在野外地方性感染現況豬鐵士古病毒經由尿液排毒之探討

Abstract

豬鐵士古病毒（Porcine Teschovirus, PTV），為單股正向無封套RNA病毒。PTV目前有13種血清型，引起離乳豬的腦脊髓炎或下痢，藉由糞口傳播。在台灣曾於2000年與2004年爆發兩次疫情，現今於台灣豬場呈現普遍的地方性感染。先前研究已確認PTV會感染腎絲球體內皮細胞與腎小管上皮，推測PTV可能從尿液排毒，但仍未有數據證實。本研究目的為偵測感染豬隻糞、尿、血液及臟器的病毒量，以釐清在地方性感染下，PTV的致病機轉及排毒狀況。本研究從7個豬場採集健康及淘汰離乳豬隻臟器及體液檢體，共7隻健康豬隻及21頭淘汰離乳豬，利用即時定量反轉錄聚合酶反應（Real-Time RT-PCR）偵測檢體中的病毒量，並使用台灣常見的PTV-7毒株序列製作標準曲線，以進行絕對定量（最低可偵測到101病毒量）。實驗結果顯示所有豬隻皆可偵測到PTV，其尿液、血液、糞便、鼻腔拭子的偵測率為100%，腎臟的偵測率約38%。依照病毒量高低可將10種檢體分為3組：腸道相關（迴腸及糞便）、血液淋巴相關（扁桃腺、鼠膝淋巴結及血漿），以及臟器與泌尿相關 (脾臟、腎臟、膀胱及尿液)。 迴腸的病毒量最高，每100毫克組織的病毒量約為106.4，約為糞便的3倍，血漿的40倍。次高為糞便，每100毫克的病毒量約為105.9，此結果與糞口病毒血症的致病機轉一致。而100微升尿液中的病毒量約為104，約為糞便病毒量的百分之一，血液病毒量的六分之一，腎臟病毒量的五倍，表示PTV容易通過絲球體進入尿液中。此10種檢體的病毒量在不同豬場及不同健康狀況豬隻皆無顯著差異。淘汰豬隻的血漿病毒量略高於健康豬隻的病毒量，但在統計上無顯著差異，顯示PTV即使在健康豬隻也會造成病毒血症，與地方性感染的結論一致。本研究確認了PTV會藉由尿液排出體外，並在糞尿混合下加速了PTV的傳播。在7個豬場皆偵測到PTV，合併持續性的病毒血症，再次印證PTV在台灣已成為重要的地方性病毒性疾病。

Porcine teschovirus (PTVs) is a non-enveloped, positive-sense, single-stranded RNA virus. At least 13 serotypes of PTVs have been identified. The PTVs can cause polioencephalomyelitis and diarrhea through fecal-oral transmission. In Taiwan, two epidemic outbreaks of PTVs occurred in years 2000 and 2004, and the endemic status of PTVs is now confirmed in herds. Previous study has demonstrated PTVs antigen and nucleic acid in glomeruli and epithelia of renal tubules, and suggested that shedding and transmission of PTVs by urine may be possible. However, this speculation remains to be verified with numerical evidence. The study aimed to clarify the shedding pathway and to further the understanding of pathogenesis in endemic infection by detecting the viral loads of urine, feces, plasma and solid tissues in naturally infected piglets. In this study, the samples were collected from 7 clinically healthy and 21 culled piglets of 7 different herds, and viral loads of each sample were quantified by means of absolute quantification in real-time RT-PCR with a detection limit of 101 copies per reaction. A standard curve was constructed by the sequence of PTV-7, which is the most common serotype in Taiwan. The results showed 100% detection rates in urine, feces, plasma and nasal swabs, and 38% in kidney. Based on the viral loads, these samples were categorized into three groups: Intestinal-related samples (feces and ileum), hematolymphoid samples (tonsil, inguinal lymph node, and plasma), visceral and fluid samples (spleen, kidney, bladder, urine and nasal swab). The viral loads of ileum were the highest, measuring 106.40 copies/100 mg tissue weight, approximately 3-times higher than those of feces (105.9 copies/100 mg), and 40-times higher than plasma (104.79 copies/100 μl). The categorization was largely consistent with the fecal-oral-viremia pathogenesis model. As speculated, PTV was present in the urine at the scale of 104 copies/100 μl fluids, which was 1% of that in feces, 17% of that in plasma but 5-times higher than in kidney. These results suggested that PTVs were easily filtered from blood to urine through glomeruli. No statistical significance was found between healthy and culled pigs, and between the 7 herds. The viral loads of plasma in culled piglets were slightly higher but no significant difference than those of healthy piglets, indicating that those outwardly healthy pigs were also infected by PTV and had viremia, consistent with the endemic situation. In conclusion, PTVs can be detected in urine, confirming the urinary shedding pathway in the naturally infected situation. The combination of urine and feces into slurry facilitate the transmission of PTVs. The detection of PTVs in different herds further confirmed the endemic situation, in which a persistent viremia was occurring. Therefore, PTVs is an important endemic disease of piglets in Taiwan.