|Abstract: ||摘要 本論文針對不同養殖環境、投餵餌料與成長階段之臺灣鮑 (Haliotis diversicolor supertexta)，進行流行病學、弧菌感染路徑、致病機制及病害防治之研究。 試驗發現弧菌為臺灣鮑養殖環境之優勢菌種，並廣泛存在於養殖設施、環境水體、投餵藻類與親源。自罹病臺灣鮑所分離之溶藻弧菌(Vibrio alginolyticus strain E2-6-2)、鮫弧菌(V. carchariae strain VC200107)與腸炎弧菌(V. parahaemolyticus strain YU2)，除分別造成附著貝苗脫落、足部肌肉囊腫、潰瘍，殼緣內側褐色物質堆積，膿包與萎縮外，並經投餵、注射與浸泡菌體試驗證實，三株弧菌皆可藉由接觸、攝食與個體間之水平傳播，對不同成長階段之臺灣鮑具致死毒性。 溶藻(strain E2-6-2)及腸炎弧菌(strain YU2)之菌體於18、23及28℃時對臺灣鮑之LD50分別為1.89×105、7.46×104、4.87×104及8.27×108、3.91×105、1.03×105 cfu/g，細胞外產物之LD50則分別為3.08、1.06、0.64及2.74、1.37、0.60 μg protein/g 體全重；鮫弧菌於23及28℃下菌體與細胞外產物之LD50則分別為2.15×107與3.05×106 cfu/g 及2.27及1.31μg protein/g 體全重，顯示臺灣鮑對病原之感受性與環境溫度呈正相關。試驗亦發現低溫因子可使溶藻弧菌分泌具單位活性較強之蛋白質分解酵素，並增進對臺灣鮑之毒性作用。 病原性溶藻弧菌(strain E2-6-2)、鮫弧菌(strain VC200107)及腸炎弧菌(strain YU2)之細胞外產物部分純化蛋白質分解酵素，皆為熱不安定型之絲胺酸型蛋白分解酵素，以SDS-PAGE估算，部分純化之蛋白分解酵素其分子量分別為33、33及94 kDa；對臺灣鮑之LD50則分別為0.31、1.70及0.41 μg protein/g 體全重。 此三株病原性菌株對novobiocin (30 μg)、tetracycline (30 μg)及furazolidone (100 μg)呈現敏感。使用不同臭氧與紫外線進行殺菌試驗，發現通入濃度15 mg /h/50 L臭氧，並搭配重複循環開啟-關閉2次下對菌株具有最佳控制效果；但過量臭氧卻易對臺灣鮑造成明顯傷害。連續照射5L/10W/min之紫外線達6小時可明顯降低環境菌量，但對具游走(swarming)特性之溶藻與鮫弧菌則無控制與提升受感染個體活存率之效果。 龍鬚菜(Gracilaria sp.)表面菌相以弧菌為主，且以溶藻弧菌為優勢菌種。20 ppm之furazolidone可有效控制菌量，碘液、雙氧水、過錳酸鉀及次氯酸鈉雖可有效抑菌，但卻易對藻體造成白化及死亡等嚴重損傷。 分離自健康臺灣鮑腸道及穩定育苗環境之各一溶藻弧菌菌株，具抑制病原菌株之能力。投餵此非病原性活菌與去活性病原菌體，可增加臺灣鮑血球細胞中之酚氧化酵素活性並降低對弧菌症之感受性，但連續投餵卻可能導致免疫疲乏。以上述2株非病原性溶藻弧菌、1株假單胞菌(Pseudomonas spp.)與紫3株色光合作用細菌(Rhodobacter sphaeroides)等菌株進行抑菌試驗，得知非病原性溶藻弧菌，對病原菌株表現明顯抑制能力，其次為假單胞菌。但由於病原菌株與具抑制性菌株特性相似，在養殖現場使用與鑑定具一定困難性，因此相關應用仍須探討；而不同來源之光合菌則無抑制效果。 由本研究結果得知，臺灣鮑之主要疾病多發生於季節交替及水溫偏高時期，而不同飼養模式與投餵管理，也使養殖環境及臺灣鮑體內之菌相與菌含量呈現明顯差異；其中使用未經過濾水源、投餵帶菌餌料與過高密度養殖，皆為造成養殖環境異常菌相與菌量之主要原因，並易引發相關感染與疾病傳播。而貝苗於附著初期發生大量落板現象，則可能來自親源垂直感染及浪板表面之異常菌相。 爲降低臺灣鮑受弧菌感染之可能性，因此謹提出下列管理員則供業者參考：1.持續監控環境菌相並降低弧菌量；2.對養殖用水及投餵藻體進行除菌處理；3.避免帶菌種源之垂直感染與4.適當使用抑制菌(益生菌)處理，如此應可提升個體活存率並達成貝苗之穩定附著及成長。|
Abstract This thesis studied on the epizootiology, infection routes, pathogenesis and disease control of vibriosis in small abalone (Haliotis diversicolor supertexta) cultured in various environments, fed on different foods and grew to different stages (sizes). Vibrio species were found to be the dominant bacterial species in the aquacultural systems for small abalone including facilities, water, seaweeds (food) and broodstocks. Vibrio alginolyticus strain E 2-6-2, V. carchariae strain VC200107 and V. parahaemolyticus strain YU2 were isolated from diseased small abalone exhibiting settlement failure (spats) and occurrence of abscess and ulcers in the mantle, occurrence of abnormal depositions of secreted brown material on the inner-shell surface, and occurrence of cysts and withering (atrophy) syndrome, respectively. In addition, the three Vibrio species could transmit horizontally as observed by the lethalities obtained from bacterial feeding, injection and immersion in the abalone at different growing stages. The LD50 values of bacterial cells and the extracellular products (ECP) of V. alginolyticus strain E 2-6-2 and V. parahaemolyticus strain YU2 in small abalone raised at temperatures 18, 23 and 28℃ were 1.89×105 cfu and 3.08 μg protein/g abalone, 7.46×104 cfu and 1.06 μg protein/g abalone, 4.87×104 cfu and 0.64 μg protein/g abalone, and 8.27×108 cfu and 2.74 μg protein/g abalone, 3.91×105 cfu and 1.37 μg protein/g abalone, 1.03×105 cfu and 0.60 μg protein/g abalone, respectively. In addition, the LD50 values of bacterial cells and the ECP of V. carchariae strain VC200107 in small abalone raised at temperature 23 and 28℃ were 2.15×107 cfu and 2.27 μg protein/g abalone, and 3.05×106 cfu and 1.31 μg protein/g, respectively. These results revealed that the susceptibility of abalone to the three Vibrio species was temperature dependent. The protease activity of the ECP produced by V. alginolyticus was enhanced when grown at low temperature and exhibited higher toxicity in the abalone. Partially purified proteases from respective ECP of V. alginolyticus, V. carchariae and V. parahaemolyticus were all belong to heat labile serine protease with molecular mass (estimated by SDS-PAGE) of 33, 33 and 94 kDa, and LD50 values of 0.31, 1.70 and 0.41 μg protein/g abalone, respectively. The three Vibrio species were all susceptible to 30 μg novobiocin, 、30 μg tetracycline and 100 μg furazolidone. The addition of 15 mg/h/50 L ozone into the water with opening and closing twice exhibited optimal bactericidal effect against vibrios. However, an over dose of ozone might cause some distinct damage to the abalone. The continuous radiation by using UV light at a dose of 5L/10W/min for 6 hr could reduce the total bacterial counts. But, this UV treatment was not effective on reducing swarming Vibrio such as V. alginolyticus and V. carchariae, and did not promote the survival rate of the abalone. The majority of the bacteria isolated from surfaces of Gracilaria sp. (seaweed for abalone) found to be Vibrio species with V. alginolyticus as the predominant species. A treatment of 20 ppm furazolidone coud reduce bacterial counts on the surfaces of Gracilaria sp. Although the treatment o iodine, hydrogen peroxide, potassium permanganate and sodium hypochlorite could inhibit bacterial counts in the surfaces of Gracilaria sp., they could also seriously damage the seaweed leading to bleaching and death. Two strains of V. alginolyticus isolated from gut of healthy small abalone and water raising spats with good survival rates were found to be inhibitory against the three pathogenic Vibrio species. The feeding of viable cells or formalin-inactivated cells of these two non-pathogenic V. alginolyticus strains could enhance the pro-phenoloxidase activity in hemocytes of abalone and reduce its susceptibility to vibriosis. However, the continuous feeding might cause the immuno-exhaustion of the abalone. In inhibitory tests, the two non-pathogenic strains of V. alginolyticus exhibited higher inhibitory activity against the three pathogenic Vibrio species, and followed by a strain of Pseudomonas sp. with no inhibitory activity exhibited by three strains of Rhodobacter sphaeroides. The present results revealed that the majority of the diseased in small abalone occurred during the change of season and in high temperature period. Significant differences of bacterial flora and bacterial counts in the environment of aquacultural system and in the small abalone employing different feeding protocols and management were determined. Of these, the use of un-filtered sea water, feeding with foods contaminated with pathogens, and culturing in high density may be the major causes leading to a further dissemination of the disease. The settlement failure of spats may be due to a vertical transmission of pathogens from broodstocks together with the presence of abnormal bacterial flora on the surfaces of substrate. Therefore, to reduce the possibility of Vibrio infection in small abalone, the following control measures were suggested for the farmer: 1. Monitor the bacterial flora of the aquacultural system and reduce the Vibrio counts continuously. 2. Eliminate bacterial counts of sea water and seaweeds to be used. 3. Avoid the vertical transmission of pathogens from broodstocks and 4. Use appropriate probiotics. Thus, it may promote survival rate of cultured small abalone and maintain stable settlement and growth of spats.