|Abstract: ||摘要:本研究探討生物因子---弧菌-Vibrio carchariae EmI82KL strain 在缺鐵條件下產生致病因子的鑑定、石斑魚體對本菌之免疫反應及試驗性疫苗之保護效果。本弧菌對鐵鰲合劑(Ethylenediamine di (O-hydroxyphenylacetic acid);EDDHA)之 MIC 值為 80 .mu.M,在 Chrome azurol S (CAS)agar plate可成長,其顏色為淡橘色,顯示其會產生Siderophore。此Siderophore 經鑑定為Hydroxamate-type。以 CM 9 agar 或另添加 EDDHA (1/5 MIC 值)之CM 9 agar分別培養本弧菌後抽取之菌體外細胞膜在 SDS-PAGE 膠片上比較發現在缺鐵條件下培養者會多出現兩條高分子量之蛋白帶,分子量分別為 78及 86 kDa。將本弧菌在 30.degree.C,24 小時條件下培養於添加有 2%鰻粉之Agar(+2% NaCl)所產生之蛋白質分解酵素活性(Protease activity)明顯高於同條件培養於 Brain heart infusion agar (BHIA,+2% NaCl),Tryptic soya agar (TSA,+2% NaCl) 或 CM 9 agar 者。以電子顯微鏡 (SEM) 觀察結果顯示本弧菌對石斑魚腸道不具明顯之附著能力。以兔子抗菌體抗血清及兔子抗細胞外產物抗血清進行石斑魚被動免疫試驗,在高達 10/sup 8/ CFU/g 魚體重之致死劑量下未造成魚死亡,顯示兩種兔子抗血清對魚皆具被動免疫效果。而以菌體經 3%福馬林在室溫處理 48 小時且經以 PBS 透析後之試驗疫苗進行腹腔注射(10/sup 8/ CFU/g 魚體重)或浸泡(10/sup 6/ CFU/ml 海水)石斑魚,在 60 天後雖然可測得魚體血清免疫反應,但若同樣以 10/sup 8/ CFU/g 魚體重進行攻擊,則魚在二週內全數死亡。此可能是魚體對菌體之免疫反應遠較兔子為差,也可能是因 TSA agar 培養之細菌菌體成份所具各保護性抗原量低或與感染部位在腸道有關,這些可能性都則仍待進一步研究。|
Abstract:The present study investigated virulence factors of biological factor---Vibrio carchariae EmI82KL strain at iron-limited condition, immune response of the groupers (Epinephelus sp.) against the bacterium and protection of the fish treated with tested bacterins against bacterial lethal challenge. The MIC value of ethylenediamine di (O-hydroxyphenylacetic acid (EDDHA, an iron-chelator was determined to be 80.mu.M. The bacterium could grow on chrome azurol S (CAS) agar plates with an orange color indicating that the strain produced a siderophore which was further identified to be a hydroxamate-type siderophore. Outer membrane proteins (OMP) were extracted from the bacterial cells grown on CM9 agar plates with (iron-limited condition) or without (iron-replete condition) the presence of 1/5 MIC value of EDDHA for comparing respective protein profile on SDS-PAGE. At iron-limited condition, two higher molecular mass OMP, 78 and 86 kDa, of the bacterial cells were additionally produced which were not present when the strain grown at iron-replete condition. An apparently higher extracellular protease activity was obtained when the bacteria grew on 2% eel meal agar (+2% NaCl) compared to those grew on brain heart infusion agar (BHIA, +2% NaCl), tryptic soya agar (TSA, +2% NaCl) or CM9 agar at 30.degree.C for 24 h. No apparent adhesion ability on grouper intestine was observed using scanning electronic microscope technology. Groupers were passively protected against bacterial lethal challenge, 10/sup 8/ CFU/g fish body weight, when intraperitoneally (i.p.) pre-injected with rabbit antisera to the bacterial cells or its extracellular products. In addition, groupers were i.p. injected or immersed with formalinized bacterial cells (treated with 3% formalin at room temperature for 48 h and then dialyzed against PBS) and maintained for 60 days prior to a lethal challenge (also at a dose of 10/sup 8/ CFU/g fish). All the fish were killed during 2 weeks observation although immune response to the bacterial antigens could be detected in fish sera after the immunization. The reason may be due to a poorer immune response elicited in the fish than that in the rabbit. Other possibilities may also exisit, i.e. lower protective antigens were present on bacterial cells when grew in vitro (TSA agar plates) or the fish intestine was the major infected site. However, these possibilities need further study to resolve.