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

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

【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DSHDVIgG,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0.01mol·L-1柠檬酸(pH6.0)缓冲液微波修复20min后,以10%马血清37℃下封闭30min,然后加入1﹕50的一抗4℃孵育过夜,最后加入1﹕100FITC标记的含0.01%伊文斯蓝的二抗37℃孵育30min。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。

【Objective】The objective of this study is to establish an indirect immunofluorescence assay （IFA） as a detection method of duck swollen head haemorrhagic disease virus （DSHDV） in paraffin-embedded tissue, subcellular antigen localization and dynamic distribution of DSHDV in vivo.【Method】DSHDV was purified by differential centrifugation and rabbit anti-DSHDV antibodies were obtained from rabbits vaccinated with purified viruses. Then the rabbit anti-DSHDV IgG was purified through DEAE-SephadexA-50 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 and clinical death ducks. PAGE was used to analyze the DSHDV nucleic acid genome characteristics.【Result】The optimum conditions of IFA were as follows： Antigen was retrieved by microwave in 0.01 mol·L^-1 citrate buffer solution （pH 6.0） for twenty minutes, and blocked with 10% horse serum at 37 for 30 ℃ min. Slices were incubated overnight at 4 with d ℃ iluted primary antibody （1﹕50）, and then incubated at 37 for 30 min with diluted ℃ FITC-labelled-secondary antibody （1﹕100） which contains 0.01% Evans blue. IFA was adopted in detection of DSHDV antigen in the liver of dead ducks. 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 abopted in detection of the virulent DSHDV antigen in different organs of artificially infected dead 28-day-old 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 antigens was not detected in the harderian gland, brain, skin and proventriculus. The antigens distributed mainly in the cytoplasm of the infected cells. The result of IFA detected the positive liver of virus isolation from the clinical dead ducks which were formaldehyde-fixed and stored for 1-6 years is positive. DSHDV genome was related to reovirus, had ten segements of dsRNA, showed “334” distribution pattern.【Conclusion】IFA is a sensitive and specific method for the detection of DSHDV in paraffin-embedded tissues. It is effective in studying the antigen locations of DSHDV. IFA can be applied in diagnosing and studying the distribution of the DSHDV in duck tissues. The DSHDV antigens distributed in the cytoplasm of the infected cells. The target organ that DSHDV mainly attacked are intestines, kidney, bursa of fabricius and thymus.