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研究生:李宗賢
研究生(外文):Tsung-Hsien Li
論文名稱:建構救援海龜之存活預測指標暨具腫瘤綠蠵龜之臨床特徵及海龜皰疹病毒角色探討
論文名稱(外文):Establishment of a Survival Prediction Index for Use in Sea Turtle Rescue and Study on the Clinical Characteristics and the Role of Chelonid Herpesvirus 5 (ChHV5) in Green Turtles (Chelonia mydas) with Tumors
指導教授:張照勤張照勤引用關係
口試委員:徐維莉陳鵬文吳永惠楊瑋誠
口試日期:2017-10-18
學位類別:博士
校院名稱:國立中興大學
系所名稱:微生物暨公共衛生學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2017
畢業學年度:106
語文別:中文
論文頁數:95
中文關鍵詞:綠蠵龜血液學纖維乳突瘤症海龜皰疹病毒第五型
外文關鍵詞:Chelonia mydasHematologyFibropapillomatosisChelonid herpesvirus 5
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海龜是海洋生態系統中重要且不可或缺的角色,但海龜的族群數量,在世界上許多區域都是逐漸減少的。海龜面臨的威脅有獵捕、混獲、船隻撞擊、海洋垃圾、海岸開發、環境汙染與傳染病。在眾多疾病中,尤其是海龜纖維乳突瘤症 (Fibropapillomatosis ; FP)更為研究者重視。海龜救傷中心進行海龜族群保育工作扮演著非常重要的角色,而獸醫師對於傷病海龜的照護扮演著極重要的角色。海龜的健康指標如生理特徵、血液學及血漿生化指數,對於傷病動物在康復期間的臨床照護而言,都是很重要的參考指標。海龜FP是一種會讓海龜虛弱的疾病,最早在於1938年在佛羅里達的綠蠵龜身上被發現。而過去研究也顯示,FP已經在世界上許多區域被發現。因為該疾病的腫瘤會對海龜生命造成威脅,FP現已被認為是一種在海龜的嚴重以及新興且廣泛的疫病。但是此疾病在亞洲的報告仍然是非常罕見的。因此在這個研究當中研究主軸主要有三部分,分述如下: 第一部分為建構可預測救援海龜存活預後的綜合性指標; 第二部分為罹患纖維乳突瘤症綠蠵龜之血液及血漿生化研究; 第三部分為臺灣綠蠵龜罹患纖維乳突瘤症之ChHV5分子流行病學研究。研究結果在第一部分發現,漂浮徵候 (Buoyancy disorders; BD)跟海龜存活有非常高的顯著性 (p<0.05)負相關。相較於存活組海龜,非存活組海龜有著較高的血漿Aminotranspherase (AST)、Creatinine kinase (CK)、Creatinine (CRE)及Uric acid (UA)濃度 (p 值皆< 0.05)。進一步經由多變項邏輯氏迴歸分析模組分析後,BD、CRE及UA這三個因子最具有顯著海龜存活預測性,因此被納入最後模式中用以計算每一隻海龜的綜合性健康指標 (Summary health index; SHI)。經Receiver operating characteristic (ROC)曲線進行以SHI來預測海龜存活與否的分析後,ROC曲線下的面積 (Area under the receiver operating characteristic curve; AUROC)為0.920 ± 0.037,以2.5244為SHI的最佳切點 (Cut-off),以此數值做為存活預測,其敏感性 (Sensitivity)及特異性 (Specificity)分別為80.0%及86.7%。因此,本研究所創建的SHI可做為一個有效的指標來評估救援海龜健康狀態,並藉此來增進獸醫師在收容中心對海龜提供及時且必要之醫療照護。研究結果在第二部分發現,相較於非腫瘤組海龜,腫瘤組海龜血漿當中有較高的麩丙酮酸轉胺脢 (Alanine aminotransferase)濃度和較低的鐵離子 (Iron)濃度 (p 值皆< 0.05),此現象可能與腫瘤海龜受血吸蟲感染有關。研究結果在第三部分發現,ChHV5的序列成功的經由PCR的方式在腫瘤海龜的腫瘤樣本中被偵測出。序列與世界上其他會導致海龜誘發腫瘤的病毒序列具有一致性的。ChHV5的RNA可經由RT-PCR的方式從FP的腫瘤樣本中被偵測出,顯示ChHV5在腫瘤當中是具有複製能力的。在本研究中,除了以分子生物學的證據來證實ChHV5確實存在腫瘤組織當中之外,我們也經由組織病理學的方式,來證實Epidermal intranuclear inclusions (EIIs)確實存在於腫瘤組織當中,此更進一步證實ChHV5在罹病海龜的腫瘤中應該是處於一個具有複製能力 (非僅為潛伏感染)的狀態。根據親緣演化樹的分析顯示,臺灣綠蠵龜的ChHV5病毒序列和Puerto Rico、Hawaii以及Sao Tome有著較高的相似度,因此,可能顯示病毒為多區域的廣泛感染。對於瀕危海龜的保育工作而言,ChHV5必須被視為一個新興的且對亞洲綠蠵龜具有威脅性的病毒。
Sea turtles are important components of ocean ecosystems; however, their numbers are in decline in many regions. Marine turtles are exposed to a number of threats, including human catch, unintended catch, boat collisions, marine debris, coastal development, environmental pollutants, and infectious diseases. Among many diseases, fibropapillomatosis (FP) is considered to be of major importance threatening sea turtle surviving. Centers for sea turtle rehabilitation play an important role in conservation of sea turtle populations, and veterinary care plays an influential role in sea turtle rehabilitation. Physiological characteristics, hematological and plasma biochemistry profiles, are useful references for clinical management in animals, especially when animals are during the convalescence period. Fibropapillomatosis (FP), a debilitating tumor disease of sea turtles, was first identified in green turtles (Chelonia mydas) in Florida in 1938. In recent decades, FP has been observed globally and is an emerging panzootic disease in sea turtles. However, few reports of FP in Asia have been published up to date. The aims of this study were :1) to establish a summarized survival prediction index for use in sea turtle rescue; 2) to compare hematology and plasma biochemistry values of green sea turtles with and without fibropapillomatosis; 3) to characterize molecular epidemiology of the chelonid herpesvirus 5 (ChHV5) from Taiwan green sea turtles with fibropapillomatosis. The results indicated that significantly negative correlation was found between buoyancy disorders (BD) and sea turtle surviving (p < 0.05). Furthermore, non-surviving sea turtles had significantly higher levels of aspartate aminotranspherase (AST), creatinine kinase (CK), creatinine and uric acid (UA) than surviving sea turtles (all p < 0.05). After further analysis by multiple logistic regression model, only factors of BD, creatinine and UA play substantial roles to predict sea turtle survival and were included in the mathematical equation for calculating a summarized health index (SHI) for each individual. Through evaluation by receiver operating characteristic (ROC) curve, the result indicated that the area under curve was 0.920 ±0.037, and a cut-off SHI value of 2.5244 showed 80.0% sensitivity and 86.7% specificity in predicting sea turtle survival. Therefore, the developed SHI could be a useful index to evaluate health status of sea turtles and to improve veterinary care at rehabilitation facilities. Significantly higher value of plasma alanine aminotransferase was identified for the turtles with FP, when compared to the turtles without FP. Significantly lower iron level was detected in the turtles with FP, when compared to the turtles without FP. Elevated value of ALT and lower iron level in turtles with FP may result from spirorchiid infection. Using FP tumors for molecular studies, ChHV5 was successfully detected by PCR in FP tumor lesions of green turtles. The sequences were found to be consistent with those of tumor-inducing viruses shown to affect sea turtles in the other parts of the world. ChHV5 RNA from the FP tissues was further detected by RT-PCR, indicating active replication of the viruses inside FP tumors. In addition to show molecular evidence of ChHV5 in FP, epidermal intranuclear inclusions were further identified in tumor lesions upon histopathological examination. This result suggests that ChHV5 should be in a transcriptionally active (i.e., non-latent) state in FP tumors of affected green turtles. The phylogenetic tree revealed that ChHV5 from the green turtles in Taiwan were closest to the ChHV5 from Hawaii, Puerto Rico, and Sao Tome. For conservation of endangered sea turtles, ChHV5 should be considered an emerging virus, which threatens sea turtles in marine waters in Asia.
中文摘要-------------------------------------------------- i
Abstract------------------------------------------------ iii
目錄------------------------------------------------------ v
表目錄--------------------------------------------------- vii
圖目錄---------------------------------------------------viii
第一章 研究背景與目的---------------------------------------- 1
第一節 臺灣的海龜種類---------------------------------------- 1
第二節 海龜在生態系統的角色----------------------------------- 2
第三節 海龜面臨的威脅---------------------------------------- 3
第四節 海龜保育--------------------------------------------- 5
第五節 海龜救傷中心------------------------------------------ 6
第六節 海龜臨床病理檢查指標----------------------------------- 7
第七節 海龜罹患纖維乳突瘤症之血液及血漿生化特徵----------------- 12
第八節 纖維乳突瘤症與ChHV5 的關係---------------------------- 16
第九節 研究目的-------------------------------------------- 19
第二章 建構可預測救援海龜存活預後的綜合性指標------------------- 21
第一節 文獻探討-------------------------------------------- 21
第二節 材料與方法------------------------------------------ 23
第三節 結果----------------------------------------------- 27
第四節 討論----------------------------------------------- 29
第五節 本研究於期刊及研討會的相關發表資料---------------------- 34
表------------------------------------------------------- 35
圖------------------------------------------------------- 41
第三章 罹患纖維乳突瘤症綠蠵龜之血液及血漿生化研究---------------- 42
第一節 文獻探討-------------------------------------------- 42
第二節 材料與方法------------------------------------------ 46
第三節 結果----------------------------------------------- 50
第四節 討論----------------------------------------------- 51
第五節 本研究於研討會的相關發表資料--------------------------- 54
表------------------------------------------------------- 55
第四章 臺灣綠蠵龜罹患纖維乳突瘤症之ChHV5 分子流行病學研究-------- 58
第一節 文獻探討-------------------------------------------- 58
第二節 材料與方法------------------------------------------ 61
第三節 結果----------------------------------------------- 64
第四節 討論----------------------------------------------- 66
第五節 本研究於期刊的相關發表資料----------------------------- 70
表------------------------------------------------------- 71
圖------------------------------------------------------- 72
第五章 總結----------------------------------------------- 74
參考文獻-------------------------------------------------- 77
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