本文選題：坦布蘇病毒感染 + 弱毒疫苗�。� 參考：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：鴨坦布蘇病毒感染(TMUV infection)是2010年我國新發(fā)的一種急性傳染性疾病。TMUV屬于黃病毒科,黃病毒屬,恩塔亞病毒群。TMUV可感染危害多個品種的鴨,主要造成5-15日齡的雛鴨食欲減退、生長緩慢和神經(jīng)癥狀,死淘率可達(dá)5%-10%,育成鴨感染后臨床癥狀輕微,死亡率較低,產(chǎn)蛋鴨的產(chǎn)蛋率大幅下降,產(chǎn)蛋率從90%降至10%以下,該病的發(fā)生給我國養(yǎng)禽業(yè)造成了巨大的經(jīng)濟損失。免疫預(yù)防是防控TMUV感染的重要措施之一,本研究利用實驗室分離到的TMUV SDS株進行傳代致弱,利用致弱毒株SDS-70株對雛鴨進行免疫,對其免疫保護效果進行了評價。本研究對從山東省某種鴨場分離的一株TMUV,采用SPF鴨胚和SPF雞胚進行交替?zhèn)鞔晒χ氯踉摱局�。對獲得的不同代次的傳代病毒進行致弱效果分析,對不同代次的弱毒株進行免疫效果比對,結(jié)果顯示SDS-70具有較好的免疫原性。對篩選到的SDS-70傳代毒株進行免疫效力的評價。7日齡櫻桃谷鴨(無TMUV母源抗體),每只腿肌注射102.46EID50的SDS-70的弱毒疫苗,20日齡進行第2次免疫,在2免后一周,利用同源強毒SDS-10對免疫組和對照組分別進行攻毒,攻毒劑量為1mL(102.9ELD50)。免疫后,觀察各組精神狀態(tài)并記錄;定期采血檢測抗體水平;定期采集免疫組與同居感染組的泄殖腔棉試子,利用熒光定量RT-PCR方法進行排毒情況的檢測;定期剖殺,采集各組織器官進行帶毒情況檢測與病理組織學(xué)研究。結(jié)果免疫組和對照組均精神狀態(tài)良好,無明顯的臨床癥狀和病理變化,無發(fā)病和死亡情況。病理組織學(xué)檢測結(jié)果顯示,脾臟出現(xiàn)出血,后期出血減輕,心肌出現(xiàn)輕微水腫現(xiàn)象,其余各組織器官均無明顯的病理學(xué)損傷,結(jié)果表明SDS-70對雛鴨無致病性,免疫性和安全性良好。攻毒試驗結(jié)果顯示SDS-70免疫過的櫻桃谷鴨對強毒SDS-10的攻擊具有100%的臨床保護力。對SDS-70弱毒疫苗進行純凈性檢測,包括支原體、細(xì)菌和外源病毒的檢測,檢測結(jié)果均為陰性,說明該弱毒疫苗純凈。將SDS-70在鴨體內(nèi)連續(xù)傳代5次,各代次的試驗鴨均無臨床癥狀,精神良好,剖檢內(nèi)臟組織器官無病理變化,收集各個代次的病料進行RT-PCR檢測,結(jié)果為陽性,表明SDS-70具有良好的穩(wěn)定性,體內(nèi)傳代后毒力無返強情況。對20、40、60、80和100代次的傳代毒株進行全基因組序列分析,與SDS原始毒株全基因組序列相比較,結(jié)果20、40、60、80和100代5個代次的傳代病毒株共出現(xiàn)了33堿基突變,包括18個導(dǎo)致氨基酸發(fā)生變化的位點和15個中性突變點。對E蛋白氨基酸序列上的N-連接的潛在糖基化位點進行預(yù)測,根據(jù)預(yù)測結(jié)果可以看出,在SDS-80代次時在E基因上出現(xiàn)了一個糖基化位點的缺失,根據(jù)氨基酸變化分析可知,該糖基化位點的缺失是由于E蛋白154位上N變?yōu)镠,改變了糖基化位點的固有結(jié)構(gòu),導(dǎo)致該位點消失。據(jù)此推測病毒毒力的改變可能是堿基的突變所導(dǎo)致的。綜上所述,SDS-70弱毒疫苗株對櫻桃谷鴨安全性、純凈性與免疫效力較好,并且毒力穩(wěn)定,可以作為TMUV弱毒疫苗候選株。
[Abstract]:Duck tanboa virus infection (TMUV infection) is a new acute infectious disease in China in 2010,.TMUV belongs to the family of the yellow virus family, the yellow virus and the ntaya virus group.TMUV can infect many varieties of ducks, which mainly cause anorexia, slow growth and neurological symptoms of 5-15 days old ducks, and the death rate can reach 5%-10%. The clinical symptoms are mild, the mortality rate is low, the laying rate of laying ducks has fallen sharply, the egg production rate has dropped from 90% to less than 10%. The occurrence of the disease has caused great economic loss to the poultry industry in China. Immune prevention is one of the important measures to prevent and control TMUV infection. This study uses the TMUV SDS strains isolated from the laboratory to make a weak passage and make use of the weak poison. The strain SDS-70 strain was immune to the ducklings, and the effect of their immune protection was evaluated. In this study, a strain of TMUV isolated from a duck farm in Shandong Province, using SPF duck embryo and SPF chicken embryo, was successfully induced by alternate passages. The results showed that SDS-70 had good immunogenicity. The evaluation of the immune efficacy of the screened SDS-70 passages was.7 day old Cherry Valley ducks (no TMUV mother antibody), 102.46EID50 SDS-70 vaccine of 102.46EID50 in each leg muscle, and second times of immunization at 20 days of age, and the same homologous strong toxic SDS-10 was used in the immune group one week after 2 Exempts. In the control group, the attack dose was 1mL (102.9ELD50). After immunization, the mental state of each group was observed and recorded; the level of antibody was detected regularly; the cloaca cotton specimens of the immune group and the cohabiting infection group were collected regularly, and the fluorescence quantitative RT-PCR method was used to detect the detoxification. Results the immune group and the histopathological study showed that both the immune group and the control group had good mental state, no obvious clinical symptoms and pathological changes, no disease and death. The results of histopathological detection showed that the spleen appeared bleeding, late bleeding, slight edema in the myocardium, and no obvious pathological pathology in the other tissues and organs. The results showed that SDS-70 had no pathogenicity, immunity and safety. The results of the attack test showed that the SDS-70 vaccinated Cherry Valley Duck had 100% clinical protection against the attack of strong toxic SDS-10. The purity test of the SDS-70 attenuated vaccine, including the detection of mycoplasma, fine bacteria and exogenous virus, was negative. It showed that the vaccine was pure. SDS-70 was passed on the duck for 5 times in the duck. All the ducks in each generation had no clinical symptoms, the spirit was good, and the visceral organs were examined without pathological changes. The samples of each generation were collected for RT-PCR detection, and the results were positive, indicating that the SDS-70 had good stability and the virulence of the body after the passage was not strong. The whole genome sequence analysis of 20,40,60,80 and 100 generation passages was carried out and compared with the whole genome sequence of the original SDS strain. The results showed that the 33 base mutations in the 20,40,60,80 and the 100 generation of 5 generations of the generation virus strains, including 18 loci causing the change of amino acids and 15 neutral points of mutation, and N on the amino acid sequence of the E protein. - the potential glycosylation site of the connection was predicted. According to the predicted results, a glycosylated site was absent at the SDS-80 generation. According to the analysis of amino acid changes, the deletion of the glycosylation site was due to the change of the intrinsic structure of the glycosylation site in the 154 position of the E protein, which changed the structure of the glycosylation site, resulting in the elimination of the site. It is concluded that the changes in virulence of the virus may be caused by the mutation of the base. To sum up, the SDS-70 attenuated vaccine strain is safe, pure and immune to Cherry Valley duck, and has a stable virulence. It can be used as a candidate for TMUV attenuated vaccine.
【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】：2017
【分類號】：S852.4

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本文編號：1937878