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Dietary nucleotides: a novel supplement in fish feeds
Aquaculture 199 Ž2001. 159–169 www.elsevier.nlrlocateraqua-online
Dietary nucleotides: a novel supplement in fish feeds 1. Effects on resistance to disease in salmonids C. Burrells a,) , P.D. Williams a , P.F. Forno b a
EWOS Technology Centre, Unit 1, Kingsthorne Park, Houstoun Industrial Estate, LiÕingston, West Lothian, EH54 5DB, UK b EWOS Chile S.A., AÕ. BenaÕente 550, Oficina 605, Edif. Torre Campanario, Puerto Montt, Chile Received 28 August 2000; received in revised form 5 February 2001; accepted 5 February 2001
Abstract To determine the effects of the inclusion of exogenous nucleotides in aquaculture diets on the resistance of fish to various challenge infections, trials were carried out and results presented. When added to normal fish feed formulations at a combined inclusion level of 0.03%, these additional nucleotides were shown to increase resistance to challenge infections with bacterial, viral and rickettsial diseases as well as ectoparasitic infestation. When fed for 3 weeks prior to challenge, the nucleotide-supplemented diet was superior Ž31% mortalities; relative percent survival: RPS s 37%. to a b-glucan-containing diet Ž43% mortalities; RPS s 12%. in reducing mortalities due to Vibrio anguillarum infection in fish fed the control diet Ž49% mortalities.. Mortalities resulting from infections with infectious salmon anaemia ŽISA. virus and Piscirickettsia salmonis were also significantly reduced ŽRPS s 25.7% and 42.1%, respectively. as were the numbers of infesting sea lice Ž Lepeophtheirus salmonis . Ž37.8% reduction in the mean number of attached lice per fish.. The mode of action of supplemental dietary nucleotides and advantages over recognised immunostimulants are discussed. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Nucleotides; Fish; Resistance; Infections
1. Introduction An increase in the susceptibility of fish to disease is a direct consequence of immunosuppression induced by physical stress associated with handling, vaccination, )
Corresponding author. Tel.: q44-1506-433688; fax: q44-1506-433774. E-mail address: [email protected] ŽC. Burrells..
0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 0 1 . 0 0 5 7 7 - 4
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grading, net changing, salt water transfer, anti-parasite bath treatments, etc., as well as pressures related to other natural events such as environmental changes and social conflicts. Whilst vaccination is the method of choice over antibiotic treatments for the control of many fish diseases, vaccines for others are unavailable or, at best, in the early stages of their development. In recent years in the aquaculture industry, increasing consideration has been given to the use of immunostimulants as adjuncts to vaccination and as a potential route to the reduction in the widespread use of antibiotics. Recently, Sakai Ž1999. reviewed the current status of research into the use of immunostimulants in fish. Mainly, substances such as glucan, chitin, lactoferrin and levamisole, as well as nutritional factors like vitamins B and C, growth hormone and prolactin are immunostimulatory because of their direct positive influence on non-specific immune elements such as phagocytic cell activity, natural killer cell activity, lysozyme levels, complement levels and total immunoglobulin ŽIg. levels. The most widely studied of these immunostimulants are the glucans, particularly yeast glucans Žb-1,3- and b-1,6-linked glucans., and their use in fish has been reviewed ŽRobertsen et al., 1994; Robertsen, 1999; Sakai, 1999.. The majority of reports have described increased resistance to mainly bacterial infections such as Vibrio anguillarum, V. salmonicida or Yersinia ruckeri ŽRobertsen et al., 1990.. LaPatra et al. Ž1998. also reported increased resistance to challenge with infectious haematopoietic necrosis ŽIHN. virus. Most studies, however, have been carried out using intraperitoneal ŽIp. administration of the glucans whilst reports on the efficacy of oral administration by dietary inclusion have been conflicting. Increases in non-specific resistance have been demonstrated to challenge-infections with V. anguillarum and V. salmonicida ŽRaa et al., 1992., Aeromonas salmonicida ŽNikl et al., 1993; Siwicki et al., 1994. and Pasteurella piscicida ŽNoya et al., 1995., but no such benefit has been shown against A. salmonicida and V. salmonicida ŽDalmo et al., 1998., Enterococcus species in turbot ŽToranzo et al., 1995. or V. anguillarum in turbot ŽOgier de Baulny et al., 1996.. There is also a paucity of information regarding the effectiveness of orally administered b-glucans against disease organisms other than bacteria. Nucleotides, precursors of DNA replication, have long been recognised as important elements in mammalian nutrition ŽUauy, 1989; Barness, 1994; Van Buren et al., 1994; Life Sciences Research Office Report, 1998.. With regard to resistance to infections, it has been shown that groups of mice fed diets supplemented with nucleotides had less mortality following challenge infection with Staphylococcus aureus ŽKulkarni et al., 1986a,b; Carver, 1994. and Candida albicans ŽCarver, 1994. compared with groups of mice fed nucleotide-free diets. This increase in resistance to infection is reported to be as a consequence of increased phagocytic activity of murine peritoneal macrophages ŽKulkarni et al., 1986a., increased T-cell dependant antibody production ŽJyonouchi, 1994., enhanced interleukin-2 ŽIL-2. production ŽCarver, 1994. and elevated bone marrow cell and peripheral neutrophil numbers ŽMatsumoto et al., 1995.. To date, there have been no reported studies of the potential of additional dietary nucleotides to enhance the non-specific disease resistance of fish. This report presents data accumulated from several individual studies at various trial sites of the effects on different challenge infections of the inclusion of additional nucleotides in aquaculture diets.
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2. Materials and methods 2.1. Diets 2.1.1. Control diet A standard commercial diet ŽEWOS VEXTRA Alphaw ; 3 mm pellets. comprising 52% protein, 20% fat, 8% carbohydrate, 10% ash, 9% moisture, 1% fibre and having a gross energy level of 21.7 MJ kgy1 was used throughout and designated as the control diet. 2.1.2. Nucleotide diet The control diet was supplemented with AOptimunB ˆ ŽChemoforma, Augst, Switzer. land at the manufacturer’s recommended rate of 0.2%. This product contained cytidine-5X-monophosphate ŽCMP., disodium uridine-5X-monophosphate ŽUMP., adenosine-5X-monophosphate ŽAMP., disodium inosine-5X-monophosphate ŽIMP., disodium guanidine-5X-monophosphate ŽGMP. and RNA at a combined inclusion level of 0.03% in the feed which was designated as the nucleotide diet. 2.1.3. b-glucan diet The control diet was supplemented with b-glucan ŽMacrogard: Vetrepharm, Fordingbridge, Hampshire. at a level of 0.2% of the total feed and designated as the glucan diet. 2.2. Trials 2.2.1. Trial 1: effect on infection with V. anguillarum This trial was carried out at the Marine Harvest ŽMH. challenge unit, Lochailort, Inverness-shire. Rainbow trout Ž Oncorhynchus mykiss . of approximately 217 g Ž"62 g. starting weight were housed in six 1 m tanks with 30 fish per tank and allocated to three groups of two replicate tanks. The fish were maintained in ozonated, UV-filtered freshwater at 168C Ž"2.08C. at a flow rate of 15 l miny1 and an average O 2 level of ) 7 mg ly1 . All fish were fed control diet for 2 weeks after which one group of two replicate tanks was fed the nucleotide diet, another group was fed the glucan diet and the third group was fed the control diet. Fish were fed at a rate of 2% body weight per day Žbw dy1 . for 3 weeks after which they were all fed the control diet. On day 23, a total of 10 fish per group Ž5 per tank. were sacrificed and head kidneys were removed for assays of macrophage activity. All remaining fish Ž2 = 25 per group. were panjet marked according to group and transferred to 4 = 1.5 m replicate tanks so that each new tank contained 12r13 fish from each group. Water flows were turned off and all fish were immersion-challenged with a broth culture containing 1 = 10 6 colony-forming units ŽCFU. of V. anguillarum ŽStrain NCIMB6 Serotype O2a. for 1 h. Resulting mortalities were removed daily and tissues removed for bacteriological culture to confirm V. anguillarum as the cause of death. No mortalities occurred after day 10 following the
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challenge and the trial was terminated on day 12. Cumulative percentage mortalities were calculated and relative percent survival ŽRPS. values were calculated as follows RPS s 100 y Ž test mortality% control mortality. = 100 . 2.2.2. Trial 2: effect on infection with infectious salmon anaemia (ISA) Õirus This study was commissioned at VESO Vikan Aquavet, The National Centre for Veterinary Contract Research and Commercial Services, Alhusstrand, Norway. Atlantic salmon Ž Salmo salar . at approximately 53–55 g were housed in six tanks Žtwo groups of three replicate tanks. with 100 fish per tank Ž0.6 m2 . and maintained in salt water Ž25‰. at 128C. One group of three tanks were fed the control diet whilst the other group was fed the nucleotide diet at a rate of 1% bw dy1 . After 2 weeks, 60 similarly sized fish were injected intraperitoneally ŽIp. with ISA virus Žstrain Glesvairr2r90. obtained from the Norwegian State Veterinary Laboratories, Oslo, Norway. The virus had been grown on salmon head kidney ŽSHK. cells and the supernatant fluid was diluted in phosphate-buffered saline, pH 7.2 to contain 5 = 10 4 tissue culture infectious doses ŽTCID. mly1 . Each fish was injected with 0.1 ml Ž5 = 10 3 TCID. and 10 of these were panjet marked and introduced into each tank as a cohabitant challenge. After a further week’s feeding with the nucleotide diet, this group of fish was fed the control feed. Mortalities in the exposed, unmarked fish were removed daily and all were examined bacteriologically for non ISA-specific causes of death. Necropsies were performed on a proportion Ž20%. of all mortalities and tissues examined visually for pathognomic changes. No mortalities occurred after day 51 following the challenge and the study was terminated at day 53. 2.2.3. Trial 3: effect on infection with Piscirickettsia salmonis A trial was commissioned at Aquatic Health Diagnostic and Research Services, Puerto Montt, Chile in which 600 coho salmon Ž O. kisutch. smolts Žapproximately 100 g. were acclimatised in UV-treated salt water at approximately 118C Ž"1.08C. in 6 = 1 m tanks Ž100 fish per tank.. The water flow was maintained at 10 l miny1 and the salinity was 25‰. Following acclimation, they were allocated to two groups of three replicate tanks each. One group was fed the control diet and the other group fed the nucleotide diet at a rate of 1% bw dy1 . After 3 weeks feeding with these diets, the fish in each tank were exposed to a challenge infection of an isolate ŽPuerto Montt; June 1988. of P. salmonis cultured for 14 days at 158C on monolayer cultures of chinook salmon embryo ŽCHSE.-214 cells. Fish were challenged by Ip injection of 0.2 ml of a suspension containing 8 = 10 4 plaque-forming units ŽPFU. of the organism which represented a 50% lethal dose ŽLD50 .. This dose was pre-determined by titration in groups of similarly sized coho salmon in which the end-point was calculated according to the method of Reed and Muench Ž1938.. Following the challenge, the fish continued to be fed control diet and mortalities were removed daily. No deaths occurred after day 34 and the trial was terminated 10 days later. Necropsies were performed on all dead fish and specificity for P. salmonis was confirmed by examination of direct impression smears stained with acridine orange, direct fluorescent antibody tests ŽIFAT. and by isolation of the organism on CHSE cultures.
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2.2.4. Trial 4: effect on infestation with sea lice (Lepeophtheirus salmonis) A study was carried out at the MH facility, Lochailort in which 160 Atlantic salmon of approximately 60 g were held in 4 = 1 m tanks Ž40 fish per tank.. The fish were maintained in ozonated, filtered seawater at a flow rate of 8 l miny1 and an O 2 level of ) 8 mg ly1 at an ambient temperature of 108C Ž"1.08C.. Day length was 12 h and fish were fed at a rate of 2% bw dy1 with either the control or nucleotide diet Žduplicate tanks per dietary group. for 3 weeks. Test groups were differentiated using alcian blue dye Ž1%. ŽSigma, Poole, UK. applied to the ventral surfaces and the groups were mixed equally between the four tanks prior to challenge. Ovigerous sea lice L. salmonis were collected from production sites during harvest operations. The oviscas were removed, rinsed and placed in 4.5 l seawater for 7 days. Temperature was maintained at 118C Ž"1.08C. with 32‰ salinity, O 2 ) 7 mg ly1 and daylight was maintained at 8 h light and 16 h dark. Copepod numbers were estimated via sub sample and added to the four replicate tanks containing the fish at 3000 copepods per m3 for 1 h in static, oxygenated seawater. All fish were fed the control diet post-challenge. At 10 days post-challenge, 10 fish from each group were gently netted and killed with a sharp blow to the head. The fins, body and gill arches were examined under 10 = magnification and numbers of L. salmonis were recorded. 2.3. Assessment of macrophage A respiratory burst B actiÕity Macrophages were prepared from head kidneys and assayed for respiratory burst activity as previously described ŽBurrells et al., 1999.. Results are expressed as an index of macrophage activity ŽIMA. obtained from the optical density at a wavelength of 620 nm ŽOD620 . per 10 5 cells obtained in the assay = 100. 2.4. Statistics Results are expressed as mean " S.E.M. and group mean differences were analysed using Student’s t-test where there were only two dietary groups and otherwise by ANOVA using the Student–Newman–Keuls technique for post hoc analysis of variance. Statistical significance was assumed at levels of ) 95% Ž P - 0.05..
3. Results 3.1. Trial 1: effect on infection with V. anguillarum All mortalities recorded during the trial were due to the challenge infection with V. anguillarum and these were observed from day 3 post-challenge ŽFig. 1.. At the conclusion of the trial Ž12 days after the challenge., the mean cumulative mortality in the group of fish fed the control diet was 49%. The cumulative mortality in the nucleotide diet fed group Ž31%. was significantly lower Ž P - 0.05. than that of the group fed the glucan diet Ž43%.. However, due to the wide range of mortalities in individual tanks in each group, significance could not be shown between the control diet
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Fig. 1. Trial 1: cumulative mortalities due to V. anguillarum in groups of rainbow trout fed the control Ž`., glucan Ž^. or nucleotide Ž=. diets prior to challenge-exposure.
fed group and the nucleotide diet fed group. In the nucleotide diet fed group, no additional mortalities were recorded after day 5 post-challenge whereas in the control and glucan diet fed groups, mortalities continued to day 9 and 10, respectively. The RPS of 37% observed in fish fed the nucleotide diet ŽFig. 2. was achieved in the absence of any direct influence on macrophage respiratory burst activity. Despite the increased macrophage activity Žnot statistically significant. induced by the glucan diet, a lower RPS value of 12% was achieved.
Fig. 2. Trial 1: the effect of different diets on macrophage activity ŽIMA. and relative percent survival ŽRPS. of rainbow trout following challenge-infection with V. anguillarum. IMA sOD620 per 10 5 cells=100.
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3.2. Trial 2: effect on infection with infectious salmon anaemia (ISA) Õirus Fig. 3 shows the cumulative mortalities resulting from the challenge infection with ISA virus in the two groups Žcontrol and nucleotide diet fed fish. of Atlantic salmon. From the time when mortalities were first noted in both groups Žday 30 following challenge. until the termination of the trial Ž53 days post-challenge. the level of mortalities in the fish fed the nucleotide diet was always lower than that of the control group. The differences in group means between days 39 and 45 post-challenge were statistically significant Ž P - 0.05.. No further mortalities were observed after day 50 and the study was terminated at day 53. At this time, the cumulated total mortality in the group fed the nucleotide diet was 35.7% compared to 48% for the fish fed the control diet ŽRPS s 25.7%., although at this time statistical significance could not be shown. 3.3. Trial 3: effect on infection with P. salmonis Mortalities due to P. salmonis in the group fed the control diet started to occur 16 days after the introduction of the infected cohabitant fish ŽFig. 4. and continued to reach a mean level of 76.8% on day 44 post-exposure. The onset of mortality in the group fed the nucleotide diet was delayed until 21 days following the challenge and reached a cumulative value of 46.9% ŽRPS s 38.9%.. Although the difference in mortality rates between the two dietary groups was marginal up to day 26 post-challenge, from day 27 until termination of the trial 44 days after exposure, differences in mortalities were statistically significant Ždays 27–29, P - 0.05; days 30–44, P - 0.005 or better..
Fig. 3. Trial 2: cumulative mortalities due to infectious salmon anaemia ŽISA. virus in groups of Atlantic salmon fed prior to challenge-exposure the control Ž`. or nucleotide Ž'. diet. Mean"S.E.M. ) Mortality significantly reduced Ž P - 0.05. between days 39 and 45 post-challenge.
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Fig. 4. Trial 3: cumulative mortalities due to infection with P. salmonis in groups of coho salmon fed prior to exposure the control Ž`. or nucleotide Ž'. diet. Mean"S.E.M.
3.4. Trial 4: effect on infestation with sea lice Ten days after exposure to L. salmonis, the mean number of lice attached to the group of fish fed the nucleotide diet Ž1.4; range 0–4. was significantly lower Ž P - 0.05. than that of the fish fed the control diet Ž2.5; range 1–5.. The difference represented a reduction in infestation rate of 37.8%. Furthermore, whereas all the 10 fish examined from the control fed group had at least one attached louse, only 7 of the 10 fish from the nucleotide fed group showed any infestation.
4. Discussion These studies have shown that supplementation of commercial salmonid diets with additional exogenous nucleotides can exert a positive influence on the resistance of Atlantic salmon, coho salmon and rainbow trout to bacterial, viral, rickettsial and ectoparasitic infections. Whilst the inclusion of b-glucan in the diet was responsible for a non-significant reduction in mortality due to infection with V. anguillarum in comparison with the control diet, the nucleotide diet was more effective. Furthermore, while mortalities in the groups fed the control or glucan diet continued for 9 and 10 days, respectively, mortalities in the nucleotide diet fed group ceased after 5 days. This would suggest an increase in resistance to infection possibly via a more rapid immune response. Macrophage activation by b-glucans has been shown to enhance the capacity of the non-specific arm of the immune system to resist bacterial infections. However, mecha-
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nisms of immune responses to viruses and parasites are different and it has been previously shown that dietary nucleotides in other species benefit many elements of the immune system. In the present study, an enhancement of macrophage killing capacity, as indicated by the assay for respiratory burst, could not be demonstrated. However, previous studies in mice have not only demonstrated increased resistance to bacterial infections ŽKulkarni et al., 1986a,b. but also, acceleration of T-cell dependant antibody production ŽJyonouchi, 1994., increased natural killer ŽNK. activity ŽCarver, 1994., increased macrophage function ŽCarver, 1994. and acceleration of bone marrow cell and peripheral neutrophil proliferation ŽMatsumoto et al., 1995.. It is suggested that whereas b-glucan-type immunostimulants primarily enhance the non-specific capacity of phagocytes, dietary nucleotides enhance the potential of the immune system in general to mount greater and more rapid specific responses when required. This hypothesis is supported by the significant reductions achieved with nucleotidesupplemented diets to viral ŽISA. and rickettsial Ž P. salmonis . challenges. The immune response to challenge with sea lice is uncertain but the reduction in infestation of fish fed the nucleotide diet could be due to a reduction in the levels of immunosuppressive elements such as cortisol and prostaglandin E 2 ŽPGE 2 .. Elevated levels of PGE 2 have previously been noted in Atlantic salmon heavily infested with sea lice. ŽWadsworth, personal communication.. The immunostimulatory effects of cell wall conponents such as b-glucans have been widely studied ŽSakai, 1999.. However, these may have certain disadvantages. The prime target for up-regulation by these products are the phagocytic cells of the immune system which provide an initial defence mechanism against bacteria. However, certain organisms such as P. salmonis, Renibacterium salmoninarum, Pas. piscicida and Edwardsiella ictaluri are intracellular and, consequently, able to evade phagocytosis ŽNelson et al., 1989; Baldwin and Newton, 1996; Gutenberger et al., 1997.. Enhanced phagocytic functions are of short duration, and although short-term oral administration can show beneficial effects, long-term feeding can be deleterious ŽYoshida et al., 1995; Sakai, 1999.. Also, responses are not directly dose-dependant. In vitro studies in which Atlantic salmon macrophages were incubated with various concentrations of yeast glucan indicated that concentrations above optimal had a negative or inhibitory effect on the cells ŽJørgensen and Robertsen, 1995.. Over-dosing and overfeeding, therefore, can lead to a suppression of stimulation. Consequently, feeds containing yeast glucans must be fed with strict regard to recommended regimes based on a ApulseB system of feeding for a pre-determined period followed by a period on a non-glucan diet after which the cycle is repeated as necessary. Immunostimulants differ from antibiotics in that they are meant to be applied prophylactically rather than therapeutically. Anticipation of known stress periods such as handling, vaccination, etc., may not be problematic but accurate anticipation of onset of disease outbreaks is less certain. Nucleotides are provided endogenously by de novo synthesis in tissues such as the liver. This, however, is an energy-expensive process and the salvage pathway is preferentially utilised when there is an exogenous source of nucleotides such as the diet ŽUauy, 1994.. Although most tissues can synthesise nucleotides de novo, other cells such as immune cells and cells in the intestine are lacking in this capacity and must depend on pre-formed nucleotides ŽQuan, 1992.. Mammalian requirements for exoge-
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nous nucleotides can vary considerably and can increase rapidly at times of rapid growth or physiological stress ŽCarver, 1994; Jyonouchi, 1994.. This may also be the case in farmed fish where the exogenous supply of nucleotides in fish feeds is sufficient for normal requirements of health and growth. However, the stressful events associated with aquaculture management Žvaccination, handling, disease, etc.. may lead to similar increases in reliance on the exogenous supply for optimal functions and responses. Supplemental dietary nucleotides applied in anticipation of these events could, therefore, ensure an adequate circulating nucleotide pool.
Acknowledgements The authors wish to thank Dr. Simon Wadsworth of Marine Harvest ŽScotland., Lochailort, Inverness-shire Žcurrently Seafish Aquaculture, Ardtoe, Argyll. for technical management of the trials with V. anguillarum and L. salmonis challenges and Sigmund Sevatdal of VESO Vikan Akvavet, Namsos, Norway for the conduct of the trial with ISA virus infection. Thanks are also due to Oscar Garate, Erwin M. Landskron and ´ Marcela Oyarzun ´ of Aquatic Health Diagnostic and Research Services, Puerto Montt, Chile for the management of the trial with P. salmonis. We also wish to thanks Joanne Good of the EWOS Technology Centre for excellent technical assistance.
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Dietary nucleotides: a novel supplement in fish feeds 1. Effects on resistance to disease in salmonids C. Burrells a,) , P.D. Williams a , P.F. Forno b a
EWOS Technology Centre, Unit 1, Kingsthorne Park, Houstoun Industrial Estate, LiÕingston, West Lothian, EH54 5DB, UK b EWOS Chile S.A., AÕ. BenaÕente 550, Oficina 605, Edif. Torre Campanario, Puerto Montt, Chile Received 28 August 2000; received in revised form 5 February 2001; accepted 5 February 2001
Abstract To determine the effects of the inclusion of exogenous nucleotides in aquaculture diets on the resistance of fish to various challenge infections, trials were carried out and results presented. When added to normal fish feed formulations at a combined inclusion level of 0.03%, these additional nucleotides were shown to increase resistance to challenge infections with bacterial, viral and rickettsial diseases as well as ectoparasitic infestation. When fed for 3 weeks prior to challenge, the nucleotide-supplemented diet was superior Ž31% mortalities; relative percent survival: RPS s 37%. to a b-glucan-containing diet Ž43% mortalities; RPS s 12%. in reducing mortalities due to Vibrio anguillarum infection in fish fed the control diet Ž49% mortalities.. Mortalities resulting from infections with infectious salmon anaemia ŽISA. virus and Piscirickettsia salmonis were also significantly reduced ŽRPS s 25.7% and 42.1%, respectively. as were the numbers of infesting sea lice Ž Lepeophtheirus salmonis . Ž37.8% reduction in the mean number of attached lice per fish.. The mode of action of supplemental dietary nucleotides and advantages over recognised immunostimulants are discussed. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Nucleotides; Fish; Resistance; Infections
1. Introduction An increase in the susceptibility of fish to disease is a direct consequence of immunosuppression induced by physical stress associated with handling, vaccination, )
Corresponding author. Tel.: q44-1506-433688; fax: q44-1506-433774. E-mail address: [email protected] ŽC. Burrells..
0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 0 1 . 0 0 5 7 7 - 4
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grading, net changing, salt water transfer, anti-parasite bath treatments, etc., as well as pressures related to other natural events such as environmental changes and social conflicts. Whilst vaccination is the method of choice over antibiotic treatments for the control of many fish diseases, vaccines for others are unavailable or, at best, in the early stages of their development. In recent years in the aquaculture industry, increasing consideration has been given to the use of immunostimulants as adjuncts to vaccination and as a potential route to the reduction in the widespread use of antibiotics. Recently, Sakai Ž1999. reviewed the current status of research into the use of immunostimulants in fish. Mainly, substances such as glucan, chitin, lactoferrin and levamisole, as well as nutritional factors like vitamins B and C, growth hormone and prolactin are immunostimulatory because of their direct positive influence on non-specific immune elements such as phagocytic cell activity, natural killer cell activity, lysozyme levels, complement levels and total immunoglobulin ŽIg. levels. The most widely studied of these immunostimulants are the glucans, particularly yeast glucans Žb-1,3- and b-1,6-linked glucans., and their use in fish has been reviewed ŽRobertsen et al., 1994; Robertsen, 1999; Sakai, 1999.. The majority of reports have described increased resistance to mainly bacterial infections such as Vibrio anguillarum, V. salmonicida or Yersinia ruckeri ŽRobertsen et al., 1990.. LaPatra et al. Ž1998. also reported increased resistance to challenge with infectious haematopoietic necrosis ŽIHN. virus. Most studies, however, have been carried out using intraperitoneal ŽIp. administration of the glucans whilst reports on the efficacy of oral administration by dietary inclusion have been conflicting. Increases in non-specific resistance have been demonstrated to challenge-infections with V. anguillarum and V. salmonicida ŽRaa et al., 1992., Aeromonas salmonicida ŽNikl et al., 1993; Siwicki et al., 1994. and Pasteurella piscicida ŽNoya et al., 1995., but no such benefit has been shown against A. salmonicida and V. salmonicida ŽDalmo et al., 1998., Enterococcus species in turbot ŽToranzo et al., 1995. or V. anguillarum in turbot ŽOgier de Baulny et al., 1996.. There is also a paucity of information regarding the effectiveness of orally administered b-glucans against disease organisms other than bacteria. Nucleotides, precursors of DNA replication, have long been recognised as important elements in mammalian nutrition ŽUauy, 1989; Barness, 1994; Van Buren et al., 1994; Life Sciences Research Office Report, 1998.. With regard to resistance to infections, it has been shown that groups of mice fed diets supplemented with nucleotides had less mortality following challenge infection with Staphylococcus aureus ŽKulkarni et al., 1986a,b; Carver, 1994. and Candida albicans ŽCarver, 1994. compared with groups of mice fed nucleotide-free diets. This increase in resistance to infection is reported to be as a consequence of increased phagocytic activity of murine peritoneal macrophages ŽKulkarni et al., 1986a., increased T-cell dependant antibody production ŽJyonouchi, 1994., enhanced interleukin-2 ŽIL-2. production ŽCarver, 1994. and elevated bone marrow cell and peripheral neutrophil numbers ŽMatsumoto et al., 1995.. To date, there have been no reported studies of the potential of additional dietary nucleotides to enhance the non-specific disease resistance of fish. This report presents data accumulated from several individual studies at various trial sites of the effects on different challenge infections of the inclusion of additional nucleotides in aquaculture diets.
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2. Materials and methods 2.1. Diets 2.1.1. Control diet A standard commercial diet ŽEWOS VEXTRA Alphaw ; 3 mm pellets. comprising 52% protein, 20% fat, 8% carbohydrate, 10% ash, 9% moisture, 1% fibre and having a gross energy level of 21.7 MJ kgy1 was used throughout and designated as the control diet. 2.1.2. Nucleotide diet The control diet was supplemented with AOptimunB ˆ ŽChemoforma, Augst, Switzer. land at the manufacturer’s recommended rate of 0.2%. This product contained cytidine-5X-monophosphate ŽCMP., disodium uridine-5X-monophosphate ŽUMP., adenosine-5X-monophosphate ŽAMP., disodium inosine-5X-monophosphate ŽIMP., disodium guanidine-5X-monophosphate ŽGMP. and RNA at a combined inclusion level of 0.03% in the feed which was designated as the nucleotide diet. 2.1.3. b-glucan diet The control diet was supplemented with b-glucan ŽMacrogard: Vetrepharm, Fordingbridge, Hampshire. at a level of 0.2% of the total feed and designated as the glucan diet. 2.2. Trials 2.2.1. Trial 1: effect on infection with V. anguillarum This trial was carried out at the Marine Harvest ŽMH. challenge unit, Lochailort, Inverness-shire. Rainbow trout Ž Oncorhynchus mykiss . of approximately 217 g Ž"62 g. starting weight were housed in six 1 m tanks with 30 fish per tank and allocated to three groups of two replicate tanks. The fish were maintained in ozonated, UV-filtered freshwater at 168C Ž"2.08C. at a flow rate of 15 l miny1 and an average O 2 level of ) 7 mg ly1 . All fish were fed control diet for 2 weeks after which one group of two replicate tanks was fed the nucleotide diet, another group was fed the glucan diet and the third group was fed the control diet. Fish were fed at a rate of 2% body weight per day Žbw dy1 . for 3 weeks after which they were all fed the control diet. On day 23, a total of 10 fish per group Ž5 per tank. were sacrificed and head kidneys were removed for assays of macrophage activity. All remaining fish Ž2 = 25 per group. were panjet marked according to group and transferred to 4 = 1.5 m replicate tanks so that each new tank contained 12r13 fish from each group. Water flows were turned off and all fish were immersion-challenged with a broth culture containing 1 = 10 6 colony-forming units ŽCFU. of V. anguillarum ŽStrain NCIMB6 Serotype O2a. for 1 h. Resulting mortalities were removed daily and tissues removed for bacteriological culture to confirm V. anguillarum as the cause of death. No mortalities occurred after day 10 following the
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challenge and the trial was terminated on day 12. Cumulative percentage mortalities were calculated and relative percent survival ŽRPS. values were calculated as follows RPS s 100 y Ž test mortality% control mortality. = 100 . 2.2.2. Trial 2: effect on infection with infectious salmon anaemia (ISA) Õirus This study was commissioned at VESO Vikan Aquavet, The National Centre for Veterinary Contract Research and Commercial Services, Alhusstrand, Norway. Atlantic salmon Ž Salmo salar . at approximately 53–55 g were housed in six tanks Žtwo groups of three replicate tanks. with 100 fish per tank Ž0.6 m2 . and maintained in salt water Ž25‰. at 128C. One group of three tanks were fed the control diet whilst the other group was fed the nucleotide diet at a rate of 1% bw dy1 . After 2 weeks, 60 similarly sized fish were injected intraperitoneally ŽIp. with ISA virus Žstrain Glesvairr2r90. obtained from the Norwegian State Veterinary Laboratories, Oslo, Norway. The virus had been grown on salmon head kidney ŽSHK. cells and the supernatant fluid was diluted in phosphate-buffered saline, pH 7.2 to contain 5 = 10 4 tissue culture infectious doses ŽTCID. mly1 . Each fish was injected with 0.1 ml Ž5 = 10 3 TCID. and 10 of these were panjet marked and introduced into each tank as a cohabitant challenge. After a further week’s feeding with the nucleotide diet, this group of fish was fed the control feed. Mortalities in the exposed, unmarked fish were removed daily and all were examined bacteriologically for non ISA-specific causes of death. Necropsies were performed on a proportion Ž20%. of all mortalities and tissues examined visually for pathognomic changes. No mortalities occurred after day 51 following the challenge and the study was terminated at day 53. 2.2.3. Trial 3: effect on infection with Piscirickettsia salmonis A trial was commissioned at Aquatic Health Diagnostic and Research Services, Puerto Montt, Chile in which 600 coho salmon Ž O. kisutch. smolts Žapproximately 100 g. were acclimatised in UV-treated salt water at approximately 118C Ž"1.08C. in 6 = 1 m tanks Ž100 fish per tank.. The water flow was maintained at 10 l miny1 and the salinity was 25‰. Following acclimation, they were allocated to two groups of three replicate tanks each. One group was fed the control diet and the other group fed the nucleotide diet at a rate of 1% bw dy1 . After 3 weeks feeding with these diets, the fish in each tank were exposed to a challenge infection of an isolate ŽPuerto Montt; June 1988. of P. salmonis cultured for 14 days at 158C on monolayer cultures of chinook salmon embryo ŽCHSE.-214 cells. Fish were challenged by Ip injection of 0.2 ml of a suspension containing 8 = 10 4 plaque-forming units ŽPFU. of the organism which represented a 50% lethal dose ŽLD50 .. This dose was pre-determined by titration in groups of similarly sized coho salmon in which the end-point was calculated according to the method of Reed and Muench Ž1938.. Following the challenge, the fish continued to be fed control diet and mortalities were removed daily. No deaths occurred after day 34 and the trial was terminated 10 days later. Necropsies were performed on all dead fish and specificity for P. salmonis was confirmed by examination of direct impression smears stained with acridine orange, direct fluorescent antibody tests ŽIFAT. and by isolation of the organism on CHSE cultures.
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2.2.4. Trial 4: effect on infestation with sea lice (Lepeophtheirus salmonis) A study was carried out at the MH facility, Lochailort in which 160 Atlantic salmon of approximately 60 g were held in 4 = 1 m tanks Ž40 fish per tank.. The fish were maintained in ozonated, filtered seawater at a flow rate of 8 l miny1 and an O 2 level of ) 8 mg ly1 at an ambient temperature of 108C Ž"1.08C.. Day length was 12 h and fish were fed at a rate of 2% bw dy1 with either the control or nucleotide diet Žduplicate tanks per dietary group. for 3 weeks. Test groups were differentiated using alcian blue dye Ž1%. ŽSigma, Poole, UK. applied to the ventral surfaces and the groups were mixed equally between the four tanks prior to challenge. Ovigerous sea lice L. salmonis were collected from production sites during harvest operations. The oviscas were removed, rinsed and placed in 4.5 l seawater for 7 days. Temperature was maintained at 118C Ž"1.08C. with 32‰ salinity, O 2 ) 7 mg ly1 and daylight was maintained at 8 h light and 16 h dark. Copepod numbers were estimated via sub sample and added to the four replicate tanks containing the fish at 3000 copepods per m3 for 1 h in static, oxygenated seawater. All fish were fed the control diet post-challenge. At 10 days post-challenge, 10 fish from each group were gently netted and killed with a sharp blow to the head. The fins, body and gill arches were examined under 10 = magnification and numbers of L. salmonis were recorded. 2.3. Assessment of macrophage A respiratory burst B actiÕity Macrophages were prepared from head kidneys and assayed for respiratory burst activity as previously described ŽBurrells et al., 1999.. Results are expressed as an index of macrophage activity ŽIMA. obtained from the optical density at a wavelength of 620 nm ŽOD620 . per 10 5 cells obtained in the assay = 100. 2.4. Statistics Results are expressed as mean " S.E.M. and group mean differences were analysed using Student’s t-test where there were only two dietary groups and otherwise by ANOVA using the Student–Newman–Keuls technique for post hoc analysis of variance. Statistical significance was assumed at levels of ) 95% Ž P - 0.05..
3. Results 3.1. Trial 1: effect on infection with V. anguillarum All mortalities recorded during the trial were due to the challenge infection with V. anguillarum and these were observed from day 3 post-challenge ŽFig. 1.. At the conclusion of the trial Ž12 days after the challenge., the mean cumulative mortality in the group of fish fed the control diet was 49%. The cumulative mortality in the nucleotide diet fed group Ž31%. was significantly lower Ž P - 0.05. than that of the group fed the glucan diet Ž43%.. However, due to the wide range of mortalities in individual tanks in each group, significance could not be shown between the control diet
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Fig. 1. Trial 1: cumulative mortalities due to V. anguillarum in groups of rainbow trout fed the control Ž`., glucan Ž^. or nucleotide Ž=. diets prior to challenge-exposure.
fed group and the nucleotide diet fed group. In the nucleotide diet fed group, no additional mortalities were recorded after day 5 post-challenge whereas in the control and glucan diet fed groups, mortalities continued to day 9 and 10, respectively. The RPS of 37% observed in fish fed the nucleotide diet ŽFig. 2. was achieved in the absence of any direct influence on macrophage respiratory burst activity. Despite the increased macrophage activity Žnot statistically significant. induced by the glucan diet, a lower RPS value of 12% was achieved.
Fig. 2. Trial 1: the effect of different diets on macrophage activity ŽIMA. and relative percent survival ŽRPS. of rainbow trout following challenge-infection with V. anguillarum. IMA sOD620 per 10 5 cells=100.
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3.2. Trial 2: effect on infection with infectious salmon anaemia (ISA) Õirus Fig. 3 shows the cumulative mortalities resulting from the challenge infection with ISA virus in the two groups Žcontrol and nucleotide diet fed fish. of Atlantic salmon. From the time when mortalities were first noted in both groups Žday 30 following challenge. until the termination of the trial Ž53 days post-challenge. the level of mortalities in the fish fed the nucleotide diet was always lower than that of the control group. The differences in group means between days 39 and 45 post-challenge were statistically significant Ž P - 0.05.. No further mortalities were observed after day 50 and the study was terminated at day 53. At this time, the cumulated total mortality in the group fed the nucleotide diet was 35.7% compared to 48% for the fish fed the control diet ŽRPS s 25.7%., although at this time statistical significance could not be shown. 3.3. Trial 3: effect on infection with P. salmonis Mortalities due to P. salmonis in the group fed the control diet started to occur 16 days after the introduction of the infected cohabitant fish ŽFig. 4. and continued to reach a mean level of 76.8% on day 44 post-exposure. The onset of mortality in the group fed the nucleotide diet was delayed until 21 days following the challenge and reached a cumulative value of 46.9% ŽRPS s 38.9%.. Although the difference in mortality rates between the two dietary groups was marginal up to day 26 post-challenge, from day 27 until termination of the trial 44 days after exposure, differences in mortalities were statistically significant Ždays 27–29, P - 0.05; days 30–44, P - 0.005 or better..
Fig. 3. Trial 2: cumulative mortalities due to infectious salmon anaemia ŽISA. virus in groups of Atlantic salmon fed prior to challenge-exposure the control Ž`. or nucleotide Ž'. diet. Mean"S.E.M. ) Mortality significantly reduced Ž P - 0.05. between days 39 and 45 post-challenge.
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Fig. 4. Trial 3: cumulative mortalities due to infection with P. salmonis in groups of coho salmon fed prior to exposure the control Ž`. or nucleotide Ž'. diet. Mean"S.E.M.
3.4. Trial 4: effect on infestation with sea lice Ten days after exposure to L. salmonis, the mean number of lice attached to the group of fish fed the nucleotide diet Ž1.4; range 0–4. was significantly lower Ž P - 0.05. than that of the fish fed the control diet Ž2.5; range 1–5.. The difference represented a reduction in infestation rate of 37.8%. Furthermore, whereas all the 10 fish examined from the control fed group had at least one attached louse, only 7 of the 10 fish from the nucleotide fed group showed any infestation.
4. Discussion These studies have shown that supplementation of commercial salmonid diets with additional exogenous nucleotides can exert a positive influence on the resistance of Atlantic salmon, coho salmon and rainbow trout to bacterial, viral, rickettsial and ectoparasitic infections. Whilst the inclusion of b-glucan in the diet was responsible for a non-significant reduction in mortality due to infection with V. anguillarum in comparison with the control diet, the nucleotide diet was more effective. Furthermore, while mortalities in the groups fed the control or glucan diet continued for 9 and 10 days, respectively, mortalities in the nucleotide diet fed group ceased after 5 days. This would suggest an increase in resistance to infection possibly via a more rapid immune response. Macrophage activation by b-glucans has been shown to enhance the capacity of the non-specific arm of the immune system to resist bacterial infections. However, mecha-
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nisms of immune responses to viruses and parasites are different and it has been previously shown that dietary nucleotides in other species benefit many elements of the immune system. In the present study, an enhancement of macrophage killing capacity, as indicated by the assay for respiratory burst, could not be demonstrated. However, previous studies in mice have not only demonstrated increased resistance to bacterial infections ŽKulkarni et al., 1986a,b. but also, acceleration of T-cell dependant antibody production ŽJyonouchi, 1994., increased natural killer ŽNK. activity ŽCarver, 1994., increased macrophage function ŽCarver, 1994. and acceleration of bone marrow cell and peripheral neutrophil proliferation ŽMatsumoto et al., 1995.. It is suggested that whereas b-glucan-type immunostimulants primarily enhance the non-specific capacity of phagocytes, dietary nucleotides enhance the potential of the immune system in general to mount greater and more rapid specific responses when required. This hypothesis is supported by the significant reductions achieved with nucleotidesupplemented diets to viral ŽISA. and rickettsial Ž P. salmonis . challenges. The immune response to challenge with sea lice is uncertain but the reduction in infestation of fish fed the nucleotide diet could be due to a reduction in the levels of immunosuppressive elements such as cortisol and prostaglandin E 2 ŽPGE 2 .. Elevated levels of PGE 2 have previously been noted in Atlantic salmon heavily infested with sea lice. ŽWadsworth, personal communication.. The immunostimulatory effects of cell wall conponents such as b-glucans have been widely studied ŽSakai, 1999.. However, these may have certain disadvantages. The prime target for up-regulation by these products are the phagocytic cells of the immune system which provide an initial defence mechanism against bacteria. However, certain organisms such as P. salmonis, Renibacterium salmoninarum, Pas. piscicida and Edwardsiella ictaluri are intracellular and, consequently, able to evade phagocytosis ŽNelson et al., 1989; Baldwin and Newton, 1996; Gutenberger et al., 1997.. Enhanced phagocytic functions are of short duration, and although short-term oral administration can show beneficial effects, long-term feeding can be deleterious ŽYoshida et al., 1995; Sakai, 1999.. Also, responses are not directly dose-dependant. In vitro studies in which Atlantic salmon macrophages were incubated with various concentrations of yeast glucan indicated that concentrations above optimal had a negative or inhibitory effect on the cells ŽJørgensen and Robertsen, 1995.. Over-dosing and overfeeding, therefore, can lead to a suppression of stimulation. Consequently, feeds containing yeast glucans must be fed with strict regard to recommended regimes based on a ApulseB system of feeding for a pre-determined period followed by a period on a non-glucan diet after which the cycle is repeated as necessary. Immunostimulants differ from antibiotics in that they are meant to be applied prophylactically rather than therapeutically. Anticipation of known stress periods such as handling, vaccination, etc., may not be problematic but accurate anticipation of onset of disease outbreaks is less certain. Nucleotides are provided endogenously by de novo synthesis in tissues such as the liver. This, however, is an energy-expensive process and the salvage pathway is preferentially utilised when there is an exogenous source of nucleotides such as the diet ŽUauy, 1994.. Although most tissues can synthesise nucleotides de novo, other cells such as immune cells and cells in the intestine are lacking in this capacity and must depend on pre-formed nucleotides ŽQuan, 1992.. Mammalian requirements for exoge-
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nous nucleotides can vary considerably and can increase rapidly at times of rapid growth or physiological stress ŽCarver, 1994; Jyonouchi, 1994.. This may also be the case in farmed fish where the exogenous supply of nucleotides in fish feeds is sufficient for normal requirements of health and growth. However, the stressful events associated with aquaculture management Žvaccination, handling, disease, etc.. may lead to similar increases in reliance on the exogenous supply for optimal functions and responses. Supplemental dietary nucleotides applied in anticipation of these events could, therefore, ensure an adequate circulating nucleotide pool.
Acknowledgements The authors wish to thank Dr. Simon Wadsworth of Marine Harvest ŽScotland., Lochailort, Inverness-shire Žcurrently Seafish Aquaculture, Ardtoe, Argyll. for technical management of the trials with V. anguillarum and L. salmonis challenges and Sigmund Sevatdal of VESO Vikan Akvavet, Namsos, Norway for the conduct of the trial with ISA virus infection. Thanks are also due to Oscar Garate, Erwin M. Landskron and ´ Marcela Oyarzun ´ of Aquatic Health Diagnostic and Research Services, Puerto Montt, Chile for the management of the trial with P. salmonis. We also wish to thanks Joanne Good of the EWOS Technology Centre for excellent technical assistance.
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