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published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

Classical Swine Fever in the Lao Peoples’ Democratic Republic: Virological, Epidemiological and Clinical studies. Classical swine fever (CSF) is a highly contagious virus infection of swine caused by classical swine fever virus (CSFV). The CSF virus is a member of the genus Pestivirus of the family Flaviviridae. Classical swine fever is believed to be endemic in Lao Peoples’ Democratic Republic (Lao PDR). Infectious diseases, including CSF, are a major constraint to pig production in developing countries such as Lao PDR. The aim of this thesis was to investigate aspects and present data regarding the nature of CSF pertinent to Lao PDR. An introduction to Lao PDR, local pig production and a review of pertinent CSF literature is presented in Chapter 1. Low levels of veterinary infrastructure have exacerbated infectious disease problems in developing countries. Chapter 2 of this thesis described the construction and refurbishment of a project laboratory in Lao PDR for the diagnosis of viral diseases, in particular CSF virus Furthermore, a diagnostic specimen submission system was adapted to the local domestic and economic conditions. Poor diagnostic facilities and lack of disease reporting systems in Lao PDR have allowed diseases to spread largely unchecked due to low levels of recognition. The process of development and assessment of appropriate diagnostic assays to the local conditions is presented and discussed in Chapter 3. ELISA and RT-PCR technologies for CSF virus detection in clinical specimens were implemented. Variations to RT-PCR methodologies were also investigated to determine the most suitable technique for the local situation. Results indicated that the RT-PCR methodology was more sensitive than ELISA for the detection of CSF virus in fresh clinical specimens. Notably, the situation was reversed when decomposed samples were tested. Methodologies for the preservation and detection of CSF virus in samples subjected to local tropical condition were also investigated. The proprietary reagent RNAlater ™ was found to be suitable for the preservation of CSF virus RNA under local conditions. Methodologies for CSF virus serology using the ELISA technique are also described. The majority of the pigs in Lao PDR are raised within village small-holder systems, with indigenous breeds being the most popular. Very little is known about the response of indigenous breed pigs to CSF virus infection. Chapter 4 described the pathogenicity of a Lao strain of CSF virus (Lao/Kham225) in both indigenous (Moo Laat) and imported pig breeds (Large white/Landrace cross-breed). Statistically significant (p = 0.05) differences in the breed-related susceptibility to CSF infection were demonstrated in clinical and haematological responses, and post-mortem pathology. The results demonstrated the course of CSF infection in the Large white/Landrace cross-breed was generally more acute than in the indigenous breed. Investigations into the epidemiology of CSF in Lao PDR are presented in Chapter 5. The distribution of CSF outbreaks during the period of mid-1997 to the end of 1999 was investigated and crude incidence results were calculated. Serological surveillance to determine the serological prevalence of CSF virus antibodies in selected regions of Lao PDR was performed during 1997 to 1999. Structured serological surveillance was performed in Vientiane Municipality, Bhorikhamxai, Khammouane and Savannakhet provinces during the survey period. Passive serological surveillance using samples from eight abattoirs in Lao PDR was also performed. Statistically significant (p = 0.05) intra- and inter-provincial differences were noted in a number of the surveys. The success of CSF vaccination via post-vaccination serology was also assessed. The results of the investigations determined that vaccination to prevent CSF infection was insufficient and post-vaccination responses were variable. Phylogenetic and phylogeographic studies to determine the genetic characteristics of Lao PDR and other regional CSF virus isolates are presented in Chapter 6. The 5’-non-coding region and the E2 gene of CSF viruses were investigated to determine genetic relationships between Lao PDR and regional isolates. Genetic typing of all field virus isolates using phylogenetic analysis techniques indicated that all viruses belonged to genogroup 2. Phylogeographic analysis of field viruses revealed a delineation of sub-genogroup allocation on a geographic basis. Members of the sub-genogroup 2.1 originated in Northern and Central regions of Lao PDR. Conversely, members of the sub-genogroup 2.2 originated in Southern and Central regions of Lao PDR. All Vietnamese viruses examined belonged to sub-genogroup 2.2. Phylogenetic analysis indicated that the Vietnamese viruses were largely distinct from Lao and Thai CSF viruses. With the exception of one virus isolate, all Thai viruses also belonged to sub-genogroup 2.2. With the exception of one Vietnamese vaccine virus, all vaccines examined belonged to genogroup 1. A general discussion of the results presented in all chapters, as well as implications for future research into this field, are presented in Chapter 7.

本研究主要在於設計一種新式生物反應器，並應用於生產豬瘟病毒疫苗。首先根據所培養細胞的生長特性與原有生物反應器之缺點，改良成新式的生物反應器，並評估此新式生物反應器適用性、效能，以及所培養豬腎細胞之生長代謝情形與豬瘟病毒力價。整個實驗過程大致分為兩個部分，第一個部分探討細胞固定化培養之最適化培養條件與生長代謝情形，第二個部分探討豬瘟病毒培養之最適化培養條件與生長代謝情形。實驗結果發現豬腎細胞(PK-15)以批次方式培養於新式生物反應器，搭配著FIBRA-CEL®載體，成功的進行擴大培養，豬腎細胞最高的生長量達到2.29×109cells/300mL的細胞量。因此，改良之新式生物反應器可提供細胞優越的生長環境，具有擴大規模培養之潛力，可藉由此簡單設備、操作容易、成本低且低能源消耗之新式生物反應器達成細胞製品之生產基座。 / In this study, the production of PK-15 cell using immobilized animal cell culture in a novel bioreactor was investigated.We evaluated the serviceability and efficiency of a design-improved novel bioreactor for the growth and metabolic states of cultured PK-15 cells and the production of HC virus. The entire experiment includes two major stages: (1) investigation of the optimal conditions and metabolic states for the growth of immobilized cells, (2) investigation of the optimal conditions for the production of HC virus. Our results showed that immobilized PK-15 cells on the fibra-cell carries in the design-improved novel bioreactor exhibited their best growth of 2.29×109 cells/300mL.The immobilized conditions for cell culture, can provide a shearing stress of growth state, easy separation of cells from the culture mediu, and a operation of continuously feeding medium, leading to possibility growth of the high density cell and a long period of production;as a result, the efficiency of producing process is promoted. Here,our design-improved novel bioreactor is expected to provide an optimal growth environment of both the cells and viruses for the production of high-yielded, stable, and consistent cellular biological preparations. Furthermore, it will also provide the basis for the production of cell products with advantages of simple-equipped, easy-to-operate, low cost, and low energy consumption. / 致謝 i 中文摘要 ------------------------------------------------------------------------ ii 英文摘要 ------------------------------------------------------------------------ iii 目錄 ------------------------------------------------------------------------ iv 表目錄 ------------------------------------------------------------------------ vi 圖目錄 ------------------------------------------------------------------------ vii 第一章 緒論------------------------------------------------------------------ 1 第二章 文獻回顧------------------------------------------------------------ 3 2-1 豬腎傳代細胞(PK-15 cell) -------------------------------------- 3 2-2 豬瘟病毒------------------------------------------------------------ 4 2-2.1 豬瘟之歷史背景--------------------------------------------------- 4 2-2.2 豬瘟病毒之特性--------------------------------------------------- 7 2-2.3 豬瘟發生原因之探討--------------------------------------------- 11 2-3 生物反應器--------------------------------------------------------- 12 第三章 實驗材料與方法--------------------------------------------------- 21 3-1 細胞------------------------------------------------------------------ 21 3-2 細胞繼代培養------------------------------------------------------ 21 3-3 細胞冷凍保存------------------------------------------------------ 22 3-4 解凍細胞培養------------------------------------------------------ 22 3-5 病毒感染------------------------------------------------------------ 23 3-6 收集病毒------------------------------------------------------------ 24 3-7 豬瘟病毒力價測試------------------------------------------------ 24 3-8 細胞滾瓶培養------------------------------------------------------ 26 3-9 生物反應器操作--------------------------------------------------- 27 3-10 載體上細胞數的測定--------------------------------------------- 33 3-11 葡萄糖的測定------------------------------------------------------ 33 3-12 培養過程中pH值測定------------------------------------------- 34 第四章 結果與討論--------------------------------------------------------- 35 4-1 測試細胞貼附的材料--------------------------------------------- 35 4-2 細胞固定時間的比較--------------------------------------------- 36 4-3 測試不同比例的載體量培養豬腎細胞------------------------ 37 4-4 測試不同接種量--------------------------------------------------- 40 4-5 測試培養基流速對豬腎細胞生長的影響--------------------- 44 4-6 測試培養基停留於培養槽時間對豬腎細胞生長的影響--- 45 4-7 測試豬腎細胞暴露空氣時間對於生長的影響--------------- 47 4-8 測試Bellocell培養豬腎細胞(PK-15)可行性----------------- 49 4-9 測試利用新式生物反應器培樣豬瘟病毒--------------------- 50 第五章 結論與建議--------------------------------------------------------- 53 參考文獻 ------------------------------------------------------------------------ 55 表目錄 表1. 兔化豬瘟疫苗與組織培養豬瘟疫苗的比較------------------ 6 表2. 急性、慢性與遲發型豬瘟比較---------------------------------- 10 表3. .Growth of Various cell Lines in bellocell-500----------------- 18 表4. Comparison of SF-9 cell Growth and BEV production in Various Laboratory bioreators------------------------------------ 19 表5. Comparison of HEK293 Cell growth and Receptor X production in Cell Factory®/20 roller bottles and BelloCell-500Bioreactor------------------------------------------ 20 表6. Reed-Muench Methods法計算方法----------------------------- 26 表7. 比較不同材料培養PK-15 cell所用的載體量---------------- 50 表8. 細胞固定時間的比較所接細胞量與載體量------------------ 51 圖目錄 圖1. Liau提出以潮汐生物反應器圖--------------------------------- 17 圖2. Operation principle of Bellocell system------------------------- 18 圖3. 新式生物反應器(novel reactor)-潮汐式生物反應器(tidal typereactor)之運作流程圖--------------------------------------- 30 圖4. 比較不同材料培養PK-15 cell ---------------------------------- 63 圖5. 比較不同時間細胞的貼附量------------------------------------ 64 圖6. 測試的不同比例載體量培養豬腎細胞生長曲線------------ 65 圖7. 測試的不同比例載體量培養豬腎細胞培養過程glucose消耗趨勢------------------------------------------------------------ 66 圖8. 測試的不同比例載體量培養豬腎細胞培養過程pH變化------------------------------------------------------------------------ 67 圖9. 測試不同接細胞量培養在10g carrier生長曲線------------- 68 圖10. 測試不同接細胞量培養在10g 載體glucose消耗趨勢----- 69 圖11. 測試不同接細胞量培養在10g carrier pH趨勢--------------- 70 圖12. 測試流速對豬腎細胞生長的影響------------------------------ 71 圖13. 測試流速對細胞影響的葡萄糖消耗--------------------------- 72 圖14. 測試流速對豬腎細胞生長影響pH值-------------------------- 73 圖15. 測試培養基停留時間對豬腎細胞生長影響------------------ 74 圖16. 測試培養基holding時間對豬腎細胞生長的影響之葡萄糖趨勢--------------------------------------------------------------- 75 圖17. 測試培養基holding時間對細胞的影響之pH值趨勢------- 76 圖18. 測試豬腎細胞暴露空氣時間對生長的影響------------------ 77 圖19. 測試豬腎細胞暴露空氣對生長的影響葡萄糖消耗趨勢--- 78 圖20. 測試豬腎細胞暴露空氣對細胞生長的影養pH值趨勢---- 79 圖21. Bellocell反應器培養豬腎細胞---------------------------------- 80 圖22. Bellocell培養豬腎細胞葡萄糖消耗趨勢---------------------- 81 圖23. Bellocell培養豬腎細胞pH值趨勢----------------------------- 82 圖24. 測試利用新式生物反應器培養豬瘟病------------------------ 83