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Studies on Non-specific Defense Mechanism of Giant Fresh Water Prawn (Macrobrachium rosenbergii)
本研究為探討淡水長腳大蝦 (Macrobrachium rosenbergii) 之非特異性 (non-specific) 免疫反應，首先分離蝦隻的血球細胞 (hemocytes) 並嘗試於體外 (in vitro) 培養，以檢測血球細胞對於病原性細菌及非病原性之乳膠顆粒 (latex beads) 的吞噬作用。試驗中得知1 mL的蝦隻血淋巴液 (hemolymph) 中約含有5×106 個血球細胞，形態上可區分為透明血球 (hyaline cell)、半顆粒血球 (semigranular cell) 及顆粒血球 (granular cell) 3種血球細胞，而血球細胞對於病原性細菌及非病原性之乳膠顆粒都會產生吞噬作用，且分別感作2及3小時所顯示之吞噬能力則明顯大於只感作1小時之試驗組。進一步再探討淡水長腳大蝦於不同環境水溫下，血球細胞對於病原性酵母菌 (Debaryomyces hansenii) 及非病原性乳膠顆粒吞噬能力之影響，試驗蝦隻經肌肉接種標示FITC螢光染劑之病原性酵母菌及乳膠顆粒，再分別飼養於17、27及35 ℃之水溫中，然後分別於接種後1、2、3、6、12及24小時抽取蝦隻的血淋巴液，以流式細胞儀 (flow cytometry) 分析血球細胞的螢光值。結果顯示27℃組蝦隻的血球細胞對於乳膠顆粒的吞噬能力明顯優於17℃低溫組。至於27℃組及35℃組相對於17℃組蝦隻血球細胞之吞噬能力比較，亦在接種後3小時呈現極顯著的差異性增強。至於接種病原性酵母菌再評估吞噬能力之試驗中，27℃組於接種6小時亦比17℃組蝦隻其血球細胞吞噬能力有顯著的差異性增強，而接種後12小時其螢光值增強，於統計上更呈現極顯著的差異 (p<0.01)。
此外，淡水長腳大蝦經餵食多醣類 (β-glucan) 後，再以流式細胞儀進一步評估是否會影響其血球細胞之吞噬活力。結果蝦隻經分別連續餵食7天及14天後，其血球細胞之吞噬活力皆呈現揚升之情形，但其中以連續餵食7天的血球細胞其吞噬活力呈現顯最著的差異性增強。而在攻毒試驗中，試驗蝦隻先以每天餵食多醣類250 µg∕prawn連續7天後，進行肌肉接種0.5 mL 之1×107cfu∕mL病原性酵母菌，再分別飼養於17、27及35℃之恆溫水槽中並連續觀察10天。結果以17℃飼養組中未餵食之對照組於接種後第6天則完全死亡，但餵食組則於第9天才呈現100％之死亡率；而27℃組中的蝦隻至接種後第10天仍有50％之存活率，但未餵食對照組之死亡率則為100％；至於35℃組中的蝦隻至接種後第10天仍有20％之存活率，但對照組則於接種後第9天完全死亡，這顯示餵食β-glucan之蝦隻雖然在不同溫度之雙重刺激下進行病原酵母菌接種，仍具有良好的保護力。
再進一步探討血球細胞於吞噬病原菌後是否產生計畫性凋亡 (apoptosis) 反應，結果顯示經吞噬病原性細菌後之各組細胞呈現計畫性凋亡反應，包括細胞質產生空泡化 (vacuolization)、核染色質濃縮 (chromatin condensation) 及核斷裂 (fragmentation) 等之情形。又萃取各組血球細胞核酸DNA 進行膠體電泳，得知吞噬病原性菌體2小時再培養12小時後，血球細胞之核酸即呈現明顯階梯片段 (DNA laddering) 的現象，若以TUNEL染色法 (terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end-labeling) 檢測計畫性凋亡之螢光反應，吞噬非病原性乳膠顆粒組與對照組其血球細胞則需培養至48小時才會有計畫性凋亡反應產生。綜合各種試驗結果得知，溫度是決定蝦隻血球細胞吞噬功能之重要因素之一，亦是影響蝦隻非特異性免疫能力之主要決定因子，而多醣類可增強淡水長腳大蝦血球細胞之吞噬活力及增強蝦隻對病原之耐受力，但是血球細胞吞噬病原性細菌後會提前產生計畫性凋亡反應。
Massive muscle necrosis caused by yeast pathogen (Debaryomyces hansenii) is among the most important diseases affecting giant freshwater prawn (Macrobrachium rosenbergii) during the wintertime in Taiwan. The objectives of this study were to investigate the influence of temperature and β-glucan on phagocytic activity of hemocytes, and to compare the effect of phagocytosed pathogens or non-pathogenic latex beads on hemocytes of giant freshwater prawn. The FITC-labeled yeast pathogen and latex beads were injected into ventral muscle of grouped prawns and maintained at different water temperature (17, 27 and 35C). The hemolymph was withdrawn directly from the heart of prawns at 1, 2, 3, 6, 12, and 24 hours after inoculation. The results showed that phagocytic activity of hemocytes at 27C and 35C groups were higher than 17C group. Moreover, the FITC-labeled latex beads and yeast pathogen were injected into experimental prawns which had been fed with β-glucan (250 µg/prawn/day) and maintained at 27C. The hemolymph was withdrawn from the hearts of the experimental prawns 3 hours post inoculation. The phagocytic activities of a total of 104 hemocytes were assayed according to the ingested cellular fluorescence using flow cytometry. The results revealed that both phagocytic activity against FITC-labeled yeast pathogen or latex beads were significantly enhanced after feeding with β-glucan for 7 and 14 days. Furthermore, after oral administration of β-glucan for one week, experimental prawns were challenged intramuscularly with yeast pathogen and then maintained in water for the survival trials at 17C, 27C and 35C. In the 17C group, mortality reached 100% on the ninth day post inoculation but was 100% on the sixth day in control group. However, the mortality was only 50% even after 10 days post inoculation at 27C, while it reached 100% on the tenth day in control group. In the 35C group, the mortality was 80% but that was 100% in control group on the ninth day.
Hemocytes were also investigated for the induction of apoptosis after phagocytosis of pathogenic yeasts, bacteria and non-pathogenic latex beads in vitro. Isolated hemocytes of M. rosenbergii were cultured at a ratio of 1:50 hemocytes to pathogen with yeasts, with bacteria Aeromonas hydrophila or Enterococcus faecium, or with latex beads at 25C for 2 h, followed by washing to remove free particles. At least two hundred hemocytes were counted to determine the phagocytosis rate, and the results showed that hemocytes engulfed latex beads at a higher rate than the aquatic pathogens. By transmission electron microscopy, the yeast- or bacterium-engulfing hemocytes displayed morphological alterations, characteristics of apoptosis, including formation of cytoplasmic vacuoles, chromatin condensation, and fragmentation of nuclei. The DNA laddering and TUNEL assays further confirmed this pathogen-induced apoptosis. Neither hemocytes treated with latex beads nor un-infected hemocytes (the control group) showed signs of apoptosis up to 48 hours in culture. In summary, the results of the studies indicated that phagocytosis of hemocytes were temperature-dependent in vivo, and both phagocytic activity and survival rate of experimental prawns against yeast pathogen were significantly improved after feeding a diet containing β-glucan for one week. Moreover, phagocytosis of pathogens, but not non-pathogenic particles, could induce apoptosis in hemocytes of the giant freshwater prawn.
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