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研究生:陳姿妤
論文名稱:台灣本土型與外來型豬瘟病毒株致病性之比較
論文名稱(外文):The Comparison of Pathogenicity on Native and Invaded strain of Classical Swine Fever Virus
指導教授:簡茂盛簡茂盛引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫病理學研究所
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:2002
畢業學年度:90
語文別:中文
論文頁數:100
中文關鍵詞:豬瘟病毒致病性病理學RT-PCR病毒分離
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摘要
豬瘟為豬隻急性發熱之高接觸性傳染性疾病,以全身臟器出血及免疫抑制現象為主徵。近年來自台灣野外所分離的豬瘟病毒株中,主要可被區分為兩個族群-即本土型與外來型 (native and invaded strain) 豬瘟病毒。為瞭解本土型 (Native strain; 94.4) 與外來型 (New invaded strain; Ping Tung) CSFV於病毒複製速率及致病性上之差異性,本實驗係採用活體內 (in vivo) 感染方式,以兩次以上回毒 (passaged in pigs) 後之毒血接種7週齡豬隻,除記錄攻毒後臨床表現外,並嘗試建立pathology score作為virulence phenotype之分析,並配合RT-PCR及病毒分離進一步分析病毒於各臟器之分佈情形。結果顯示94.4株攻毒組並不呈現出全身各臟器明顯出血及脾臟梗塞的病灶,但屏東株攻毒組不但臨床症狀如發燒,病程較快且持續較久,而其他典型豬瘟病變也較早出現,且在病變程度上亦較為嚴重,特別是非化膿性腦炎之病變更比94.4株明顯。由此可見豬瘟之特徵性病變中,其各項病變強度及出現頻度會依不同病毒株而有相當程度上之差異。此外,在病毒增殖之速率上,94.4株及屏東株攻毒組若以nested RT-PCR於第2天即可由血液偵測出病毒核酸,而第4天以nested RT-PCR即可自扁桃腺、脾臟、胸腺、腎臟以及骨髓偵測到病毒核酸。以IFA法亦能在腦組織及腎臟中測得病毒,但屏東株攻毒組之病毒量皆高於94.4株。綜合上述臨床症狀、病理學變化、RT-PCR及病毒分離結果顯示,目前台灣田間所流行之豬瘟野外毒如屏東株應是屬於中間強毒,而感染後之致病性亦較本土型94.4株強。為進一步探討豬瘟病毒之致病機制,經利用正常豬肺臟巨噬細胞 (porcine alveolar macrophage; PAM) 做初代培養後再感染病毒,另一方面則灌洗攻毒豬之PAM,經培養後測定細胞培養液之病毒力價,結果證實PAM確為豬瘟病毒感染及複製之標的細胞之一,並且可因持續性感染PAM而導致TNF-a mRNA表現量降低,然而PAM所媒介之炎症反應在調節免疫系統上所扮演之角色仍未明瞭,應有更進一步探討的必要性。總而言之,因外來型豬瘟病毒 (屏東分離株) 之致病性及複製速率皆優於本土型豬瘟病毒 (94.4分離株),因此推論外來型屏東株因具活體內複製優勢,導致兩型病毒株間之消長現象,造成台灣地區豬隻皆以感染屏東株為主的因素之一。

Classical swine fever (CSF) is a highly contagious viral disease affecting swine and generating leucopoenia and immunosuppression. At least two major genotypes, named native strain such as 94.4 isolate and new invaded strain such us Ping-Tung isolate of classical swine fever virus (CSFV), have been identified in Taiwan. The aim of this study was to evaluate the differences of pathogenicity and replication kinetics between two genotypes of CSFV in vivo. In order to minimize the influence of virus virulence following the passage of virus in cell culture, the new invaded Ping-Tung and native 94.4 isolates were repeatedly replicated in pigs for the recovery as well as amplification of viruses. The whole blood samples were collected at the peak of disease after inoculation. The pathological scores based on clinical signs, pathological lesions and histopathological examination were calculated to evaluate the differences of the pathogenicity between viruses. Moreover, RT-PCR, virus isolation and IFA were applied to assess the distributions and titers of viruses in organs during the course of infection. The results demonstrated that no significant hemorrhage and spleen infarct were noticed even after a four-time passage of the native 94.4 in pigs. However, the Ping-Tung isolate caused clinical signs more severe including fever duration, hemorrhage, and encephalitis during the period of infection. The results of nested RT-PCR also indicated that the new invaded Ping-Tung isolate had higher replication rate than the native 94.4 in tonsil, spleen, thymus, kidney and bone marrow in vivo. In addition, the porcine alveolar macrophages (PAM) were collected and used to evaluate virus titer and cytokine profiles after inoculation. The results revealed that CSFV could infect and replicate in PAM and also downregulate the m-RNA of TNF-a in pigs. In conclusion, the new invaded Ping-Tung isolate surpassed the native 94.4 in pathogenicity and in replication, that might contribute to the reason for why domination of new invaded strain in fields in Taiwan.

中文摘要 ---------------------------------------------------------------------------------- I
英文摘要 ---------------------------------------------------------------------------------- III
目錄 ---------------------------------------------------------------------------------------- V
表次 ---------------------------------------------------------------------------------------- VII
圖次 ---------------------------------------------------------------------------------------- VIII
第一章 緒言 ---------------------------------------------------------------------------- 1
第二章 文獻探討 ----------------------------------------------------------------------- 3
第一節 豬瘟之歷史背景 ------------------------------------------------------------- 3
第二節 豬瘟病毒之特性 --------------------------------------------------------------- 3
2.2.1 豬瘟病毒基因與蛋白之研究 -------------------------------------------- 4
2.2.2 豬瘟病毒基因型 (genetic typing) 與分子流行病學之研究--------- 5
2.2.3 豬瘟病毒毒力 (virulence) 之研究 ------------------------------------- 7
第三節 豬瘟病毒感染之臨床症狀及病理學變化 ------------------------------- 8
2.3.1 臨床症狀 -------------------------------------------------------------------- 8
2.3.2 病理學變化 ----------------------------------------------------------------- 9
第四節 豬瘟病毒之診斷 -------------------------------------------------------------- 12
第五節 豬瘟病毒感染之致病機制 -------------------------------------------------- 15
2.5.1 豬瘟病毒對造血系統之影響 -------------------------------------------- 16
2.5.2 豬瘟病毒對免疫系統之影響 -------------------------------------------- 17
2.5.3 豬瘟病毒與內皮細胞之關係 -------------------------------------------- 19
2.5.4 細胞激素與疾病之關係 -------------------------------------------------- 21
第三章 實驗設計與材料方法 -------------------------------------------------------- 23
第一節 實驗設計與實驗流程圖 ----------------------------------------------------- 23
第二節 實驗材料 ----------------------------------------------------------------------- 27
3.2.1 細胞 -------------------------------------------------------------------------- 27
3.2.2 豬瘟病毒之增殖及病毒力價之測定 ------------------------------------ 27
3.2.3 實驗動物 --------------------------------------------------------------------- 28
3.2.4 組織均質化 (Homogenization) ------------------------------------------- 29
3.2.5 豬肺泡巨噬細胞 (PAM) 之收集 --------------------------------------- 29
3.2.6 豬瘟多價抗體之製備 ----------------------------------------------------- 30
3.2.7 致裂原 (mitogen) ----------------------------------------------------------- 30
第三節 實驗方法 ---------------------------------------------------------------------- 31
3.3.1 豬瘟病毒分離 -------------------------------------------------------------- 31
3.3.2 血液學檢查 ------------------------------------------------------------------ 31
3.3.3 病理學檢查 ------------------------------------------------------------------ 31
3.3.4 間接免疫螢光染色法 (IFA) ---------------------------------------------- 32
3.3.5 反轉錄聚合酵素鏈鎖反應 (RT-PCR) ---------------------------------- 33
3.3.6 細胞激素之檢測 ----------------------------------------------------------- 36
3.3.7 豬瘟病毒活體外 (in vivo) 感染PAM及PK-15 --------------------- 37
第四章 結果 ----------------------------------------------------------------------------- 38
第一節 體溫變化 ----------------------------------------------------------------------- 38
第二節 臨床症狀 ----------------------------------------------------------------------- 39
第三節 血球細胞數之變化 ----------------------------------------------------------- 41
第四節 病理學檢查 -------------------------------------------------------------------- 42
  4.4.1 肉眼病變 -------------------------------------------------------------------- 42
4.4.2 組織病理學變化 ----------------------------------------------------------- 45
第五節 豬瘟病毒於組織臟器之分布情形 ----------------------------------------- 49
4.5.1 應用反轉錄聚合酵素鏈鎖反應檢測豬瘟病毒核酸------------------ 49
4.5.2 病毒分離 (Virus isolation) 及測定 (titration) ------------------------ 50
第六節 豬瘟病毒於豬肺泡巨噬細胞 (PAM) 之感染情形 --------------------- 52
第五章 討論 ---------------------------------------------------------------------------- 80
參考文獻 --------------------------------------------------------------------------------- 92
表次
Table 1. Checklist for the determination of the gross pathology score in CSF experiment ---------------------------------------------------------------------------- 53
Table 2. Checklist for the determination of the histopathology score in CSF experiment ---------------------------------------------------------------------------- 54
Table 3. Primers used for RT-PCR for detecting CSFV, porcine housekeeping and cytokine genes ------------------------------------------------------------------ 55
Table 4. Clinical observation on different passages of CSFV inoculation test ---- 56
Table 5. Determination of viremia by RT-PCR and viral detection in organs from the first passage of the 94.4 and Ping-Tung inoculation ---------------- 57
Table 6. Determination of viremia by RT-PCR and viral detection in organs from the second passage of the 94.4 and Ping-Tung inoculaion -------------- 57
Table 7. The viral titers in various organs after CSFV inoculation ----------------- 58
Table 8. The duration of disease after pigs inoculated with PT and 94.4 of CSFV ---------------------------------------------------------------------------------- 59
圖次
Fig. 1: Fluctuation of body temperature in pigs after inoculated with various passages of the CSFV 94.4 isolate ------------------------------------------------ 60
Fig. 2: Fluctuation of body temperature in pigs after inoculated with various passages of the CSFV Ping-Tung isolate ----------------------------------------- 60
Fig. 3: Fluctuation of body temperature in pigs after inoculated with the 94.4 and Ping -Tung isolates ------------------------------------------------------------- 61
Fig. 4: Frequency of different clinical signs in pigs inoculated with the 94.4, PT, and 94.4 + PT viruses --------------------------------------------------------------- 61
Fig. 5: White blood cell counts in pigs after inoculated with different passages of the CSFV 94.4 ------------ ------------------------------------------------------- 62
Fig. 6: White blood cell counts in pigs after inoculated with different passages of the CSFV PT isolate ------------------------------------------------------------- 62
Fig. 7: White blood cell counts in pigs after co-inoculated with the 94.4 and PT isolates of CSFV --------------------------------------------------------------------- 63
Fig. 8: Gross lesions in pigs inoculated with the 94.4, PT, and 94.4+PT isolates of CSFV ------------------------------------------------------------------------------- 64
Fig. 9: Frequency of specific lesions in pigs inoculated with the 94.4, PT, and 94.4 + PT of CSFV ------------------------------------------------------------------ 66
Fig. 10: Microscopic lesions in pigs inoculated with the 94.4, PT, and 94.4+PT of CSFV ------------------------------------------------------------------------------- 67
Fig. 11: Average of pathology score on pigs inoculated with different passages of the 94.4 ---------------------------------------------------------------------------- 70
Fig. 12: Average of pathology score on pigs inoculated with different passages of the PT ------------------------------------------------------------------------------ 70
Fig. 13: The pathology score on pigs co-inoculated with the 94.4 and PT of CSFV ---------------------------------------------------------------------------------- 71
Fig. 14-1: The result of nested RT-PCR for CSFV in pigs infected with the PT isolate during the viremia stage tion ---------------------------------------------- 72
Fig. 14-2: The result of RT-PCR for CSFV in pigs infected with the PT isolate during the viremia stage ------------------------------------------------------------ 72
Fig. 15-1: The result of nested RT-PCR for CSFV in different organs from a pig infected with 94.4 isolate on dpi 4 ------------------------------------------------ 73
Fig. 15-2: The result of RT-PCR for CSFV in different organs from a pig infected with PT isolate on dpi 21------------------------------------------------- 73
Fig. 16: Results of indirect immunofluorscence assay for CSFV antigen in PK-15 cells --------------------------------------------------------------------------- 74
Fig. 17: The viral titers in different organs at the day 4, 7, 14 and 21 after inoculation with the third passage of CSFV 94.4 ------------------------------- 75
Fig. 18: The viral titers in different organs at the day 4, 7, 14 and 21 after inoculation with the third passage of CSFV Ping- Tung ----------------------- 75
Fig. 19: The viral titers in different organs at the day 13 and 21 after inoculation with Ping- Tung + 94.4. The virus titers were the highest in the spleen ----- 76
Fig. 20: Kinetics of CSFV replication in porcine alveolar macrophages (PAM) - 77
Fig. 21: Kinetics of CSFV replication in PK-15 cells -------------------------------- 77
Fig. 22: Effect of CSFV on the synthesis of pro-inflammatory cytokine IL-1α, IL-6 and TNF-α of porcine alveolar macrophage (PAM) analyzed by RT-PCR ------------------------------------------------------------------------------- 78
Fig. 23: Genetic typing of classical swine fever virus in Taiwan ------------------- 79

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