於淘汰豬隻中偵測豬鐵士古病毒並區分血清型 及建構qRT-PCR偵測組織內病毒絕對含量

Detection of Porcine Teschovirus and Serotyping of the PTVs in Culled Post-weaned Pigs, and Development of qRT-PCR to Quantitate PTVs Load in Tissues

胡書佳(Shu-Chia Hu)

指導教授 ： 王汎熒

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10315

摘要

豬鐵士古病毒(Porcine teschovirus, PTV)為Picornaviridae科Teschovirus屬，為一直徑約25~30 nm球形、無封套、正向單股的RNA病毒。PTV迄今共有11種血清型，並和許多疾病相關。於1930 至1950年代，由強毒力的PTV-1感染導致豬隻的高死亡率、非化膿性的腦脊髓炎(鐵線病，Teschen disease)造成嚴重的經濟損失。如今，高毒力的鐵線病已逐漸被低毒力的鐵芬病(Talfan disease)所取代。而台灣自2000年首次於田間豬隻中分離出PTV-1(首次疫情報告)，此後PTV分離率便逐年逐漸增加，顯示出PTV於台灣田間普遍汙染的情形。本實驗的目的為藉由反轉錄-聚合酶鏈鎖反應(RT-PCR)及巢式聚合酶鏈鎖反應(nested PCR)檢測淘汰豬隻體內15種不同臟器(腦的前、中、後區域、第一頸椎C1、肺、肝、腎、脾、扁桃腺、鼠蹊淋巴結、空腸及迴腸的腸系膜淋巴結、空腸、迴腸、及結腸)的病毒分布，並篩選一組臟器以利實驗室診斷。另一目的則是探討各臟器病變與PTV呈現的相關性。第三目的為觀察PTV血清型的分布。第四目的是藉由即時聚合酶鏈鎖反應(qRT-PCR)定量病毒於不同組織之間的分布。在30頭淘汰離乳豬隻當中有96.7% (29/30)呈現PTV陽性，臟器中PTV偵測最高的為腸道(結腸: 65.5%、空腸: 62.1%、迴腸: 55.2%)、其次為淋巴器官(迴腸淋巴結: 79.3%、鼠蹊淋巴結: 58.6%)、腦及內臟的偵測率則相似。統計顯示，在腦的後段（含部份大腦、小腦及腦幹）非化膿性腦炎的出現與PTV的出現有明顯的相關性 (P = 0.054)；空腸的絨毛萎縮與淋巴球浸潤與PTV的出現也有相關性 (P = 0.139)。較適合用於偵測的器官為腦的前、中區域、腎、脾、扁桃腺、迴腸淋巴結、迴腸。由30頭淘汰離乳豬隻當中的20頭共檢出5種PTV血清型，分別為PTV-1、-4、-6、-7、-11，其中4頭有兩種血清型同時存在於不同臟器。最常見的血清型為PTV-7 (≒ 60%) 及 PTV-6 (≒ 30%)。qRT-PCR具有比nested PCR高的敏感性，在各臟器當中以迴腸的病毒量顯著最高 (P = 0.005~0.045)，其含量約104.5±1.52 copy/μg RNA。

關鍵字

豬鐵士古病毒 ； 血清型 ； 絕對定量

並列摘要

Porcine teschoviruses (PTVs) belong to genus Teschovirus within the family Picornaviridae. The virions are spherical, 25 to 30 nm in diameter, nonenveloped with a linear plus sense ssRNA genome. Hitherto, PTVs have 11 serotypes and are associated with a variety of clinical diseases. The virulent PTV-1 strains were associated with highly fatal, nonsuppurative encephalomyelitis of pigs (Teschen disease) causing considerable economic loss during 1930-1950s. Nowaday, the highly virulent Teschen strains have been replaced by less virulent Talfan strain and spread worldwide. In Taiwan, PTV-1 was first isolated from pigs in year 2000 (virgin epidemic), and the isolation rates of PTVs from porcine herds had increased yearly. One aim of this study was to investigate in the detail the PTV distribution in a set of 15 different organs by RT-PCR/nested PCR in culled postweanling pigs, and to select a set of representative organs for diagnostic purpose. Another aim was to correlate the histopathological changes with the detection of PTV. The third aim was to investigate the variety of serotypes of PTV present in Taiwan. The fourth aim was to quantitate the virus load (RNA copy number per μg RNA) in tissues by qRT-PCR. A set of 15 organs, including cranial, medial, caudal portion of brain, C1, lung, liver, kidney, spleen, tonsil, inguinal LN, mesentery LN near jejunum and ileum, jejunum, ileum, and colon in 30 culled postweanling pigs were collected. The positive rate of PTVs detection was 96.7% (by heads), and most commonly detected in intestine (colon: 65.5%, jejunum: 62.1%, and ileum 55.2%), followed by lymphoid organs (ileac LN: 79.3%, inguinal LN: 58.6%), and similar rates in brain and visceral organs. Based on the above results, a set of organs including cranial and medial portion of cerebrum, kidney, spleen, tonsil, ileac LN, ileum, and inguinal LN was considered representative for diagnosis purpose. The presence of nonsuppurative encephalitis in the caudal brain (include part of cerebrum, cerebellum and brain stem) had prominent correlation with the simultaneous detection of PTV (P = 0.054). The villi atrophy and lymphoid infiltration in the jejunum also had a marginal correlaction with the detection of PTV (P = 0.139). A total of 5 serotype namely PTV-1, -4, -6, -7, -11 were identified from 20 animals which had finished genotyping, and 4/20 animals had two serotypes co-existed the same animal but in different organs. The most prevalent serotypes were PTV-7 (≒ 60%) and PTV-6 (≒ 30%). The qRT-PCR had higher sensitivity than nested PCR, and showed that ileum has the significantly highest viral load, equal to104.5±1.52 copy/μg RNA, of all organs (P = 0.005~0.045).

並列關鍵字

porcine teschovirus ； serotyping ； absolute quantitate

參考文獻

Borg, I., Rohde, G., Loseke, S., Bittscheidt, J., Schultze-Werninghaus, G., Stephan, V., Bufe, A., 2003, Evaluation of a quantitative real-time PCR for the detection of respiratory syncytial virus in pulmonary diseases. Eur Respir J 21, 944-951.

Buitrago, D., Cano-Gomez, C., Aguero, M., Fernandez-Pacheco, P., Gomez-Tejedor, C., Jimenez-Clavero, M.A., 2010, A survey of porcine picornaviruses and adenoviruses in fecal samples in Spain. J Vet Diag Invest 22, 763-766.

Bustin, S., 2000, Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25, 169-193.

Bustin, S., Mueller, R., 2005, Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis. Clin Sci 109, 365-379.

Chittick, W.A., Stensland, W.R., Prickett, J.R., Strait, E.L., Harmon, K., Yoon, K.J., Wang, C., Zimmerman, J.J., 2011, Comparison of RNA extraction and real-time reverse transcription polymerase chain reaction methods for the detection of Porcine reproductive and respiratory syndrome virus in porcine oral fluid specimens. J Vet Diag Invest 23, 248-253.