本文選題：豬流行性腹瀉 + Vero細(xì)胞��； 參考：《華南農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：豬流行性腹瀉(PED)是由冠狀病毒屬的豬流行性腹瀉病毒(PEDV)引起的一種以仔豬水樣腹瀉、嘔吐、脫水為主要特征的接觸性腸道傳染病。豬流行性腹瀉是世界上流行最廣泛的豬病之一。近年,豬流行性腹瀉作為烈性傳染病,開始瘋狂肆虐我國廣大養(yǎng)殖戶。我國的廣東、廣西、江西、湖南、福建、浙江、安徽、江蘇、湖北、河北和山東等省(自治區(qū))均有豬流行性腹瀉的發(fā)現(xiàn)。PED在我國的流行面逐漸擴(kuò)大,對26個省市自治區(qū)的調(diào)查表明PED的總死亡率占36種疾病總死亡率的1.74%。該病多發(fā)于冬春季節(jié),但就廣東地區(qū)這個全年高濕度的環(huán)境,PED呈高發(fā)趨勢,全年均可發(fā)生,7天齡內(nèi)的新生仔豬在發(fā)生腹瀉后3?4天,通常因脫水導(dǎo)致衰竭而死亡,病死率高達(dá)100%。目前,在廣東粵西地區(qū),豬流行性腹瀉呈高發(fā)趨勢,一旦進(jìn)入冬春季節(jié),該病都是大規(guī)模爆發(fā),病死率高。雖然可以采取疫苗接種方式來控制此病流行,但經(jīng)典的CV777株疫苗免疫后效果并不理想,接種過疫苗的豬場仍然有此病的流行。本研究主要分為下面三個部分:1、PEDV-YX株的分離鑒定。本研究從廣東粵西9個疑似發(fā)生腹瀉疫情的規(guī)�；i場采集180份病料樣本。通過在Vero細(xì)胞培養(yǎng)基中添加胰酶的方法孵育、選取1%接毒量接種Vero單層細(xì)胞,進(jìn)行病毒分離純化,從中分離出9株未知病毒HS1、HS2、HS3、HS4、YC1、YC2、YC3、YC4和KP,經(jīng)膠體金快速診斷技術(shù)、RT-PCR擴(kuò)增、基因序列比對,確診均為豬流行性腹瀉病毒(PEDV)。通過豬體回歸試驗(yàn),選取臨床癥狀最明顯,毒力最強(qiáng)的YC1毒株作為研究對象,命名為PEDV-YX株。在胰酶總濃度為7.5ug/ml的作用下,盲傳到第8代,能觀察到細(xì)胞圓縮、聚集,空泡增多等明顯的細(xì)胞病變。2、對PEDV-YX株生物學(xué)特性、中和試驗(yàn)及其TCID50測定研究。對PEDV-YX株生物學(xué)特性、中和試驗(yàn)及其TCID50研究表明,該分離株對5-Brd U不敏感,為具有囊膜的RNA病毒;對乙醚、氯仿敏感;在pH為3.0的環(huán)境下易失活;對熱敏感,在50℃條件下作用1h,病毒易失活;設(shè)計(jì)特異性引物,進(jìn)行RT-PCR擴(kuò)增,電泳檢測得到與預(yù)期長度一致的條帶;與PEDV抗血清中和效價為120.23;PEDV-YX F10的TCID50為10-5.67/0.1ml。3、PEDV-YX株氫氧化鋁膠滅活疫苗制備及不同免疫劑量、不同免疫途徑對抗體水平的影響研究。PEDV-YX株F10經(jīng)0.3%的甲醛溶液滅活,以病毒液樣40ml+PBS40ml+20ml鋁膠佐劑的培苗方案,配制豬流行性腹瀉滅活疫苗。以口服免疫、后海穴注射免疫為免疫途徑,2ml、1ml、0.5ml不同免疫劑量,研究該滅活疫苗的抗體消長規(guī)律。當(dāng)免疫劑量相同時,后海穴注射免疫比口服免疫抗體效價更高,且抗體維持時間更長;當(dāng)免疫途徑相同時,免疫2ml的抗體整體水平比免疫1ml的更高,免疫0.5ml的中和抗體水平最低。通過分離廣東粵西地區(qū)PEDV的流行性毒株,為了解該地區(qū)流行性毒株的特性及流行情況,建立PEDV快速準(zhǔn)確的診斷方法及新型疫苗的研究提供理論依據(jù),從而為制定有效的防控措施,促進(jìn)該地區(qū)養(yǎng)豬業(yè)的穩(wěn)定發(fā)展做好鋪墊。
[Abstract]:Porcine epidemic diarrhea (PED) is a kind of contact intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV) of the coronavirus, which is characterized by diarrhea, vomiting and dehydration of piglets. Porcine epidemic diarrhea is one of the most popular swine diseases in the world. In recent years, swine epidemic diarrhea, as a strong infectious disease, began to ravage me wildly. Guangdong, Guangxi, Jiangxi, Hunan, Hunan, Hunan, Fujian, Zhejiang, Anhui, Jiangsu, Hubei, Hebei and Shandong and other provinces (autonomous regions) have been found to have epidemic diarrhea in China. The prevalence of.PED in China is gradually expanded, and the total mortality of PED in 26 provinces and autonomous regions accounts for the incidence of the total mortality of 36 diseases. Yu Dongchun season, but in Guangdong area this year high humidity environment, PED is high trend, can occur throughout the year, 7 days of age of newborn piglets in 3 to 4 days after diarrhea, usually due to dehydration and death, the mortality rate is up to 100%., in the west of Guangdong Province, swine epidemic diarrhea is high trend, once into the winter and spring season, The disease is a large outbreak, with high mortality. Although the vaccine can be taken to control the epidemic, the classic CV777 vaccine is not ideal after immunization. The vaccinated piggery still has the epidemic. This study is divided into three parts: 1, isolation and identification of PEDV-YX strain. This study from Guangdong, Guangdong, 9 180 samples of disease materials were collected in a large-scale pig farm suspected of diarrhea. By incubating the trypsin in the Vero cell culture medium, 1% single cells were inoculated and purified. 9 unknown viruses, HS1, HS2, HS3, HS4, YC1, YC2, YC3, YC4 and KP were isolated from the Vero monolayer. Increase, gene sequence alignment, confirmed as porcine epidemic diarrhea virus (PEDV). Through the pig body regression test, select the most obvious clinical symptoms, the most virulent YC1 strain as the research object, named PEDV-YX strain. Under the effect of total trypsin concentration of 7.5ug/ml, blind to the eighth generation, can observe the cell roundness, aggregation, vacuoles and so on obvious Cytopathic.2, biological characteristics, neutralization test and TCID50 determination of PEDV-YX strain. Biological characteristics of PEDV-YX strain, neutralization test and TCID50 study showed that the isolate was insensitive to 5-Brd U, was a RNA virus with capsule, sensitive to ether, chloroform, and easily inactivated in a pH 3 environment; it was sensitive to heat and acted at 50 degrees centigrade. 1H, the virus was easily inactivated; specific primers were designed for RT-PCR amplification. Electrophoresis was used to detect the expected length of the strip; neutralization titer with PEDV antiserum was 120.23; TCID50 of PEDV-YX F10 was 10-5.67/0.1ml.3, aluminum hydroxide gel inactivated vaccine of PEDV-YX strain and different immunization doses, and the effect of different immune pathways on antibody level .PEDV-YX strain F10 was inactivated by 0.3% Formaldehyde Solution, and the pig epidemic diarrhea inactivated vaccine was prepared by the solution of virus like 40ml+PBS40ml+20ml aluminum gum adjuvant. Immunization was taken orally, the immunization of Houhai acupoint immunization, 2ml, 1ml, 0.5ml in different immune doses to study the law of antibody growth of the inactivated vaccine. When the immune dose was the same, Houhai The titer of acupoint injection is higher than that of oral immunization antibody, and the time of antibody maintenance is longer. When the immune pathway is simultaneously, the overall level of the antibody to the immune 2ml is higher than that of the immune 1ml, and the neutralizing antibody level of the immune 0.5ml is the lowest. By separating the epidemic strains of PEDV in the western Guangdong region, the characteristics and epidemic of the epidemic strains in this area are understood. It provides a theoretical basis for the establishment of a rapid and accurate diagnostic method of PEDV and a new vaccine, so as to lay the groundwork for the formulation of effective prevention and control measures and the promotion of the stable development of the pig industry in this area.

【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S858.28

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