發(fā)布時間：2018-02-28 10:27

  本文關鍵詞： 羊種布魯氏菌 細菌分布 免疫組化 原位雜交 動態(tài)變化　出處：《中國農(nóng)業(yè)大學》2015年博士論文　論文類型：學位論文

【摘要】：布魯氏菌病(Brucellosis)是由布魯氏菌(Brucella)引起的人畜共患病,在世界范圍內(nèi)造成了巨大經(jīng)濟損失。布魯氏菌主要寄生于網(wǎng)狀淋巴組織,對生殖、神經(jīng)和骨骼系統(tǒng)造成嚴重的免疫系統(tǒng)病理反應,并常伴有菌血癥。因布魯氏菌種型復雜,受到菌株、劑量和宿主遺傳背景等因素的影響較大,防控過程中迫切需要了解不同菌株與宿主相互作用的過程及其后果。 為研究羊種布魯氏菌感染的自然發(fā)病史及對動物組織器官的致病作用,本研究用羊種布魯氏菌參考株16M人工接種10月齡綿羊并觀察接種動物臨床表現(xiàn),于接種后第7、15、30、60、90、120、180d隨機挑取感染組公母綿羊各1只擊暈后放血致死解剖,記錄剖檢變化,同時采集血液和各組織器官樣品各兩份以用于病原學檢測以及病理組織學、免疫組織化學(IHC)和病原體原位雜交(ISH)檢測,試圖通過動物感染后抗體和病原體的動態(tài)變化及分布解讀臨床患病動物的自然發(fā)病史,為羊種布魯氏菌病防控和凈化提供參考。 研究結果顯示,非妊娠綿羊人工感染后大約48h會出現(xiàn)一過性低燒和厭食,隨后臨床癥狀消失,表現(xiàn)為亞臨床狀態(tài)。感染早期被擊暈后放血致死的動物,在大體解剖病變上僅見肝臟、體表淋巴結以及脾臟輕微或中度腫大,其他組織器官未見明顯變化。病理組織學檢測發(fā)現(xiàn),富含網(wǎng)狀內(nèi)皮系統(tǒng)的免疫器官病變最明顯,其中第7d病變最嚴重,隨后逐漸減輕,感染后180d時基本恢復正常。用免疫組化(IHC)技術對上述組織樣品檢測結果顯示,脾臟、肩前淋巴結、腮腺淋巴結和頜下淋巴結在感染早期階段可見布魯氏菌抗原,陽性信號主要分布于巨噬細胞和中性粒細胞的胞漿中。以原位雜交(ISH)試驗對上述組織中存在的布魯氏菌特異性DNA的檢測發(fā)現(xiàn),陽性信號廣泛分布于心臟、肝臟、脾臟、肺臟、腎臟、淋巴結、睪丸、十二指腸、空腸、回腸、真胃、膀胱等器官組織中,感染7d時檢出率最高,陽性信號最強,感染后120d仍然可從頜下淋巴結組織中檢出。其中,陽性細胞主要包括巨噬細胞、中性粒細胞、各種上皮細胞以及淋巴細胞。 細菌分離結果顯示,感染后7d布魯氏菌即可形成全身性感染,隨后逐漸被清除,120d后所采集的組織樣品無布魯氏菌被檢出,膀胱和肌肉樣品在整個試驗期內(nèi)均未檢出布魯氏菌。整個感染過程中抗體滴度定期檢測發(fā)現(xiàn),感染后7d可檢測到特異性凝集抗體,15d抗體滴度最高,隨后開始下降,240d時抗體陰性。 上述研究結果初步揭示了非妊娠狀態(tài)綿羊和性成熟公羊感染羊種布魯氏菌的自然史,即病理學變化和病原分布主要集中于富含網(wǎng)狀內(nèi)皮系統(tǒng)的器官和組織,感染后7d即可在所有的靶器官和組織內(nèi)發(fā)現(xiàn)明顯病變和病原分布,并隨時間推移感染的器官組織中病變程度和組織病原分布及其細菌數(shù)量逐漸減弱,感染120d后基本消失。對采集樣品進行四種抗原檢測方法比較分析顯示,ISH檢測結果與經(jīng)典的細菌分離技術相比敏感性更高,經(jīng)技術方法優(yōu)化后可望開發(fā)為布魯氏菌抗原檢測的“金標準”。 本研究通過對布魯氏菌人工感染綿羊抗體滴度、細菌分離、免疫病理學和分子病理學檢測技術的對比分析,初步闡釋了羊種布魯氏菌感染后的抗體消長規(guī)律、病原分布動態(tài)變化規(guī)律及各器官組織病理學動態(tài)變化,將為羊種布魯氏菌病防控措施的制定提供重要基礎數(shù)據(jù)。
[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.

【學位授予單位】：中國農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：S858.26

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本文編號：1546910