坦布苏病毒在种鸭中垂直传播及其对种鸭的致病性研究

发布时间：2018-06-29 02:32

本文选题：坦布苏病毒 + 种鸭　；参考：《山东农业大学》2015年硕士论文

【摘要】：坦布苏病毒感染(Tembusu virus infection),是我国在2010年新出现的由坦布苏病毒(Tembusu virus,TMUV)引起的一种传染性疾病,该病主要引起产蛋鸭产蛋下降以及雏鸭出现头颈震颤、四肢麻痹等神经症状。目前该病在我国养鸭集中的地区均有发生,给养鸭业造成了巨大的损失(Su,et al.2011)。TMUV属于黄病毒科(Flaviviridae)、黄病毒属(Flavivirus)、恩塔亚病毒群(Ntaya virus group)(Cao,et al.2011)。本试验就TMUV能否在种鸭中垂直传播及TMUV对种鸭的致病性进行研究,为该病的控制提供可靠的流行病学依据。1、自然感染病例种鸭、种蛋、鸭胚、雏鸭中TMUV检测2013年,山东省聊城、泰安、广饶地区共4个种鸭场相继发生TMUV自然感染。收集发病鸭产的种蛋,共125枚。从中随机抽取35枚种蛋,无菌收取卵黄膜样品。将其余90枚种蛋(自然感染组)全部孵化,按照孵化场中樱桃谷种鸭蛋孵化要求设置孵化参数。同时,取20枚健康樱桃谷种鸭蛋作为对照组进行孵化。将不同来源的种蛋分开孵化。每天照蛋一次,挑出未受精蛋和死亡鸭胚,及时无菌收取卵黄膜或胚脑、肝、脾等样品。对出壳雏鸭在1日龄剖杀,取脑、肝、脾等组织。所有收取的样品置于-80oC保存。通过尿囊腔途径接种9日龄健康鸭胚进行病毒分离,采用半套式RT-PCR检测方法对收获的尿囊液进行病毒检测。结果显示,自然感染组总受精率为82.22%(74/90),总孵化率为62.16%(46/74);对照组的受精率和孵化率分别为95%(19/20)和84.21%(16/19)。在孵化过程中,28枚(31.1%)鸭胚死亡,剖检可见胚脑充血、出血、水肿,心脏和肝脏出血。病毒检测结果显示,种蛋、死亡鸭胚和雏鸭中TMUV检出率分别为51.43%(18/35)、60.71%(17/28)和10.87%(5/46)。此外,挑出的16枚未受精种蛋中有2枚检测为TMUV阳性(12.5%)。总计,自然感染组的TMUV总检出率为33.6%(42/125)。对照组的种蛋、鸭胚和雏鸭检测均为TMUV阴性。首次从死亡鸭胚中分离到一株TMUV,命名为TMUV-SDDE株,该分离株的ELD50为10-2.50/0.2 mL。用PCR扩增TMUV-SDDE的E基因,测序。序列分析结果显示,TMUV-SDDE与其他TMUV分离株的同源性高达96%-99.5%。经动物回归试验,可以从攻毒雏鸭中重新分离到该病毒。根据本试验结果,我们推测TMUV可能经种蛋发生垂直传播。2、tmuv人工感染动物模型的建立基于上述试验结果,我们通过人工感染tmuv阴性的健康樱桃谷种鸭建立动物模型,进行tmuv能否垂直传播及tmuv对种鸭致病性的研究。将38周龄的樱桃谷种鸭随机分为a、b、c、d、e五组。a、b、c三组,每组15只母鸭、4只公鸭,公母鸭混群饲养。a组母鸭和b组公鸭静脉接种tmuv-sdsg株(eld50=10-2.37/0.2ml),2.5ml/只,a组公鸭和b组母鸭不接种病毒。c组作为对照组,接种等量生理盐水。d和e组各20只母鸭,d组静脉接种2.5ml的tmuv-sdsg株,e组接种等量生理盐水。各组隔离饲养。收集a、b、c三组种蛋,分别孵化,记录种蛋受精和孵化情况。出壳后的1日龄雏鸭,随机剖杀其中的一部分,取脑、肝、脾等。其余的雏鸭饲养于spf隔离罩中,至15日龄剖杀,取脑、肝、脾等。收取饲养过程中死亡雏鸭的上述组织。对收集的各组织进行处理后,接种鸭胚进行病毒分离和半套式rt-pcr检测。结果显示,a组种蛋的受精率和孵化率分别82.2%(74/90)和62.2%(46/74),b组种蛋的受精率和孵化率分别为78.0%(67/86)和65.7%(44/67),而c组种蛋的受精率和孵化率分别为94%(94/100)和85.1%(80/94)。a组43枚和b组37枚鸭胚在孵化过程中死亡。剖检可见,胚体出血、卵黄吸收不良、卵黄稀薄、肝脏或心脏出血。有的鸭胚颈部水肿、出血,脑水肿、充血。a组4只和b组7只弱雏死亡,剖检可见脑水肿、或有出血点,卵黄吸收不良。病毒检测结果显示,a组种蛋、死亡鸭胚、1日龄雏鸭、15日龄雏鸭和死亡弱雏中tmuv检出率分别为60%,67.44%,35.48%,16.67%和100%。b组相应的tmuv检出率分别为50%,51.35%,46.88%,10%和71.43%。c组各样品未检测到tmuv。将阳性扩增片段进行序列分析,结果与tmuv-sdsg株同源性在98.7%-100%。从种蛋、鸭胚到孵出的雏鸭均能检测到tmuv,表明tmuv可以通过感染的种鸭经鸭胚传给下一代,即tmuv可以在种鸭中发生垂直传播。3、tmuv对种鸭的致病性研究d和e组,每组随机标记出10只,分别在1、4、7、10、13、16、19dpi采血一次,采集泄殖腔棉拭子样品一次,用间接elisa方法检测鸭血清中抗体水平,用细胞因子检测试剂盒检测ifn-γ和il-4水平,用半套式rt-pcr检测攻毒鸭排毒情况。每组另外10只,分别在3、7、11、15、19dpi随机剖杀两只,无菌采集脑、肝脏、脾脏、卵泡膜等组织,制作病理切片,用荧光定量pcr检测各组织中tmuv载量。结果显示,攻毒后,攻毒组种鸭产蛋下降(约由80%至60%),排白色稀便。攻毒组种鸭抗体水平从1dpi后开始升高;至10dpi左右,达到最高水平;之后逐渐下降。IL-4和IFN-γ表达水平均在1dpi后开始上升,分别在13dpi和16dpi达到高峰,之后开始下降。对照组种鸭的抗体阴性,IFN-γ和IL-4表达水平无明显变化。从4dpi开始能够检测到排毒,一直持续到试验结束时(19dpi)检测仍为阳性。在3dpi时,仅脑、胰脏、卵泡膜检测为TMUV阳性;在7dpi时,卵泡膜、脑、胰脏、脾脏、肝脏、十二指肠检测为TMUV阳性;从11dpi开始,各组织均检测为TMUV阳性,其中卵泡膜、脑、胰脏、肝脏病毒载量较高。上述结果表明,TMUV感染种鸭后能刺激机体产生体液免疫和细胞免疫;病毒主要侵害种鸭的卵泡、脑、胰脏、肝脏等组织器官;攻毒后种鸭可经泄殖腔排毒,且持续时间较久,有利于TMUV在鸭群中的传播。
[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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.32

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