本文選題：坦布蘇病毒 + 種鴨��； 參考：《山東農(nóng)業(yè)大學》2015年碩士論文

【摘要】：坦布蘇病毒感染(Tembusu virus infection),是我國在2010年新出現(xiàn)的由坦布蘇病毒(Tembusu virus,TMUV)引起的一種傳染性疾病,該病主要引起產(chǎn)蛋鴨產(chǎn)蛋下降以及雛鴨出現(xiàn)頭頸震顫、四肢麻痹等神經(jīng)癥狀。目前該病在我國養(yǎng)鴨集中的地區(qū)均有發(fā)生,給養(yǎng)鴨業(yè)造成了巨大的損失(Su,et al.2011)。TMUV屬于黃病毒科(Flaviviridae)、黃病毒屬(Flavivirus)、恩塔亞病毒群(Ntaya virus group)(Cao,et al.2011)。本試驗就TMUV能否在種鴨中垂直傳播及TMUV對種鴨的致病性進行研究,為該病的控制提供可靠的流行病學依據(jù)。1、自然感染病例種鴨、種蛋、鴨胚、雛鴨中TMUV檢測2013年,山東省聊城、泰安、廣饒地區(qū)共4個種鴨場相繼發(fā)生TMUV自然感染。收集發(fā)病鴨產(chǎn)的種蛋,共125枚。從中隨機抽取35枚種蛋,無菌收取卵黃膜樣品。將其余90枚種蛋(自然感染組)全部孵化,按照孵化場中櫻桃谷種鴨蛋孵化要求設置孵化參數(shù)。同時,取20枚健康櫻桃谷種鴨蛋作為對照組進行孵化。將不同來源的種蛋分開孵化。每天照蛋一次,挑出未受精蛋和死亡鴨胚,及時無菌收取卵黃膜或胚腦、肝、脾等樣品。對出殼雛鴨在1日齡剖殺,取腦、肝、脾等組織。所有收取的樣品置于-80oC保存。通過尿囊腔途徑接種9日齡健康鴨胚進行病毒分離,采用半套式RT-PCR檢測方法對收獲的尿囊液進行病毒檢測。結果顯示,自然感染組總受精率為82.22%(74/90),總孵化率為62.16%(46/74);對照組的受精率和孵化率分別為95%(19/20)和84.21%(16/19)。在孵化過程中,28枚(31.1%)鴨胚死亡,剖檢可見胚腦充血、出血、水腫,心臟和肝臟出血。病毒檢測結果顯示,種蛋、死亡鴨胚和雛鴨中TMUV檢出率分別為51.43%(18/35)、60.71%(17/28)和10.87%(5/46)。此外,挑出的16枚未受精種蛋中有2枚檢測為TMUV陽性(12.5%)�？傆�,自然感染組的TMUV總檢出率為33.6%(42/125)。對照組的種蛋、鴨胚和雛鴨檢測均為TMUV陰性。首次從死亡鴨胚中分離到一株TMUV,命名為TMUV-SDDE株,該分離株的ELD50為10-2.50/0.2 mL。用PCR擴增TMUV-SDDE的E基因,測序。序列分析結果顯示,TMUV-SDDE與其他TMUV分離株的同源性高達96%-99.5%。經(jīng)動物回歸試驗,可以從攻毒雛鴨中重新分離到該病毒。根據(jù)本試驗結果,我們推測TMUV可能經(jīng)種蛋發(fā)生垂直傳播。2、tmuv人工感染動物模型的建立基于上述試驗結果,我們通過人工感染tmuv陰性的健康櫻桃谷種鴨建立動物模型,進行tmuv能否垂直傳播及tmuv對種鴨致病性的研究。將38周齡的櫻桃谷種鴨隨機分為a、b、c、d、e五組。a、b、c三組,每組15只母鴨、4只公鴨,公母鴨混群飼養(yǎng)。a組母鴨和b組公鴨靜脈接種tmuv-sdsg株(eld50=10-2.37/0.2ml),2.5ml/只,a組公鴨和b組母鴨不接種病毒。c組作為對照組,接種等量生理鹽水。d和e組各20只母鴨,d組靜脈接種2.5ml的tmuv-sdsg株,e組接種等量生理鹽水。各組隔離飼養(yǎng)。收集a、b、c三組種蛋,分別孵化,記錄種蛋受精和孵化情況。出殼后的1日齡雛鴨,隨機剖殺其中的一部分,取腦、肝、脾等。其余的雛鴨飼養(yǎng)于spf隔離罩中,至15日齡剖殺,取腦、肝、脾等。收取飼養(yǎng)過程中死亡雛鴨的上述組織。對收集的各組織進行處理后,接種鴨胚進行病毒分離和半套式rt-pcr檢測。結果顯示,a組種蛋的受精率和孵化率分別82.2%(74/90)和62.2%(46/74),b組種蛋的受精率和孵化率分別為78.0%(67/86)和65.7%(44/67),而c組種蛋的受精率和孵化率分別為94%(94/100)和85.1%(80/94)。a組43枚和b組37枚鴨胚在孵化過程中死亡。剖檢可見,胚體出血、卵黃吸收不良、卵黃稀薄、肝臟或心臟出血。有的鴨胚頸部水腫、出血,腦水腫、充血。a組4只和b組7只弱雛死亡,剖檢可見腦水腫、或有出血點,卵黃吸收不良。病毒檢測結果顯示,a組種蛋、死亡鴨胚、1日齡雛鴨、15日齡雛鴨和死亡弱雛中tmuv檢出率分別為60%,67.44%,35.48%,16.67%和100%。b組相應的tmuv檢出率分別為50%,51.35%,46.88%,10%和71.43%。c組各樣品未檢測到tmuv。將陽性擴增片段進行序列分析,結果與tmuv-sdsg株同源性在98.7%-100%。從種蛋、鴨胚到孵出的雛鴨均能檢測到tmuv,表明tmuv可以通過感染的種鴨經(jīng)鴨胚傳給下一代,即tmuv可以在種鴨中發(fā)生垂直傳播。3、tmuv對種鴨的致病性研究d和e組,每組隨機標記出10只,分別在1、4、7、10、13、16、19dpi采血一次,采集泄殖腔棉拭子樣品一次,用間接elisa方法檢測鴨血清中抗體水平,用細胞因子檢測試劑盒檢測ifn-γ和il-4水平,用半套式rt-pcr檢測攻毒鴨排毒情況。每組另外10只,分別在3、7、11、15、19dpi隨機剖殺兩只,無菌采集腦、肝臟、脾臟、卵泡膜等組織,制作病理切片,用熒光定量pcr檢測各組織中tmuv載量。結果顯示,攻毒后,攻毒組種鴨產(chǎn)蛋下降(約由80%至60%),排白色稀便。攻毒組種鴨抗體水平從1dpi后開始升高;至10dpi左右,達到最高水平;之后逐漸下降。IL-4和IFN-γ表達水平均在1dpi后開始上升,分別在13dpi和16dpi達到高峰,之后開始下降。對照組種鴨的抗體陰性,IFN-γ和IL-4表達水平無明顯變化。從4dpi開始能夠檢測到排毒,一直持續(xù)到試驗結束時(19dpi)檢測仍為陽性。在3dpi時,僅腦、胰臟、卵泡膜檢測為TMUV陽性;在7dpi時,卵泡膜、腦、胰臟、脾臟、肝臟、十二指腸檢測為TMUV陽性;從11dpi開始,各組織均檢測為TMUV陽性,其中卵泡膜、腦、胰臟、肝臟病毒載量較高。上述結果表明,TMUV感染種鴨后能刺激機體產(chǎn)生體液免疫和細胞免疫;病毒主要侵害種鴨的卵泡、腦、胰臟、肝臟等組織器官;攻毒后種鴨可經(jīng)泄殖腔排毒,且持續(xù)時間較久,有利于TMUV在鴨群中的傳播。
[Abstract]:Tembusu virus infection (tanbsu virus infection) is a new infectious disease caused by Tembusu virus (TMUV) in China in 2010. The disease mainly causes the decline of egg laying ducks, as well as the nervous symptoms such as head and neck tremor and paralysis of the ducks. Su, et al.2011).TMUV belongs to the family yellows (Flaviviridae), the yellow virus (Flavivirus), the Ntaa virus group (Ntaya virus group) (Cao, et al.2011). According to.1, the natural infection cases of duck, seed egg, duck embryo and duckling were detected by TMUV in 2013, 4 species of duck farms in Liaocheng, Tai'an and Guangrao of Shandong province were infected successively. 125 eggs were collected from the disease ducks, 35 eggs were extracted randomly and the yolk membrane samples were collected aseptic. The rest 90 eggs (natural infection group) were all At the same time, the hatching parameters were set according to the hatching requirements of cherry seed eggs in the hatchery. At the same time, 20 healthy cherry eggs were hatched as the control group. The eggs of different sources were incubated separately. The eggs were taken once a day, unfertilized eggs and dead duck embryos were picked out, and samples of yolk membrane or embryo brain, liver and spleen were collected and collected in time. The duck was killed at 1 days, taking brain, liver, spleen and other tissues. All the samples collected were stored in -80oC. The 9 day old healthy duck embryos were inoculated through the anantral pathway to isolate the virus. The reap was detected by a half set of RT-PCR methods. The results showed that the total fertilization rate was 82.22% (74/90) and the total hatching rate was 62.16%. (46/74); the fertilization rate and hatching rate in the control group were 95% (19/20) and 84.21% (16/19). During the incubation, 28 (31.1%) duck embryos died, and the embryo brain congestion, bleeding, edema, heart and liver hemorrhage were detected. The results of virus detection showed that the detection rates of TMUV were 51.43% (18/35), 60.71% (17/28) and 10.87% (5/) in the eggs, the dead duck embryos and the ducks. 46). In addition, 2 of the 16 unfertilized eggs were detected as TMUV positive (12.5%). Overall, the total detection rate of TMUV in the natural infection group was 33.6% (42/125). The eggs of the control group, duck embryos and ducks were all TMUV negative. A TMUV was isolated from the dead duck embryo for the first time, named TMUV-SDDE strain, and the ELD50 of the isolate was 10-2.50/0.2 mL.. The E gene of TMUV-SDDE was amplified by PCR and sequenced. The sequence analysis showed that the homology of TMUV-SDDE and other TMUV isolates reached 96%-99.5%. through animal regression test and could be re isolated from the ducklings. According to the results of the experiment, we speculated that TMUV might be vertical transmission of.2 and the construction of a tmuv artificial infection model. Based on the above test results, we set up an animal model through artificial infection of tmuv negative healthy Cherry Valley duck to study the vertical transmission of tmuv and the pathogenicity of tmuv to the species of duck. The 38 week old Cherry Valley ducks were randomly divided into a, B, C, D, e five groups.A, B, C three groups, 15 ducks in each group, 4 ducks, and male and female ducks to feed the.A group ducks. And B group ducks were inoculated with tmuv-sdsg strain (eld50=10-2.37/0.2ml), 2.5ml/ only, a group ducks and B group ducks did not inoculate the virus.C group as the control group, inoculated the same amount of normal saline.D and E group 20 female ducks, D group inoculated 2.5ml tmuv-sdsg strain, the group inoculated the same amount of physiological saline. The 1 day old ducklings of 1 days of age after the shell were randomly killed, including brain, liver, spleen, etc. the rest of the ducklings were reared in the shroud to 15 days of age to kill, take the brain, liver, spleen, etc. to collect the above tissues of the ducklings in the feeding process and inoculate the duck embryos to carry out the virus points. The fertilization rate and hatchability of the a eggs were 82.2% (74/90) and 62.2% (46/74) respectively. The fertilization rate and hatchability of the B group eggs were 78% (67/86) and 65.7% (44/67), respectively, while the fertilization rate and hatchability of the C group eggs were 94% (94/100) and 85.1% (80/94).A group 43 and 37 duck embryos in the B group during incubation. Death. It was found that the embryo was bleeding, the yolk was poorly absorbed, the yolk was thin, the liver or the heart bleed. Some ducks' embryos were edema, bleeding, brain edema, the.A group 4 and the B group died, the brain edema, or the bleeding point, and the yolk inabsorption. The test results showed that the a group eggs, the dead duck embryos, 1 day old ducks, 15 day old chicks were found. The detection rates of tmuv in ducks and deaths were 60%, 67.44%, 35.48%, 16.67% and 100%.b were 50%, 51.35%, 46.88%, 10% and 71.43%.c were not detected by tmuv. to sequence the positive amplified fragment. The homology of tmuv-sdsg strain was detected in 98.7%-100%. from seed eggs, duck embryos to hatched ducks. Tmuv showed that tmuv could pass through the infected duck embryo to the next generation by duck embryo, that is, tmuv could spread.3 vertically in the duck. Tmuv was used to study the pathogenicity of the duck in D and E group. 10 rats were randomly labeled in each group. The sample of the cloaca swab was collected once in 1,4,7,10,13,16,19dpi and the sample of the cloaca swab was collected once, and the duck blood was detected by indirect ELISA method. The level of ifn- gamma and IL-4 was detected by cytokine detection kit. Half set of RT-PCR was used to detect the detoxification of ducks. The other 10 rats in each group were killed at random in 3,7,11,15,19dpi, and the tissues of brain, liver, spleen and follicular membrane were collected asepsis, and the pathological sections were made, and the tmuv load in each tissue was detected by fluorescence quantitative PCR. The results showed that after attack, the eggs of duck in the attack group dropped (about 80% to 60%), and the level of the duck antibody in the attack group began to rise from 1dpi to about 10dpi and reached the highest level, and then the level of.IL-4 and IFN- gamma gradually declined after 1dpi and reached the peak after 13dpi and 16dpi, and then began to decline. The antibody of the group duck was negative, and there was no obvious change in the expression of IFN- gamma and IL-4. From the beginning of 4dpi, detoxification was detected, and the test was still positive at the end of the test (19dpi). At 3dpi, only the brain, pancreas, and follicular membrane were positive for TMUV; in 7dpi, the follicle membrane, the brain, pancreas, spleen, liver, duodenum were positive for TMUV, from 11dpi. 11dpi At the beginning, all the tissues were detected as TMUV positive, in which the follicular, brain, pancreas, and liver virus load was higher. The results showed that TMUV infected the duck to stimulate body humoral immunity and cell immunity, and the virus mainly infringed the follicular, brain, pancreas, liver and other groups of the duck. After attack, the duck could be detoxification through the cloaca and duration. For a long time, it is beneficial to the spread of TMUV in the duck group.
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.32

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