间接免疫荧光检测石蜡切片中鸭肿头出血症病毒及抗原定位方法的初步建立

 【目的】建立间接免疫荧光（IFA）检测石蜡切片中鸭肿头出血症病毒（DSHDV）的方法，为DSHDV感染的实验室诊断、DSHDV在感染鸭组织细胞中的亚细胞定位和动态研究提供有效的检测手段。【方法】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DEAE-SephadexA-50柱层析纯化出兔抗DSHDV IgG，建立IFA检测石蜡切片中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及临床样品进行检测。应用PAGE电泳分析DSHDV基因组特性。【结果】IFA最佳条件为：切片在0.01 mol•L-1 柠檬酸（pH 6.0）缓冲液微波修复20 min后，以10%马血清37℃下封闭30 min，然后加入1﹕50的一抗4℃孵育过夜，最后加入1﹕100 FITC标记的含0.01%伊文斯蓝的二抗37℃孵育30 min。IFA检测DSHDV感染死亡鸭肝脏石蜡切片为阳性，而检测鸭瘟、禽流感病毒(H5N1)、鸭病毒性肝炎、鸭疫里默氏杆菌感染死亡鸭肝脏石蜡切片为阴性。IFA检测28日龄人工感染DSHDV死亡鸭的不同组织器官，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；哈德氏腺、脑、皮肤、腺胃为阴性。阳性组织中病毒抗原主要分布在细胞浆。经甲醛固定保持1～6年的临床死亡鸭的病毒分离阳性肝脏，IFA检测结果也呈阳性。DSHDV具有呼肠孤病毒特征，有10条分节段核酸，呈“334”分布。【结论】建立的检测石蜡组织切片中DSHDV抗原的IFA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。DSHDV抗原存在感染细胞浆，肠道、肾脏、法氏囊和胸腺是DSHDV侵害的主要靶器官。

Preliminary Establishment of the Indirect Immunofluorescence Assay for Detecting DSHDV in Paraffin-Embedded Tissues and Antigen Localization

【Objective】To establish an Indirect Immunofluorescence assay (IFA) technique for the detection of Duck Swollen Head Haemorrhagic Disease Virus (DSHDV) in paraffin-embedded tissue,and provide an effective examination method for the diagnosis, subcellular antigen localization and dynamic distribution in vivo of DSHDV.【Method】Through differential centrifugation purified DSHDV,DSHDV-specofic antibodies were obtained from rabbits vaccinated with purified virus, and then purified through DEAE-SephadexA-50 columns. An IFA was established to detect the DSHDV antigen in paraffin-embedded tissue. then DSHDV-antigen was detected in each organ tissues of 28-day-old ducks which were experimentally infected with DSHDV.【Result】The optimum conditions of this IFA were as follows: Antigen was retrieved by microwave in 0.01 mmol/L citrate buffer solution(PH6.0) for twenty minutes, and blocked with 10% horse serum for 30min at 37℃.Slices were incubated overnight at 4℃ with diluted primary antibody(1:50) ,and then incubated for 30 min at 37℃ with diluted FITC-labelled-secondary antibody(1:100)which contain 0.01% Evans blue. The IFA was applied in detecting the DSHDV antigen in liver tissues of dead ducks, and the results were positive in DSHDV infected dead ducks and negative in dead ducks infected by Duck Plague Virus, Duck viral hepatitis Virus and Riemerella Anatipestifer. The IFA was also applied in detecting the virulent DSHDV antigen in different organs 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 and rectum, while viral antigen was not detected in the harderian gland、brain、skin and proventriculus. The antigen distributes mainly in the cytoplasm of the infected cells. 【Conclusion】The establishment of IFA is a sensitive and specific method in detecting DSHDV antigen in paraffin-embedded tissue, and it’s a good method in diagnosis and antigen location of DSHDV. IFA can application in the experimentally diagnosis and study on the antigen distribution dynamics in duck tissue cells.