种鸭生殖系统新城疫病毒检测及鸭新城疫病毒垂直感染动物试验
摘要
鸭新城疫是由新城疫病毒(Newcastle disease virus, NDV)引起的一种急性、败血性传染病,该病可造成鸭的大量死亡,对养鸭业危害严重。加强对该病的诊断方法和传播方式的研究对预防和控制鸭新城疫的发生,保护和促进养鸭业的健康发展具有重要意义。本研究建立了两种新城疫病毒的检测方法,即鸭源新城疫病毒RT-PCR诊断方法和间接免疫荧光染色方法,并应用这两种方法对人工感染种鸭生殖系统新城疫病毒进行了检测,同时进行了鸭新城疫病毒垂直感染动物试验。本研究内容包括四部分：一、鸭源新城疫病毒RT-PCR诊断方法的建立与应用
根据GenBank中新城疫F基因序列设计、合成了1对引物扩增相对保守的727 bp的目的片段,建立了鸭源新城疫病毒病的RT-PCR诊断方法。该方法能从鸭源新城疫病毒SDFCH株扩增到727bp的特异性片段,而鸭瘟病毒、H9N2亚型流感病毒、鸭肝炎病毒扩增结果为阴性,敏感性试验最低检出量的cDNA浓度为3pg/μL。表明所建立的RT-PCR方法特异性强、敏感性高。应用该方法对山东不同地区20株疑似鸭新城疫病毒分离株进行检测,结果15份为阳性；对150份疑似新城疫病鸭的感染病变组织进行检测,结果125例为阳性。
二、鸭源新城疫病毒间接免疫荧光染色检测方法的建立
以抗新城疫病毒F蛋白的单克隆抗体为一抗,FITC-羊抗小鼠IgG为二抗,同时优化固定液、黏片剂、洗涤液、抗体稀释度及孵育时间、封片剂等试验条件并进行标本自发荧光对照、荧光抗体对照、吸收试验、替代试验等特异性试验,建立了间接免疫荧光染色方法。结果显示,感染NDV发病死亡鸭的输卵管不同部位、肝脏、肾脏、肺脏、脾脏、胰腺、脑、心脏等组织切片IFA检测发出明亮的黄绿色荧光,呈阳性。正常健康对照组种鸭鸭脏器组织切片IFA检测未发出黄绿色荧光,呈阴性。对鸭瘟病毒、禽流感病毒、鸭疫里默氏杆菌、Ⅰ型鸭肝炎病毒感染致死鸭的病变组织检测均无黄绿色荧光,呈阴性。结果表明建立的间接免疫荧光染色检测方法特异性强。
三、人工感染种鸭生殖系统新城疫病毒的检测
应用本实验室分离的一株经鸭胚传递的新城疫病毒SDFCH株,对42只无新城疫病毒感染的28周龄樱桃谷种鸭进行人工感染试验,试验分A、B、C 3组,A组母鸭攻毒组,B组公鸭攻毒组, C组为对照组。攻毒4 d后,每隔2 d每组各扑杀2只,采集母鸭卵巢、输卵管,公鸭采集睾丸、输精管。对上述组织进行病毒分离鉴定,并利用已建立的RT-PCR方法进行F基因的扩增与同源性分析,确定在上述组织器官中是否存在鸭源新城疫病毒SDFCH株。同时用已建立间接免疫荧光染色方法检测输卵管内新城疫病毒及病毒在输卵管的分布情况。结果发现RT-PCR方法能在种鸭生殖系统各个组织中重新分离到新城疫病毒SDFCH株,输卵管子宫部和蛋白分泌部纤毛柱状上皮的纤毛细胞和分泌细胞内有阳性荧光信号存在。
四、鸭源新城疫病毒垂直感染动物试验
应用本实验室分离的一株经鸭胚传递的新城疫病毒SDFCH株,对68只无新城疫病毒感染的28周龄樱桃谷种鸭进行人工感染试验。攻毒采集母鸭卵巢、输卵管、未产出的种蛋,以及产出后及时消毒的种蛋,出壳后的雏鸭胎粪、脑、肺等。公鸭采集睾丸、输精管、精液。结果H.E.染色后病理组织学变化,表现为输卵管毛细血管出血,黏膜脱落,IFA检测固有层淋巴细胞浸润等输卵管纤毛柱状上皮的纤毛细胞和分泌细胞内有阳性信号存在,从母鸭及公鸭生殖系统各组织、胚蛋以及80%雏鸭中能分离和检测到新城疫病毒SDFCH株。结果显示,鸭源新城疫病毒能存在于母鸭输卵管粘膜上皮细胞及公鸭精液中,并随蛋白分泌、卵黄的形成、精液的排泄而进入种蛋中,发生垂直感染,可能是造成鸭胚死亡及1日龄雏鸭发生新城疫的重要原因,关于该病垂直传播的发生机制还有待于进一步研究。
Duck-origin Newcastle disease is an acute septic infectious disease that caused by duck-origin Newcastle disease virus. The disease is mainly characterized by diarrhea, neurological symptoms, focal glandular gastric bleeding or ulcers, intestinal mucosal bleeding and necrosis of the pancreas or the spleen. It is generally believed that NDV is disseminated by horizontal transmission via alimentary canal, respiratory tract and conjunctiva in direct or indirect ways. But the study our laboratory had done shows that duck-origin Newcastle disease virus exist vertical infection which had lead to the neurosis phenomenon such as strophocephaly and circling of ducklings. In order to verify this phenomenon, duck-origin Newcastle disease virus, stain SDFCH, which were isolated from ducklings'brain of neurosis phenomenon, was made use of to challeng 28-week-old Cherry Valley ducks which were without Newcastle disease infection, then animal model of vertical infection of duck-origin NDV was established. This study contains four parts:
Part 1:Establishment and application of RT-PCR diagnostic method for duck-origin Newcastle disease
The fusion (F) gene fragment (727 bp) of Newcastle disease was amplified by RT-PCR diagnostic method using a pair of primers, which was designed according to F gene sequence of chicken Newcastle disease virus, followed by specificity test and sensitivity test. Then 20 suspected strains of duck paramyxovirus and 150 samples of infectious or diseased tissues of ducks paramyxovirus were detected by the RT-PCR method. The specificity test showed that the method could specially amplify the 727bp gene-specific fragment of duck paramyxovirus SDFCH strain, while the amplification results of duck plague virus, H9N2 subtype avian influenza virus, duck hepatitis virus were negative. Sensitivity test showed that the minimum detectable amount of cDNA concentration was 3 pg/μL.15 from the 20 isolated strains were paramyxovirus (positive) while 125 from the 150 tissue samples were positive. The established RT-PCR diagnostic method appeared to be fast, accurate, highly sensitive and specific for the purpose.
Part 2:Establishment of the indirect immuno-fluorescence assay for Newcastle disease virus
First of all, monoclonal antibody anti-fusion protein was used as 1st antibody, immunoglobulin G (IgG) labeled by fluorescein isothiocyanate (FITC) was used as 2nd antibody, at the same time, experimental condition, such as stationary liquid, stiky liquid, eluant, dilution and incubation time of antibody, mounting liquid were optimized, followed by a series of specificity tests, then the indirect fluorescence assay (IFA) was established. The results showed that histological section of NDV infected organs were IFA positive, including parts of fallopian tube, liver, kidney, lung, spleen, abdominal salivary gland, brain and heart, while the histological section of control group of healthy ducks were IFA negative. Meantime, histological section of other virus were IFA negative, duck plague virus, avain influenza virus, riemerella anatipestifer and duck hepatitis virus-I included, which showed that the established IFA method was highly specific for the purpose.
Part 3:Detection of Newcastle disease virus in the reproductive system of ducks by artificial infection
Artificial infection experiment was done to 42 twenty-eight weeks old Cherry Valley ducks which were without Newcastle disease infection. The ducks were divided into three groups namely A, B and C. In group A, female ducks were challenged by duck-origin Newcastle disease virus, strain SDFCH, In group B female ducks were challenged by duck-origin Newcastle disease virus, strain SDFCH, group C was the normal control group. After being challenged for 4 days, two ducks were killed every other day. And ovarian tissue, follicles and fallopian tube were collected from female ducks while testis vas deferens and semen were collected from drakes. Then the virus was re-isolated and F gene was amplified and analyzed by established RT-PCR method to verify the existence of the Newcastle disease virus SDFCH in these organs. And the distribution of the virus in oviduct was detected with the indirect immunofluorescence assay that was established by anti-Newcastle disease virus F protein monoclonal antibody. The result showed that strain SDFCH of the Newcastle disease virus had been detected from tissues of reproductive system of the duck and drake with RT-PCR method. Positive signal had been detected in ciliated cells of the ciliated columnar epithelium of oviduct and secretory cells by IFA.
Part 4:Animal experiment of Newcastle disease virus'vertical infection
Artificial infection test was done to 68 twenty-eight weeks old Cherry Valley ducks which were without Newcastle disease infection. After being challenged for 4 days, two ducks were killed every other day. And ovarian tissue, follicles, fallopian tube, eggs not having outputted and eggs disinfected promptly after outputting were collected from female ducks. Meconium, brain and lung were collected from ducklings. Testis vas deferens and semen were collected from drakes. Then the virus was re-isolated and F gene was amplified and analyzed by established RT-PCR method to verify the existence of the Newcastle disease virus SDFCH in these organs. And the indirect immuno-fluorescence assay (IFA) to locate antigen of Newcastle disease virus in the oviduct. Positive signal had been detected in ciliated cells of the ciliated columnar epithelium of oviduct and secretory cells by IFA. Strain SDFCH of the Newcastle disease virus had been detected from tissues of genital system of the duck and drake and 80% ducklings with RT-PCR method. The results suggest that some strains of duck-origin Newcastle disease virus could be found in oviduct epithelial cells of ducks and semen of drakes, and they might enter into the egg with protein secretion, yolk formation and sperm excretion to the occurrence of vertical infection. It might be one of the reasons of causing embryo death and outbreak of Newcastle disease in one day old ducklings, but the exact mechanism of the vertical transmission needs further research.
根据GenBank中新城疫F基因序列设计、合成了1对引物扩增相对保守的727 bp的目的片段,建立了鸭源新城疫病毒病的RT-PCR诊断方法。该方法能从鸭源新城疫病毒SDFCH株扩增到727bp的特异性片段,而鸭瘟病毒、H9N2亚型流感病毒、鸭肝炎病毒扩增结果为阴性,敏感性试验最低检出量的cDNA浓度为3pg/μL。表明所建立的RT-PCR方法特异性强、敏感性高。应用该方法对山东不同地区20株疑似鸭新城疫病毒分离株进行检测,结果15份为阳性；对150份疑似新城疫病鸭的感染病变组织进行检测,结果125例为阳性。
二、鸭源新城疫病毒间接免疫荧光染色检测方法的建立
以抗新城疫病毒F蛋白的单克隆抗体为一抗,FITC-羊抗小鼠IgG为二抗,同时优化固定液、黏片剂、洗涤液、抗体稀释度及孵育时间、封片剂等试验条件并进行标本自发荧光对照、荧光抗体对照、吸收试验、替代试验等特异性试验,建立了间接免疫荧光染色方法。结果显示,感染NDV发病死亡鸭的输卵管不同部位、肝脏、肾脏、肺脏、脾脏、胰腺、脑、心脏等组织切片IFA检测发出明亮的黄绿色荧光,呈阳性。正常健康对照组种鸭鸭脏器组织切片IFA检测未发出黄绿色荧光,呈阴性。对鸭瘟病毒、禽流感病毒、鸭疫里默氏杆菌、Ⅰ型鸭肝炎病毒感染致死鸭的病变组织检测均无黄绿色荧光,呈阴性。结果表明建立的间接免疫荧光染色检测方法特异性强。
三、人工感染种鸭生殖系统新城疫病毒的检测
应用本实验室分离的一株经鸭胚传递的新城疫病毒SDFCH株,对42只无新城疫病毒感染的28周龄樱桃谷种鸭进行人工感染试验,试验分A、B、C 3组,A组母鸭攻毒组,B组公鸭攻毒组, C组为对照组。攻毒4 d后,每隔2 d每组各扑杀2只,采集母鸭卵巢、输卵管,公鸭采集睾丸、输精管。对上述组织进行病毒分离鉴定,并利用已建立的RT-PCR方法进行F基因的扩增与同源性分析,确定在上述组织器官中是否存在鸭源新城疫病毒SDFCH株。同时用已建立间接免疫荧光染色方法检测输卵管内新城疫病毒及病毒在输卵管的分布情况。结果发现RT-PCR方法能在种鸭生殖系统各个组织中重新分离到新城疫病毒SDFCH株,输卵管子宫部和蛋白分泌部纤毛柱状上皮的纤毛细胞和分泌细胞内有阳性荧光信号存在。
四、鸭源新城疫病毒垂直感染动物试验
应用本实验室分离的一株经鸭胚传递的新城疫病毒SDFCH株,对68只无新城疫病毒感染的28周龄樱桃谷种鸭进行人工感染试验。攻毒采集母鸭卵巢、输卵管、未产出的种蛋,以及产出后及时消毒的种蛋,出壳后的雏鸭胎粪、脑、肺等。公鸭采集睾丸、输精管、精液。结果H.E.染色后病理组织学变化,表现为输卵管毛细血管出血,黏膜脱落,IFA检测固有层淋巴细胞浸润等输卵管纤毛柱状上皮的纤毛细胞和分泌细胞内有阳性信号存在,从母鸭及公鸭生殖系统各组织、胚蛋以及80%雏鸭中能分离和检测到新城疫病毒SDFCH株。结果显示,鸭源新城疫病毒能存在于母鸭输卵管粘膜上皮细胞及公鸭精液中,并随蛋白分泌、卵黄的形成、精液的排泄而进入种蛋中,发生垂直感染,可能是造成鸭胚死亡及1日龄雏鸭发生新城疫的重要原因,关于该病垂直传播的发生机制还有待于进一步研究。
Duck-origin Newcastle disease is an acute septic infectious disease that caused by duck-origin Newcastle disease virus. The disease is mainly characterized by diarrhea, neurological symptoms, focal glandular gastric bleeding or ulcers, intestinal mucosal bleeding and necrosis of the pancreas or the spleen. It is generally believed that NDV is disseminated by horizontal transmission via alimentary canal, respiratory tract and conjunctiva in direct or indirect ways. But the study our laboratory had done shows that duck-origin Newcastle disease virus exist vertical infection which had lead to the neurosis phenomenon such as strophocephaly and circling of ducklings. In order to verify this phenomenon, duck-origin Newcastle disease virus, stain SDFCH, which were isolated from ducklings'brain of neurosis phenomenon, was made use of to challeng 28-week-old Cherry Valley ducks which were without Newcastle disease infection, then animal model of vertical infection of duck-origin NDV was established. This study contains four parts:
Part 1:Establishment and application of RT-PCR diagnostic method for duck-origin Newcastle disease
The fusion (F) gene fragment (727 bp) of Newcastle disease was amplified by RT-PCR diagnostic method using a pair of primers, which was designed according to F gene sequence of chicken Newcastle disease virus, followed by specificity test and sensitivity test. Then 20 suspected strains of duck paramyxovirus and 150 samples of infectious or diseased tissues of ducks paramyxovirus were detected by the RT-PCR method. The specificity test showed that the method could specially amplify the 727bp gene-specific fragment of duck paramyxovirus SDFCH strain, while the amplification results of duck plague virus, H9N2 subtype avian influenza virus, duck hepatitis virus were negative. Sensitivity test showed that the minimum detectable amount of cDNA concentration was 3 pg/μL.15 from the 20 isolated strains were paramyxovirus (positive) while 125 from the 150 tissue samples were positive. The established RT-PCR diagnostic method appeared to be fast, accurate, highly sensitive and specific for the purpose.
Part 2:Establishment of the indirect immuno-fluorescence assay for Newcastle disease virus
First of all, monoclonal antibody anti-fusion protein was used as 1st antibody, immunoglobulin G (IgG) labeled by fluorescein isothiocyanate (FITC) was used as 2nd antibody, at the same time, experimental condition, such as stationary liquid, stiky liquid, eluant, dilution and incubation time of antibody, mounting liquid were optimized, followed by a series of specificity tests, then the indirect fluorescence assay (IFA) was established. The results showed that histological section of NDV infected organs were IFA positive, including parts of fallopian tube, liver, kidney, lung, spleen, abdominal salivary gland, brain and heart, while the histological section of control group of healthy ducks were IFA negative. Meantime, histological section of other virus were IFA negative, duck plague virus, avain influenza virus, riemerella anatipestifer and duck hepatitis virus-I included, which showed that the established IFA method was highly specific for the purpose.
Part 3:Detection of Newcastle disease virus in the reproductive system of ducks by artificial infection
Artificial infection experiment was done to 42 twenty-eight weeks old Cherry Valley ducks which were without Newcastle disease infection. The ducks were divided into three groups namely A, B and C. In group A, female ducks were challenged by duck-origin Newcastle disease virus, strain SDFCH, In group B female ducks were challenged by duck-origin Newcastle disease virus, strain SDFCH, group C was the normal control group. After being challenged for 4 days, two ducks were killed every other day. And ovarian tissue, follicles and fallopian tube were collected from female ducks while testis vas deferens and semen were collected from drakes. Then the virus was re-isolated and F gene was amplified and analyzed by established RT-PCR method to verify the existence of the Newcastle disease virus SDFCH in these organs. And the distribution of the virus in oviduct was detected with the indirect immunofluorescence assay that was established by anti-Newcastle disease virus F protein monoclonal antibody. The result showed that strain SDFCH of the Newcastle disease virus had been detected from tissues of reproductive system of the duck and drake with RT-PCR method. Positive signal had been detected in ciliated cells of the ciliated columnar epithelium of oviduct and secretory cells by IFA.
Part 4:Animal experiment of Newcastle disease virus'vertical infection
Artificial infection test was done to 68 twenty-eight weeks old Cherry Valley ducks which were without Newcastle disease infection. After being challenged for 4 days, two ducks were killed every other day. And ovarian tissue, follicles, fallopian tube, eggs not having outputted and eggs disinfected promptly after outputting were collected from female ducks. Meconium, brain and lung were collected from ducklings. Testis vas deferens and semen were collected from drakes. Then the virus was re-isolated and F gene was amplified and analyzed by established RT-PCR method to verify the existence of the Newcastle disease virus SDFCH in these organs. And the indirect immuno-fluorescence assay (IFA) to locate antigen of Newcastle disease virus in the oviduct. Positive signal had been detected in ciliated cells of the ciliated columnar epithelium of oviduct and secretory cells by IFA. Strain SDFCH of the Newcastle disease virus had been detected from tissues of genital system of the duck and drake and 80% ducklings with RT-PCR method. The results suggest that some strains of duck-origin Newcastle disease virus could be found in oviduct epithelial cells of ducks and semen of drakes, and they might enter into the egg with protein secretion, yolk formation and sperm excretion to the occurrence of vertical infection. It might be one of the reasons of causing embryo death and outbreak of Newcastle disease in one day old ducklings, but the exact mechanism of the vertical transmission needs further research.
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