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研究生:杜昀姍
研究生(外文):Tu, Yun-Shan
論文名稱:苗栗地區石虎、犬及貓之食肉目小病毒親緣關係之探討
論文名稱(外文):The phylogenetic relationship of carnivore protoparvovirus 1 between domestic dogs, cats and leopard cats (Prionailurus bengalensis) in Miaoli
指導教授:陳貞志陳貞志引用關係
指導教授(外文):Chen, Chen-Chin
口試委員:陳怡寧林昭男
口試委員(外文):Chen, Yi-NingLin, Chao-Nan
口試日期:2019-01-21
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:野生動物保育研究所
學門:生命科學學門
學類:生態學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:52
中文關鍵詞:貓小病毒犬小病毒跨物種傳播胺基酸突變
外文關鍵詞:Feline panleukopenia virusCanine parvovirusCross species transmissionAmino acid mutation
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石虎族群在台灣數量少以及面臨人類活動的威脅,除此之外,疾病的感染可能增加滅絕的風險,研究發現台灣野生石虎有感染小病毒 (CP1)病毒株的案例,而小病毒可感染數種食肉動物,因此共域的食肉動物有機會相互傳染疾病,在苗栗地區石虎與流浪犬貓的活動範圍重疊,而野生貓科動物感染小病毒後具高死亡率。為瞭解小病毒是否在石虎與犬貓中傳播,本研究應用屏東科技大學野生動物疾病生態研究室所採集之苗栗地區石虎及流浪犬貓樣本,並 (1)分析石虎與犬貓所分離小病毒的病毒株分布; (2)檢視小病毒基因序列的胺基酸突變狀況; (3)比較石虎與犬貓所分離小病毒的親緣關係; (4)以分子鐘推算序列間演化的時間差異。本研究提出假說為:若CP1已在物種間傳播,且病毒未長時間與特定宿主共同適應,因未受到特定物種宿主的選擇,所以病毒在不同物種的差異會很小,且序列間的分化時間相近。因此若分離出的CP1基因序列無呈現物種間的差異,或是分化時間相近,則推測CP1已在石虎與犬貓間互相感染。本研究分析42隻石虎樣本和30隻犬貓樣本,使用PCR檢測發現苗栗地區石虎感染貓小病毒 (feline panleukopenia virus; FPV)、犬小病毒 (canine parvovirus; CPV-2)的2a、
2b、2c型等,其中以CPV-2a的比例最高。特定位點胺基酸的突變於石虎及流浪犬貓間皆可見,兩者間並無特異性。樣本中有15隻石虎樣本和6隻犬貓樣本使用PCR將VP2基因增幅至1380bp,其中石虎與犬貓間有相同的序列,而親緣分析結果顯示石虎的小病毒並無獨立分群,且與犬貓中的小病毒分化時間皆小於4年。從上述結果支持本研究假說,因此推測小病毒在石虎與犬貓間傳播。若要保育石虎已不能只關注單一物種,應納入共域食肉動物,以及流浪動物的控管,更全面的進行疾病監測與管理,才能建立有效的防疫機制。
The population of leopard cats (Prionailurus bengalensis) in Taiwan was facing several threats, such a low population abundance and various environmental hazard and human activities. In addition, previous studies indicate that wild leopard cats were commonly infected with carnivore protoparvovirus 1 (CP1) CP1 has the potential of cross species transmission between sympatric carnivores, such as domestic dogs and cats, and inducing high mortality in wild felids. In Miaoli, the home range of leopard cat is general overlap with domestic dogs and cats. In order to figure out that whether CP1 was transmitted between cats, dogs and leopard cats. The objectives of this study included: (1) analyze the CP1 strains which isolation from cats, dogs and leopard cats; (2) analysis of the amino acid mutation at the specific site; (3) to figure out the phylogenetic relationship of CP1 isolated from wild leopard cats, and
domestic dogs and cats; (4) using molecular clock to estimate divergence times between CP1 isolated from leopard cats, dogs and cats. The hypothesis was that if CP1 is currently transmitted between different species, the phylogeny of CP1 would be closed to each other. We collected 42 individuals of wild leopard cats distributed around Miaoli County. In addition, we collected rectal swabs and blood samples from 22 stray dogs and 8 stray cats in the same sampling area of leopard cats. We adopted PCR for screening CP1 in samples. We identified the infection of variants of canine parvovirus type 2a, 2b ,2c (CPV-2a, CPV-2b, CPV-2c) and feline panleukopenia virus (FPV) in leopard cats, and major type was CPV-2a. The partial VP2 gene of 1380bp was sequenced from 15 leopard cats and 6 domestic dog and cats, and we found a sequence from leopard cat was same as domestic. Phylogenetic analysis indicated that there wasn’t independent cluster in leopard cats. And the divergence times was less than 4 years. According to the phylogenetic analysis, we conclude that CP1 is currently transmitted between sympatric carnivores.
摘要 I
Abstract III
謝誌 V
目錄 VI
表目錄 VIII
圖目錄 IX
壹、 前言 1
貳、 文獻回顧 3
一、 分類 3
二、 VP2基因特性 3
三、 演化與胺基酸變異 4
四、 親緣分析方法運用在流行病學分析 5
五、 台灣犬小病毒流行史 5
參、 材料與方法 7
一、 樣本來源 7
二、 去氧核糖核酸 (Deoxyribonucleic acid; DNA)萃取 7
三、 檢測 7
四、 VP2基因 8
五、 分型 10
六、 親緣分析 10
肆、 結果 11
一、 樣本 11
二、 感染狀況 11
三、 VP2基因序列分析 11
四、 胺基酸突變 12
五、 親緣樹 13
伍、 討論 15
一、 感染狀況 15
二、 胺基酸突變 16
三、 親緣分析 17
陸、 結論 19
柒、 參考文獻 20
附錄一、石虎樣本採集資訊 43
附錄二、犬貓樣本採集資料 48
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