- 學位論文
犬瘟熱感染犬隻臨床檢體之病毒核酸偵測、定量及 H 基因之序列分析
Nucleic acid detection, quantification and sequence analysis of canine distemper virus from clinical specimens
摘要
犬瘟熱病毒(canine distemper virus；CDV)屬於Paramyxoviridae科，Morbillivirus屬，感染犬隻後會引起呼吸道、消化道以及神經症狀，尤其對 3-6 月齡幼犬引發高發生率及高死亡率。雖然透過疫苗注射的接種本病普遍可獲得良好的控制，但國內外仍時有病例發生且近年來有增加之趨勢，本病的早期症狀與犬舍咳很難區分，因此造成疾病早期診斷上的困難。為建立快速與敏感之診斷本實驗藉由 RT-PCR 結合巢式聚合酶鏈鎖反應 (Nested PCR) 針對臨床感染疑似之病例進行檢測。在本實驗共收集了 440 個臨床上懷疑 CDV 感染病例共 661 個檢體，檢體種類包含全血、眼結膜、口、鼻拭子以及尿液和直腸拭子。檢測結果發現 Nested PCR 之陽性率為37.7% (166/440) 比 RT-PCR 17.0% (75/489) 增加 20.7% 診斷的有效性 (P<0.001)。統計在感染季節分佈方面，各月份均有 CDV 感染之病例，其中以晚秋及冬季發生率較高(P<0.001)。為建立一個有效的檢體採樣選擇，將 Nested PCR 診斷為陽性的 166 個病例中具有多重檢體的 62 個病例做進一步分析，發現檢出率以眼結膜抹片 100.0% (11/11) 為最高，比血液 64.4% (38/59) 高出甚多(P<0.05)。在Real-time PCR 定量 27個病例各臨床檢體的病毒量，結果發現臨床檢體中犬瘟熱病毒的定量最高可達1.8 x 1010 copies/mL，而單一病例檢體間病毒量的差異可高達 109倍。在與臨床症狀相關性分析方面，於眼結膜拭子、鼻腔拭子、口腔拭子、直腸拭子及尿液等檢體均可定出相當高之病毒量，證明病毒可經由呼吸道、消化道及泌尿道排毒。而不論在病毒血症或非病毒血症的病例，大多數的鼻腔拭子具有較高的病毒量。另外有 1/3 (9/27) 血液檢測為陰性之病例，於其它檢體中仍可測得相當高的病毒量，可見血液並不是最佳的檢測檢體。本實驗結合62 個具有多重檢體的陽性病例分析與病毒定量之結果，犬瘟熱感染犬隻臨床檢體之採樣須考量採樣當時病犬之臨床症狀，才可達到最佳之檢出率。臨床上具全身性症狀之犬隻其眼結膜抹片及眼、鼻分泌液檢體具有較高的檢出率，另外患犬若有消化道症狀其直腸拭子亦有相當高的檢出率，具神經症狀之犬隻則以尿液及眼結膜抹片檢體檢出率較高，若患犬有發熱及流涎之症狀其血液與唾液檢體亦是不錯的考量，而利用RT-nPCR可達到最佳的檢出效果。 H 基因序列分析結果顯示，目前 CDV 台灣株間基因相似度介於 99.1%-99.5% 之間，與鄰近國家日本 1992 年犬 Hammam 分離株的基因相似度則介於 98.9 %-99.0% 之間皆屬於 Asia-1 基因型。
並列摘要
Canine distemper virus (CDV) infection in dogs can result in subclinical infection, gastrointestinal signs, and/or respiratory signs, frequently with central nervous system (CNS) involvement, high morbidity and mortality. Recently, the incidence of canine distemper (CD) both in unvaccinated and vaccinated dogs seemed increasing in Taiwan. In order to understand more about the current CDV infection in Taiwan, a rapid and sensitive diagnotic test for CD using a RT-PCR combined with nested PCR was applied to 440 dogs clinically suspected with CDV infection. 660 clinical specimens including CSF, whole blood, nasal swab, ocular swab, rectal swab and urine were collected from 440 dogs. The results showed that Nested PCR (37.7%, 166/440) increase 20.7% in the efficiency of the diagnosis than RT-PCR (17.0%, 75/489) (P<0.001). The seasonal distribution of the infection was mainly seen in late autumn to winter (P<0.001). Detection rate of conjunctival scraping (100.0%, 11/11) had a significant elevation compared with whole blood (64.4%, 38/59) (P<0.05). Quantification of the viral RNA concentration in clinical specimens from 27 cases by real-time PCR revealed that the highest concentration was as high as 1.8 x 1010 copies/mL. Different specimens from the same animal could vary up to 109 times. Nasal swab, ocular swab, oral swab and rectal swab had higher viral load, indicating that virus was shed in respiratory tract, digestive tract and urinary tract correlated to their clinical syndrome. Nasal swab was found to have the highest viral copies in cases with or without viremia. Therefore, the blood is not the best choice for clinical diagnosis. The result of H gene sequence analysis showed, that Taiwanese CDV is in the lineage of CDV Asia-1.