地方品种JS鸡多发肿瘤病的病原学探究

发布时间：2018-06-15 01:14

本文选题：JS鸡 + 肿瘤病　；参考：《山东农业大学》2017年硕士论文

【摘要】：马立克病(MD)、禽白血病(AL)和网状内皮组织增生病(RE)是鸡的三大肿瘤病,近年,由于国外品种的引进、品种间的杂交以及家禽业集约化饲养的不断发展,MD、AL和RE的感染情况趋于复杂化,发病率也越来越高。通过流行病学调查发现,在国外品种和我国众多地方品种中,三种肿瘤性疾病存在单一或多重感染的情况。2010年JS鸡被上海世博会指定为“安全准入产品”,2013年被评为“省名牌产品”。自2015年以来,对JS鸡进行三次疾病的诊断,发现其在不同日龄特别是产蛋期不断发生肿瘤病,肿瘤的频发对JS鸡的日常生产和种群繁育造成了很大影响。为探讨该品种易发肿瘤病的原因,本研究对送检疑似发生肿瘤病的JS鸡进行临床观察与剖检,并将病变组织制作病理切片;肝、肾等病变器官混合研磨,研磨液接种CEF进行细胞培养,应用间接免疫荧光试验(IFA)在培养10 d的CEF(期间传代一次)上检测REV,细胞上清采用酶联免疫吸附反应(ELISA)检测ALV-p27抗原。提取研磨后固体及细胞培养物的DNA,用针对REV(LTR、env)、ALV(A、B、C、D、E和J亚型的6对引物)以及MDV(meq、132bp,鉴别野毒与疫苗毒)进行PCR检测。同时从发病鸡群采集病鸡血液分离MDV、ALV,对获得的MDV、ALV进行动物回归实验进一步验证。剖检发现JS鸡心、肝、肾有白色局灶性或弥漫性肿瘤结节,初步判定发生肿瘤病;切片观察到肝脏发生淋巴细胞浸润,心脏有明显的淋巴细胞瘤,肺脏、肾脏静脉发生髓样细胞增生;PCR检测研磨液接种CEF进行培养的细胞和组织中的病原,结果显示,组织中MDV阳性率为50%(2/4),ALV阳性率为25%(1/4),REV阴性。细胞中ALV阳性率为75%(3/4),REV阴性。测序显示MDV为野毒,ALV为J亚型;ELISA检测细胞上清中ALV-p27抗原,阳性率为25%(1/4);IFA检测REV为阴性。检测结果表明,JS鸡肿瘤病主要由MDV和ALV-J引起。动物回归试验:MDV和ALV-J分离株人工接种1日龄SPF鸡,5日龄自血液中分离到MDV和ALV-J,用鉴别引物进行PCR扩增和IFA(鉴别MDV野毒的H19单抗)检测,并将PCR扩增的片段进行测序。结果显示该MDV为野毒株,ALV为J亚型,表明MDV和ALV-J分离株可感染SPF鸡,具有完整的生物学活性。本研究从病原学方面对JS鸡易发肿瘤病的原因进行探究,确定了MDV野毒和ALV-J是引发肿瘤病的病原,这一研究结果对JS鸡肿瘤病的防制提供了科学依据。
[Abstract]:Marek's disease (MDD), Avian Leukemia (ALL) and reticuloendotheliosis (REE) are the three major oncology diseases in chickens. In recent years, due to the introduction of foreign species, The cross between breeds and the continuous development of intensive breeding in poultry industry the infection of MDL AL and RE tends to be complicated and the incidence of disease is increasing. Through epidemiological investigation, it is found that, among foreign varieties and many local varieties in China, JS chickens were designated as "safe entry products" by the Shanghai World Expo in 2010 and "provincial famous brand products" in 2013. Since 2015, the disease of JS chicken has been diagnosed three times, and it is found that the disease occurs continuously at different ages, especially in laying stage, and the frequent occurrence of tumor has a great influence on the daily production and population breeding of JS chicken. In order to investigate the causes of the disease, the JS chickens suspected to have tumor diseases were observed and dissected, and pathological sections were made, and liver, kidney and other diseased organs were mixed and ground. CEF was inoculated with grinding fluid for cell culture. Indirect immunofluorescence assay (IFA) was used to detect the rev at CEF (once during passage) for 10 days. The supernatant was detected for ALV-p27 antigen by enzyme linked immunosorbent assay (ELISA). The DNA of the ground solid and cell cultures was extracted and detected by PCR using 6 pairs of primers for the subtypes of ALVN and J) and MDV meqfen 132bp for identification of wild and vaccine viruses. At the same time, the blood samples were collected from the infected chickens to isolate the MDV and ALV, which were further verified by animal regression test. JS chicken heart, liver and kidney were found to have white focal or diffuse tumor nodules, which were preliminarily diagnosed as oncology, and lymphocytic infiltration was observed in the liver, and obvious lymphocytoma and lung were observed in the heart. Myeloid cell proliferation in renal vein was detected by PCR. The results showed that the positive rate of MDV in the tissue was 50 / 2 / 4 / 4. The positive rate of MDV was 25 / 4 / 4. The positive rate of ALV in cells was 75 / 4 / 4. The results of sequencing showed that MDV was wild virus ALV and ALV was subtype J Elisa to detect ALV-p27 antigen in supernatant. The positive rate of MDV was 25 / 4 / 4 and the REV was negative by IFA. The results showed that MDV and ALV-J were the main causes of JS tumor disease. MDV and ALV-Js were isolated from blood of SPF chickens at 5 days old after inoculation with strain 1: MDV and ALV-J by animal regression test. PCR amplification and IFA (H19 McAb for identification of MDV wild virus) were carried out with identification primers, and the amplified fragments were sequenced. The results showed that the MDV was a wild virus strain ALV was subtype J which indicated that MDV and ALV-J isolates could infect SPF chickens and had complete biological activity. In this study, the etiology of JS chicken oncology was studied, and MDV wild virus and ALV-J were found to be the causative agents of JS chicken oncology. The results provided scientific basis for the prevention and control of JS chicken oncology.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31

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