)牛支原體的主要原因。同時牛支原體在引起病理變化的過程中可以為其它致病菌大開門戶。本課題首先對中國大部分地區(qū)分離的菌株進(jìn)行了鑒定和流行病學(xué)調(diào)查,然后對純化得到的牛支原體進(jìn)行后續(xù)試驗:在此基礎(chǔ)上研發(fā)了牛支原體滅活疫苗和建立抗體間接ELISA檢測方法。本實驗室過去8年時間里對國內(nèi)12個省30個規(guī)模化奶牛場進(jìn)行了流行病學(xué)調(diào)查。采用牛支原體間接ELISA、直接培養(yǎng)法和特異性PCR檢測,進(jìn)行了牛支原體感染情況調(diào)查及分析,結(jié)果顯示,血清抗體陽性率為52.31%(441/843);病料陽性率為55%(33/60);乳樣陽性率為34.22%(373/1090);并且從中我們收集了具有地方代表性的55株牛支原體菌種,進(jìn)行了 16SrDNA測序比對,比對結(jié)果表明在16SrDNA水平上沒有發(fā)生變異,這個結(jié)果為以后牛支原體免疫預(yù)防提供了重要依據(jù)。從分離到的55株牛支原體中選取5株牛支原體進(jìn)行培養(yǎng)和特性研究,我們從牛支原體的生化特征、血清生長抑制、營養(yǎng)及代謝、生長和培養(yǎng)特征、藥物敏感等實驗,并采用PCR、CCU顏色變化單位、CFU菌落計數(shù)單位等方法獲取實驗數(shù)據(jù),并選取優(yōu)勢菌株傳代培養(yǎng),經(jīng)過傳代培養(yǎng),實現(xiàn)對牛支原體的馴化,傳代中牛支原體活菌數(shù)可達(dá)到10111cfu/mL,并且可以維持穩(wěn)定,到目前為止,牛支原體共在體外傳代200代次。以牛支原體全菌體蛋白的超聲波裂解物作為包被抗原,建立檢測牛支原體抗體的間接ELISA方法。通過正交篩選確定蛋白包被量為4 μg/mL,包被液為批pH9.6的碳酸鹽緩沖液,包被最佳溫度和時間為4℃ 16 h,封閉液為1%濃度的脫脂乳,最佳封閉時間和溫度為37℃2 h,待檢血清最佳稀釋度為1:100,反應(yīng)溫度和時間37℃ 1 h,二抗稀釋度為1:4000,最佳反應(yīng)溫度和時間為37℃1h。通過實驗確定陰性血清的臨界值為OD450=0.346,該方法與口蹄疫、副結(jié)核、布魯菌病、牛病毒性腹瀉、牛傳染性鼻氣管炎及結(jié)核的陽性血清均無交叉反應(yīng),具有極強(qiáng)的特異性。將陽性血清倍比稀釋,1:512倍稀釋時OD450仍大于臨界值,證明該方法具有很好的敏感性。組內(nèi)和組間變異系數(shù)均小于5%,證明該方法重復(fù)性較好,利用該方法與加拿大Biovet公司生產(chǎn)的牛支原體間接ELISA試劑盒對150份臨床樣本進(jìn)行檢測,結(jié)果顯示兩者的符合率為95%。以上結(jié)果可以說明我們建立的間接ELISA方法可為該病的快速診斷、流行病學(xué)調(diào)查及免疫牛只的抗體檢測提供一種手段。用分離純化、傳代培養(yǎng)的牛支原體在在家兔上進(jìn)行毒力鑒定,癥狀與牛感染牛支原體的癥狀高度相似;通過病理組織切片、間接免疫熒光、特異性PCR、分離培養(yǎng)等方法驗證其為牛支原體感染。確定牛支原體在體外連續(xù)傳代培養(yǎng)過程中毒力明顯減弱但仍具有致病性,可作為疫苗備選株。將第160代次的牛支原體滅活后與鋁膠鹽佐劑1:1混合做成滅活疫苗對家兔進(jìn)行免疫試驗,第28 d時對疫苗組和對照組家兔進(jìn)行攻毒試驗,疫苗組不發(fā)病,解剖后肺臟沒有病變,而對照組高熱,解剖后肺臟有明顯的病變。家兔免疫試驗證明牛支原體滅活疫苗在家兔體內(nèi)可產(chǎn)生較高水平的抗體,并可產(chǎn)生確實的保護(hù)作用。在家兔免疫試驗的基礎(chǔ)上我們制定了牛支原體滅活疫苗免疫程序,并在內(nèi)蒙古某牧場進(jìn)行了臨床實驗,選取了 210頭60日齡以內(nèi)的小公牛進(jìn)行牛支原體滅活疫苗本動物免疫試驗,疫苗中活菌含量為1011cfu/mL,免疫計量為1mL/頭。通過牛支原體滅活疫苗免疫后,到第60 d免疫組發(fā)病率下降20.7%,分菌率下降35%,PCR陽性率下降50%,對照組發(fā)病率上升17%,分菌率上升8.3%,PCR陽性率下降25%。在兩次免疫后疫苗所產(chǎn)生的抗體滴度最高可達(dá)1:256,80%的小牛抗體滴度達(dá)到1:128及以上,到第60 d時仍可檢測到抗體,有67%的小�？贵w滴度達(dá)到1:64及以上,此次臨床實驗前后經(jīng)歷一年時間,共進(jìn)行了三個批次的實驗,在不斷摸索中最終確定最佳免疫時間、免疫日齡及免疫程序,并初步建立了疫苗免疫后抗體消漲規(guī)律。
[Abstract]:Mycoplasma bovine has not been paid enough attention to, and it has not been included in the list of major diseases. But the loss of Mycoplasma in cattle has been very huge. Many reports have shown that the Mycoplasma disease is distributed worldwide and the pathogenicity of bovine mycoplasma is very strong, which is mainly caused by cow milk. Pneumonia, which is also the main reason for people to understand and discover Mycoplasma bovine. At the same time, Mycoplasma bovine can open the door for other pathogenic bacteria in the process of pathological changes. First, this subject has conducted identification and epidemiological investigation of isolated strains in most areas of China, and then carried out follow-up tests on the purified Mycoplasma bovine: On this basis, the bovine mycoplasma inactivated vaccine and the establishment of indirect ELISA for the establishment of antibody were developed. In the past 8 years, the laboratory conducted an epidemiological survey on 30 dairy farms in 12 provinces in China. The infection of Mycoplasma bovine was investigated by indirect ELISA, direct culture and specific PCR detection. The results showed that the positive rate of serum antibody was 52.31% (441/843), the positive rate of the disease was 55% (33/60), the positive rate of milk sample was 34.22% (373/1090), and we collected 55 mycoplasma strains with local representativeness and performed the 16SrDNA sequencing comparison, which showed that there was no variation on the 16SrDNA level compared with the results, and this result was the result. In the future, the immune prevention of Mycoplasma bovine provided an important basis. From the isolation of 55 mycoplasma from 55 strains of mycoplasma, 5 mycoplasma from bovine mycoplasma were selected for culture and characteristics. We studied the biochemical characteristics of Mycoplasma bovine, serum growth inhibition, nutrition and metabolism, growth and culture characteristics, drug sensitivity, and the use of PCR, CCU color change units, and CFU colony. Counting unit and other methods were used to obtain the experimental data, and the dominant strains were selected and cultured to be cultured to domesticate Mycoplasma bovine. The number of Mycoplasma bovine viable bacteria in the passage can reach 10111cfu/mL, and it can maintain stability. So far, Mycoplasma bovine have been passed in vitro for 200 generations. An indirect ELISA method for detecting mycoplasma antibody was established by using the lysate as the envelope antigen. Through orthogonal screening, the protein package was determined to be 4 mu g/mL and the clad liquid was a batch pH9.6 carbonate buffer. The best temperature and time were 4, 16 h, and the closed liquid was 1% concentration of skimmed milk. The optimum closing time and temperature were 37, 2 h, and the serum was tested most. The best dilution was 1:100, the reaction temperature and time were 37 C 1 h, the two anti dilution was 1:4000, the optimum reaction temperature and time was 37 1h., and the critical value of the negative serum was OD450=0.346. The method was not cross against the foot and mouth disease, the accessory tuberculosis, brucellosis, bovine viral diarrhea, bovine infectious rhinotracheitis and the positive serum of tuberculosis. It has very strong specificity. Dilute the positive serum double ratio and OD450 more than the critical value at 1:512 times dilution, which proves that the method has good sensitivity. The coefficient of variation within and between groups is less than 5%, which proves that the method is more repeatable, and 150 copies of the bovine mycoplasma indirect ELISA kit produced by the Canadian Biovet company are used in this method. The clinical samples were tested and the results showed that the coincidence rate of the two was more than 95%.. The results showed that the indirect ELISA method established by us could provide a means for the rapid diagnosis of the disease, the epidemiological investigation and the antibody detection of immunized cattle. The symptoms of Mycoplasma bovine infected cattle were highly similar; the Mycoplasma bovine infection was confirmed by pathological tissue section, indirect immunofluorescence, specific PCR, and isolation and culture. The virulence of Mycoplasma oxabi was obviously weakened but still pathogenicity in the process of continuous subculture in vitro, which could be used as a vaccine candidate. The 160th generation of bovine mycoplasma was used. After the inactivation of the body, the vaccine was mixed with the aluminum gel salt adjuvant 1:1 to make an inactivated vaccine against the rabbit. At twenty-eighth D, the vaccine group and the control group were attacked by the vaccine group. The vaccine group was not sick and the lungs were diseased after dissection. The control group had high fever and the lung had obvious pathological changes after dissection. The rabbit immunization test proved that the bovine mycoplasma inactivated vaccine was in the home. In rabbits, a high level of antibodies can be produced and can produce a certain protective effect. On the basis of the immune test in rabbits, we formulated the immunization program for the inactivated vaccine of Mycoplasma bovine, and carried out a clinical experiment in a pasture in Inner Mongolia, and selected 210 heads of bulls within 60 days of age to be vaccinated with the inactivated vaccine of bovine mycoplasma. The living bacteria content in the vaccine was 1011cfu/mL and the immuno metrology was 1mL/ head. After immunization with the inactivated vaccine of Mycoplasma bovine, the incidence of the sixtieth D immunization group decreased by 20.7%, the rate of bacteria division decreased by 35%, the positive rate of PCR decreased by 50%, the incidence of the control group increased by 17%, the rate of bacteria separation increased by 8.3%, and the PCR positive rate decreased the antibody titer produced by the two post immunization vaccine. The titer of the calf antibody up to 1:256,80% reached 1:128 and above, and the antibody was still detected at sixtieth D, and 67% of the calf antibody titer reached 1:64 and above. After a year, three batch experiments were carried out before and after the clinical trial. The rule of antibody elimination after vaccination was preliminarily established.

【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.23

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