| Summary: | 碩士 === 國立中興大學 === 獸醫微生物學研究所 === 84 === Vibrio anguillarum 其 pJM1(65-kb) 質體所衍生的攝鐵系統(
plasmidmediated iron uptake system) 在流行病學及其致病性均有密切
的關係。為了探討水生動物之主要弧菌病原是否有 pJM1(65-kb) 質體及
其衍生的攝鐵系統，以及相互間的影響乃進行一系列的試驗。從 79 株弧
菌分離菌株進行質體之萃取發現其中 8 株分離株具有大分子的質體，其
分子量介於 30-140 kb 之間，這八株弧菌分別為 V. anguillarum(5/11)
、V. tubiashii(1/4)、V.parahaemolyticus 和 V.harveyi(1/6)。再利
用限制酵素(RFLP)與隨機聚合酵素連鎖反應 (RAPD-PCR ) 進行質體之分
析，發現此八株具質體的弧菌菌種之間，質體有很大的差異性。為了探討
V.anguillarum、V.tubiashii、V.parahaemolyticus、V.harveyi 四菌種
是否亦具有 pJM1 質體之攝鐵系統，於是針對 pJM1 質體基因設計四組引
子，並以 PCR 與南方雜交反應進行偵測，結果只有 V.anguillarum 才有
pJM1 攝鐵系統。為了更進一步瞭解具 pJM1 質體之 V.anguillarum 於限
鐵生長環境下之影響，因此將具 pJM1 質體之V.anguillarum 與不具
pJM1 質體之其他弧菌菌株分別進行限鐵生長試驗。結果發現各弧菌限鐵
最低抑制濃度 ( MIC ) 值以具質體 V.tubiashii (VT-3-22)(MIC) 值最
高。當 V.anguillarum (13810) 或 V.tubiashii (VT-3-22) 分別與含質
體之 V.parahaemolyticus (38-1) 或不具質體之 V.
parahaemolyticus(10806) 混合培養於限鐵環境中，發現彼此間會有相互
影響生長之現象。經利用 autometed fluorescent DNA sequencing 之方
法，進行各弧菌菌株染色體衍生之攝鐵調節 (ferric uptake regulator;
fur ) 基因之偵測，結果顯示彼此間的同源性介於 80 － 100% 之間。
Vibrio anguillarum, the etilolgical agent of Vibriosis
in aquaticanimals, carries a 65 kb plasmid (pJM1) which can
secrete highly specificiron chelators called siderophore-
anguibactin to transport the ferriciron-anguibactin complex into
the bacterial cells. In our laboratory,several plasmids with
similar large molecular size were recently identifiedin V.
anguillarum (5/11), V. parahaemolyticus (1/7), V.
tubiashii(1/4), andV. harveyi (1/6) from 79 Vibrio spp. which
were isolated from the diseasedaquatic animals. Restriction
fragment length polymorphism (RFLP) and randomamplified
polymorphic DNA (RAPD) PCR methods were applied to identify
theplasmid profiles among those different Vibrio isolates. The
resultsindicated that there were significant molecular variation
of plasmidprofiles among those different Vibrio isolates using
restriction enzymesdigestion and 10-mer random primers for RPAD
amplification. Four specificPCR primers were also desinged to
detect whether different plasmids maycontain all or partial of
iron uptake system gene of pJM1 plasmid as foundin V.
anguillarum. However, only pJM1 (or pJM1-like) but no other
plasmidswas identified to possess the unique iron uptake system
genes using PCR,nested PCR and southern hybridization. Although
no iron uptake genes werefound in those different plasmids, some
influence were still noticed withrespect to the growth rate
under normal and limited iron condition. Ofwhich, V. tubiashii
(VT-3-22) is more resistant to higher limited
ironconcentrations. When V. anguillarum (13810) or V. tubiashii
(VT-3-22)cultured along with V. parahaemolyticus with plasmid or
non-plasmidstrain (38-1 or 10806), each of those Vibrio strains
showed consequentialinfluence of growth rate in each group. The
sequence alanysis usingautomatic fluorescent DNA sequencing
method indicated that the homologyof the fur gene among those
Vibrio spp. in between 80% to 100%.
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