Detection of Porcine Teschovirus in Tissues of Experimentally Infected Pigs

豬鐵士古病毒屬於Picornaviridae病毒科，早期認為是Enterovirus，但之後獨立為Teschovirus病毒屬，目前有11個血清型，其中以PTV-1強毒株於1929年捷克鐵線鎮造成豬隻嚴重腦脊髓灰質炎，致死率高，稱為鐵線病。之後1957年弱毒株於英國他爾番地區豬隻發生，神經症狀輕微，因此稱為他爾番病，目前弱毒株已擴散至全球。台灣於西元2000年九月南投出現首例，且於當年苗栗、嘉義、台中等地區皆有病例報告，截至目前該病毒已普遍存在台灣的豬群。本研究目的為使用免疫組織化學染色法(Immunohistochemistry, IHC)偵測病原於感染組織中分佈，組織來源為人工鼻內感染台灣本土分離株之SPF豬，並且依臨床症狀、病變、及相同個體各臟器間及不同感染天數之差異，探討致病機轉。臨床症狀，於接種後第二天動物出現下痢症狀且持續到第五天，而第四天出現咳嗽症狀，而神經症狀方面於14隻豬隻中僅有2隻分別於第四和第五天出現不等程度的後肢麻痹。組織病理檢查顯示所有實驗動物皆出現輕微到中度間質性肺炎，而檢測11隻動物中有6隻於脊髓及背根神經節出現不等程度單核球細胞圍管合併髓鞘水腫或神經元細胞變性壞死。IHC結果PTV-1訊號出現於扁桃、空腸、迴腸、結腸之固有層、腸系膜淋巴結、肺臟、脾臟，於表現神經症狀動物之腰椎、頸椎、薦椎、及胸椎也有PTV-1訊號。藉由細胞型態及PTV-1訊號分佈相似於不同白血球抗體訊號分佈，證明在大多數器官內，感染細胞為T淋巴球及巨噬細胞。神經組織內感染細胞主要為血管內皮，星狀細胞，及少量T淋巴球。此結果顯示病毒感染神經組織以血行途徑為主，然而，由腸道逆行至中樞神經途徑之可能性仍無法排除。病理機轉模式推估消化道的固有層及脾臟之邊緣區(Marginal zone)及紅髓，在維持體內持續病毒血症及糞便排毒導致病毒於豬場中循環或重複感染扮演重要角色，但PTV-1如何僅趨向於脊髓及背根神經節之機制仍未完全明瞭須待進一步研究。The porcine teschovirus (PTV) belongs to genus Teschovirus of the Picornaviridae family. It was classified as Enterovirus genus before genotyping and reclassified it into 11 serotypes. The virulent PTV-1 strain is associated with Teschen disease, which is a polioencephalomyelitis with high mortality first occurred in Czechoslovakia in year 1929. After year 1957, the lower virulent strain and milder forms of polioencephalomyelitis had occurred in the Talfan district of England, and thus named Talfan disease. The lower virulent strains have since spread all over the world. In Taiwan, the 1st report was at Nantou district in September of year 2000, and had since been reported from Miaoli, Chiayi, and Taichung districts. The purpose of this study was to detect the presence of PTV-1 antigen in tissues, by immunohistochemistry (IHC), of pigs inoculated intranasally with a PTV-1 Taiwan isolate to correlate clinical signs and lesions and to further the understanding of pathogenesis. The observed clinical signs were mainly diarrhea at days postinfection (DPI) 2-5, and cough appeared at DPI 4, and 2 out of 14 pigs presented variably degrees of hind leg paralysis at DPI 4 and 5 respectively. Histologically, all experimental pigs presented mild to moderate interstitial pneumonia, and 6 out of 11 pigs presented perivascular mononuclear cuffing with myelin edema in white matter of cord, and neuronal degeneration in dorsal root ganglion. By IHC, PTV-1 signals were present in tonsil, lamina propia of jejunum, ileum, colon, mesenteric lymph node, lung and spleen, and those have nervous signs were in lumbar, cervical, sacral, and thoracic cords. Based on cellular morphology and a similar PTV-1 signal distribution with leukocyte marker distribution, infected cells were most likely T lymphocyte and macrophages in most organs. Infected cells in the spinal cord were mainly endothelial cells and astrocytes, and minorly T lymphocytes. Based on these findings a hematogenous route of invasion to CNS was most possible, however, a possibility of retrograde axonal transport from the intestines also likely existed. A model of pathogenesis was proposed, wherein lamina propia of intestines and marginal zone and red pulp of spleen play critical roles in sustaining viremia and fecal shedding to facilitate recirculation of the virus within the herd. The neurotropism to certain parts of the CNS remained unclear.ACKNOWLEDGEMENT 2文摘要 IBSTRACT II錄 …………………………………………………………………………………IVist of Tables VIIist of Figures VIIIhapter I INTRODUCTION 1hapter II LITERATURE REVIEW 2.1 Porcine teschovirus-1 2.1.1 History 2.1.2 Morphology, Genomic Organization and Expression 2.1.3 Taxonomy and Classification 3.1.4 Pathogenesis 4.1.5 Clinical signs 5.1.6 Lesions 6.1.7 Porcine teschovirus-1 in Taiwan 7.2 Pathogenesis of neurotropic virus- the poliovirus model 7.3 Immunohistochemistry: classical and modified 9.3.1 Antibody types 10.3.2 Direct immunohistochemistry 10.3.3 Indirect immunohistochemistry 11.3.4 Modified Immonihistochemistry 12hapter III MATERIALS AND METHODS 14.1 Animal tissue samples 14.2 Antibodies 15.3 Classical indirect immunohistochemistry 15.4 Modified indirect immunohistochemistry 16.5 Analysis results 16hapter IV RESULTS 18.1 Histological examination of HE stained tissue sections 18.1.1 Respiratory system 18.1.2 Digestive system 18.1.3 Nervous system 19.1.4 Immune system 20.1.5 Urinary and circulation systems 20.2 Examination of immunohistochemically stained tissue sections 20.2.1 Distribution of antigen-positive cells and their identification in each organ 21.2.2 Quantification of the number of antigen-positive cells in 3 fields with 200X magnification in each organ of each pig 23.2.3 Quantification of IHC scores in parts of spleen and tonsil 25.2.4 Correlation of IHC results with clinical signs and histological lesions 26hapter V Discussion and Conclusion 29hapter VI Sources and manufactures 37ables 38igures 45eferences 5