Modulation of B-glucan of saccharomyces cerevisiae on swine immune competence against classical swine fever virus infection

中文摘要 病毒感染後因毒力及與宿主抵抗力交互關係間之消長或平衡，導致宿主恢復、死亡或持續性感染之現象，所牽涉之因子複雜，其中宿主的免疫狀態扮演一重要之角色。本實驗利用免疫調節劑 b-glucan調控宿主之免疫系統，探討其對豬瘟中間毒（S-59）感染之影響。選取15頭健康、三品種（LYD）六週齡豬隻供實驗用，在免疫促進組係以口服酵母菌（Saccharomyces cerevisiae）之細胞壁，做為免疫誘發物；在免疫抑制組則以肌肉注射合成葡萄糖性類固醇-dexamethasone（1mg / kg體重），以模擬臨床緊迫因子對宿主免疫系統之影響。連續25天處置後，以S-59中間毒2 x 106 PFU肌肉注射攻毒，於攻毒後第0、7、14、21、28、42及56天抽血檢測，計算白血球總數、以流式細胞儀分析淋巴次族群之變化、並利用抗原ELISA、reverse transcriptase-polymerase chain reaction （RT-PCR）方法偵測病毒血症及以END法檢測豬瘟中和抗體產生情形。而於攻毒後第14、28、56天分三次淘汰豬隻，進行病理學檢查及利用RT-PCR偵測各實質臟器病毒抗原，以確認病毒分佈之情形。結果顯示：與對照組比較，以S. cerevisiae長期給予可增加豬隻白血球總數、淋巴細胞總數，CD4+ 淋巴細胞族群絕對值上昇，並減輕豬瘟感染所造成之臨床症狀及病理病變。以dexamethasone處理，則使CD4+ 淋巴細胞族群絕對值下降，且加重豬瘟感染之臨床症狀及病理病變。故利用免疫促進劑及良好的飼養管理，以減少豬隻產生緊迫，可降低疾病對宿主之傷害。The immune status of the host defense system plays an important role in classical swine fever infection. The experiment was aimed to evaluate the immunomodulator, b-glucan, in the regulation of porcine immune system against CSF virus (S-59 strain) infection. Fifteen 6-week-old, healthy, LYD piglets were grouped as(1)immune-promoting group, pigs fed with the cell wall of Saccharomyces cerevisiae as immune inducer ; (2) immune-inhibiting group, piglets were subsequently injected with dexamethasone ( 1 mg/kg b.w., IM ) to mimic the stress effect on immune system, and (3) control. Virus challenge (S-59, 2 x 106 PFU, IM ) was conducted after 25 days of treatment with S. cerevisiae or dexamethasone . On 0, 7, 14, 21, 28, 42, and 56 days post infection (d.p.i.), blood samples were collected for WBC count and phenotypic changes in subpopulation of lymphocyte that was analyzed by flow cytometry. Moreover, antigen ELISA and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed to detect the presence of viremia, while the neutralization antibody of experimental pigs after CSF virus challenge was assayed by END method. On 14, 28, and 56 d.p.i., piglets were sacrificed for pathological examination and viral loading and distribution. The results showed that comparing to the control group, the numbers of WBC, lymphocyte, particular in CD4+ lymphocytes in long term treatment of S. cerevisiae was increased. Meanwhile, clinical signs and pathological lesions immune-promoting group were mild and restricted after CSF virus challenge. In contrast, dexamethasone decreased the absolute numbers of CD4+ lymphocytes and aggravated the clinical signs and pathological lesions pigs after CSF virus challenge. These results indicated that proper management along with the application of immunomodulator would enhance host defense and thereby reduce the injury of infectious.目 錄 頁次 中文摘要 -------------------------------------------------------------- I 英文摘要 -------------------------------------------------------------- II 目錄 -------------------------------------------------------------------- III 表次 -------------------------------------------------------------------- Ⅳ 圖次 -------------------------------------------------------------------- Ⅴ 第一章 緒言 -------------------------------------------------------- 1 第二章 文獻探討 -------------------------------------------------- 3 第一節 b-glucan ------------------------------------------------- 3 第二節 豬瘟 ----------------------------------------------------- 12 第三章 材料與方法 ----------------------------------------------- 28 第四章 結果 -------------------------------------------------------- 35 第五章 討論 -------------------------------------------------------- 55 參考文獻 -------------------------------------------------------------- 64 表 次 頁次 Table 1 The components of the reagent of RT-PCR reaction. ---- 40 Table 2 Piglets were sacrificed for pathological examinationafter CSFV inoculation. -------------------------------------- 41 圖 次 頁次 Figure 1 The variation in total amount of WBC. ----------------- 42 Figure 2 Dot plot of leukocyte ( A ) and leukocyte stained with monoclon antibody 74-22-15 ( B ). ---------------- 43 Figure 3 The change in absolute value of lymphocyte after CSFV（S-59） challenge. ------------------------------- 44 Figure 4 The change in absolute value of granulocyte after CSFV（S-59） challenge. ------------------------------- 45 Figure 5 The change in absolute value of monocyte after CSFV（S-59） challenge. ------------------------------- 46 Figure 6 The change in subpopulation of lymphocyte（CD4＋）after CSFV（S-59） challenge. ------------------------- 47 Figure 7 The change in subpopulation of lymphocyte（IgM＋）after CSFV（S-59） challenge. ------------------------- 48 Figure 8 The change in subpopulation of lymphocyte（CD8＋）after CSFV（S-59） challenge. ------------------------- 49 Figure 9 Piglets were examined the gross lesion after 14 days of CSFV inoculation. -------------------------------- 50 Figure 10 CSF viremia detected by antigen ELISA. -------------- 51 Figure 11 Using RT-PCR to detect CSF viremia. ------------------ 52 Figure 12 Using RT-PCR to detect the distribution of viral nucleic acid within organs. ------------------------------- 53 Figure 13 Using END to detect the production of neutralizating antibody after CSFV inoculation. ------ 5