比較吳郭魚來源分離之莢膜型及不含莢膜型無乳鏈球菌之病原性

本實驗室於2010年接獲尼羅紅魚（Oreochromis sp.）的病例，魚隻臨床症狀為死前迴游等神經症狀及體表出血情形，臟器未見明顯異常，從血液及臟器分離到鏈球菌，經indian ink染色證實具有莢膜，16S rDNA gene鑑定為無乳鏈球菌Streptococcus agalactiae。但同時在其他吳郭魚神經症狀及體表出血的病例中，我們亦分離到無莢膜的S. agalactiae，故本研究將比較有莢膜及無莢膜的S. agalactiae在吳郭魚（O. niloticus, tilapia）的病原性，包括二者之生存條件、貼附及侵入吳郭魚細胞的能力、半致死劑量、宿主免疫清除力上的差異，並鑑定S. agalactiae莢膜血清型。本實驗收集台灣各地由尼羅紅魚、吳郭魚、數種鱸魚等魚種來源之S. agalactiae分離株，以S. agalactiae莢膜抗血清Ia及Ib進行血清型鑑定，結果顯示收集到的37株S. agalactiae莢膜血清型多為type Ia及Ib。Ia共24株，皆為無莢膜、β溶血性；Ib共13株，有莢膜佔5株，為γ溶血，無莢膜8株，為β溶血性。魚種方面，在吳郭魚、尼羅紅魚、鱸魚、烏魚皆可分離到Ia及Ib型，顯示無感染魚種差異。由收集到的分離株中，挑選一株有莢膜株990906及無莢膜株990823進行實驗，進行生存條件包括培養基（BHIA、TSA、THA）、鹽度（0.1%~10%）、pH值（4.5~12）、溫度（28℃、32℃、35℃、37℃、40℃、43℃）及比較兩株菌於最適合溫度（25℃、28℃）下的生長曲線，結果顯示兩株菌可生長於常見三種培養基； 990823的最適鹽度在3.5%，高於990906的範圍（0.1~1%），而其最適pH值在7~8.5，一樣高於990906的範圍（6~8）；最高耐受溫度以990823較高可耐到40℃，990906僅可耐高溫到35℃；最適生長溫度（25℃、28℃）下的生長曲線以990823生長速率較快且OD值較高。兩株菌在貼附及侵入吳郭魚細胞（卵巢細胞株及腦細胞）的能力部分，在未經攻毒回魚體，毒力回復前，以無莢膜株990823對吳郭魚卵巢細胞株及吳郭魚腦細胞有較明顯的貼附及侵入能力，有莢膜株990906未見侵入吳郭魚卵巢細胞；而毒力回復後，有莢膜株990906對吳郭魚腦細胞的貼附、侵入能力有明顯提升；半致死劑量部分，兩株菌於高濃度（6 × 107、107、6 × 106 cfu/mL）組攻毒魚隻皆於攻毒前3天達到50％以上的死亡率，攻毒有莢膜株990906的高濃度組於48小時後開始有魚隻大量死亡，無莢膜株於攻毒後72小時有魚隻大量死亡，計算結果兩株菌濃度相似，無莢膜株990823稍高於990906；吞噬能力部分，有莢膜株990906對吳郭魚吞噬細胞（周邊血液單核細胞、頭腎巨噬細胞）有較強的抗吞噬能力，與吞噬細胞作用後能於細胞內存活較長時間，而巨噬細胞吞噬了有莢膜株990906後於觀察時間內存活的細胞數比吞了無莢膜株990823後存活的細胞數來得少。

關鍵字

無乳鏈球菌；莢膜；生長曲線；貼附及侵入能力；吞噬能力

並列摘要

In 2010, capsulated Streptococcus agalactiae have been isolated from Red Tilapia’s（Oreochromis sp.）blood, which presented erratic swimming and hemorrhage in skin and fins. Other non-capsulated S. agalactiae have been isolated from other Nile Tilapia（O. niloticus）cases. The goal was to compare the difference between capsulated and non capsulated S. agalactiae in pathogenecity such as adhesion and invasion ability to tilapia cells, anti-phagocytosis ability to tilapia’s macrophages, median lethal dose（LD50）. 37 strains of S. agalactiae have been isolated from Red tilapia, Nile tilapia and many species of bass. We identified the capsular polysaccharide serotype about all isolates and their were 24 isolates belonged to type Ia, all of them were non capsulated and β-hemolytic. Other 13 isolates belonged to type Ib including capsulated, γ-hemolytic isolates （5 isolates）and non capsulated, β-hemolytic isolates（8 isolates）. They were no significant difference in host species. Two isolates grew on BHIA, TSA and THA plate. 990823’s optimal salinity was 3.5%, higher than 990906 (0.1~1%). 990823’s optimal pH was 7~8.5, also higher than 990906 (pH 6~8). The tolerance temperature of non capsulated isolate 990823 was 40℃, higher than capsulated isolate 990906 (35℃). In addition, the growth curve of 990823 grew faster than 990906. In adhesion and invasion test, non capsulated isolate 990823 had higher ability to adhere and invade to tilapia ovary cell line (TO) and tilapia brain cell (TB) than capsulated isolate 990906. After virulence evolution, the invasion of capsulated isolate 990906 to TB was higher than non capsulated isolate 990823. In LD50, the results of three high concentration（6 × 107、107、6 × 106 cfu/mL）groups showed that fish death started at 48hr post-injection（p.i）, and 72hr p.i in 990823. However, cumulative mortality of fish was higher in non capsulated isolated 990823. In phagocytosis assay, capsulated isolate 990906 had higher ability to suppress phagocytosis and survival rate both in peripheral blood mononuclear cell （PBMC）and head kidney macrophage（HKM）.