【作者】 张舍郁；

【导师】 汪铭书； 程安春；

【作者基本信息】 四川农业大学 ， 预防兽医， 2007， 硕士

【摘要】 本文开展了间接免疫荧光(Indirect Immunofluorescence Assay，IFA)检测石蜡切片与冰冻切片中鸭病毒性肿头出血症病毒(Duck Swollenhead HaemorrhagicDisease Virus，DSHDV)方法建立，应用IFA检测DSHDV在实验感染鸭体内侵染规律等系列研究，获如下结果：1 IFA检测石蜡切片与冰冻切片中鸭病毒性肿头出血症病毒方法建立及其比较用差速离心纯化DSHDV，将纯化DSHDV免疫家兔制备兔抗DSHDV高免血清，并以High Q阴离子柱层析纯化出兔抗DSHDV IgG，建立间接免疫荧光(IFA)检测石蜡切片与冰冻切片中DSHDV的方法；利用建立的IFA对28日龄鸭人工感染DSHDV死亡鸭不同组织器官以进行检测。IFA检测DSHDV感染死亡鸭石蜡切片肝脏为阳性，检测鸭瘟病毒、禽流感病毒(H5N1)、鸭病毒性肝炎病毒、鸭疫里默氏杆菌感染死亡鸭肝脏呈阴性；IFA检测28日龄人工感染DSHDV死亡鸭的不同组织器官石蜡切片，心、肝、脾、胰、肺、肾、法氏囊、食管、气管、胸肌、胸腺、十二指肠、空肠、回肠、盲肠和直肠为阳性或强阳性；该法具有直观、特异性强的优点，应用于DSHDV在感染鸭组织细胞中的亚细胞定位具有良好效果，可用于DSHDV感染的实验室诊断、病原在鸭组织细胞中分布研究。IFA检测28日龄人工感染DSHDV死亡鸭的不同组织器官冰冻切片，心、肝、脾、胰、肺、肾、法氏囊、食管、气管、胸肌、胸腺、哈德氏腺、十二指肠、空肠、回肠、盲肠和直肠为阳性或强阳性；该法敏感性高于石蜡切片且能快速出结果，可用于临床快速诊断研究。2 DSHDV在实验感染鸭体内侵染规律的研究2．1 DSHDV实验感染鸭临床病理学发展规律采用肌注、口服和滴鼻三种途径感染28日龄北京鸭。感染鸭发病症状：病鸭初期精神沉郁，不愿走动，随着病程的加剧，卧地不起，食欲减退，被毛凌乱，个别鸭流血泪，鼻腔流血性分泌物(90％)。并开始出现特征性症状：头部肿胀(100％)，眼结膜充血、出血呈红色，排灰白色或草绿色稀粪甚至血便。眼观病理变化：心冠脂肪有少量出血斑。头部颈部下黄色胶冻样水肿(80％)。胸腺出血严重，表面和切面有大小不等坏死点(70％)。肝脏呈橙黄色或土黄色，表面有大小不等的出血斑(85％)。心包有淡黄色积液(50％)。肾脏弥漫性出血。气管环状出血，滴鼻组多见(70％)。法氏囊严重出血。食道弥漫性出血形成条状出血带，口服组多见。泄殖腔黏膜初期有出血，后期坏死(50％)。脾肿大且呈深黑色，其上有白色坏死灶(50％)。2．2 DSHDV在实验感染鸭体内侵染规律2．2．1肌注组侵染规律经肌注感染90min后最早能从肾脏中检出病毒抗原；感染后6d仅不能从皮肤、脑、气管、哈德氏腺和腺胃中检测出抗原，该时间段也是病毒达到分布的高峰时期；从不同的时间的检测情况可以看出，经肌注感染病毒在鸭体内分布最为广泛的时间段为感染后12h～6d。2．2．2口服组侵染规律DSHDV经口服途径感染初期肾脏、肠道、胸腺、法氏囊的阳性信号较强，而肺脏、心脏阳性信号弱。感染中期大多数组织都检测到了阳性信号，其中肾脏、肠道阳性信号有所增强，脾脏、肌肉、气管开始检测到阳性；哈德氏腺、脑、皮肤、腺胃在感染的整个过程都无阳性信号出现。2．2．3滴鼻组侵染规律DSHDV经滴鼻感染后4h最早能从肾脏肾小管上皮细胞浆中检出病毒抗原；感染后12h，能够从肝脏、肾脏、十二指肠和回肠中检出病毒抗原：感染后3d，仅不能从脑、腺胃、皮肤、哈德氏腺中检测出抗原。所以经过滴鼻途径感染DSHDV后24h～6d时间段是病毒达到分布的高峰时期；从不同的时间的检测情况可以看出，经滴鼻感染病毒在鸭体内分布最为广泛的时间段为感染后3d～6d。综上所述，鸭胚是DSHDV增殖的良好宿主平台，运用建立的IFA对人工感染DSHDV后石蜡切片与冰冻切片中病毒在鸭体内的分布规律进行研究表明：IFA检测28日龄鸭人工感染DSHDV死亡鸭的心、肝、脾、胰、肺、肾、法氏囊、食管、气管、胸肌、胸腺、哈德氏腺、十二指肠、空肠、回肠、盲肠和直肠为阳性；脑、皮肤、腺胃为阴性；阳性组织中病毒抗原主要分布在心脏心肌膜心肌纤维的肌浆中，肝脏肝小叶间的结缔组织和肝索区，胰腺外分泌部腺泡上皮细胞和腺泡心细胞的胞浆中，肾脏肾小管上皮细胞浆，法氏囊粘膜上皮细胞浆，食管黏膜肌层，十二指肠、空肠、直肠、回肠、盲肠肠绒毛上皮细胞浆、肠腺细胞浆中。肠道、肾脏、法氏囊和胸腺是DSHDV侵害的主要靶器官。更多还原

【Abstract】 This article has established IFA for the Detection of DSHDV in Paraffin-embedded Tissues & Frozen Section. The series studies has applied in detecting the infection characteristics of virus in Ducks. Attains following result:1 A comparative study for DSHDV detection in Paraffin-embedded tissues and frozen section by IFADSHDV was purified by differential centrifugation and rabbit anti-DSHDV antibodies were obtained from rabbits be vaccinated with purified viruses. Then the rabbit anti-DSHDV IgG were purified through High-Q chromotography. IFA was established and used to detect DSHDV antigen in paraffin-embedded tissues of 28-day-old death ducks which experimentally infected with DSHDV. The results were positive in dead ducks infected with DSHDV and negative in dead ducks infected with Duck Plague Virus、Avian influenza virus (H5N1)、Duck viral hepatitis virus and Riemerella Anatipestifer. IFA was also applied in detection of the DSHDV antigen in different organs of artificially infected dead 28 old days ducks, and the antigen was detected in the heart、liver、spleen、pancreas、lung、kidney、bursa of fabricius、esophagus、trachea、muscle、thymus、duodenum、jejunum、ileum、cecum and rectum. The establishment of IFA to the detection DSHDV in paraffin-embedded tissues had good specificity, can be applied in diagnosing and studying the distribution of the DSHDV in duck tissues. IFA was also applied in detection of the DSHDV antigen in Frozen Section of artificially infected dead 28 old days ducks, and the viral antigen was detected in the heart、liver、spleen、pancreas、lung、kidney、bursa of fabricius、esophagus、trachea、muscle、thymus、duodenum、jejunum、ileum、cecum、harderian gland and rectum. IFA to the detection DSHDV in Frozen Section’s sensitivity is higher than in the paraffin-embedded tissues can be applied in clinical diagnosing.2 The study of the Infection Characteristics of DSHDV in Ducks2.1 The law of development of clinicopathologic by DSHDV infected28-day-old beijing ducks which experimentally infected with DSHDV by inject、oral、intranasally. The infected ducks Symptom: Initial period ducks spirit strongly fragrant, not willing to take a walk, Along with course of an illness aggravating, Lies does not get up, anorexia, feather disorderly, some ducks flows tears of blood, nose flows blood secretion (90%). Starts to appear characteristic value symptom: swollen head (100%), conjunctiva hyperaemia or hemorrage, diarrhea with green dejecta even blood excrement. Observations of pathology transformations: Heart crown fat had hemorrhages, frozen type secretion fluid from Subcutaneous (80%). Thymus haemorrhage and the surface and the section had necrosis spots (70%). Yellow and swollen liver with haemorrhage (85%). Heart had yellow accumulates fluid (50%). kidney diffused bleeds. Trachea ring bleeding sees in intranasally (70%). Bursa of fabricius had bleeding. Esophagus diffused hemorrhage forms the strip shape hemorrhage sees in oral. Cloaca mucosal bleed, later ecrosis. Splenomegaly pesents duck blank, above had massive white necrosis stove (50%).2.2 The Infection Characteristics of DSHDV in Ducks2.2.1 The Infection Characteristics in inject groupDSHDV can be detected from kidney 90mpi by injected; DSHDV can be detedted from all tissues excepte for skin、brain、trachea、harderian gland and proventriculus at 6dpi, This time section also is the virus achieves the distribution peak time. from the different time detection situation we can see the distribution rate of inject inoculation beteen 12h～6dpi.2.2.2 The Infection Characteristics in oral groupDSHDV infected by orally, kidney、Intestines、bursa of fabricius、thymus had strong positive, lung、heart had weak positive. Infection intermediate stage, most of tissues were detected positive, in among kidney and intestines had strengthens positive, spleen、muscle、trachea begins detected positive. During the whole process harderian gland、brain、skin and proventriculus were negative.2.2.3 The Infection Characteristics in intranasal groupDSHDV can be detected from kidney in the cytoplasm of renal tubular epithelial cells 4hpi; DSHDV can be detedted from liver, kidney, duodenum, ileum 12hpi; DSHDV can’t be detected only in brain、proventriculus、skin and harderian gland 3dpi. So the the virus achieves the distribution peak time of intranasally inoculation beteen 24h～6dpi. from the different time detection situation we can see the distribution rate of intranasally inoculation beteen 3d～6dpi.In summary, The duck embryo is good host platform which DSHDV multiplies, Used establishment of IFA for the Detection distributed rule of DSHDV in Paraffin-embedded Tissues & Frozen Section: IFA was detected of the DSHDV antigen in Paraffin-embedded Tissues & Frozen Section of artificially infected dead 28 old days ducks, and the viral antigen was detected in the heart、liver、spleen、pancreas、lung、kidney、bursa of fabricius、esophagus、trachea、muscle、thymus、duodenum、jejunum、ileum、cecum、harderian gland and rectum. brain、skin and proventriculus are negative. DSHDV mainly located in the cytoplasm of myocardial fibroblasts, sinus hepaticus in liver, cytoplasm of centroacinar cells in pancreas, cytoplasm of renal tubular epithelial cells in kidney, cytoplasm of epidermic cell of bursa cloacalis, cytoplasm of intestinal epithelial cells in duodenum、jejunum、ileum、cecum and rectum. The target organ that DSHDV mainly attacked were intestines、kidney、bursa of fabricius and thymus.更多还原