羊布鲁氏菌感染绵羊的抗体、病原及组织病变动态的研究

发布时间：2018-02-28 10:27

本文关键词： 羊种布鲁氏菌细菌分布免疫组化原位杂交动态变化　出处：《中国农业大学》2015年博士论文　论文类型：学位论文

【摘要】：布鲁氏菌病(Brucellosis)是由布鲁氏菌(Brucella)引起的人畜共患病,在世界范围内造成了巨大经济损失。布鲁氏菌主要寄生于网状淋巴组织,对生殖、神经和骨骼系统造成严重的免疫系统病理反应,并常伴有菌血症。因布鲁氏菌种型复杂,受到菌株、剂量和宿主遗传背景等因素的影响较大,防控过程中迫切需要了解不同菌株与宿主相互作用的过程及其后果。为研究羊种布鲁氏菌感染的自然发病史及对动物组织器官的致病作用,本研究用羊种布鲁氏菌参考株16M人工接种10月龄绵羊并观察接种动物临床表现,于接种后第7、15、30、60、90、120、180d随机挑取感染组公母绵羊各1只击晕后放血致死解剖,记录剖检变化,同时采集血液和各组织器官样品各两份以用于病原学检测以及病理组织学、免疫组织化学(IHC)和病原体原位杂交(ISH)检测,试图通过动物感染后抗体和病原体的动态变化及分布解读临床患病动物的自然发病史,为羊种布鲁氏菌病防控和净化提供参考。研究结果显示,非妊娠绵羊人工感染后大约48h会出现一过性低烧和厌食,随后临床症状消失,表现为亚临床状态。感染早期被击晕后放血致死的动物,在大体解剖病变上仅见肝脏、体表淋巴结以及脾脏轻微或中度肿大,其他组织器官未见明显变化。病理组织学检测发现,富含网状内皮系统的免疫器官病变最明显,其中第7d病变最严重,随后逐渐减轻,感染后180d时基本恢复正常。用免疫组化(IHC)技术对上述组织样品检测结果显示,脾脏、肩前淋巴结、腮腺淋巴结和颌下淋巴结在感染早期阶段可见布鲁氏菌抗原,阳性信号主要分布于巨噬细胞和中性粒细胞的胞浆中。以原位杂交(ISH)试验对上述组织中存在的布鲁氏菌特异性DNA的检测发现,阳性信号广泛分布于心脏、肝脏、脾脏、肺脏、肾脏、淋巴结、睾丸、十二指肠、空肠、回肠、真胃、膀胱等器官组织中,感染7d时检出率最高,阳性信号最强,感染后120d仍然可从颌下淋巴结组织中检出。其中,阳性细胞主要包括巨噬细胞、中性粒细胞、各种上皮细胞以及淋巴细胞。细菌分离结果显示,感染后7d布鲁氏菌即可形成全身性感染,随后逐渐被清除,120d后所采集的组织样品无布鲁氏菌被检出,膀胱和肌肉样品在整个试验期内均未检出布鲁氏菌。整个感染过程中抗体滴度定期检测发现,感染后7d可检测到特异性凝集抗体,15d抗体滴度最高,随后开始下降,240d时抗体阴性。上述研究结果初步揭示了非妊娠状态绵羊和性成熟公羊感染羊种布鲁氏菌的自然史,即病理学变化和病原分布主要集中于富含网状内皮系统的器官和组织,感染后7d即可在所有的靶器官和组织内发现明显病变和病原分布,并随时间推移感染的器官组织中病变程度和组织病原分布及其细菌数量逐渐减弱,感染120d后基本消失。对采集样品进行四种抗原检测方法比较分析显示,ISH检测结果与经典的细菌分离技术相比敏感性更高,经技术方法优化后可望开发为布鲁氏菌抗原检测的“金标准”。本研究通过对布鲁氏菌人工感染绵羊抗体滴度、细菌分离、免疫病理学和分子病理学检测技术的对比分析,初步阐释了羊种布鲁氏菌感染后的抗体消长规律、病原分布动态变化规律及各器官组织病理学动态变化,将为羊种布鲁氏菌病防控措施的制定提供重要基础数据。
[Abstract]:Brucellosis (Brucellosis) (Brucella) caused by Brucella zoonosis, caused huge economic losses in the world. Brucella mainly parasitize on reproductive, reticular lymphatic tissue, nerve and skeletal system cause serious pathological immune system response, and often accompanied by bacteremia. Because of complex type strains of Brucella strain,, effect of dose and host genetic background and other factors greatly, there is an urgent need to understand the process and consequences of different strains and host interactions in the process of prevention and control.
For the study of Brucella melitensis infection incidence of natural history and pathogenic effects on animal tissues and organs in the study of Brucella melitensis 16M reference strains inoculated with 10 month old sheep inoculated animal and observe the clinical manifestations, in the group of male and female sheep 1 stun after bleeding to death 7,15,30,60,90120180d anatomy of the randomly selected infection after vaccination, autopsy records at the same time change, collecting blood and tissues samples of two copies each for pathogen detection and histopathology, immunohistochemistry and in situ hybridization (IHC) pathogen detection (ISH), attempts to natural disease history animal infection after interpretation of dynamic changes and distribution of pathogens in animal antibody and clinical, to provide the reference for the sheep the prevention and control of brucellosis and purification.
The results showed that the non pregnant sheep after artificial infection about 48h will appear a fever and anorexia, then the clinical symptoms disappeared and showed subclinical infection. Early after being stunned bled to death animal in anatomic lesions was observed in liver and spleen, superficial lymph node with mild or moderate swelling, obvious change no other tissues. The pathological detection showed that the most obvious immune organ disease in the reticuloendothelial system, in which the 7d lesion is the most serious, then gradually reduce the infection after 180d returned to normal. By immunohistochemistry (IHC) technique shows that the spleen tissue sample test results, the shoulder lymph nodes, the lymph node of the parotid gland and submandibular lymph nodes in the early phase of infection of Brucella antigen visible cytoplasm, the positive signal was mainly distributed in macrophages and neutrophils. In situ hybridization (ISH) test on the Detection of Brucella specific DNA in the organization, the positive signal is widely distributed in the heart, liver, spleen, lung, kidney, lymph node, testis, duodenum, jejunum, ileum, stomach, bladder and other organs and tissues, the highest detection rate of 7D infection, the positive signal is the strongest, 120d can still be infected the detection from the submandibular lymph node tissues. The positive cells including macrophages, neutrophils, various epithelial cells and lymphocytes.
The results of bacterial isolates showed 7d after infection of Brucella can form a systemic infection, then gradually cleared and collected 120d after tissue samples without Brucella were detected, and the bladder muscle samples in the whole experimental period were not detected. The detection of anti Brucella found regular titers during infection, 7d post infection can be detected agglutination antibody, 15d antibody titer, then began to decline, 240d antibody negative.
The preliminary results revealed the natural history of pregnant sheep and mature rams infected with Brucella melitensis, namely the pathological changes and the pathogens are mainly concentrated in the rich in the reticuloendothelial system organs and tissues, 7d after infection can all target organs and tissues was found in lesions and pathogen distribution, severity and tissue pathogen the distribution and quantity of bacteria and over time infected organs gradually weakened and disappeared after 120d infection. Four kinds of antigen detection method for the sample comparative analysis showed that the separation technology of bacterial ISH detection results compared with classical higher sensitivity, the technology of the optimized method can be developed as the gold standard for detection of Brucella antigen ".
The separation of bacteria based on the artificial infection of sheep brucella antibody titers, comparative analysis of detection technology of immunopathology and molecular pathology, a preliminary explanation of the Brucella melitensis infection after antibody dynamics, dynamic change rule and the dynamic change of the distribution of pathogenic organ pathology, will provide an important basis for formulating measures for sheep data the prevention and control of brucellosis.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S858.26

【参考文献】