JOURNAL OF THE WORLD AQUACULTURE SOCIETY Vol. 38, No. 1 March, 2007 Immune Response and Resistance to Stress and Edwardsiella ictaluri Challenge in Channel Catfish, Ictalurus punctatus, Fed Diets Containing Commercial Whole-Cell Yeast or Yeast Subcomponents THOMAS L. WELKER1, CHHORN LIM, MEDIHA YILDIRIM-AKSOY, RICHARD SHELBY, AND PHILLIP H. KLESIUS Aquatic Animal Health Research Unit, Agricultural Research Service, United States Department of Agriculture, 990 Wire Road, Auburn, Alabama 36832 USA Abstract Dietary supplementation of yeast or yeast subcomponents (YYS) as commercial preparations of b-glucan (MacroGardÒ; Biotec-Mackzymal, Tromsø, Norway; and Betagard AÒ; Aqua-In-Tech, Inc., Seattle, WA, USA), mannan oligosaccharide (Bio-MosTM Aqua Grade; Alltech, Nicholasville, KY, USA), or whole-cell Saccharomyces cerevisiae (Levucell SB20Ò; Lallemand Animal Nutrition, Milwaukee, WI, USA) at the manufacturer’s recommended levels was evaluated on the physiological performance of juvenile channel catfish, Ictalurus punctatus. Fish were fed YYS diets for 4 wk, followed by 2 wk of control diet. Fish were sampled at the end of each feeding period (4 and 6 wk) to measure hematological and immune parameters and growth and to determine the effects of dietary bglucan on resistance to Edwardsiella ictaluri infection and to low-water stress (6 wk). Supplementation of YYS in diets did not affect growth performance, hematology, or immune function. Survival from E. ictaluri infection was from 5 to 17.5% higher in fish fed YYS diets than in the control group, but the increases were not significant. Some improvement in stress resistance was observed in YYS-fed catfish after exposure to low-water stress. Stress reduction in fish fed diets supplemented with yeast subcomponents has been reported previously, but thus far, no explanation has been proposed for this effect. The present study and the previously published research suggest that dietary YYS supplementation does not appear to improve resistance of channel catfish to E. ictaluri. or b-1,3 and b-1,4 bonds. b-glucans with b-1,3 and b-1,6 bonding are most commonly found in the cell walls of yeast and mycelial fungi (Verlhac et al. 1998). The health benefits of b-glucans have been studied in many different animals (Raa 2000), with most research in fish focusing on their immune-enhancing properties. The influence of glucans on immune function and disease resistance in fish has been reviewed (Sakai 1999), but the effects of dietary b-glucan on channel catfish, Ictalurus punctatus, have been inconsistent. In channel catfish, Chen and Ainsworth (1992) reported that catfish injected with yeast glucans showed increased resistance to Edwardsiella ictaluri. However, dietary supplementation of b-glucan, while enhancing nonspecific immune function, did not appear to increase resistance to E. ictaluri (Ainsworth et al. 1994; Duncan and Klesius 1996). The supplementation of mannan oligosaccharides in diets of fish has also shown promise in stimulating the immune response and In recent years, a number of chemical agents, polysaccharides, plant extracts, and some nutrients have been included in fish diets as immunostimulants (Sakai 1999; Gannam and Schrock 2001). There is a growing body of evidence suggesting that immunostimulants added to diets can improve the ability of fish to respond to disease challenge. Immunostimulants can stimulate various components of the cellular and humoral immune systems and may act as adjuvants to increase vaccine effectiveness (Sakai 1999). Diets supplemented with immunostimulants, such as the yeast subcomponents b-glucan and mannan oligosaccharide, are also being promoted in aquaculture as a means of overcoming the immunosuppressive effects of stress that commonly occur in intensive fish production, even though few published reports support the claims. b-glucans are polysaccharides composed of glucose molecules linked by b-1,3 and b-1,6 1 Corresponding author. Ó Copyright by the World Aquaculture Society 2007 24 IMMUNE RESPONSE AND RESISTANCE TO STRESS AND E. ICTALURI CHALLENGE IN CHANNEL CATFISH increasing resistance to disease (Yoshida et al. 1995; Staykov et al. 2005); however, more research needs to be conducted to determine the effects of dietary supplementation of mannan oligosaccharides on fish health. The goal of immunostimulation in fish is to promote not only a greater and more effective immune response to infectious agents but also overcome the immunosuppressive effects of stress (Jeney et al. 1997). Some immunostimulants, such as bovine lactoferrin (Kakuta 1998) and vitamin C (Henrique et al. 1998), have shown promise in increasing stress resistance, although results have been highly variable and dependent upon the type of stressor and fish species examined. b-glucan as a stress-reducing agent, however, has received little attention. An increase in stress resistance has been observed in Nile tilapia, Oreochromis niloticus (Cain et al. 2003) and rainbow trout, Oncorhynchus mykiss (Jeney et al. 1997) fed b-glucansupplemented diets. In contrast, Li et al. (2005) observed no difference in the stress responsiveness of juvenile red drum, Sciaenops ocellatus, fed a control or brewers-yeast-supplemented diet. Although b-glucan has shown promise in increasing stress resistance in some fish species (Jeney et al. 1997; Cain et al. 2003), its effect has not been examined in channel catfish. Enteric septicemia (ESC) of channel catfish, caused by E. ictaluri (Hawke 1979), is one of the leading causes of economic loss in the catfish industry and is responsible for about $60 million in lost revenue each year (Shoemaker et al. 2002). From 1986 to 1996, ESC was the most reported infectious disease in the southeastern USA (Plumb 1999). Increases in infectivity and mortality of channel catfish from ESC have largely been attributed to stressrelated reductions in immune function (Klesius et al. 2003; Sink and Strange 2004). Dietary prophylaxis with immunostimulants, such as b-glucan, prior to exposure of channel catfish to stressful practices or at times of increased disease susceptibility may prove effective in preventing infection from E. ictaluri and other pathogens. Our objective was to examine the performance of several commercial sources of 25 b-glucan, at the manufacturer’s recommended dietary level for aquatic organisms, on immune function, resistance to stress, and survival following E. ictaluri infection in juvenile channel catfish. We hypothesized that addition of yeast or yeast subcomponents (YYS) to channel catfish diets would increase resistance to stress and increase immune function, leading to increased resistance to ESC. Materials and Methods Experimental Fish Juvenile channel catfish (NWAC 103) raised from yolk-sac fry to juveniles on a commercial fry diet were acclimated to laboratory conditions and fed the basal experimental diet without YYS for approximately 4 wk. At the end of the acclimation period, 45 fish were randomly selected and stocked in each of twenty 57-L aquaria. Total weight of fish in each aquarium was 476.1 6 0.3 g (mean 6 SEM), with an average individual weight of 10.6 6 0.1 g. The aquaria were supplied with flow-though, dechlorinated, city water heated with a central water heater at an initial rate of 0.5 L/min and increased gradually to about 0.8 L/min by the sixth week of the trial. Water flow rates were checked and adjusted twice daily to ensure proper water exchange. The water was continuously aerated, with compressed air diffused through air stones, and the photoperiod was maintained on a 12:12 h light : dark schedule. Dissolved oxygen and temperature in three randomly chosen aquaria were measured daily using a YSI model 58 Oxygen Meter (Yellow Springs Instruments, Yellow Springs, OH, USA). During the trial, water temperature averaged 27.3 6 0.1 C and dissolved oxygen averaged 4.9 6 0.4 mg/mL. Feed and Feeding A nutritionally complete practical basal diet formulated to contain approximately 32% crude protein, 5.6% crude lipid, and 2877 kcal of digestible energy/kg based on feedstuff values reported in NRC (1993) (Table 1) was supplemented with commercially available YYS products: MacroGardÒ (Biotec-Mackzymal, Tromsø, 26 WELKER ET AL. TABLE 1. Composition of basal diet. Ingredient Percent in diet Menhaden fish meal Soybean meal Corn meal Wheat middlings Carboxymethyl cellulose Cod liver oil Dicalcium phosphate Mineral premixa Vitamin premixb Celufil (nonnutritive binder) 8.0 45.0 16.3 22.5 3.0 3.0 1.0 0.5 0.5 0.2 a Mineral premix (mg/kg diet unless otherwise stated): cobalt chloride hexahydrate, 0.2; zinc sulfate heptahydrate, 659.6; iron sulfate pentahydrate, 199.0; manganese sulfate monohydrate, 77.0; copper chloride, 4.7; potassium iodide, 6.5; and sodium selenite, 0.2. b Vitamin premix (mg/kg diet unless otherwise stated): vitamin A-acetate, 4000 IU; vitamin D3, 2000 IU; vitamin K, 10; a-tocopherol acetate, 50; thiamin, 10; riboflavin, 12; pyridoxine, 10; pantothenic acid, 32; nicotinic acid, 80; folic acid, 5; biotin, 0.2; cyanocobalamine, 0.01; choline chloride, 400; L-ascorbyl acid-2-polyphosphate (15% vitamin C activity), 75. crude protein content of the basal diet was 33.4 6 0.2%. Each dietary treatment was randomly assigned to four aquaria. Fish were fed to apparent satiation twice daily (between 0730–0830 and 1500–1600 h) for a total period of 4 wk. The amount of feed eaten was recorded daily by calculating the differences in the weight of feed before the first and after the last feeding. After 4 wk on experimental diets, fish were sampled to measure hematological and immunological parameters and growth. Thereafter, all YYS groups were switched to the control diet, and feeding was continued for a period of 2 wk, at the end of which fish were again sampled (6-wk sampling) to measure immunological and hematological parameters and tested for resistance to stress and E. ictaluri infection as outlined below. No feeding was carried out on disease challenge or sampling days. All the aquaria were cleaned thoroughly once every other week. On cleaning days, fish were fed only in the afternoon. Growth Measurements TM Norway), Bio-Mos Aqua Grade (Alltech, Nicholasville, KY, USA), Betagard AÒ (AquaIn-Tech, Inc., Seattle, WA, USA), and Levucell SB20Ò Saccharomyces cerevisiae (Lallemand Animal Nutrition, Milwaukee, WI, USA), supplied by the manufacturers. MacroGard and Betagard A are polysaccharides of b-1,3/1,6-linked glucose molecules extracted from the cell wall of the yeast S. cerevisiae, while BioMos Aqua Grade is a mannan oligosaccharide derived from S. cerevisiae. Levucell SB20 is comprised of live, whole-cell S. cerevisiae. YYS were supplemented in the basal diet at the expense of celufil (nonnutritive binder) at the manufacturer’s recommended concentrations (MacroGard, 1 g/kg; Bio-Mos Aqua Grade, 2 g/kg; Betagard A, 0.1 g/kg; and Levucell SB20, 0.1 g/kg). Each diet was mixed thoroughly and processed into 3-mm-diameter pellets as described by Lim et al. (1996). Diets were dried at room temperature (23 C) to a moisture content of approximately 10%, ground in a S-500 Disc Mill (Glen Mills, Inc., Glenmill, NJ, USA), and sieved with a No. 14 US standard sieve. The particles retained in the sieve were stored at 20 C in sealed plastic bags until used. Determined Fish in each aquarium were counted and group weighed after 4 wk feeding of experimental diets and again after 2 wk on control diet (6-wk sampling), following 24 h of feed deprivation. Weight measurements and fish counts were used for estimation of weight gain, feed efficiency ratio (FER; wet weight gain/dry feed intake), and survival. Hematological and Immunological Assays At the 4- and 6-wk samplings, blood was sampled from the caudal vasculature of three anesthetized (120 mg/L tricaine methanesulfonate or MS-222; Argent Chemical Laboratories, Redmond, WA, USA) fish per aquarium with an air-dried, heparinized (500 U sodium heparinate/mL) tuberculin syringe. Hematocrit of each fish was determined in duplicate using microhematocrit method (Brown 1988). Red blood cells (RBC) and white blood cell (WBC) counts were performed in duplicate for each sample by diluting whole blood and counting in a Spencer Bright Line hemacytometer as described by Barros et al. (2002). Hemoglobin was analyzed using a kit from Pointe Scientific, Inc. (Canton, MI, USA), and values were adjusted IMMUNE RESPONSE AND RESISTANCE TO STRESS AND E. ICTALURI CHALLENGE IN CHANNEL CATFISH by cyanomethemoglobin correction factor for channel catfish as described by Larsen (1964). The remaining whole blood was centrifuged at 1000 g for 5 min, and plasma was stored frozen at 80 C for subsequent assays of bactericidal, lysozyme, and spontaneous hemolytic complement (SH50) activities. Plasma lysozyme activity was determined according to the method of Litwack (1955) as modified by Sankaran and Gurnani (1972). The assay is based on lysis of lysozyme-sensitive Gram-positive bacterium Micrococcus lysodeikticus (Sigma, St. Louis, MO, USA) by lysozyme present in the plasma. A suspension of 0.25 mg/mL freeze-dried Micro. lysodeikticus was prepared immediately before use by dissolving in sodium phosphate buffer (0.04 M Na2HPO4, pH 6.0). Plasma (10 mL/well in duplicate) was placed in a microtiter plate and 250 mL of bacterial cell suspension added to each well. Hen egg white lysozyme was used as an external standard. The initial and final (after 20-min incubation at 35 C) absorbances of the samples were measured at 450 nm. The rate of reduction in absorbance of samples was converted to lysozyme concentration (mg/mL) using a standard curve. Spontaneous hemolytic complement activity (SH50) was calculated using the method reported by Sunyer and Tort (1995). Sheep red blood cells (SRBC) in Alsever’s solution (Remel, Inc., Lenexa, KS, USA) were washed four times in cold phosphate-buffered saline (PBS+) solution (0.85% PBS, 0.1% gelatin, 0.15 mM CaCl2, 0.5 mM MgCl2) and adjusted to 5 3 107 cells/mL in cold PBS+. Twenty-five microliters of channel catfish plasma to be tested for complement activity was serially diluted in round-bottom microtiter plates in PBS+, and 25 mL of SRBC was added to each well. Distilled water was substituted for channel catfish plasma in one column to provide 100% hemolysis for a positive control. An additional 100 mL of PBS+ was added to all wells to increase well volume and aid in ease of pipette transfer of lysates. A second plate was assayed in tandem, except that no SRBC were added. Values obtained from this plate were subtracted from corresponding complement activity samples and used to account for any spontaneous hemolysis 27 of channel catfish RBC during blood sampling and handling. The plates were incubated at room temperature (23 C) for 1 h. After incubation, plates were centrifuged at 200 g and the supernatant pipetted into a new microplate. Hemolysis was evaluated spectrophotometrically at 570 nm and converted to percent hemolysis based on distilled water controls. The 50% lysis point (SH50) was calculated by linear regression of each serum sample and expressed as the log dilution. Bactericidal activity was determined as described by Kampen et al. (2005), with modifications for plasma. Twenty microliters of sample plasma or Hank’s Balanced Salt Solution (HBSS) (Gibco Laboratories, Grand Island, NY, USA) for positive controls was added to duplicate wells of a round-bottom, 96-well microtiter plate. Twenty microliters of a 24-h culture of E. ictaluri adjusted to 0.5 optical density (OD) at 540 nm was added to sample wells. Plates were incubated at room temperature (23 C) for 2.5 h on a platform shaker at 50 g (190 rpm). After incubation, plates were centrifuged at 2000 g for 10 min and the supernatant discarded. The remaining pellets were resuspended in 50 mL of brain–heart infusion (BHI) broth (Difco Laboratories, Sparks, MD, USA) and incubated for 3 h at room temperature on a platform shaker at 50 g (190 rpm). To each well, 25 mL of 3-(4,5-dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide (MTT) (2.5 mg/mL) (Sigma) was added and incubated for 10 min to allow the formation of formazan. Plates were again centrifuged at 2000 g for 10 min, the supernatant discarded, and the precipitate dissolved in 200 mL of dimethyl sulfoxide (DMSO). The absorbance of the dissolved formazan was read at 560 nm. Bactericidal activity was calculated as the decrease in number of viable E. ictaluri cells by subtracting the absorbance of samples from that of controls and reported as absorbance units. At the end of the 4-wk experimental feeding period, five fish from each aquarium were intraperitoneally (IP) injected with 250 mL of squalene (Sigma) as an attractant to obtain peripheral leucocytes (Yildirim et al. 2003). After squalene injection, fish were transferred to a new set of 28 WELKER ET AL. aquaria and switched to the control diet. Seven or 8 d later, fish were anesthetized with MS222 and IP injected with 10 mL of ice-cold, sterile, 0.85% PBS solution using a 20-gauge needle attached to a three-way valve. Then, PBS was removed along with the squalene-elicited exudate cells into a 50-mL centrifuge tube. The peritoneal fluid of fish from the same tank was combined and centrifuged at 300 g for 10 min. The supernatant was discarded, and the pellets were resuspended in 1 mL of calcium-, magnesium-, and sodium-free HBSS without phenol red (Gibco). Samples were stained with Yokayama’s solution for determination of the number of leucocytes in samples using a hemacytometer. Isolated channel catfish peripheral leucocytes were adjusted in 0.85% PBS solution, and the respiratory burst of phagocytic cells, based on superoxide anion (O2 ) production, was measured by nitroblue tetrazolium (NBT) assay according to the method of Secombes (1990), with some modifications. Quadruplicate monolayers of 5 3 106-adherent cells per well were prepared by placing 100-mL cell suspensions in a 96-well microplate. After incubation for 2 h at room temperature, the wells were washed with HBSS to remove the nonadherent cells. Adherent leucocyte monolayers were incubated with NBT (0.2% w/v in saline) solution to stimulate the respiratory burst activity for 1 h at room temperature in a humidified chamber. After removal of medium from the wells, 100% methanol was added to stop the reaction and washed twice with 70% methanol and allowed to air-dry. The reduced formazan within adherent cells was solubilized by adding 120 mL of 2 M potassium hydroxide (KOH) and 140 mL DMSO to each well. After incubation on a horizontal platform shaker 25 g (100 rpm) for 15 min, the OD of samples was measured at 620 nm using KOH/ DMSO as a reference. Stress Resistance Six fish were transferred from each aquarium to a new set of aquaria at the end of the 4-wk experimental feeding period for the stress challenge. Fish were fed the control diet and acclimated for 7 d. After the acclimation period, three baseline fish per aquarium were anesthe- tized, and the whole blood was collected and plasma obtained as described for hematological and immunological measurements. After baseline sampling, water depth in aquaria was reduced from approximately 26.5 to 6.5 cm by inserting a shortened standpipe. Fish were exposed to low water conditions for 30 min. Aeration and water inflow were maintained during the exposure period. Blood was again sampled from three fish at the end of the 30 min as previously described. Baseline fish and fish sampled after stress exposure were euthanized in a 200 mg/L solution of MS-222 directly after blood sampling. Plasma was collected for determination of cortisol, glucose, lactate, and nitrite concentrations. Cortisol was measured using an ELISA kit manufactured by DRG International, Inc. (Mountainside, NJ, USA). Glucose and lactate concentrations were determined by kits (Pointe Scientific, Inc.) following the manufacturer’s instructions. Nitrite is a stable end product of nitric oxide (NO) metabolism in vertebrates (AlaghbandZadeh et al. 1996). In the neuroendocrine system of mammals, NO influences the secretion of various endocrine hormones, such as corticotropin releasing hormone, adrenocorticotropic hormone, and vasopressin (Kishimoto et al. 1996), and is therefore intimately connected to the hypothalamic–pituitary–adrenal axis, which is activated in response to stress in mammals. Plasma nitrite, as an indirect measure of NO synthesis, has been shown to increase in rats subjected to immobilization stress (Kishimoto et al. 1996). Plasma nitrite concentrations were measured using a Griess Reagent System kit from Promega, Inc. (Madison, WI, USA). The Griess Reagent System is based on a chemical reaction that uses sulfanilamide and N-1-naphthylethylenediamine dihydrochloride (NED) under acidic conditions. Fifty microliters of sample plasma was added to duplicate wells of a 96-well microtiter plate, and 50 mL of PBS was used as a negative control. A nitrite standard reference curve was constructed by performing a twofold serial dilution in PBS of a 100 mM solution of the standard provided with the kit. The range of the standard curve was 1.56–100 mM nitrite. To each well, 50 mL of the sulfanilamide solution was added IMMUNE RESPONSE AND RESISTANCE TO STRESS AND E. ICTALURI CHALLENGE IN CHANNEL CATFISH and the plate incubated at room temperature in the dark for 10 min. Fifty microliters of the NED solution was added to all wells and again incubated in the dark for 10 min. The absorbance of samples was read with a microplate reader at a wavelength of 560 nm. The nitrite concentration in samples was calculated from the standard curve. Disease Challenge Edwardsiella ictaluri (AL-93-75) from an outbreak of ESC was used to challenge channel catfish by immersion. Frozen stock culture of E. ictaluri was grown in BHI broth for 24 h at 27 C. The concentration of the culture after 24 h of growth was estimated at 1 3 1010 colonyforming units (CFU)/mL. Plasma collected at the 6-wk sampling for use in immune assays (n 5 3 fish per aquarium) was used to determine prechallenge antibody titers – all sampled fish were negative for E. ictaluri. The number of channel catfish remaining in the original aquaria at the end of 6 wk was adjusted to 20 and challenged in aquaria by addition of the E. ictaluri culture at a rate to produce 1 3 107 CFU/mL. During immersion challenge, water flow but not aeration was halted for 1 h. The final challenge concentration was approximately 9.1 3 106 CFU/mL, determined using a spiral autoplater and Qcount (Spiral Biotech, Norwood, MA, USA) after challenge. Fish continued to be fed twice daily with the control diet, and mortality was recorded twice a day for 21 d. At the end of the challenge trial (Day 22), blood was sampled from the caudal vasculature of four surviving fish in each aquarium by syringe, allowed to clot for 4 h at 4 C, and centrifuged at 1000 g. Serum was collected and stored frozen at 80 C for subsequent determination of postchallenge agglutinating antibody titer against E. ictaluri. Necropsies were performed, and anterior kidney tissue from dead fish was cultured to confirm death as a result of infection with E. ictaluri (Klesius 1992). Agglutinating antibody titer against E. ictaluri (AL-93-75) in pre- and postchallenge serum was determined by modifying the method of Chen and Light (1994). E. ictaluri was grown for 24 h in BHI broth at 28 C and killed in 1% 29 formalin. The cells were centrifuged at 3000 g for 15 min, and the resulting pellet was suspended and washed thrice in sterile PBS. The bacterial concentration was adjusted to an OD of 0.8 at 540 nm. Each well of a 96-, roundbottomed microtiter plate was plated with 15 mL of sterile PBS; then, 15 mL of plasma was added to the first well of each row and mixed. Twofold serial dilutions were then made by adding 15 mL of diluted plasma into the remaining wells. An equal volume (15 mL) of bacterial suspension was added to each well, making the initial dilution of the plasma 1:4. Positive plasma from E. ictaluri-infected fish and negative (PBS) were used as assay controls. The plates were covered with plastic film and incubated at room temperature for 16 h. The agglutination endpoint was established as the last dilution where cell agglutination was visible. Statistical Analysis YYS source (MacroGard, Bio-Mos Aqua Grade, Betagard A, Levucell SB20, and control diets) and sampling date (4 and 6 wk) were fixed effects in this study. Growth, hematological, and immunological parameters and percent survival to E. ictaluri infection were analyzed by twoway ANOVA (except for cell counts performed only at 4 wk). For the low-water stress challenge, the effects of YYS source and sampling time (baseline and poststress) on measured stress parameters were also analyzed by twoway ANOVA. No differences in baseline stress values were observed among diets. Therefore, poststress values for stress parameters were analyzed by one-way ANOVA. Measured values from individual fish were averaged for each aquarium (experimental unit) for use in statistical analyses. Pair-wise comparisons between means for main effects were made using the Tukey–Kramer method by examining mean contrasts with the associated confidence interval at P 5 a/c, where c 5 number of pair-wise comparisons and a 5 0.05. A significance level of a 5 0.05 was used for all statistical analyses. Results Plasma lysozyme, bactericidal, and spontaneous hemolytic complement activities were not 30 WELKER ET AL. affected (P . 0.05) by dietary YYS supplementation (Table 2). The respiratory burst of phagocytes (NBT assay) measured at 6 wk was unaffected by diet. There were significant differences in the immune parameters measured at Weeks 4 and 6. At 6 wk, lysozyme (P # 0.05) and bactericidal (P . 0.05) activities were lower, but SH50 values were significantly higher (P # 0.05) than at 4 wk (Table 2). YYS source did not have a significant effect on survival (%) following E. ictaluri infection (P . 0.05) (Table 3). Antibody titers against E. ictaluri 21 d postchallenge were also unaffected by dietary treatments (Table 3). YYS source significantly affected the plasma cortisol and lactate responses (P # 0.05) but not the glucose or nitrite response (P . 0.05) to low-water stress. Fish fed the control diet had significantly elevated (P # 0.05) cortisol concentrations than those fed the Bio-Mos Aqua Grade. Lactate concentrations were higher in the control fish than in the fish fed the Levucell SB20- and Betagard A-supplemented diets. For all treatments, low-water stress produced significant increases (P # 0.01) in plasma cortisol, glucose, lactate, and nitrite relative to prestress baseline values (Table 4). RBC and WBC counts were not significantly affected (P . 0.05) by addition of YYS to diets (Table 5). Hematocrit was significantly higher in MacroGard group (P # 0.05) than in the control group at 4 wk, but no differences were observed at the end of Week 6. No effect of YYS was seen on hemoglobin at either sampling time. Overall, hematocrit and hemoglobin values were significantly elevated (P # 0.05) at 6 wk compared to 4 wk (Table 5). Dietary YYS supplementation did not significantly affect (P . 0.05) the total weight gain at 4 or 6 wk, FER at 4 or 6 wk, or feed intake at 6 wk (Table 6). However, feed intake was significantly higher for channel catfish fed Betagard A compared to those fed Levucell SB20 (P # 0.05). Survival of the fish in this study was near 100% and not correlated with the experimental diets (P . 0.05). Discussion No effect of dietary YYS on immune parameters or survival following E. ictaluri challenge was observed in the present study. Of note, survival following ESC infection was from 5 to 17.5% higher in fish fed b-glucan-supplemented diets than in the control group; however, these increases were not significant. Considerable variation exists in the published effects of dietary YYS on immune function and disease resistance. Channel catfish have generally shown some increase in nonspecific immune function but not a corresponding increase in resistance TABLE 2. Mean 6 SEM lysozyme, spontaneous hemolytic complement (SH50), and plasma BA activities and respiratory burst (NBT) of phagocytes in channel catfish fed diets containing b-glucans from various commercial sources.a,b Lysozyme (mg/mL) SH50 (U/mL) BAc b-glucan source 4 wk 6 wk 4 wk 6 wk Control Bio-Mos Aqua Grade MacroGard Betagard A Levucell SB20 Date averagee 7.23 ± 0.48 5.31 ± 0.24 8.04 ± 0.96 9.10 ± 2.16 6.47 6.88 7.04 6.79 6.88 5.25 5.87 4.95 5.06 5.29 7.32 8.57 9.56 9.16 8.53 ± ± ± ± ± 0.45 0.58 0.70 0.32 0.22* ± ± ± ± ± 0.50 0.64 0.53 0.19 0.20* ± ± ± ± ± 0.90 2.36 1.67 1.04 0.62* 9.25 14.57 11.53 12.31 11.35 ± ± ± ± ± 2.05 3.09 2.77 2.64 1.12* 4 wk NBTd (OD) 6 wk 6 wk 0.190 ± 0.024 0.161 ± 0.013 0.427 ± 0.017 0.186 0.172 0.148 0.191 0.178 ± ± ± ± ± 0.019 0.049 0.033 0.011 0.011 0.102 0.139 0.176 0.144 0.144 ± ± ± ± ± 0.011 0.020 0.045 0.025 0.012 0.355 0.452 0.428 0.368 0.406 ± ± ± ± ± 0.060 0.012 0.042 0.029 0.016 BA 5 bactericidal, OD 5 optical density, NBT 5 nitroblue tetrazolium. a No significant differences (P . 0.05) were observed among dietary treatment means. b Fish were sampled after 4 wk on experimental diets containing b-glucan and after an additional 2 wk on control diet (6-wk sampling). c Plasma BA activity reported as absorbance units. d NBT test was determined only after 6 wk. e Values are averages for each sampling date. Values for the same parameter at 4 and 6 wk with asterisks are significantly different. 31 IMMUNE RESPONSE AND RESISTANCE TO STRESS AND E. ICTALURI CHALLENGE IN CHANNEL CATFISH TABLE 3. Mean 6 SEM survival (%) and agglutinating antibody titer to Edwardsiella ictaluri in channel catfish fed diets containing b-glucans from various commercial sources.a,b b-glucan source Control Bio-Mos Aqua Grade MacroGard Betagard A Levucell SB20 Survival (%) Antibody titer (log10) 75.0 87.5 80.0 92.5 85.0 3.13 2.88 3.08 3.21 3.18 ± ± ± ± ± 7.5 2.5 10.0 2.5 2.9 ± ± ± ± ± 0.17 0.19 0.06 0.16 0.05 a No significant differences (P . 0.05) were observed among treatment means. b Survival and antibody titer were measured 21 d postimmersion challenge. to E. ictaluri when fed diets supplemented with YYS. Duncan and Klesius (1996) found increased macrophage and neutrophil migration and phagocytosis in channel catfish fed a diet containing 0.2% b-glucan but not in those fed 2.7% S. cerevisiae. However, neither diet increased survival following E. ictaluri infection. Ainsworth et al. (1994) observed increased antibody titers to E. ictaluri in channel catfish fed 0.1% b-glucan, but these increases did not translate into increased survival following ESC. In contrast, Chen and Ainsworth (1992) have reported increased antibody titers and survival to E. ictaluri infection in channel catfish when a mixture of b-glucan and baker’s yeast was administered IP. The authors hypothesized that the increased protection was related to increased phagocytic and bactericidal ability of phagocytes and neutrophils, which have receptor sites specific for b-1,3 glucan (Ainsworth et al. 1994). Binding of b-glucan to the receptor site enhances phagocytosis by activation of the alternative complement pathway or stimulation of the 5-lipoxygenase eicosanoid pathway (Chen and Ainsworth 1992). The positive results reported by Chen and Ainsworth (1992), however, were in channel catfish receiving b-glucan by IP injection and not from the diet. Enhanced immune function does not always translate to increased disease protection. In channel catfish, E. ictaluri can survive and multiply in macrophages (Shotts et al. 1986). YYS-enhanced chemotaxis and phagocytosis without increased killing of E. ictaluri by phagocytes would not necessarily result in increased protection (Duncan and Klesius 1996). No mechanism has thus far been proposed to explain the stress-reducing effects of YYS. In the present study, significantly lower levels of cortisol and lactate were observed in Bio-Mos Aqua Grade- and Levucell SB20-fed channel catfish, respectively, after low-water stress. Cain et al. (2003) also observed a reduction in the cortisol but not in glucose or chloride responses to handling stress in Nile tilapia fed a 0.2% bglucan diet. Significant reductions in cortisol were observed 2 h and 1 wk posttransportation stress in rainbow trout fed b-glucan at a rate of 0.1% of diet (Jeney et al. 1997). Li et al. (2005), on the other hand, found no difference in the cortisol response of juvenile red drum fed a control or brewers-yeast-supplemented diet and hypothesized that this was as a result of the extreme variation among individual fish within the same treatment. As Cain et al. (2003) cautioned, care TABLE 4. Mean 6 SEM plasma cortisol, glucose, lactate, and nitrite levels in channel catfish fed diets containing b-glucans from various commercial sources and after (poststress) 30 min of low-water stress.1,2 b-glucan source Control Bio-Mos Aqua Grade MacroGard Betagard A Levucell SB20 Baseline3 Poststress3 Cortisol (ng/mL) 100.2 63.8 72.9 73.4 78.5 14.3 77.8 ± ± ± ± ± ± ± 14.1a 12.9b 15.5ab 13.0ab 14.8ab 4.9* 13.8* Glucose (mg/dL) 80.2 63.9 75.6 84.9 64.5 35.7 73.8 ± ± ± ± ± ± ± 3.6 4.0 4.2 5.2 2.8 1.1* 3.0* Lactate (mg/dL) 57.1 38.9 38.3 34.4 33.2 31.0 40.4 ± ± ± ± ± ± ± 15.4a 14.3ab 13.4ab 11.9b 12.3b 8.0* 12.9* Nitrite (mM) 5.42 2.60 4.61 4.33 3.53 0.44 4.14 ± ± ± ± ± ± ± 0.72 0.36 0.76 0.70 0.74 0.05* 0.55* Means in the same column with different superscript letters are significantly different (P , 0.05). Stress tolerance was measured after the 2-wk control feeding period (6 wk). 3 Baseline and poststress means (averaged across treatments) in the same column and marked with asterisks are significantly different. 1 2 32 WELKER ET AL. TABLE 5. Mean 6 SEM WBC count, RBC count, hematocrit, and hemoglobin of channel catfish fed diets containing b-glucans from various commercial sources.1,2 WBC3 (105/mL) b-glucan source Control Bio-Mos Aqua Grade MacroGard Betagard A Levucell SB20 Date average4 RBC3 (106/mL) 4 wk 2.53 2.69 5.02 4.68 2.83 3.55 ± ± ± ± ± ± Hematocrit (%) 4 wk 0.79 0.64 1.97 1.86 0.88 0.70 1.94 1.46 1.62 1.60 1.81 1.60 ± ± ± ± ± ± 4 wk 0.20 0.17 0.18 0.17 0.15 0.08 38.6 40.1 43.1 42.3 40.1 40.8 ± ± ± ± ± ± Hemoglobin (g/dL) 6 wk 1.0a 0.7ab 1.5b 0.6ab 1.0ab 0.6* 44.7 44.4 46.5 47.3 44.2 45.4 4 wk ± ± ± ± ± ± 0.7 0.2 1.4 0.9 1.0 0.5* 7.45 7.38 7.87 8.23 7.57 7.70 ± ± ± ± ± ± 0.15 0.14 0.27 0.41 0.23 0.13* 6 wk 8.98 8.38 9.55 9.12 9.17 9.04 ± ± ± ± ± ± 0.38 0.37 0.10 0.09 0.40 0.15* RBC 5 red blood cells, WBC 5 white blood cells. 1 Means in the same column with different superscript letters are significantly different (P , 0.05). 2 Fish were sampled after 4 wk on experimental diets containing b-glucan and after an additional 2 wk on control diet (6-wk sampling). 3 Cell counts were only conducted at 4 wk. 4 Values are averages for each sampling date. Average values for the same parameter at 4 and 6 wk marked with asterisks are significantly different. should be taken when interpreting results of stress experiments, especially with regards to cortisol concentrations. We similarly observed rather high standard errors for mean cortisol and lactate concentrations. The focus of most studies examining the effects of YYS dietary supplementation in fish has been on immune enhancement and disease resistance and not on growth. Because of the potential immunosuppressive effects of longterm YYS feeding (Sakai 1999; Couso et al. 2003), the duration of the majority of studies on YYS has often been too short (4 wk or less) to adequately determine an effect on growth performance. In the present study, dietary supplementation with commercial YYS had no substantial effects on growth performance. In agreement with the results of our study, growth rate was also unaffected in Asian catfish, Clarias batrachus, fed 0.1% b-glucan for 1, 2, or 3 wk (Kumari and Sahoo 2006) and in juvenile red drum fed 2% brewers yeast for 6 wk (Li et al. 2005). Likewise, juvenile hybrid striped bass, Morone chrysops female 3 M. saxatilis male, fed 1 or 2% brewers yeast diets for 4 or 7 wk did not exhibit significant changes in growth (Li and Gatlin 2004). The feeding of b-glucan supplemented at 50, 100, or 200 mg/kg diet for the extended period of 14 wk also did not produce significant differences in growth in Nile tilapia (Whittington et al. 2005). Conversely, bglucan feeding provided a significant increase in TABLE 6. Mean 6 SEM weight gain, DM feed intake, FER, and survival of channel catfish fed diets containing b-glucans from various commercial sources.1,2 Feed intake (g DM/fish) Weight gain (g/fish) b-glucan source 4 wk Control 15.6 ± 0.2 Bio-Mos Aqua Grade 16.2 ± 0.3 MacroGard 16.5 ± 0.6 Betagard A 17.0 ± 0.2 Levucell SB20 15.7 ± 0.2 6 wk 23.8 25.5 25.1 27.4 25.3 ± ± ± ± ± 0.9 1.2 1.0 0.6 1.3 4 wk 22.3 22.9 22.9 23.5 21.8 ± ± ± ± ± 0.5ab 0.3ab 0.5ab 0.1b 0.2a 6 wk 39.3 40.0 39.9 41.1 39.2 Survival (%) FER3 ± ± ± ± ± 0.9 0.9 0.9 0.7 0.7 4 wk 0.70 0.71 0.72 0.72 0.72 ± ± ± ± ± 0.01 0.01 0.01 0.02 0.01 6 wk 0.61 0.64 0.63 0.67 0.64 ± ± ± ± ± 0.01 0.02 0.02 0.01 0.02 6 wk 96.8 97.4 98.1 98.1 97.4 ± ± ± ± ± 0.6 0.0 0.6 0.6 0.0 DM 5 dry matter, FER 5 feed efficiency ratio. 1 Means in the same column with different superscript letters are significantly different (P , 0.05). 2 Fish were sampled after 4 wk on experimental diets containing b-glucan and after an additional 2 wk on control diet (6-wk sampling). Data measured at 6 wk were cumulative for the study period. 3 FER: wet weight gain/dry feed intake. IMMUNE RESPONSE AND RESISTANCE TO STRESS AND E. ICTALURI CHALLENGE IN CHANNEL CATFISH growth of snapper, Pagrus auratus, after 56 and 84 d of feeding during winter at ambient temperatures, but no effect on growth was observed during summer months at optimal growth temperature, suggesting that b-glucan may only affect growth of snapper reared under suboptimal growth temperatures (Cook et al. 2003). Misra et al. (2006) also observed significant increases in growth of Labeo rohita fingerlings, fed 250 and 500 mg/kg b-glucan for 56 d. Feeding of mannan oligosaccharides has proven effective in promoting growth in fish. Bio-Mos Aqua Grade fed for 60 d produced a significant improvement in growth performance of rainbow trout, Oncorhynchus mykiss, and common carp, Cyprinus carpio (Staykov et al. 2005). There is considerable variation in the effect of dietary supplementation of YYS on growth, which is likely dependent upon the type of YYS, fish species, and feeding duration. In the current study, we followed the manufacturer’s recommended dietary concentrations for aquatic species, but the effective dose concentration of YYS in diet can vary based on fish species and feeding duration. Several studies have suggested that short-duration feeding of immunostimulants, followed by a period of control diet feeding, produces optimal effects on immune function enhancement and disease resistance (Chen and Ainsworth 1992; Bagni et al. 2000; Couso et al. 2003; Bridle et al. 2005). We fed diets supplemented with YYS for 4 wk, followed by 2 wk of control diet feeding. There may be a balance between feeding duration and dietary YYS concentration that must be met in order to achieve increased immunity and disease resistance. This balance probably varies considerably with fish species, age or size, nutritional status, physiological condition, and YYS source. In the current study, feeding of experimental diets for different durations or altering dietary concentrations of the tested YYS sources under the same feeding regimen may have resulted in enhanced immunity. Conclusions In this study, supplementation of channel catfish diets with YYS did not produce increases in immune function or resistance to E. ictaluri. 33 Published research indicates that high variability exists in the efficacy of dietary YYS to boost immunity. Much of the variability can probably be explained by differences in source and dietary concentration of YYS, feeding regimen, and fish species. The current study suggests that dietary b-glucan supplementation does not improve resistance of channel catfish to ESC. Even though dietary YYS can have positive effects on immune function in channel catfish as reported in previously published research, a corresponding increase in disease resistance was not conferred. Some fish pathogens, such as E. ictaluri, may not be affected by immune parameters enhanced by YYS administration. We also observed a significant decrease in stress responsiveness in channel catfish fed YYS compared to controls. This has been reported previously, but thus far, the mechanism in which YYS affect fish stress has not been explained. Acknowledgments The authors would like to thank Micah Simmons, Mark Smith, Craig Shoemaker, Paige Mumma, Rashida Eljack, Todd Threadgill, and Justin Brock, all of the Aquatic Animal Health Research Unit (AAHRU), United States Department of Agriculture (USDA), Agricultural Research Service (ARS), Auburn, AL, for assistance in this project. 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