- 學位論文
台灣地區乳牛鼻腔接種牛腸病毒致病機制之研究
Investigation of pathogenesis towards bovine enterovirus in Taiwan via intra-nasal inoculation in dairy cattle
摘要
牛腸病毒(Bovine enterovirus, BEV)屬於微小核糖核酸病毒科(Picornaviridae)，腸病毒屬(Enterovirus)中Enterovirus E或Enterovirus F。近年來，台灣許多乳牛牧場中的下痢牛隻單獨檢測出牛腸病毒的數量相當多，顯示牛腸病毒可能已經為造成台灣乳牛下痢的重要病原，故本研究目的為分析於台灣下痢乳牛分離之牛腸病毒基因序列，判定是否為新型別病毒，並將病毒實驗接種於牛隻探討病毒對牛隻之致病機制，期望確認牛腸病毒與牛隻疾病之間的關聯，提供牛隻產業對牛腸病毒的預防及治療方式。本研究採集台灣乳牛牧場中下痢牛隻糞便檢測牛腸病毒，利用細胞培養方式分離病毒，並進行基因序列分析。動物攻毒實驗將三隻十五週齡荷仕登牛利用鼻腔內接種病毒(實驗組)，一隻牛未接種病毒(控制組)，實驗期間每日觀察牛隻臨床症狀兩次，牛隻分別在攻毒後五天、七天及十四天施行安樂死並進行病理解剖及組織病理學診斷，將收集之樣本進行血液學及分子生物學檢測，最後將檢測結果進行分析及比較。本次病毒株命名為Taiwan-2022，據基因定序結果判定為Enterovirus F。攻毒實驗期間兩隻實驗組牛隻出現發燒及精神沉鬱症狀，肉眼及組織病理學病變於實驗組牛隻的肺臟出現氣管旁淋巴濾泡增生、化膿性支氣管炎及間質性肺炎，一隻實驗組牛隻另外具有心臟、腎臟、肝臟及膽囊病變，即時聚合酶連鎖反應除了實驗組牛隻的糞便樣本檢測出病毒外，在組織及血液樣本皆未檢測出病毒。將實驗組和控制組牛隻進行比較後顯示牛腸病毒會引起牛隻肺臟免疫反應並造成後續病變，繼而引發二次性細菌感染。此外病毒會定植於牛隻腸道中並伺機引起下痢等症狀，當牛隻感染牛腸病毒，下痢症狀建議進行支持療法及施打抗生素預防大腸桿菌過度繁殖，呼吸道症狀則建議施打抗生素來預防或治療二次性細菌感染，且將來可以研發疫苗來預防病毒感染。未來可以透過原位雜合技術確認病毒於組織分布情形。
並列摘要
Bovine enterovirus (BEV) belongs to the species Enterovirus E or Enterovirus F, genus Enterovirus and family Picornaviridae. Recently, A lot of samples was detected BEV in dairy cattle in Taiwan. It shows that BEV is an important virus, which might be a primary cause of diarrhea. The objective of this study is to analyze BEV isolated from diarrhea dairy cattle in Taiwan and evaluate pathogenesis of BEV after inoculated in calves. It is expected to confirm the relationship between bovine enterovirus and cattle disease, and to provide the cattle industry with preventive and therapeutic method. This study collected feces from diarrhea cattle in Taiwan dairy farms to detect bovine enterovirus. The virus was isolated by cell culture, and analyzed genome sequence. Three calves were inoculated with BEV via intra-nasal route (infected group) and another one calf was no treatment (control group). The clinical signs were observed twice a day at the experiment period. Necropsy and histopathology were carried out after the calves were inoculated on 5, 7 and 14 days. The collected samples were tested by hematology and molecular biology, and the test results were analyzed and compared. The result of genome sequencing analysis determined that the isolation belongs to Enterovirus F and is named as Taiwan-2022. For the clinical signs, two calves had fever and depression at the period of the experiment. After euthanasia, the infected calves had peri-bronchial lymphoid hyperplasia, purulent bronchitis and interstitial pneumonia, and one infected calf also had heart, kidney, liver and gallbladder lesions in gross lesions and histopathology. For the real-time PCR, the infected calves were detected BEV in fecal samples, but no BEV was detected in the tissue and blood samples. Compare to the infected and control calves, it shows that BEV may cause an immune response and lesions in lungs. In addition, BEV may colonizes the intestine and wait for the opportunity to cause symptoms such as diarrhea. Supportive therapy and antibiotics could be used to prevent E. coli overgrowth when cattle have diarrhea. For respiratory symptoms, antibiotics could be used to prevent or treat secondary bacterial infection. The development of vaccine is a choice to prevent BEV infections. In situ hybridization can be used to confirm viral distribution in the future.