水產生物科技產業開發計畫

• Main Author: 楊惠郎
• Format: Report
• Language: Chinese; English
• Published: 2006
• Subjects: Detroit; Midtlyng; Nordmo; St. Louis; Steine; atlantic cod; Atlantic salmon; Gadus morhua; Salmo salar
• Online Access: http://ir.lib.ncku.edu.tw/handle/987654321/140171; http://ir.lib.ncku.edu.tw/bitstream/987654321/140171/-1/index.html

計畫編號:95農科-6.1.2-科-a3 執行機構:成功大學生物科技研究所 研究期間:2006-02~2006-12 為迅速產品商品化，及協助產業，建立國際競爭力，必需完成具經濟效益之量產產程，以降低生產成本。在技術移轉前極需進行下列基盤工作：第一年度：整合健康種苗及關鍵技術：健康魚苗先導工廠規劃完成後，希達到下列工作目標：製程的放大：規模以每批次生產二十萬尾、全年生產100萬尾為目標，並建立標準作業程序。 1. 訓練儲備人才，移轉廠商。 2. 簡化產程作業步驟。 3. 降低生產成本。 4. 乾淨之初期餌料試量產設施及方法。 5. 試量產健康魚苗產品。 6. 建立具經濟規模之健康魚苗標準生產作業程序(SOP)。 一、 健康魚苗： 1.1 SPF養殖環境監控-完成“SFP養殖場環境監控作業，及檢驗工作手冊”一本(第一年第一季完成)。 1.2 高品質，無病毒及病菌的孵化卵的品管及處理方法：完成“魚卵品管，及檢驗工作手冊”一本(第一年第二季完成)。 1.3 魚苗疾病防治：完成“種苗場疾病預防產品使用，品管，及檢驗工作手冊”一本(第一年第三季完成)。 1.4 高品質之初期餌料：完成“生物餌料製造，品管，及檢驗工作手冊”一本(第一年第四季完成)。 1.5 完成“石斑健康種苗生產手冊”一本(第一年第四季完成)。 二、 疫苗關鍵技術： 2.1 穩定菌種及病毒種種原-完成“疫苗種原鑑定，保存，及品管操作手冊”一本(第一年第二季完成)。 2.2 口服疫苗生產方法的改進-完成“疫苗製造，安全性，有效性測試及品管操作手冊”一本(第一年第四季完成)。 2.3 健康種苗產品試用結果評估-每半年提出養殖業者產品使用結果報告一篇，作為產品績效的評估。 三、 市場調查：完成國際市場調查報告一份，完成國內外產品策略計畫書一份。 Cobia (Rachycentron canadum), a warm water fish recently commercially cultured in Taiwan, has encountered severe mortalities with survival rates often below 20%. The major causative pathogens were Vibrio alginolyticus, V. parahaemolyticus and Photobacterium damselae subsp. piscicida. We prepared a combined three inactivated bacterins antigen preparation and vaccinated cobia. This mixture of bacterins was safe, and the immune response in cobia stimulated specific antibody in one week that remained for at least six weeks until the end of the aquarium trial. Its efficacy in protecting fish was evaluated in aquarium and field trials. In the aquarium challenge, the vaccine gave a relative percentage survival of 93.8%, 91.1% and 84.7% after challenge with V. alginolyticus, V. parahaemolyticus and Photobacterium damselae subsp. piscicida, respectively. In two farm trials using two batches of fish from different hatcheries, one vaccination gave a survival rate of 86-92%. A single vaccination of three combined inactivated bacteria into cobia elicited specific antibodies, and protected fish in both the laboratory aquarium challenge and a farm trial. Key words: Inactivated bacterin, efficacy and safety, Vibrio alginolyticus, Vibrio parahaemolyticus, Photobacterium damselae subsp. piscicida. 1. Introduction: Cobia (Rachycentron canadum) is a pelagic fish common in tropical and subtropical oceans (Fricke 1999; Vaught and Nakamura, 1989). It is the sole species of the genus, belonging to the Rachycentridae, order Perciformes. Its common names include large black kingfish, ling, crab eater, sergeant fish and lemon fish. Culture of cobia in marine cages was developed in Taiwan a decade ago. Since then, cobia farming has received worldwide attention because of the excellent meat quality, with fat content often reaching 25% in the belly, and growth rate to 6 kg in the first year (Su et al., 2000). However, by the third year of production massive mortalities occurred, especially during the early grow-out period when fish were less than 4 months old and below 500 g. The major pathogens of cobia in Taiwan are bacteria. Liu et al. (2003) reported Photobacterium damselae, Rajan et al. (2001) and Liu et al. (2004) isolated Vibrio. alginolyticus from diseased cobia. The aim of this study was to investigate the use of a combination of three inactivated bacterins of local bacterial isolates to induce specific antibody and stimulate a protective immunity in challenge tests in a laboratory aquarium system and in cage farms. 2. Material and methods: 2.1. Bacteria antigen preparation: Local strains of Vibrio alginolyticus (PFVAG-101, our collection number), Vibrio parahaemolyticus (PFVP-06) and Photobacterium damselae subsp. piscicida (PFPP-02) were used for the preparation of inactive bacterins. Bacterins were prepared from fresh cultures of bacteria raised from frozen stock under the following conditions: Vibrio spp. were grown in trypticase soy agar (TSB; Difco, Detroit, MI, USA) with 2% NaCl at 25 ℃ for 16 hours, Photobacterium damselae subsp. piscicida was incubated in brain heart infusion (BHI, Difco) agar with 2% NaCl at 25 ℃ for 24 hours. One colony was selected from each agar plate and inoculated to its specific broth medium and grown to OD600nm = 1. After harvest, the bacteria suspension was inactivated by adding formalin to a final concentration of 3% and incubating overnight. The inactivation of bacteria was confirmed by plating 0.1 ml of bacterial suspension on BHI agar with 2% NaCl and incubating at 25 ℃ for 24 h. Absence of bacteria indicated inactivation. Formalin was removed by three cycles of washing with 0.15 M phosphate buffered saline (PBS), pH 7.2 at 4 ℃ and centrifugating at 4500 x g for 5 min. The final bacterial pellet was suspended in pre-chilled saline, and the concentration of the suspension was adjusted to OD600nm = 1. The three bacterial suspensions were combined in equal quantities to which was added an equal volume of commercial oil-based adjuvant (Fu-Bo Inc., Taipei, Taiwan). The mixture was homogenized with an Uultrason (Vibra cell ultrasonicater, Sonics & Materials Inc., Newtown, Ct, USA) to a final emulsion particle size of 10 nm, and stored at 4 ℃ until use. 2.2. Fish for safety study and efficacy trial: Cobias used in this study were obtained from southern Taiwan at 40 days post-hatching (DPH) when they were approximately 5 g and 8 cm long. Fish were held in 200 l FRP (Fiberglass Reinforced Plastics) tanks supplied with filtered, aerated, UV treated regular seawater and fed commercial dry pellets (Chuenshin Co., Ping-Tung, Taiwan), at 3% body weight, twice daily. Fish health was monitored weekly by morphological, histopathological and microbiological analyses for 50 days (90 DPH) at which time fish weighed on average 50 g and were approximately 20 cm long. Their health status was stable and they were considered suitable for safety and efficacy tests. 2.3. Safety test of vaccine: Three experimental groups: vaccine, adjuvant only, and PBS control, each with 20 cobias, were used to evaluate the safety of the combined antigen preparation and adjuvant. Fish were implanted with visible alphanumeric tags in three different colors (Northwest Marine Technology, Inc., WA, USA) for group identification. Fish were starved 24 h before injection, anesthetized by immersion for 2 min in fresh sea water containing 300 ppm 2-phenoxy-ethanol (Sigma, St. Louis, USA), and injected intraperitoneally (IP) with 0.1 ml of the respective solutions. The vaccine group was injected with a mixture 10x the designed dosage of 1mg fish-1 of antigen, 20?慊 g-1 fish mixed with adjuvant; the adjuvant group was injected with a mixture of PBS mixed (v/v = 1:1) with adjuvant, and the control group was injected with PBS only. Fish were then distributed into four 200 l FRP tanks, each tank containing 5 fish from each of the three groups. Fish were observed for mortality, abnormal swimming behavior, and appetite for 60 days post vaccination (PV). They were then killed for pathological and histopathological examination. 2.4. Efficacy trial: Fish vaccination: Fish were obtained as for the safety test (section 2.2) and distributed randomly into two groups. The vaccine was injected intraperitoneally by the same process as in the safety test. In the vaccine group, fish were injected with the antigen preparation (0.1 mg fish-1) premixed with adjuvant. The control group fish were injected with PBS premixed with adjuvant. Then fish were held in 15 ton [you refer to volume in other references to these tanks. I do not think you mean ton which is a measure of weight!] FRP tanks supplied with filtered, aerated, UV treated regular sea water. Feeding and management was as described in the safety test. Blood samples were collected randomly from 30 fish of each group at weeks 1, 2, 4 and 6 PV, for antiserum analysis. At the end of week 6, 60 fish from each group were transferred to laboratory tanks for the aquarium challenge test, and the remainder were transferred to the fish farms for the field trial. 2.5. Efficacy trial: Antiserum analysis: ELISA plates were prepared, and anti-bacterium antibody titer was analyzed following the method of Magnadottir et al. (1999). The micro titer plate was prepared using protein extracted from freshly grown bacteria as the coating antigen. Cobia blood samples were drawn from the caudal vein, coagulated at 25 ℃ for 2 h, and centrifuged at 4500 xg, at 25 ℃ for 10 min to obtain the serum. The serum was then diluted 1:50 with PBS, and 0.1 ml of each diluted sample was used for ELISA assay. A secondary antibody was prepared by immunizing rabbit with a liquid chromatography-purified cobia immunoglobulin (cobia Ig) following the method of Watts et al. (2001). Commercial goat anti-rabbit antibodies alkaline phosphatase conjugate (Bethyl Laboratories, Montgomery, TX, USA) was used as the tertiary antibody. The secondary and tertiary antibodies were diluted 1:1000 immediately before use. Chromogen (0.1 ml of p-nitrophenyl phosphate [1 mgml-1; pNPP, Sigma Chemical Co., St. Louis, MO, USA] in 10% diethanolamine buffer, pH 9.8) was added to each reaction well, and color allowed to develop for 30 min at 37 ℃. It was measured in a micro titer plate reader (Multiskan RC, Labsystems, Helsinki, Finland) at 405 nm. Student's t-test was used to evaluate the statistical significance of the data. 2.6. Efficacy trial: Laboratory aquarium challenge: One hundred and twenty fish were distributed into 6 tanks, each tank containing 10 vaccine fish and 10 control fish. Fish were challenged in replicate with V. alginolyticus, V. parahaemolyticus or P. damselae subsp. piscicida, respectively. Bacteria used for the challenge experiment were prepared freshly from a frozen stock with fresh culture medium as described earlier. Bacteria in exponential growth phase were concentrated by centrifugation, resuspended in growth medium, and adjusted to a density of 1 x 108 CFU ml.-1. Fish were challenged by IP injection as described earlier with the predetermined LD60 of Vibrio spp. (1 x 106 CFU g-1 of fish) and LD99 of Photobacterium (1 x 106 CFU g-1 of fish ), and the fish were observed for 35 days. Dead fish were recorded, collected, and examined to confirm cause of death. Samples from kidney, liver spleen and wounds were cultured to identify Vibrio spp, and Photobacterium identified by a specific PCR plus a TCBS-selective agar method (Rajan et al., 2003). 2.7. Efficacy trial: Field trial: Efficacy of the vaccination in cobia was assessed under cobia cage farming operation. To evaluate the effect of location and individual farming practices, trials were conducted at two different farms in southern Taiwan with a double blind protocol (trial A and B). Each trial had two experimental groups, a vaccine group and a control group. The number of fish used was based on cage size to maintain the farming density at 1.5% of the weight at the end of production over the total cage volume (W/V). All fish were tagged, as described previously, and transferred to the marine cage farms when at least 4 weeks PI. The maintenance of fish followed the routine of each farm. Dead fish were removed routinely from the cage bottom every one or two days and counted. The performance of the vaccine was monitored by examining these fish using the methodology described previously for the laboratory challenge test. According to clinical records of cobia in Taiwan, the most disease prone period is the first four months when fish are below 500g weight. Average mortality is 70%. Therefore, the efficacy of immunization was evaluated at four months and again at 8 months, when fish reached market weight of 6-8 kg. At the end of the 4th month, fish were netted, counted (by tag scanning) by farm workers to determine the number of surviving fish in each group, and the Relative Percentage Survival (RPS) calculated as described by Amend (1981). 3. Results: 3.1. Safety of the vaccine: The safety of the vaccine was evaluated by injection of a dosage tenfold that used in the treatment study (1 mg per fish). All fish survived with no abnormality in swimming behavior and no observed morphological or pathological changes during the 60 day observation. Fish in the vaccine group and in the adjuvant group consumed about 15% less food in week 1, as also reported by Midtlyng (1994); but then resumed normal intake. Fish in vaccine and adjuvant groups developed adhesions in the abdominal cavity at the injection site (data not shown). Fish injected with PBS were not affected, indicating that the adhesion and lost of appetite were possibly related to the injection of oil based adjuvant. 3.2. Development of serum specific antibody after immunization: The ELISA plate using whole cell extract of V. alginolyticus, V. parahaemolyticus, and P. damselae subsp. piscicida, separately as coating antigens was employed to measure the development of specific serum antibody after vaccination. In fish of the vaccine group, anti- V. alginolyticus and anti- V. parahaemolyticus increased to almost 0.7 during week 1 PV. It increased to 0.8 between weeks 2 and 4 PV and plateaued until at least the end of week 6. Fish serum titers against P. damselae subsp. piscicida rose to 0.5 in week 1 PV, and reached 0.7 in week 6 (Fig. 1). All serum titer measurements were significantly different from those of the control groups based on Student's t-test (P < 0.05). The experiment was terminated at the end of week 6, when the fish grew too large (150 g) to be maintained in the laboratory aquarium. 3.3. The efficacy test in the laboratory challenge: In order to evaluate the efficacy of vaccination accurately and rapidly, a reproducible laboratory challenge was developed. Fish were challenged with an injection of 106 CFUg-1 of fish, the LD60 for V. alginolyticus and V. parphaemolyticus and the LD99 for P. damselae subsp. piscicida. Control fish challenged with V. alginolyticus, died from day 6 to day 12 after injection. Those challenged with V. parahaemolyticus died from day 8 to day 20, with 45% and 50% of fish surviving, respectively. None of the vaccinated fish died (Fig. 2). In fish challenged with P. damselae subsp. piscicida mortalities began on day 9, increased rapidly from day 11 to day 14, and continued until day 18; mortalities ceased before the end of day 20. Vaccinated fish died from day 9 through day 12. The mean survival rate for the vaccinated group was 85% (Fig. 2). Cause of death in all cases was determined to be the challenge bacteria. Two additional challenge trials have been completed and results of these trials were similar. The mean RPS of the efficacy tests from three trials was 93.8% for V. alginolyticus, 91.1% for V. parahaemolyticus and 84.7% for P. damselae subsp. piscicida (Table 1). Vaccination of cobia therefore stimulated protective immunity in cobia in the laboratory aquarium challenge. 3.4. Development of protective immunity in the field trial: The efficacy of the vaccine in the field was evaluated in two cage farms, one in Peng Hu Island (trial A) and one in Ping Tung (trial B). In field trial A, fish were transferred to the cage in Ping Tung on February, 2003, when the average water temperature was 22 ℃. Two groups of 750 fish were used. First mortalities were in the control fish on day 2, and continued to day 13. The pathogen was identified as V. alginolyticus and the mortality was about 6%; there were no mortalities in vaccinated fish during the same period. The second mortalities, also in control fish, were caused by P. damselae subsp. piscicida. Three major mortality peaks in the infection period occurred from day 19 to 27, day 31 to 49, and day 54 to 69. The mortality was 10%, 13% and 17%, respectively. In the vaccinated fish there was one major mortality peak from day 14 to 24 with mortality about 7%. At the end of the 4 month observation period, the cumulative survival rate of the control group was 47% (356 fish left), and 92% in vaccinated group (697 fish left) with an RPS of 85% (Table 2). Trial B was conducted in October 2003, at Peng-Fu Island, when the average water temperature was 24 ℃. Approximately 2500 fish were used in each of two groups. The pattern was similar to that of trial A. For the control group, there were three major peaks of mortality from day 3 to 21, from day 80 to 90, and from day 100 to 107. The mortality was 9%, 45% and 20%, respectively, and the major pathogen identified was P. damselae subsp. piscicida. Mortality in vaccinated fish was about 11%, with all mortalities occurring before day 53. The overall survival rate at the end of fourth month was 19% for the control group and 89% for the vaccinated group (Fig 3b) with a corresponding RPS of 86% (Table 2). The fish in trial A and trial B were grown to market size (6 kg) by the end of 8 months and harvested. No significant mortality was observed from the 4th month to the 8th month. The survival rate of the vaccinated group was similar in both trials, indicating that vaccination could stimulate protective immunity in cobia for at least the first 4 months of life when fish are most sensitive to pathogens. 4. Discussion: According to regional veterinary clinical records and the literature, the major bacterial pathogens of cobia in southern Taiwan, where most of the marine cages are located, (Rajan et al., 2001; Liu et al., 2004) are V. alginolyticus, V. parahaemolyticus and Photobacterium damselae subsp. piscicida. These bacteria have been reported as pathogens for various other fish and shellfish; for example Vibrio alginolyticus in tiger prawn Penaeus monodon (Lee et al., 1996; Ruangpan et al., 1991) and in sea mullet Mugil cephalus (Burke et al., 1981). V. parahaemolyticus has been reported in sole Solea senegalensis (Zorrilla et al., 2003) and P. damselae subsp. piscicida in white perch (Morone americana; Janssen et al., 1968). Other bacterial pathogens such as Streptococcus and Aeromonas have been reported by local veterinarians, but these bacteria have been infrequently isolated from diseased fish. Vaccination of cold water fish such as salmonids has proven successful and is already common practice. However, there is no report on the effect of vaccination on warm water fish species. In this study, a combination of bacterins from three major bacterial pathogens isolated from cobia farms in Taiwan was administered to immunize cobia, and the immune response was measured. A single vaccination with bacterial antigen induced a specific antibody response that protected cobia in the laboratory aquarium challenge and on the farm. Several bacterial components have been tried in vaccine formulation. Research has shown that some vaccines using only whole bacteria provided good protection (Midtlyng, 1996; Ross and Klontz, 1965). Others have suggested that additional components such as extracellular products (ECPs) could enhance immune protection (Santos et al. 1991). ECPs have been added to vaccines for Aeromonas, Photobacterium, and Vibrio (Ellis, 1991; Arnesen et al., 1993; Bonet et al., 1994; Bricknell et al., 1997; Magarinos et al., 1992). With a P. damselae subsp. piscicida vaccine, the culture media and culture conditions have been reported to influence efficacy. Bakopoulos et al., (2003) showed that protection rate can vary with the growth medium and suggested that this phenomenon might be due to the synthesis of some undefined ECP product. Hirono et al. (1997) found that potent antigens of P. damselae subsp. piscicida were produced only under special growth conditions, but the identity and character of the effective antigens remain unknown. Although our vaccine was formulated from whole bacterins, without the addition of ECP, it was effective in the laboratory challenge and field trials. An initial evaluation of the benefit of adding ECPs from V. alginolyticus, V. parahaemolyticus and P. damselae subsp. piscicida to our vaccine formulation indicated that ECP was toxic in grouper cell culture (in vitro) and to live cobia (in vivo) with a lethal dose of about 15 µg g-1 of fish weight (data not shown). Ellis (1991), working on Aeromonas, reported a similar effect. The cobia challenge protocol was established by modifying several reported procedures (Gudmundsdottir et al., 2003; Magarinos et al., 1994; Midtlyng et al., 1996; Romalde et al., 1997). The challenge dose of the pathogens that caused 60-99% mortalities was used (Reitan and Secombes, 1997; Nordmo, 1997) and was reproducible in several challenge trials. For convenience, a dosage of 106 bacteria that usually caused 60% mortality (LD60) in the two Vibrio species and 99% mortality (LD99) in the P. damselae subsp. piscicida group was employed for our challenge trial. Methods of infection, such as injection, scraping the skin, and immersion have been evaluated in cobia. Although immersion may mimic the natural infection route, it was difficult to reproduce and impractical to perform in the laboratory. The injection route in the aquarium trial, although not reflecting the natural route of infection, was more reproducible and provided a reliable indication for subsequent farm trials (Nordomo, 1997). In addition to the two cage trials, an addition trial was performed using 15,000 vaccinated cobia in July 2004 at Peng-Fu Island when water temperature averaged 27 ℃. During the initial four month observation period the survival rate in the vaccinated group reached 86.3%, while the survival rate of fish in surrounding cages averaged 15%. These results indicate that vaccination can protect cobia in the field environment in different farming locations. The ELISA assay can be used to predict the potency of protective immunity before fish are transferred to a marine cage and for monitoring health during the farming period. Some reports indicate that antibody titers in Atlantic cod (Gadus morhua L.) (Arnesen et al., 2002) might not a good indicator of protection, however, antibody titer measured by ELISA was useful in evaluating the efficacy of vaccines in Atlantic salmon (Salmo salar L.) and Atlantic halibut (Hippoglossus hippoglossus L.) (Reitan and Secombes, 1997; Bricknell et al., 1997; Gudmundsdottir et al., 2003; Hirono et al., 1997; Midtlyng et al., 1996; Olivier et al., 1985; Santos et al., 1991). Our ELISA data (unpublished) indicated that the immune response in vaccinated cobia correlated well with the protection efficacy. We observed that cobia could produce antibody within one week of injection (about 200 days) in our laboratory aquarium, which is a shorter time than is seen in salmon (Steine et al., 2001). The antibody titer remained elevated for at least 6 weeks, when our laboratory evaluation was terminated. In farm cages, most vaccinated fish and control group survivors can live up to 8 months with one vaccination effective in maintaining high antibody titers. In our three years of experience in studying methods of controlling infections in cobia in Taiwan, we found that coupling an effective vaccination with ELISA to monitor the condition of fish provided a useful disease prevention system for cobia farming. The effectiveness of repeat vaccination was also evaluated. Cobia receiving a single vaccination performed better than those given a booster vaccination. This observation was confirmed with replicated experiments, but the reason for this result remains unknown. In summary, this study using a combination of three bacterins to vaccinate cobia demonstrated that a single dose of the vaccine was sufficient to induce an immune response and prevent disease in cobia. Vaccination of cobia contributes to the understanding of vaccination in the health management of warm water fish farming. Since this is the first time immunization of warm water fish was achieved, further work is needed to develop a commercial product. Further research should address the optimal antigen formulation, vaccination dose, and age for fingerling vaccination.