臺灣博碩士論文加值系統

摘 要魚類非特異性防禦機制，主要是以吞噬細胞來清除入侵的病原菌與
異物。本實驗利用所分離與初代培養之吳郭魚(Oreochromis aureus)與虹
鱒(Oncorhynchus mykiss)之血液、脾臟及頭腎吞噬球，進行吞噬不同致
病性之Vibrio parahemolyticus、V. anguillarum、V. vulnificus、
Aeromonas hydrophila、Streptococcus spp.等水中常在病原。經由流體
細胞儀分析結果顯示，在比較血液、脾臟和頭腎吞噬細胞對相同病原的吞
噬能力上，以血液吞噬球呈現最強的吞噬能力，其次為脾臟與頭腎吞噬細
胞。而吞噬球於接觸病原後120分鐘，可將大部份的細菌攝入而開始進行
分解消化，但血清調理作用並無明顯影響其對相同細菌之吞噬能力，唯有
吳郭魚脾臟吞噬細胞經加入256倍稀釋之魚類血清調理後，吞噬能力有明
顯增強。此外，當吳郭魚脾臟吞噬細胞對吞噬V. parahemolyticus (Vp2)
活菌的量亦高於吞噬被熱不活化的細菌量。經探討不同致病性病原是否影
響魚類吞噬細胞之吞噬能力，結果顯示脾臟及頭腎吞噬細胞吞噬不同毒力
之細菌時，以病原能明顯產生溶血素與胞外產物並對吞噬球具較高細胞毒
性者，所被吞噬的量較多，且對細胞毒性愈大的細菌存活於吞噬球內的能
力亦較強。此外，魚體內是一個限鐵的環境，將上述病原菌培養於含有不
同濃度螯鐵物質2,2'-dipyridyl的LB broth中以模擬魚體內限鐵環境。其
中A. hydrophila (A2)及V. anguillarum (Va2)的溶血活性及細胞毒性隨
著2,2'-dipyridyl 濃度的上升而有明顯增加的現象，除可造成吞噬球大
小及顆粒性的改變外，並可能影響吞噬球吞噬細菌後的殺菌功能。而虹鱒
及吳郭魚頭腎吞噬球經加入對細胞毒性較強的A. hydrophila (A2)之細胞
外產物後，可明顯增加吞噬細胞主動吞噬其他較低毒力病原的菌量。反之
，若加入細胞毒性較弱的A. hydrophila (A1) 所產生的細胞外產物，對
吞噬細胞吞噬其他病原的數量則無影響。

AbstractThe phagocytes are believed to play the major role in
fish non-specific defense against most aquatic pathogens and
irritants. In this study, the phagocytes of tilapia (Oreochromis
nilotica) and rainbow trout (Oncorhynchus mykiss) isolated from
blood, head kidney and spleen were used to compare the
phagocytosis and eradication abilities against several aquatic
pathogens such as Vibrio parahaemolyticus, V. anguillarum, V.
vulificus, Aeromonas hydrophilia and Streptococcus spp. using
flow cytometry analysis. In general, the blood phagocytes had
better phagocytosis capability than spleen and head kidney
phagocytes in engulfing the same pathogen. The results also
demonstrated that bacteria opsonized with or without fish serum
in advance did not influence most phagocytes to uptake and
eradicate same bacteria after 120 minutes incubation, but
tilapia spleen phagocytes could enhance their activity when
bacteria opsonized with 1:256 fold diluted fish serum. In
addition, the number of bacteria (V. parahaemolyticus) engulfed
by tilapia spleen phagocytes would obviously decrease if
bacteria were heat inactivated before interacting with
phagocytes.Moreover, all phagocytes showed higher capability of
phagocytosis against most toxigenic bacteria especially those
could produce hemolysins and extracellular products (ECPs) to
induce significant cytotoxicity of phagocytes. In order to
characterize the influence of bacterial toxicity, bacteria were
cultured in LB broth containing different concentration of
2,2'-dipyridyl to simulate the pathogens growing at iron-
limiting condition while interacting with phagocytes in fish.
The results showed both A. hydrophilia (A2) and V. anguillarum
(Va2) apparently increased their hemolytic and cytotoxic
efficiency against all phagocytes under iron starvation.
Furthermore, the ECPs could induce granulation and size
variation of phagocytes and affect their eradication ability.
However, ECPs of A. hydrophilia (A2) did obviously enhance
rainbow trout and tilapia head kidney phagocytes for uptaking
other less or non-pathogenic bacteria. In contrast, ECPs of A.
hydrophilia (A1), without any cellular cytotoxicity against
phagocytes, showed no influence the capability of phagocytosis.