淡水长臂大虾酵母菌感染症之病原监定病原性与快速诊断方法之研究.docVIP

淡水長臂大蝦酵母菌感染症之病原鑑定、病原性與快速診斷方法之研究Identification, Pathogenicity and Rapid Diagnostic Method of Yeast Infection in Giant Freshwater Prawn Macrobrachium rosenbergii 研究生： 陳育津 Chen, Yu-Chin 陳石柱 本省養殖淡水長臂大蝦 Macrobrachium rosenbergii 經常於冬季低水溫期發生酵母菌感染症，臨床上呈肌肉與血淋液白濁，組織病理學下各組織臟器呈變性壞死並有多量的酵母菌浸潤。由於此疾病於臨床上之鑑定結果分歧，故本實驗由所分離的菌株進行生化鑑定套組API ID 32C及26S rDNA D1/D2 domain序列的PCR技術增幅與定序來進行鑑定。鑑定結果中25株分離株和2株分讓株為Metschnikowia bicuspidata Genbank Accession No. U44822，相似度97-98% ，1株分讓株為Saccharomyces cerevisiae和1株分離株為Candida albicans 分別與Genbank Accession No. U53879和AF156536的相似度均為100% 。另將本次定序結果中為M. bicuspidata之菌株與標準株所增幅出約520 bp的D1/D2 domain片段產物，選用兩種限制酶BslI和XhoI進行切割，並自行設計針對M. bicuspidata病原菌的特異性引子對 MB-F和MB-R 。經採集病蝦之各組織臟器檢測其所含酵母菌量後，發現血淋巴液、肝胰腺、鰓和肌肉等組織的平均含菌量分別為9.0×109，4.9×109，8.0×108，4.5×108 CFU/gm。由定序鑑定證實本省淡水長臂大蝦感染之酵母菌計有M. bicuspidata、S. cerevisiae和C. albicans三種；而主要病原性酵母菌為M. bicuspidata。分別以健康蝦隻進行病原性之研究，結果顯示三種不同酵母菌的半致死劑量 LD50 分別為3.8×103、2.0×103和4.3×103 CFU/prawn。本研究中利用自行設計的特異性引子對之PCR反應和兩種特定限制酶之Restricted fragment length polymorphism技術，已建立了最快速又正確地診斷、鑑定泰國蝦酵母菌M. bicuspidata感染症的方法。淡水長臂大蝦 ; 酵母菌感染症 ; 快速診斷方法In Taiwan, yeast infection of giant freshwater prawns Macrobrachium rosenbergii is an outbreak often during the low water temperature periods in winter. The clinical signs of infection are white cloudiness of muscle tissue and hemolymph. Histopathological examinations of affected tissues revealed extensive necrosis associated with large numbers of yeast aggregates. In previous neports, the results of clinical diagnosis are differences. In this study, we identified the isolate yeast by API ID 32C, by PCR assay and 26S rDNA D1/D2 domain sequencing. The sequences of 26S rDNA from 25 isolates and 2 share strains gave 97-98% sequence identity to as Metschnikowia bicuspidata GenBank Accession No. U44822 . One share strain was identified to as Saccharomyces cerevisiae and one isolate was Candida albicans, both their sequences of 26S rDNA D1/D2 domain gave 100% sequence identity to Saccharomyces cerevisiae G