Arginine and vitamin E improve the immune response after a Salmonella challenge in broiler chicks X. Liu,* J. A. Byrd,† M. Farnell,‡ and C. A. Ruiz-Feria*1 *Poultry Science Department, Texas A&M University, College Station 77843-2472; †USDA-ARS, Southern Plains Agricultural Research Center, College Station TX 77845; and ‡Poultry Science Department, Mississippi State University, Mississippi State 39762
Key words: immune response, Salmonella, arginine, vitamin E, mannanoligosaccharide 2014 Poultry Science 93:882–890 http://dx.doi.org/10.3382/ps.2013-03723 of antimicrobial in food-producing animals for growth promotion and prophylaxis may lead to the development of microbial strains resistant to antibiotic therapy (Bach Knudsen, 2001; Harrison et al., 2013). Therefore, it is essential to find alternatives to antibiotics in foodproducing animals. One way to improve immune response and avian health is through the use of nutritional supplements (Klasing, 1998; Kidd, 2004). Arginine, an essential amino acid for avian species, and vitamin E (VE), an important antioxidant, have been shown to influence both the humoral- and cell-mediated immune responses of birds. In chickens, an Arg-deficient diet has been associated with poor development of the thymus and spleen, which are key organs of the immune system (Kwak et al., 1999). Arginine supplementation modulates or boosts humoral and cellular immune response, and improves nitric oxide (NO) production and the acute phase inflammatory response following lipopoly-
INTRODUCTION Consumption of contaminated poultry products is a major cause of human salmonellosis, with most of the cases being attributed to Salmonella enterica serovar Enteritidis or Typhimurium (Beal et al., 2006). On the other hand, intensive poultry production practices may increase susceptibility to disease due to high stocking densities, recycled litter, and environmental conditions that are sometimes less than optimal. To reduce the incidence of disease and improve growth, antibiotics are used in commercial animal production; drug makers sold 13.5 million kg of antibiotics for use in food-producing animals in 2011 (FDA, 2013). However, there are increasing concerns that the subtherapeutic use ©2014 Poultry Science Association Inc. Received October 30, 2013. Accepted December 18, 2013. 1 Corresponding author: [email protected]
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ment, birds fed the AVE diet had higher MOB than birds fed CTL+ or the AVM diet at 7 d PI, whereas 9 d PI birds fed the AVM diet had the highest MOB. In experiment 2, birds fed the AVE diet had higher MOB, HOB, and LPR than birds in the other treatments 7 and 14 d PI, except at 7 d PI, when MOB was not different among treatments. Birds fed the AVM diet had the highest IgA antibody titer, and a higher IgM antibody titer than the CTL+ birds. In experiment 1, Salmonella Typhimurium content in the ceca was lower in birds fed the AVM diet compared with birds fed the CTL− diet 3 d PI, but later on (10 and 17 d PI), and in experiment 2 (7, 14, and 21 d PI), Salmonella Typhimurium concentrations were not different among treatments. Thus, Arg and VE improved immune response after a Salmonella Typhimurium challenge in young chicks, and although they did not reduce Salmonella Typhimurium concentrations in the ceca, they may improve bacterial resistance against other pathogens in commercial growing conditions.
ABSTRACT Two experiments were conducted to evaluate the effects of Arg, vitamin E (VE), and mannanoligosaccharide (MOS) on the immune response and clearance of Salmonella in broiler chickens. In each experiment, 1-d-old chicks (n = 160) were randomly distributed into 4 groups: antibiotic-free diet (negative control, CTL−), antibiotic-supplemented diet (positive control, CTL+), antibiotic free-diet plus Arg and VE (AVE), or antibiotic-free diet plus Arg, VE, and MOS (AVM). Birds were orally challenged with 106 cfu of a novobiocyn and nalidixic acid-resistant Salmonella enterica serovar Typhimurium strain at d 7 (experiment 1) or at d 3 (experiment 2). Heterophil- (HOB) and monocyte- (MOB) oxidative burst and lymphocyte proliferation (LPR), antibody titers, and Salmonella content in the ceca were measured at several intervals postinfection (PI). In experiment 1, both AVM and AVE decreased HOB compared with the controls 5 and 9 d PI, but increased LPR 9 d PI. In the same experi-
IMMUNE FUNCTION AFTER SALMONELLA CHALLENGE
antibody titers, and Salmonella concentrations in the ceca, after an experimental challenge in young broiler chickens.
MATERIALS AND METHODS Birds and Treatments Two experiments were conducted. One-day-old broiler chicks (Cobb 500) were obtained from a local hatchery and housed in a biosafety level 2 facility at the USDA-Southern Plains Agriculture Research Center in College Station, Texas. Chicks were grown on fresh pine shavings and brooded following standard temperature regimens, which gradually decreased from 32 to 24°C, and under a 16L:8D cycle throughout the studies. All birds were fed a corn-soybean meal-based diet formulated to meet or exceed all of the NRC (1994) requirements. The basal diet contained 23% of CP, 3,200 kcal of ME/kg, 1.54% Arg, and 40.3 IU of VE/kg. A completely randomized design with 4 dietary treatments (40 birds/treatment) was used, as follows: basal diet free of antibiotics (negative control diet, CTL−), basal diet plus 40 mg of bacitracin/kg of feed (positive control, CTL+), basal diet free of antibiotics supplemented with Arg (0.8%, wt/wt) and 40 IU of VE/kg of feed (AVE; for a total content of 2.34% Arg and 80 IU of VE/kg of feed), or AVE diet plus 0.2% (wt/wt) MOS (AVM; BioMos, Alltech Co., Lexington, KY). All the experimental procedures were approved by the Institutional Animal Care Committee.
Bacterial Challenge and Cecal Counts A primary poultry isolate of Salmonella enterica serovar Enteritidis or Typhimurium was obtained from the National Veterinary Services Laboratory (Ames, IA) and selected for resistance to novobiocin and nalidixic acid, and maintained in media containing 25 μg of novobiocin and 20 μg of nalidixic acid/mL. Portions (1 to 2 mL) of cultures grown overnight at 37°C in tryptic soy broth (Difco Laboratories, Detroit, MI) were used as inocula for challenging broilers. At d 7 (experiment 1) or d 3 (experiment 2), all the chicks were orally challenged with 106 cfu of Salmonella Typhimurium (1 mL/chick). Ten birds per treatment were euthanized by cervical dislocation at 3, 10, and 14 d after challenge (experiment 1) or 7, 14, and 21 d after challenge (experiment 2). Cecal contents (0.25 g) were aseptically collected and deposited in 2.25 mL of PBS (pH 6.5). Samples were thoroughly mixed, serially diluted in PBS, and then spread plated onto XLT4 Agar (BD Diagnostics, Sparks, MD) containing 20 μg of nalixidic acid/mL and 25 μg of novobiocin/mL, at a final dilution of 1:100, 1:1,000, and 1:10,000. Plates were incubated for at least 24 h at 37°C, and the number of Salmonella cfu per gram was determined. Salmonella colony numbers were log-transformed before being analyzed. For qualitative enrichment, cecal contents were
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saccharide (LPS) injections, and NO is an important component of the macrophage defense against Salmonella (Moncada et al., 1991; Xie et al., 1994). Vitamin E has been shown to improve the immune response in birds by enhancing macrophage phagocytic function, decreasing prostaglandin E2 production, increasing IL-1 secretion by macrophages, and enhancing IL-2 production and T-cell proliferation (Moriguchi et al., 1993). Erf et al. (1998) reported that the T-helper/T-cytotoxic lymphocyte ratio and the percentages of T-helper lymphocytes in the spleen and thymus were higher in broilers fed diets with high levels of VE. Prebiotics are nondigestible feed ingredients that beneficially affect the host by selectively stimulating the growth or metabolic activity of a limited number of intestinal microorganisms (Gibson and Roberfroid, 1995; Macfarlane et al., 2006). Mannanoligosaccharides (MOS), a prebiotic derived from yeasts cells, have been shown to have antimicrobial properties, modulate intestinal microbial populations, and stimulate immune response in several animal species, including poultry (Spring et al., 2000; Fairchild et al., 2001; Fernandez et al., 2002; Denev et al., 2005). Spring et al. (2000) suggested that MOS reduced Salmonella colonization in the ceca of chickens by adsorbing bacteria and keeping them from adhering to the gut wall. We previously reported that MOS supplementation increased the populations of beneficial bacteria (bifidobacteria and lactobacilli), and reduced Escherichia coli shedding in the litter (Baurhoo et al., 2007a), showing the capacity of MOS to modulate intestinal microbial populations. More recently, it was reported that birds fed MOS had an increased innate immune response (upregulation of IL-3, and upregulation of signal transducer and activator of transcription 2 gene, which increases macrophage phagocytic activity) than birds fed virginiamycin after a challenge with Salmonella LPS (Baurhoo et al., 2012). Although the effects of Arg, VE, and prebiotics, on immune response have been investigated, their combined effects on the immune response and resistance to bacterial challenges have not been evaluated. We have documented that the combination of high levels of Arg and VE have complementary or synergistic effects on the immune response against several immune challenges, suggesting that the combination of Arg and VE may improve the health of broilers and potentially eliminate the need for antimicrobials (Abdukalykova and RuizFeria, 2006; Abdukalykova et al., 2008; Ruiz-Feria and Abdukalykova, 2009; Perez-Carbajal et al., 2010; ChanDíaz et al., 2012). We have also reported that MOS supplementation improved intestinal integrity and reduced E. coli colonization in the ceca of broiler chickens (Baurhoo et al., 2007b). Thus, we hypothesized that the concurrent supplementation of Arg, VE, and prebiotics (MOS) will improve the immune response and will reduce Salmonella levels in the ceca. The objectives of the present experiments were to evaluate the effects of Arg, VE, and MOS on monocyte and heterophil oxidative burst, lymphocyte proliferation in vitro, serum
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enriched in Rappaport-Vassiliadis Broth (Difco) for 24 h at 37°C, and were then struck on XLT4 agar plates. After 24 h of incubation at 42°C, the plates were read as positive or negative. Cecal contents that were negative at a 100-fold dilution on XLT4 agar plates but were positive on the enriched solution were assigned a 1.50 log10 Salmonella/g of cecal content (Byrd et al., 1998; Kubena et al., 2001; Kogut et al., 2010).
Blood Collection
Isolation of Heterophils and Monocytes The polymorphonuclear and mononuclear cell fractions were isolated from peripheral blood as described previously (Kogut et al., 1995; Stringfellow et al., 2011). Briefly, the blood samples were mixed with a solution of 1% methylcellulose (Sigma-Aldrich, St. Louis, MO) dissolved in Roswell Park Memorial Institute (RPMI) 1640 media (Mediatech Inc., Herndon, VA) at a 1:1.5 ratio and centrifuged at 37 × g for 15 min at 4°C. The supernatant was removed and mixed with an equal volume of calcium and magnesium free Hank’s balanced salt solution (Mediatech Inc.). This suspension was layered over a 1.077/1.119 Histopaque (Sigma-Aldrich) gradient and centrifuged at 235 × g for 60 min at 4°C. Following centrifugation, the interfaces containing monocytes or heterophils were collected, washed, and resuspended with RPMI. The cells were counted using a Neubauer hemacytometer and the concentration adjusted to 4 × 106 heterophils or monocytes/mL and kept on ice until used. Cell viability (>95%) was determined using a trypan blue solution (Sigma-Aldrich) at a 1:1 ratio.
Oxidative Burst Assay Oxidative burst activity of heterophils and monocytes was measured using a Wallac fluorescent plate reader (Perkin Elmer, Boston, MA), using DCF-DA (Molecular Probes Inc., Eugene, OR) as reactive oxygen species indicator, as described previously (Xie et al., 2002; Stringfellow et al., 2011). Briefly, heterophils and monocytes were preincubated with an agonist (2 μg of phorbol-12-myristate-13 acetate (Calbiochem, La Jolla, CA) or an equivalent volume of RPMI during 30 min at 42°C in a heated orbital shaker plate (Thermo-Forma, Marietta, OH). Then, 125 μL of DCF-DA
Lymphocyte Proliferation A modified cell proliferation assay was conducted to evaluate cell-mediated immunity (Pauly et al., 1973). After isolation, heterophils and monocytes were deposited into the plates. Then equal volumes of the lymphocyte suspension and concanavalin A were added in each of 6 wells of 96-well flat-bottomed tissue culture plates. Negative controls were maintained with RPMI only and cell suspension without concanavalin A. Plates were then incubated in a water-jacketed 5% CO2 incubator at 42°C for 24 h. Fifteen microliters of stock 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (5 mg/mL) was added to each well and plates were incubated for 4 h. The color change was measured at 450 nm by a colorimetric plate reader (Sunrise, Tecan, Austria).
ELISA The concentration of IgA, IgG, and IgM isotypes from 19-d-old chickens (16 d after challenge) were measured in serum samples (8/treatment, experiment 2). Blood samples (2 mL) were obtained by venipuncture and allowed to clot at room temperature for 4 h, samples were then centrifuged at 400 × g for 8 min at 4°C and the serum was collected and stored at −80°C until assayed. The serum samples were thawed at 4°C to calculate antibody concentrations using chicken IgA, IgG, and IgM ELISA kits (Bethyl Laboratories, Montgomery, TX) according to the manufacturer’s instructions. Briefly, flat-bottomed microtiter plates were coated for 60 min with capture antibody (goat anti-chicken IgG-Fc or IgM or IgA affinity purified) and coating buffer (0.05 M carbonate-bicarbonate, pH 9.6). Plates were washed 3 times with wash solution (50 mM Tris buffered saline, 0.14 M NaCl, 0.05% Tween 20, pH 8.0), and wells were incubated with blocking (postcoat) solution containing 50 mM Tris buffered saline, 0.14 M NaCl, 1% BSA, at pH 8.0 during 30 min, then rinsed 3 times with wash solution. The calibrator (chicken reference serum) and sample/conjugate diluent (50 mM Tris buffered saline, 0.14 M NaCl, 1% BSA, 0.05% Tween 20, pH 8.0) were used as standards, whereas serum samples, thawed at 4°C overnight, were diluted at 1:1,000 in sample/conjugate diluent. Then, they were incubated in wells for 60 min, and washed 5 times with wash solution. Horseradish peroxidase detection antibody (goat anti-chicken IgG-Fc or IgA or IgM) diluted in sample/conjugate diluent was added to wells, incubated for 60 min, and rinsed 5 times with wash solution. Enzyme substrate (3,3c,5,5c-tetramethylbenzidine peroxidase substrate and peroxidase solution B) was added and incubated
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Blood samples were taken from a jugular vein of chickens 5 and 9 d (experiment 1) or 7 and 14 d (experiment 2) after challenge. In each experiment, 10 birds were sampled before being euthanized for Salmonella counting, until 180 mL of blood were collected from each treatment using EDTA (EMD Chemicals Inc., Gibbstown, NJ) as an anticoagulant. Blood samples were separated evenly into four 50-mL tubes and kept on ice until use for oxidative burst and lymphocyte proliferation assays.
(0.2 mg/mL) was added to monocytes and heterophils, mixed thoroughly, and aliquotted into a clear 96-well flat-bottomed plate. Oxidative burst was then measured at an excitation/emission wavelength of 485/535 nm.
IMMUNE FUNCTION AFTER SALMONELLA CHALLENGE
had intermediate MOB values (Figure 1). In experiment 2, the MOB was not different among treatments 7 d postinfection; however, 14 d postinfection birds fed the AVE diet had the highest MOB (P < 0.05) compared with birds in the other treatments (Figure 1). The heterophil oxidative burst (HOB) in experiment 1 was lowest in birds fed the AVE diet, and highest in birds fed the CTL− diet, 5 d postinfection (P < 0.05; Figure 2). At 9 d postinfection, birds fed the control diets (CTL− or CTL+) had significantly higher HOB than birds fed AVE or AVM diet (Figure 2). In experiment 2, birds fed the CTL− or the AVE diet had higher (P < 0.05) HOB than birds fed the CTL+ or the AVM diet 7 d postinfection; however, 14 d postinfection birds fed the AVE diet had the highest HOB (P < 0.05). The lymphocyte proliferation (LPA) was significantly lower in birds fed the AVE diet compared with the other treatments 5 d postinfection in experiment 1; however, 9 d postinfection birds fed the AVE or AVM diet had significantly higher LPA than birds fed the CTL− or the CTL+ diets (Figure 3). In experiment 2, birds fed the AVE diet had the highest (P < 0.05) LPA at both times (7 and 14 d postinfection); in the same experiment, birds fed the CTL+ diet had significantly higher LPA than birds fed the CTL− diet 7 d postinfection, but by d 14 postinfection there were no differences in LPA among birds fed the CTL−, CTL+, or AVM diet.
for 15 min (IgA or IgM) or 30 min (IgG). Finally, 2 M H2SO4 was used to stop the 3,3c,5,5c-tetramethylbenzidine reaction. The absorbance, measured at 450 nm with a microtiter plate reader (Wallac Victor-2 1420 Multilabel Counter), was used to calculate the immunoglobulins (IgG, IgA, or IgM) concentration with a 4 parameter logistic curve-fit.
Statistical Analysis Data were analyzed as a completely randomized design using a one-way ANOVA using the SigmaStat software (Jandel Scientific, 1994). Means were separated using the Student-Newman-Keuls method, and significance was declared at P < 0.05. All data are presented as the least squares means ± SE.
RESULTS Immune Function Parameters In experiment 1, the monocyte oxidative burst (MOB) was higher in birds fed the CTL− or the AVE diet than in birds fed the CTL+ or AVM diet 5 d (P < 0.05) postinfection (Figure 1); however, 9 d postinfection, the MOB was highest (P < 0.05) in birds fed the AVM diet, and lowest (P < 0.05) in birds fed the CTL+ diet, whereas birds fed the CTL− or AVE diet
Figure 2. In vitro heterophil oxidative burst (n = 4 replicates per column) isolated from the pooled peripheral blood of broiler chickens (n = 10 per treatment) at 5 and 9 (experiment 1) or at 7 and 14 (experiment 2) d postinfection with 106 cfu of Salmonella Typhimurium (birds were orally challenged at d 7 in experiment 1 or at d 3 in experiment 2). Chickens were fed an antibiotic-free diet (CTL−), an antibiotic-free diet plus 40 mg of bacitracin/kg of feed (CTL+), a CTL− plus 0.8% Arg and 40 IU of VE/kg of feed (AVE), or an AVE diet plus 0.2% mannanoligosaccharides (AVM). Heterophils were stimulated with phorbol-12-myristate-13-acetate or cultured in an equal volume of Roswell Park Memorial Institute 1640 media as a negative control. Columns with different letters (a–c) within sampling time are statistically different (P < 0.05).
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Figure 1. In vitro oxidative burst of monocytes (n = 4 replicates per column) isolated from the pooled peripheral blood of broiler chickens (n = 10 per treatment) at 5 and 9 (experiment 1) or at 7 and 14 (experiment 2) d postinfection with 106 cfu of Salmonella Typhimurium (birds were orally challenged at d 7 in experiment 1 or at d 3 in experiment 2). Chickens were fed an antibiotic-free diet (CTL−), an antibiotic-free diet plus 40 mg of bacitracin/kg of feed (CTL+), a CTL− plus 0.8% Arg and 40 IU of VE/kg of feed (AVE), or an AVE diet plus 0.2% mannanoligosaccharides (AVM). Monocytes were stimulated with phorbol-12-myristate-13-acetate or cultured in an equal volume of Roswell Park Memorial Institute 1640 media as a negative control. Columns with different letters (a–c) within sampling time are statistically different (P < 0.05).
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LIU ET AL. Table 1. Effects of arginine, vitamin E, and mannanoligosaccharides on serum antibody titers 16 d after an experimental challenge with 106 cfu of Salmonella Typhimurium in broiler chickens1 Treatment2,3 Isotype IgM IgG IgA
CTL− 17.1ab
121.3 ± 1,249.3 ± 229.5 127.1 ± 38.1b
CTL+ 9.2b
AVE
97.6 ± 936.0 ± 143.3 97.3 ± 10.9b
15.6ab
116.2 ± 943.6 ± 81.3 138.3 ± 37.3b
AVM 152.3 ± 11.0a 674.3 ± 65.0 310.3 ± 68.7a
a,bValues
with different superscripts within the same row are different (P < 0.05). of 8 observations ± SEM. 2CTL−, antibiotic-free diet; CTL+, diet with 40 mg of bacitracin/kg of feed; AVE, antibiotic-free diet plus 0.8% l-Arg, and 40 IU of vitamin E/kg of feed; AVM, AVE diet supplemented with 0.2% mannanoligosaccharides (Biomos, Alltech Inc., Nicholasville, KY). 3Birds were orally challenged at d 3 (experiment 2). 1Mean
Salmonella Concentrations in the Ceca
The amount of circulating IgM was significantly higher in birds fed the AVM diet than in birds fed the CTL+ diet, but similar to the IgM concentrations of birds fed the CTL− or the AVE diet (Table 1). The amount of IgG was not affected by dietary treatment. Conversely, birds fed the AVM diet had the highest (P < 0.05) concentrations of circulating IgA, with no differences among the other 3 treatments.
In experiment 1, the amount of recovered Salmonella (cfu) was significantly lower in birds fed the AVM diet than in birds fed the CTL− diet 3 d postinfection, but was not different from the Salmonella concentrations recovered in birds fed the CTL+ or AVE diet. However, 10 and 17 d postinfection, there was no effect of dietary treatment on Salmonella recovery in the ceca. In experiment 2, there were no effects of treatment on Salmonella at any measuring time (7, 14, and 21 d postinfection).
DISCUSSION
Figure 3. In vitro proliferation of lymphocytes (n = 4 replicates per column) isolated from the pooled peripheral blood of broiler chickens (n = 10 per treatment) at 5 and 9 (experiment 1) or at 7 and 14 (experiment 2) d postinfection with 106 cfu of Salmonella Typhimurium (birds were orally challenged at d 7 in experiment 1 or at d 3 in experiment 2). Chickens were fed an antibiotic-free diet (CTL−), an antibiotic-free diet plus 40 mg of bacitracin/kg of feed (CTL+), a CTL− plus 0.8% Arg and 40 IU of VE/kg of feed (AVE), or an AVE diet plus 0.2% mannanoligosaccharides (AVM). Lymphocytes were stimulated with concanavalin A or cultured in an equal volume of Roswell Park Memorial Institute 1640 media as a negative control. Columns with different letters (a–c) within sampling time are statistically different (P < 0.05).
In previous studies we have documented that the supplementation of Arg and VE has complementary or synergistic effects on the immune response of broilers after vaccination with immune bursal disease virus or after challenges with Eimeria spp. (Abdukalykova et al., 2008; Ruiz-Feria and Abdukalykova, 2009; PerezCarbajal et al., 2010; Chan-Díaz et al., 2012). We have also documented that birds fed MOS had an improved intestinal integrity, an increase in beneficial bacteria in the ceca, and reduced shedding of E. coli after challenge (Baurhoo et al., 2007a,b). Thus, we hypothesized that the concurrent supplementation of Arg, VE, and MOS, will improve the innate and acquired immune function and will reduce Salmonella colonization in the ceca of broiler chickens after a challenge with Salmonella Typhimurium. In experiment 1, when the birds were challenged at 7 d of age, the effects of Arg and VE (AVE) or the combination of AVE and MOS (AVM) on the activity of MOB were not very consistent, but feeding AVE improved the MOB 5 d after challenge, and feeding the AVM diet improved the MOB 9 d after challenge compared with the CTL+ treatment. Also, birds fed the CTL− diet had consistently higher (P < 0.05) MOB than birds fed the CTL+ diet. Thus, the higher MOB in birds fed the CTL− and the AVE diet 5 d after challenge could be due to a higher immune stimulation in the absence of antimicrobial agents or prebiotics; in other words, the presence of antibiotics or prebiotics
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Humoral Immunity
IMMUNE FUNCTION AFTER SALMONELLA CHALLENGE
significantly higher MOB, HOB, and LPR, than birds in the other treatments. It has been reported that neonatal poultry exhibit a transient susceptibility to infectious disease during the first week of life and this is attributed to a deficient host defense mechanism (Kogut et al., 2012), in particular a functional inefficiency of heterophils and monocytes (He et al., 2008); thus, it is of great significance that the supplementation of Arg and VE increased the oxidative burst of both monocytes and heterophils when the challenge was done at an early age. Also, the LPR was consistently improved in birds fed the AVE diet when the challenge was done at an early age. The effects of VE on the immune response are mediated by direct effects on lymphocyte cytokine expression and modulation of immune cell proliferation (Li-Weber et al., 2002; Leshchinsky and Klasing, 2003; Babu and Raybourne, 2008), whereas the effects of Arg on the immune response to Salmonella have been attributed to a higher production of NO for host defense (MacFarlane et al., 1999). Also, macrophages from chickens selected for high antibody response produce more NO (Guimarães et al., 2011). However, the effects of Arg alone can be deleterious because an overproduction of NO may lead to increased oxidative stress and immune suppression (Huang et al., 1996; MacFarlane et al., 1999; Xie et al., 2008). Thus, the complementary effects of Arg and VE may be explained by the antioxidant effects of VE on the overproduction of NO elicited by the immune challenge. In these experiments, we did not test Arg and VE individually because we have documented their complementary effects in several immune challenge models (Abdukalykova and Ruiz-Feria, 2006; Abdukalykova et al., 2008; Ruiz-Feria and Abdukalykova, 2009; Perez-Carbajal et al., 2010; Chan-Díaz et al., 2012). Also in these experiments it was evident that further supplementing an AVE diet with MOS did not further improve immune response in experiment 1 (except MOB 9 d after challenge), and even reversed the positive effects of Arg and VE on immune response in experiment 2. The reason for this could be attributed to the mode of action of MOS, which binds gram-negative bacteria expressing the type 1 fimbrae, including Salmonella, reducing its adherence to intestinal epithelial cells (Spring et al., 2000) and immune activation. Recently, Baurhoo et al. (2012) reported that birds fed MOS and challenged with Salmonella LPS showed gene expression downregulation of Toll-like receptor-2, gallinacin-1 α, and CXC1, an IL-8 receptor that binds the IL-8 chemoattractant expressed by macrophages, whereas Wigley et al. (2006) reported that Salmonellaresistant lines of chickens presented a higher expression of IL-6, IL-8, and IL-18, and Kogut (2002) reported the importance of IL-8 as a major chemotactic factor to increase heterophil recruitment to the site of Salmonella infection; thus, it appears that downregulation of IL-8 by MOS may explain the effects seen in these experiments. Our results agree with reports indicating that prebiotics do not have an effect on monocyte function
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may reduce the interaction of microbes with the gastric immune system, resulting in a downregulation of the MOB capacity. However, birds fed the AVM diet had the highest MOB 9 d after challenge, which could be attributed to the immunomodulatory effects of MOS (Ferket, 2004; Janardhana et al., 2009). Because monocytes are important in phagocytosis and activation of the acquired immune response through the production of cytokines, our results show that Arg and VE supplementation (AVE) are better than AVM diets, and that the presence of a prebiotic may delay the activation of MOB capacity. Birds fed the AVE or the AVM diet had consistently lower HOB than birds fed the control diets 9 d after challenge in experiment 1. Genovese et al. (2013) reported that chickens with less functional heterophils are more susceptible to infection, including Salmonella Enteritidis, than those with highly functional heterophils. Thus, under these conditions the supplementation of AVE or AVM does not seem to have beneficial effects in term of heterophil function. Conversely, birds fed the AVE or the AVM diet had a significantly higher lymphocyte proliferation (LPR) reaction than birds fed the CTL diets 9 d after challenge. Thus, when birds are challenged with Salmonella Typhimurium at 7 d of age and are fed diets supplemented with Arg and VE (AVE), or a combination of Arg, VE, and MOS (AVM) they show a reduced (P < 0.05) HOB, and higher LPR (P < 0.05) than birds fed the control diets, although the differences were modest and may not have a major biological significance (Figures 2 and 3). Also, it was evident that further supplementing an AVE diet with MOS did not have additive effects on immune function under the conditions of this experiment. The limited effects of the dietary treatments on immune function could be explained by the fact that birds were challenged at 7 d of age, when the normal microflora was already established, and under these circumstances the Salmonella challenge was not enough to elicit a strong immune response. Redmond et al. (2011) found no effect of dietary β-glucans or ascorbic acid on heterophil function after a Salmonella Typhimurium challenge, and they attributed this result to the lack of other stressors or disease, suggesting that under most challenging environments the effects of prebiotics and antioxidants would be more evident. Thus, to avoid the effects of an established microflora, in experiment 2 the birds were challenged at 3 d of age, and the effects of the concurrent supplementation of Arg and VE on immune response were more consistent, and in agreement with our previous findings (Abdukalykova et al., 2008; Ruiz-Feria and Abdukalykova, 2009; Perez-Carbajal et al., 2010; Chan-Díaz et al., 2012). Seven days after challenge, MOB was not different among treatments, whereas at this time birds fed the AVE diet had significantly higher HOB than birds fed the CTL+ or the AVM diet, and higher (P < 0.05) LPR than birds fed the other diets. Furthermore, 14 d after challenge birds fed the AVE diet had consistently
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LIU ET AL. Table 2. Effects of Arg, vitamin E, and mannanoligosaccharides on cecal Salmonella Typhimurium (cfu, log10), and on number of Salmonella-positive samples (out of 10 samples, in parentheses) after an experimental challenge with 106 cfu of Salmonella Typhimurium in broiler chickens1 Treatment2 Days after challenge3 Experiment 1 3 10 17 Experiment 2 7 14 21
CTL−
CTL+
AVE
AVM
3.0 ± 0.5a (8) 0.8 ± 0.4 (1) 0.5 ± 0.4 (1)
2.1 ± 0.5ab (5) 1.5 ± 0.4 (1) 0.7 ± 0.3 (0)
1.6 ± 0.5ab (3) 0.2 ± 0.2 (0) 1.0 ± 0.6 (2)
0.8 ± 0.4b (1) 0.7 ± 0.4 (0) 0.6 ± 0.4 (1)
4.4 ± 0.6 (10) 1.5 ± 0.5 (7) 0.3 ± 0.3 (1)
4.0 ± 1.1 (10) 1.9 ± 0.6 (2) 0 ± 0 (0)
4.4 ± 1.1 (10) 1.9 ± 0.6 (7) 0.8 ± 0.4 (0)
4.4 ± 1.14 (9) 0.9 ± 0.6 (7) 0.4 ± 0.4 (0)
a,bValues
(Bunout et al., 2002) or leukocyte proliferation (Janardhana et al., 2009). In experiment 2, the dietary treatments did not affect antibody titers of IgG, but the antibody titers of IgM were significantly higher in birds fed the AVM diet compared with birds fed the CTL+ diet, and IgA titers were highest (P < 0.05) in birds fed the AVM diet. These results are in contrast with the effects of MOS on the innate immune response and LPR discussed above. It is well documented that infection with Salmonella leads to increased levels of IgG, IgM, and IgA antibodies (Withanage et al., 2005). However, the role of humoral immunity on Salmonella clearance is not well understood. Beal et al. (2006) demonstrated that although Salmonella infection in chickens elicited high concentrations of antibodies, B cells did not play an essential role in clearance of the primary or secondary infection. More recently, Nanton et al. (2012) suggested that B cells play an indirect role in protection against Salmonella through the development of T cell immunity after secondary infection. Thus, the higher concentrations of IgA in birds fed the AVM diet may be beneficial in a secondary challenge, or may reduce horizontal transmission of Salmonella during growing. However, in these experiments the dietary treatments did not affect Salmonella recovery in the ceca after challenge. Only in experiment 1, when birds were challenged at d 7, birds fed the AVM diet had lower (P < 0.05) concentrations of Salmonella than birds fed the CTL− diet, but not different from the ceca Salmonella levels of birds fed the AVE or the CTL+ diet 3 d after challenge (Table 2). At 10 and 17 d after challenge, the recovery of Salmonella was very low and not different among treatments. In experiment 2, when the challenge was performed at an earlier age (d 3) Salmonella recovery was high in all treatments 7 d after challenge (4–4.4 logs), was reduced by more than half by 14 d after challenge (0.9–1.9 logs), and was very low by 21 d after challenge (0–0.8 logs), but there were
no significant differences among treatments. Therefore, the higher immune response elicited by Arg and VE in experiment 2 was not correlated with a lower Salmonella recovery in the ceca compared with birds fed the control diets; this could be attributed to a low dose of Salmonella in the challenge, or to the lack of other stresses in otherwise healthy birds. Also, it has been suggested that the expression of cytokines and chemokines during primary Salmonella infection will result in the modulation of CD4 T cell response to improve resistance against secondary infection (Withanage et al., 2005; Nanton et al., 2012); accordingly, the higher HOB and MOB found in this experiment, along with our previous findings on the effects of Arg and VE on T cell differentiation (Abdukalykova et al., 2008) may be important in the development of acquired immunity after a secondary challenge. Further research is warranted on this subject. In the same way, the higher concentrations of IgA in birds fed the AVM diet did not correlate with lower concentrations of Salmonella in birds fed the AVM diet. The effects of prebiotics on Salmonella prevention have been variable. Agunos et al. (2007) reported reductions in fecal Salmonella 16 and 19 d after a challenge with 2 × 107 cfu of Salmonella when birds were fed β1–4-mannobiose, whereas Revolledo et al. (2009) did not find any effect on ceca Salmonella in birds fed β-glucans. In the same way, Burkey et al. (2004) and Calveyra et al. (2011) reported no effects of MOS on Salmonella shedding in pigs experimentally infected with Salmonella Typhimurium. In summary, our results show that supplementing diets with Arg and VE improve the innate and acquired immune response in chicks challenged with Salmonella, but only when the challenge occurred early in their life (3 d old). We also found that further supplementing the diet with MOS did not improve the effects of Arg and VE, except in the production of IgA. Although the dietary treatments did not reduce Salmonella con-
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with different superscripts within the same row are different (P < 0.05). of 10 observations ± SEM (number of birds tested positive, out of 10, after Rappaport-Vassiliadis broth media enrichment for 24 h). 2CTL−, antibiotic-free diet; CTL+, diet with 40 mg of bacitracin/kg of feed; AVE, antibiotic-free diet plus 0.8% l-Arg, and 40 IU of vitamin E/kg of feed; AVM, AVE diet supplemented with 0.2% mannanoligosaccharides (Biomos, Alltech Inc., Nicholasville, KY). 3Birds were orally challenged at d 7 (experiment 1) or d 3 (experiment 2). 1Mean
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centrations in the ceca, the better immune response of birds fed Arg and VE may benefit birds grown under commercial conditions, where they face more stressful conditions and are exposed to several microbial challenges. Further studies are needed to evaluate the effects of Arg and VE on Salmonella clearance after a secondary Salmonella challenge.
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Abdukalykova, S., and C. A. Ruiz-Feria. 2006. Arginine and vitamin E improve the cellular and humoral immune response of broiler chickens. Int. J. Poult. Sci. 5:121–127. Abdukalykova, S. T., X. Zhao, and C. A. Ruiz-Feria. 2008. Arginine and vitamin E modulate the subpopulations of T lymphocytes in broiler chickens. Poult. Sci. 87:50–55. Agunos, A., M. Ibuki, F. Yokomizo, and Y. Mine. 2007. Effects of dietary β1–4 mannobiose in the prevention of Salmonella enteritidis infection in broilers. Br. Poult. Sci. 48:331–341. Babu, U. S., and R. B. Raybourne. 2008. Impact of dietary components on chicken immune system and Salmonella infection. Expert Rev. Anti Infect. Ther. 6:121–135. Bach Knudsen, K. E. 2001. Development of antibiotic resistance and options to replace antimicrobials in animal diets. Proc. Nutr. Soc. 60:291–299. Baurhoo, B., P. Ferket, C. H. Ashwell, J. de Oliveira, and X. Zhao. 2012. Cell walls of Saccharomyces cerevisiae differentially modulate innate immunity and glucose metabolism during late systemic inflammation. PLoS ONE 7:e30323. http://dx.doi. org/101371/journal.pone.0030323. Baurhoo, B., A. Letellier, X. Zhao, and C. A. Ruiz-Feria. 2007b. Cecal populations of Lactobacilli and Bifidobacteria and E. coli populations after in vivo challenge in birds fed diets with purified lignin or mannanoligasaccharides. Poult. Sci. 86:2509–2516. Baurhoo, B., L. Phillip, and C. A. Ruiz-Feria. 2007a. Effects of purified lignin and mannanoligosaccharides on intestinal integrity and microbial populations in the ceca and litter of broiler chickens. Poult. Sci. 86:1070–1078. Beal, R. K., P. Wigley, C. Powers, P. A. Barrow, and A. L. Smith. 2006. Cross-reactive cellular and humoral immune responses to Salmonella enterica serovars Typhimurium and Enteritidis are associated with protection to heterologous re-challenge. Vet. Immunol. Immunopathol. 114:84–93. Bunout, D., S. Hirsh, M. P. De la Maza, C. Munoz, F. Haschke, P. Steenhout, P. Klassen, G. Barrera, V. Gattas, and M. Petermann. 2002. Effects of prebiotics on the immune response to vaccination in the elderly. J. Parent. Enter. Nutr. 26:372–376. Burkey, T. E., S. S. Dritz, J. C. Nietfeld, B. J. Johnson, and J. E. Minton. 2004. Effect of dietary mannanoligosaccharide and sodium chlorate on the growth performance, acute-phase response, and bacterial shedding of weaned pigs challenged with Salmonella enterica serotype Typhimurium. J. Anim. Sci. 82:397–404. Byrd, J. A., D. E. Corrier, J. R. Deloach, D. J. Nisbet, and L. H. Stanker. 1998. Horizontal transmission of Salmonella Typhimurium in broiler chicks. J. Appl. Poult. Res. 7:75–80. Calveyra, J. C., M. G. Nogeira, J. D. Kich, L. L. Biesus, R. Vizzotto, L. Berno, A. Coldebella, L. Lopes, N. Mores, G. J. Lima, and M. Cardoso. 2011. Effect of organic acids and mannanoligosaccharide on excretion of Salmonella typhimurium in experimentally infected growing pigs. Res. Vet. Sci. 93:46–47. Chan-Díaz, D. J., A. Pro-Martínez, C. A. Ruíz-Feria, J. M. CucaGarcía, C. Narciso-Gaytan, M. E. Ramírez-Guzmán, J. GallegosSánchez, E. Sosa-Montes, and J. C. Rodriguez-Castillo. 2012. Effects of arginine and vitamin E supplemented diets on the immunological response of broiler chickens. Tropical and Subtropical Agroecosystems 15:367–374. Denev, S. A., I. Dinev, I. Nikiforov, and V. Koinarski. 2005. Effects of mannan oligosaccharides on composition of cecal microflora and performance of broiler chickens. Pages 351–353 in 15th Eur. Symp. Poult. Nut., Balatonfüred, Hungary. World’s Poult. Sci. Assoc., Budapest, Hungary.
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LIU ET AL. Redmond, S. B., P. Chuammitri, C. B. Andreasen, D. Palic, and S. L. Lamont. 2011. Proportion of circulating chicken heterophils and CXCLi1 expression in response to Salmonella enteriditis are affected by genetic line and immune modulating diet. Vet. Immunol. Immunopathol. 140:323–328. Revolledo, L., C. S. A. Ferreira, and A. J. P. Ferreira. 2009. Prevention of Salmonella Typhimurium colonization and organ invasion by combination treatment in broiler chicks. Poult. Sci. 88:734–743. Ruiz-Feria, C. A., and S. Abdukalykova. 2009. Arginine and vitamin E improve antibody titres to infectious bursal disease virus (IBDV) and sheep red blood cells in broiler chickens. Br. Poult. Sci. 50:291–297. Spring, P., C. Wenk, K. A. Dawson, and K. E. Newman. 2000. The effects of dietary mannanoligosaccharides on cecal parameters and the concentrations of enteric bacteria in the ceca of Salmonella-challenge broiler chicks. Poult. Sci. 79:205–211. Stringfellow, K., D. Caldwell, J. Lee, M. Mohnl, R. Beltran, G. Schatzmayr, S. Fitz-Coy, C. Broussard, and M. Farnell. 2011. Evaluation of probiotic administration on the immune response of coccidiosis-vaccinated broilers. Poult. Sci. 90:1625–1658. Wigley, P., S. Hulme, L. Rothwell, N. Bumstead, P. Keiser, and P. Barrow. 2006. Macrophages isolated from chickens genetically resistant or susceptible to systemic salmonellosis show magnitudinal and temporal differential expression of cytokines and chemokines following Salmonella enterica challenge. Infect. Immun. 74:1425–1430. Withanage, G. S. K., P. Wigley, P. Kaiser, P. Mastrochi, H. Brooks, C. Powers, R. Beal, P. Barrow, and I. McConnell. 2005. Cytokine and chemokine responses associated with clearance or a primary Salmonella enterica serovar Typhimurium infection in the chicken and in protective immunity to rechallenge. Infect. Immun. 73:5173–5182. Xie, H., G. R. Huff, W. E. Huff, J. M. Balog, and N. C. Rath. 2002. Effects of ovotransferrin on chicken macrophages and heterophilgranulocytes. Dev. Comp. Immunol. 26:805–815. Xie, Q. W., Y. Kashiwabar, and C. Nathan. 1994. Role of transcription factor NF-kB/Rel in induction of nitric oxide synthase. J. Biol. Chem. 269:4707–4708. Xie, X. Q., Y. Shinozawa, J. Sasaki, K. Takuma, S. Akaishi, S. Yamanouchi, T. Endo, R. Nomura, M. Kobayashi, D. Kudo, and N. Hojo. 2008. The effects of arginine and selective inducible nitric oxide synthase inhibitor on pathophysiology of sepsis in a CLP model. J. Surg. Res. 146:298–303.
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Kubena, L. F., J. A. Byrd, C. R. Young, and D. E. Corroer. 2001. Effect of tannic acid on cecal volatile fatty acids and susceptibility to Salmonella typhimurium colonization in broiler chicks. Poult. Sci. 80:1293–1298. Kwak, H., R. E. Austic, and R. R. Dietert. 1999. Influence of dietary arginine concentration on lymphoid organ growth in chickens. Poult. Sci. 78:1536–1541. Leshchinsky, T. V., and K. C. Klasing. 2003. Profile of chicken cytokines induced by lipopolysaccharides is modulated by dietary α-tocopheryl acetate. Poult. Sci. 82:1266–1273. Li-Weber, M., M. Giaisi, M. K. Treiber, and P. H. Krammer. 2002. Vitamin E inhibits IL-4 gene expression in peripheral blood T cells. Eur. J. Immunol. 32:2401–2408. Macfarlane, S., G. T. Macfarlane, and J. H. Cummings. 2006. Review article: Prebiotics in the gastrointestinal tract. Aliment. Pharmacol. Ther. 24:701–714. MacFarlane, A. S., M. G. Schwacha, and T. K. Eisenstein. 1999. In vivo blockage of nitric oxide with aminoguanidine inhibits immunosuppression induced by an attenuated strain of Salmonella typhimurium, potentiates Salmonella infection, and inhibits macrophage and polymorphonuclear leukocyte influx into the spleen. Infect. Immun. 67:891–898. Moncada, S., R. M. Palmer, and E. A. Higgs. 1991. Nitric oxide: Physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43:109–142. Moriguchi, S., H. Miwa, M. Okamura, K. Maekawa, Y. Kishino, and K. Maeda. 1993. Vitamin E is an important factor in T cell differentiation in thymus of F344 rats. J. Nutr. Sci. Vitaminol. (Tokyo) 39:451–463. Nanton, M. R., S. S. Way, M. J. Schlomchik, and S. J. McSorley. 2012. Cutting edge: B cells are essential for protective immunity against Salmonella independent of antibody secretion. J. Immunol. 189:5503–5507. NRC. 1994. Nutrient Requirements of Poultry. Natl. Res. Counc., Natl. Acad. Sci., Washington, DC. Pauly, J. L., J. E. Sokal, and T. Han. 1973. Whole blood culture techniques for functional studies of lymphocyte reactivity to mitogens, antigens, and homologous lymphocytes. J. Lab. Clin. Med. 82:500–512. Perez-Carbajal, C., D. Caldwell, M. Farnell, K. Stringfellow, G. Casco, S. Pohl, A. Pro-Martinez, and C. A. Ruiz-Feria. 2010. Immune response of broiler chickens fed different levels of arginine and vitamin E to a coccidiosis vaccine and Eimeria challenge. Poult. Sci. 89:1870–1877.
Key words: immune response, Salmonella, arginine, vitamin E, mannanoligosaccharide 2014 Poultry Science 93:882–890 http://dx.doi.org/10.3382/ps.2013-03723 of antimicrobial in food-producing animals for growth promotion and prophylaxis may lead to the development of microbial strains resistant to antibiotic therapy (Bach Knudsen, 2001; Harrison et al., 2013). Therefore, it is essential to find alternatives to antibiotics in foodproducing animals. One way to improve immune response and avian health is through the use of nutritional supplements (Klasing, 1998; Kidd, 2004). Arginine, an essential amino acid for avian species, and vitamin E (VE), an important antioxidant, have been shown to influence both the humoral- and cell-mediated immune responses of birds. In chickens, an Arg-deficient diet has been associated with poor development of the thymus and spleen, which are key organs of the immune system (Kwak et al., 1999). Arginine supplementation modulates or boosts humoral and cellular immune response, and improves nitric oxide (NO) production and the acute phase inflammatory response following lipopoly-
INTRODUCTION Consumption of contaminated poultry products is a major cause of human salmonellosis, with most of the cases being attributed to Salmonella enterica serovar Enteritidis or Typhimurium (Beal et al., 2006). On the other hand, intensive poultry production practices may increase susceptibility to disease due to high stocking densities, recycled litter, and environmental conditions that are sometimes less than optimal. To reduce the incidence of disease and improve growth, antibiotics are used in commercial animal production; drug makers sold 13.5 million kg of antibiotics for use in food-producing animals in 2011 (FDA, 2013). However, there are increasing concerns that the subtherapeutic use ©2014 Poultry Science Association Inc. Received October 30, 2013. Accepted December 18, 2013. 1 Corresponding author: [email protected]
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ment, birds fed the AVE diet had higher MOB than birds fed CTL+ or the AVM diet at 7 d PI, whereas 9 d PI birds fed the AVM diet had the highest MOB. In experiment 2, birds fed the AVE diet had higher MOB, HOB, and LPR than birds in the other treatments 7 and 14 d PI, except at 7 d PI, when MOB was not different among treatments. Birds fed the AVM diet had the highest IgA antibody titer, and a higher IgM antibody titer than the CTL+ birds. In experiment 1, Salmonella Typhimurium content in the ceca was lower in birds fed the AVM diet compared with birds fed the CTL− diet 3 d PI, but later on (10 and 17 d PI), and in experiment 2 (7, 14, and 21 d PI), Salmonella Typhimurium concentrations were not different among treatments. Thus, Arg and VE improved immune response after a Salmonella Typhimurium challenge in young chicks, and although they did not reduce Salmonella Typhimurium concentrations in the ceca, they may improve bacterial resistance against other pathogens in commercial growing conditions.
ABSTRACT Two experiments were conducted to evaluate the effects of Arg, vitamin E (VE), and mannanoligosaccharide (MOS) on the immune response and clearance of Salmonella in broiler chickens. In each experiment, 1-d-old chicks (n = 160) were randomly distributed into 4 groups: antibiotic-free diet (negative control, CTL−), antibiotic-supplemented diet (positive control, CTL+), antibiotic free-diet plus Arg and VE (AVE), or antibiotic-free diet plus Arg, VE, and MOS (AVM). Birds were orally challenged with 106 cfu of a novobiocyn and nalidixic acid-resistant Salmonella enterica serovar Typhimurium strain at d 7 (experiment 1) or at d 3 (experiment 2). Heterophil- (HOB) and monocyte- (MOB) oxidative burst and lymphocyte proliferation (LPR), antibody titers, and Salmonella content in the ceca were measured at several intervals postinfection (PI). In experiment 1, both AVM and AVE decreased HOB compared with the controls 5 and 9 d PI, but increased LPR 9 d PI. In the same experi-
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antibody titers, and Salmonella concentrations in the ceca, after an experimental challenge in young broiler chickens.
MATERIALS AND METHODS Birds and Treatments Two experiments were conducted. One-day-old broiler chicks (Cobb 500) were obtained from a local hatchery and housed in a biosafety level 2 facility at the USDA-Southern Plains Agriculture Research Center in College Station, Texas. Chicks were grown on fresh pine shavings and brooded following standard temperature regimens, which gradually decreased from 32 to 24°C, and under a 16L:8D cycle throughout the studies. All birds were fed a corn-soybean meal-based diet formulated to meet or exceed all of the NRC (1994) requirements. The basal diet contained 23% of CP, 3,200 kcal of ME/kg, 1.54% Arg, and 40.3 IU of VE/kg. A completely randomized design with 4 dietary treatments (40 birds/treatment) was used, as follows: basal diet free of antibiotics (negative control diet, CTL−), basal diet plus 40 mg of bacitracin/kg of feed (positive control, CTL+), basal diet free of antibiotics supplemented with Arg (0.8%, wt/wt) and 40 IU of VE/kg of feed (AVE; for a total content of 2.34% Arg and 80 IU of VE/kg of feed), or AVE diet plus 0.2% (wt/wt) MOS (AVM; BioMos, Alltech Co., Lexington, KY). All the experimental procedures were approved by the Institutional Animal Care Committee.
Bacterial Challenge and Cecal Counts A primary poultry isolate of Salmonella enterica serovar Enteritidis or Typhimurium was obtained from the National Veterinary Services Laboratory (Ames, IA) and selected for resistance to novobiocin and nalidixic acid, and maintained in media containing 25 μg of novobiocin and 20 μg of nalidixic acid/mL. Portions (1 to 2 mL) of cultures grown overnight at 37°C in tryptic soy broth (Difco Laboratories, Detroit, MI) were used as inocula for challenging broilers. At d 7 (experiment 1) or d 3 (experiment 2), all the chicks were orally challenged with 106 cfu of Salmonella Typhimurium (1 mL/chick). Ten birds per treatment were euthanized by cervical dislocation at 3, 10, and 14 d after challenge (experiment 1) or 7, 14, and 21 d after challenge (experiment 2). Cecal contents (0.25 g) were aseptically collected and deposited in 2.25 mL of PBS (pH 6.5). Samples were thoroughly mixed, serially diluted in PBS, and then spread plated onto XLT4 Agar (BD Diagnostics, Sparks, MD) containing 20 μg of nalixidic acid/mL and 25 μg of novobiocin/mL, at a final dilution of 1:100, 1:1,000, and 1:10,000. Plates were incubated for at least 24 h at 37°C, and the number of Salmonella cfu per gram was determined. Salmonella colony numbers were log-transformed before being analyzed. For qualitative enrichment, cecal contents were
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saccharide (LPS) injections, and NO is an important component of the macrophage defense against Salmonella (Moncada et al., 1991; Xie et al., 1994). Vitamin E has been shown to improve the immune response in birds by enhancing macrophage phagocytic function, decreasing prostaglandin E2 production, increasing IL-1 secretion by macrophages, and enhancing IL-2 production and T-cell proliferation (Moriguchi et al., 1993). Erf et al. (1998) reported that the T-helper/T-cytotoxic lymphocyte ratio and the percentages of T-helper lymphocytes in the spleen and thymus were higher in broilers fed diets with high levels of VE. Prebiotics are nondigestible feed ingredients that beneficially affect the host by selectively stimulating the growth or metabolic activity of a limited number of intestinal microorganisms (Gibson and Roberfroid, 1995; Macfarlane et al., 2006). Mannanoligosaccharides (MOS), a prebiotic derived from yeasts cells, have been shown to have antimicrobial properties, modulate intestinal microbial populations, and stimulate immune response in several animal species, including poultry (Spring et al., 2000; Fairchild et al., 2001; Fernandez et al., 2002; Denev et al., 2005). Spring et al. (2000) suggested that MOS reduced Salmonella colonization in the ceca of chickens by adsorbing bacteria and keeping them from adhering to the gut wall. We previously reported that MOS supplementation increased the populations of beneficial bacteria (bifidobacteria and lactobacilli), and reduced Escherichia coli shedding in the litter (Baurhoo et al., 2007a), showing the capacity of MOS to modulate intestinal microbial populations. More recently, it was reported that birds fed MOS had an increased innate immune response (upregulation of IL-3, and upregulation of signal transducer and activator of transcription 2 gene, which increases macrophage phagocytic activity) than birds fed virginiamycin after a challenge with Salmonella LPS (Baurhoo et al., 2012). Although the effects of Arg, VE, and prebiotics, on immune response have been investigated, their combined effects on the immune response and resistance to bacterial challenges have not been evaluated. We have documented that the combination of high levels of Arg and VE have complementary or synergistic effects on the immune response against several immune challenges, suggesting that the combination of Arg and VE may improve the health of broilers and potentially eliminate the need for antimicrobials (Abdukalykova and RuizFeria, 2006; Abdukalykova et al., 2008; Ruiz-Feria and Abdukalykova, 2009; Perez-Carbajal et al., 2010; ChanDíaz et al., 2012). We have also reported that MOS supplementation improved intestinal integrity and reduced E. coli colonization in the ceca of broiler chickens (Baurhoo et al., 2007b). Thus, we hypothesized that the concurrent supplementation of Arg, VE, and prebiotics (MOS) will improve the immune response and will reduce Salmonella levels in the ceca. The objectives of the present experiments were to evaluate the effects of Arg, VE, and MOS on monocyte and heterophil oxidative burst, lymphocyte proliferation in vitro, serum
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enriched in Rappaport-Vassiliadis Broth (Difco) for 24 h at 37°C, and were then struck on XLT4 agar plates. After 24 h of incubation at 42°C, the plates were read as positive or negative. Cecal contents that were negative at a 100-fold dilution on XLT4 agar plates but were positive on the enriched solution were assigned a 1.50 log10 Salmonella/g of cecal content (Byrd et al., 1998; Kubena et al., 2001; Kogut et al., 2010).
Blood Collection
Isolation of Heterophils and Monocytes The polymorphonuclear and mononuclear cell fractions were isolated from peripheral blood as described previously (Kogut et al., 1995; Stringfellow et al., 2011). Briefly, the blood samples were mixed with a solution of 1% methylcellulose (Sigma-Aldrich, St. Louis, MO) dissolved in Roswell Park Memorial Institute (RPMI) 1640 media (Mediatech Inc., Herndon, VA) at a 1:1.5 ratio and centrifuged at 37 × g for 15 min at 4°C. The supernatant was removed and mixed with an equal volume of calcium and magnesium free Hank’s balanced salt solution (Mediatech Inc.). This suspension was layered over a 1.077/1.119 Histopaque (Sigma-Aldrich) gradient and centrifuged at 235 × g for 60 min at 4°C. Following centrifugation, the interfaces containing monocytes or heterophils were collected, washed, and resuspended with RPMI. The cells were counted using a Neubauer hemacytometer and the concentration adjusted to 4 × 106 heterophils or monocytes/mL and kept on ice until used. Cell viability (>95%) was determined using a trypan blue solution (Sigma-Aldrich) at a 1:1 ratio.
Oxidative Burst Assay Oxidative burst activity of heterophils and monocytes was measured using a Wallac fluorescent plate reader (Perkin Elmer, Boston, MA), using DCF-DA (Molecular Probes Inc., Eugene, OR) as reactive oxygen species indicator, as described previously (Xie et al., 2002; Stringfellow et al., 2011). Briefly, heterophils and monocytes were preincubated with an agonist (2 μg of phorbol-12-myristate-13 acetate (Calbiochem, La Jolla, CA) or an equivalent volume of RPMI during 30 min at 42°C in a heated orbital shaker plate (Thermo-Forma, Marietta, OH). Then, 125 μL of DCF-DA
Lymphocyte Proliferation A modified cell proliferation assay was conducted to evaluate cell-mediated immunity (Pauly et al., 1973). After isolation, heterophils and monocytes were deposited into the plates. Then equal volumes of the lymphocyte suspension and concanavalin A were added in each of 6 wells of 96-well flat-bottomed tissue culture plates. Negative controls were maintained with RPMI only and cell suspension without concanavalin A. Plates were then incubated in a water-jacketed 5% CO2 incubator at 42°C for 24 h. Fifteen microliters of stock 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (5 mg/mL) was added to each well and plates were incubated for 4 h. The color change was measured at 450 nm by a colorimetric plate reader (Sunrise, Tecan, Austria).
ELISA The concentration of IgA, IgG, and IgM isotypes from 19-d-old chickens (16 d after challenge) were measured in serum samples (8/treatment, experiment 2). Blood samples (2 mL) were obtained by venipuncture and allowed to clot at room temperature for 4 h, samples were then centrifuged at 400 × g for 8 min at 4°C and the serum was collected and stored at −80°C until assayed. The serum samples were thawed at 4°C to calculate antibody concentrations using chicken IgA, IgG, and IgM ELISA kits (Bethyl Laboratories, Montgomery, TX) according to the manufacturer’s instructions. Briefly, flat-bottomed microtiter plates were coated for 60 min with capture antibody (goat anti-chicken IgG-Fc or IgM or IgA affinity purified) and coating buffer (0.05 M carbonate-bicarbonate, pH 9.6). Plates were washed 3 times with wash solution (50 mM Tris buffered saline, 0.14 M NaCl, 0.05% Tween 20, pH 8.0), and wells were incubated with blocking (postcoat) solution containing 50 mM Tris buffered saline, 0.14 M NaCl, 1% BSA, at pH 8.0 during 30 min, then rinsed 3 times with wash solution. The calibrator (chicken reference serum) and sample/conjugate diluent (50 mM Tris buffered saline, 0.14 M NaCl, 1% BSA, 0.05% Tween 20, pH 8.0) were used as standards, whereas serum samples, thawed at 4°C overnight, were diluted at 1:1,000 in sample/conjugate diluent. Then, they were incubated in wells for 60 min, and washed 5 times with wash solution. Horseradish peroxidase detection antibody (goat anti-chicken IgG-Fc or IgA or IgM) diluted in sample/conjugate diluent was added to wells, incubated for 60 min, and rinsed 5 times with wash solution. Enzyme substrate (3,3c,5,5c-tetramethylbenzidine peroxidase substrate and peroxidase solution B) was added and incubated
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Blood samples were taken from a jugular vein of chickens 5 and 9 d (experiment 1) or 7 and 14 d (experiment 2) after challenge. In each experiment, 10 birds were sampled before being euthanized for Salmonella counting, until 180 mL of blood were collected from each treatment using EDTA (EMD Chemicals Inc., Gibbstown, NJ) as an anticoagulant. Blood samples were separated evenly into four 50-mL tubes and kept on ice until use for oxidative burst and lymphocyte proliferation assays.
(0.2 mg/mL) was added to monocytes and heterophils, mixed thoroughly, and aliquotted into a clear 96-well flat-bottomed plate. Oxidative burst was then measured at an excitation/emission wavelength of 485/535 nm.
IMMUNE FUNCTION AFTER SALMONELLA CHALLENGE
had intermediate MOB values (Figure 1). In experiment 2, the MOB was not different among treatments 7 d postinfection; however, 14 d postinfection birds fed the AVE diet had the highest MOB (P < 0.05) compared with birds in the other treatments (Figure 1). The heterophil oxidative burst (HOB) in experiment 1 was lowest in birds fed the AVE diet, and highest in birds fed the CTL− diet, 5 d postinfection (P < 0.05; Figure 2). At 9 d postinfection, birds fed the control diets (CTL− or CTL+) had significantly higher HOB than birds fed AVE or AVM diet (Figure 2). In experiment 2, birds fed the CTL− or the AVE diet had higher (P < 0.05) HOB than birds fed the CTL+ or the AVM diet 7 d postinfection; however, 14 d postinfection birds fed the AVE diet had the highest HOB (P < 0.05). The lymphocyte proliferation (LPA) was significantly lower in birds fed the AVE diet compared with the other treatments 5 d postinfection in experiment 1; however, 9 d postinfection birds fed the AVE or AVM diet had significantly higher LPA than birds fed the CTL− or the CTL+ diets (Figure 3). In experiment 2, birds fed the AVE diet had the highest (P < 0.05) LPA at both times (7 and 14 d postinfection); in the same experiment, birds fed the CTL+ diet had significantly higher LPA than birds fed the CTL− diet 7 d postinfection, but by d 14 postinfection there were no differences in LPA among birds fed the CTL−, CTL+, or AVM diet.
for 15 min (IgA or IgM) or 30 min (IgG). Finally, 2 M H2SO4 was used to stop the 3,3c,5,5c-tetramethylbenzidine reaction. The absorbance, measured at 450 nm with a microtiter plate reader (Wallac Victor-2 1420 Multilabel Counter), was used to calculate the immunoglobulins (IgG, IgA, or IgM) concentration with a 4 parameter logistic curve-fit.
Statistical Analysis Data were analyzed as a completely randomized design using a one-way ANOVA using the SigmaStat software (Jandel Scientific, 1994). Means were separated using the Student-Newman-Keuls method, and significance was declared at P < 0.05. All data are presented as the least squares means ± SE.
RESULTS Immune Function Parameters In experiment 1, the monocyte oxidative burst (MOB) was higher in birds fed the CTL− or the AVE diet than in birds fed the CTL+ or AVM diet 5 d (P < 0.05) postinfection (Figure 1); however, 9 d postinfection, the MOB was highest (P < 0.05) in birds fed the AVM diet, and lowest (P < 0.05) in birds fed the CTL+ diet, whereas birds fed the CTL− or AVE diet
Figure 2. In vitro heterophil oxidative burst (n = 4 replicates per column) isolated from the pooled peripheral blood of broiler chickens (n = 10 per treatment) at 5 and 9 (experiment 1) or at 7 and 14 (experiment 2) d postinfection with 106 cfu of Salmonella Typhimurium (birds were orally challenged at d 7 in experiment 1 or at d 3 in experiment 2). Chickens were fed an antibiotic-free diet (CTL−), an antibiotic-free diet plus 40 mg of bacitracin/kg of feed (CTL+), a CTL− plus 0.8% Arg and 40 IU of VE/kg of feed (AVE), or an AVE diet plus 0.2% mannanoligosaccharides (AVM). Heterophils were stimulated with phorbol-12-myristate-13-acetate or cultured in an equal volume of Roswell Park Memorial Institute 1640 media as a negative control. Columns with different letters (a–c) within sampling time are statistically different (P < 0.05).
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Figure 1. In vitro oxidative burst of monocytes (n = 4 replicates per column) isolated from the pooled peripheral blood of broiler chickens (n = 10 per treatment) at 5 and 9 (experiment 1) or at 7 and 14 (experiment 2) d postinfection with 106 cfu of Salmonella Typhimurium (birds were orally challenged at d 7 in experiment 1 or at d 3 in experiment 2). Chickens were fed an antibiotic-free diet (CTL−), an antibiotic-free diet plus 40 mg of bacitracin/kg of feed (CTL+), a CTL− plus 0.8% Arg and 40 IU of VE/kg of feed (AVE), or an AVE diet plus 0.2% mannanoligosaccharides (AVM). Monocytes were stimulated with phorbol-12-myristate-13-acetate or cultured in an equal volume of Roswell Park Memorial Institute 1640 media as a negative control. Columns with different letters (a–c) within sampling time are statistically different (P < 0.05).
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LIU ET AL. Table 1. Effects of arginine, vitamin E, and mannanoligosaccharides on serum antibody titers 16 d after an experimental challenge with 106 cfu of Salmonella Typhimurium in broiler chickens1 Treatment2,3 Isotype IgM IgG IgA
CTL− 17.1ab
121.3 ± 1,249.3 ± 229.5 127.1 ± 38.1b
CTL+ 9.2b
AVE
97.6 ± 936.0 ± 143.3 97.3 ± 10.9b
15.6ab
116.2 ± 943.6 ± 81.3 138.3 ± 37.3b
AVM 152.3 ± 11.0a 674.3 ± 65.0 310.3 ± 68.7a
a,bValues
with different superscripts within the same row are different (P < 0.05). of 8 observations ± SEM. 2CTL−, antibiotic-free diet; CTL+, diet with 40 mg of bacitracin/kg of feed; AVE, antibiotic-free diet plus 0.8% l-Arg, and 40 IU of vitamin E/kg of feed; AVM, AVE diet supplemented with 0.2% mannanoligosaccharides (Biomos, Alltech Inc., Nicholasville, KY). 3Birds were orally challenged at d 3 (experiment 2). 1Mean
Salmonella Concentrations in the Ceca
The amount of circulating IgM was significantly higher in birds fed the AVM diet than in birds fed the CTL+ diet, but similar to the IgM concentrations of birds fed the CTL− or the AVE diet (Table 1). The amount of IgG was not affected by dietary treatment. Conversely, birds fed the AVM diet had the highest (P < 0.05) concentrations of circulating IgA, with no differences among the other 3 treatments.
In experiment 1, the amount of recovered Salmonella (cfu) was significantly lower in birds fed the AVM diet than in birds fed the CTL− diet 3 d postinfection, but was not different from the Salmonella concentrations recovered in birds fed the CTL+ or AVE diet. However, 10 and 17 d postinfection, there was no effect of dietary treatment on Salmonella recovery in the ceca. In experiment 2, there were no effects of treatment on Salmonella at any measuring time (7, 14, and 21 d postinfection).
DISCUSSION
Figure 3. In vitro proliferation of lymphocytes (n = 4 replicates per column) isolated from the pooled peripheral blood of broiler chickens (n = 10 per treatment) at 5 and 9 (experiment 1) or at 7 and 14 (experiment 2) d postinfection with 106 cfu of Salmonella Typhimurium (birds were orally challenged at d 7 in experiment 1 or at d 3 in experiment 2). Chickens were fed an antibiotic-free diet (CTL−), an antibiotic-free diet plus 40 mg of bacitracin/kg of feed (CTL+), a CTL− plus 0.8% Arg and 40 IU of VE/kg of feed (AVE), or an AVE diet plus 0.2% mannanoligosaccharides (AVM). Lymphocytes were stimulated with concanavalin A or cultured in an equal volume of Roswell Park Memorial Institute 1640 media as a negative control. Columns with different letters (a–c) within sampling time are statistically different (P < 0.05).
In previous studies we have documented that the supplementation of Arg and VE has complementary or synergistic effects on the immune response of broilers after vaccination with immune bursal disease virus or after challenges with Eimeria spp. (Abdukalykova et al., 2008; Ruiz-Feria and Abdukalykova, 2009; PerezCarbajal et al., 2010; Chan-Díaz et al., 2012). We have also documented that birds fed MOS had an improved intestinal integrity, an increase in beneficial bacteria in the ceca, and reduced shedding of E. coli after challenge (Baurhoo et al., 2007a,b). Thus, we hypothesized that the concurrent supplementation of Arg, VE, and MOS, will improve the innate and acquired immune function and will reduce Salmonella colonization in the ceca of broiler chickens after a challenge with Salmonella Typhimurium. In experiment 1, when the birds were challenged at 7 d of age, the effects of Arg and VE (AVE) or the combination of AVE and MOS (AVM) on the activity of MOB were not very consistent, but feeding AVE improved the MOB 5 d after challenge, and feeding the AVM diet improved the MOB 9 d after challenge compared with the CTL+ treatment. Also, birds fed the CTL− diet had consistently higher (P < 0.05) MOB than birds fed the CTL+ diet. Thus, the higher MOB in birds fed the CTL− and the AVE diet 5 d after challenge could be due to a higher immune stimulation in the absence of antimicrobial agents or prebiotics; in other words, the presence of antibiotics or prebiotics
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Humoral Immunity
IMMUNE FUNCTION AFTER SALMONELLA CHALLENGE
significantly higher MOB, HOB, and LPR, than birds in the other treatments. It has been reported that neonatal poultry exhibit a transient susceptibility to infectious disease during the first week of life and this is attributed to a deficient host defense mechanism (Kogut et al., 2012), in particular a functional inefficiency of heterophils and monocytes (He et al., 2008); thus, it is of great significance that the supplementation of Arg and VE increased the oxidative burst of both monocytes and heterophils when the challenge was done at an early age. Also, the LPR was consistently improved in birds fed the AVE diet when the challenge was done at an early age. The effects of VE on the immune response are mediated by direct effects on lymphocyte cytokine expression and modulation of immune cell proliferation (Li-Weber et al., 2002; Leshchinsky and Klasing, 2003; Babu and Raybourne, 2008), whereas the effects of Arg on the immune response to Salmonella have been attributed to a higher production of NO for host defense (MacFarlane et al., 1999). Also, macrophages from chickens selected for high antibody response produce more NO (Guimarães et al., 2011). However, the effects of Arg alone can be deleterious because an overproduction of NO may lead to increased oxidative stress and immune suppression (Huang et al., 1996; MacFarlane et al., 1999; Xie et al., 2008). Thus, the complementary effects of Arg and VE may be explained by the antioxidant effects of VE on the overproduction of NO elicited by the immune challenge. In these experiments, we did not test Arg and VE individually because we have documented their complementary effects in several immune challenge models (Abdukalykova and Ruiz-Feria, 2006; Abdukalykova et al., 2008; Ruiz-Feria and Abdukalykova, 2009; Perez-Carbajal et al., 2010; Chan-Díaz et al., 2012). Also in these experiments it was evident that further supplementing an AVE diet with MOS did not further improve immune response in experiment 1 (except MOB 9 d after challenge), and even reversed the positive effects of Arg and VE on immune response in experiment 2. The reason for this could be attributed to the mode of action of MOS, which binds gram-negative bacteria expressing the type 1 fimbrae, including Salmonella, reducing its adherence to intestinal epithelial cells (Spring et al., 2000) and immune activation. Recently, Baurhoo et al. (2012) reported that birds fed MOS and challenged with Salmonella LPS showed gene expression downregulation of Toll-like receptor-2, gallinacin-1 α, and CXC1, an IL-8 receptor that binds the IL-8 chemoattractant expressed by macrophages, whereas Wigley et al. (2006) reported that Salmonellaresistant lines of chickens presented a higher expression of IL-6, IL-8, and IL-18, and Kogut (2002) reported the importance of IL-8 as a major chemotactic factor to increase heterophil recruitment to the site of Salmonella infection; thus, it appears that downregulation of IL-8 by MOS may explain the effects seen in these experiments. Our results agree with reports indicating that prebiotics do not have an effect on monocyte function
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may reduce the interaction of microbes with the gastric immune system, resulting in a downregulation of the MOB capacity. However, birds fed the AVM diet had the highest MOB 9 d after challenge, which could be attributed to the immunomodulatory effects of MOS (Ferket, 2004; Janardhana et al., 2009). Because monocytes are important in phagocytosis and activation of the acquired immune response through the production of cytokines, our results show that Arg and VE supplementation (AVE) are better than AVM diets, and that the presence of a prebiotic may delay the activation of MOB capacity. Birds fed the AVE or the AVM diet had consistently lower HOB than birds fed the control diets 9 d after challenge in experiment 1. Genovese et al. (2013) reported that chickens with less functional heterophils are more susceptible to infection, including Salmonella Enteritidis, than those with highly functional heterophils. Thus, under these conditions the supplementation of AVE or AVM does not seem to have beneficial effects in term of heterophil function. Conversely, birds fed the AVE or the AVM diet had a significantly higher lymphocyte proliferation (LPR) reaction than birds fed the CTL diets 9 d after challenge. Thus, when birds are challenged with Salmonella Typhimurium at 7 d of age and are fed diets supplemented with Arg and VE (AVE), or a combination of Arg, VE, and MOS (AVM) they show a reduced (P < 0.05) HOB, and higher LPR (P < 0.05) than birds fed the control diets, although the differences were modest and may not have a major biological significance (Figures 2 and 3). Also, it was evident that further supplementing an AVE diet with MOS did not have additive effects on immune function under the conditions of this experiment. The limited effects of the dietary treatments on immune function could be explained by the fact that birds were challenged at 7 d of age, when the normal microflora was already established, and under these circumstances the Salmonella challenge was not enough to elicit a strong immune response. Redmond et al. (2011) found no effect of dietary β-glucans or ascorbic acid on heterophil function after a Salmonella Typhimurium challenge, and they attributed this result to the lack of other stressors or disease, suggesting that under most challenging environments the effects of prebiotics and antioxidants would be more evident. Thus, to avoid the effects of an established microflora, in experiment 2 the birds were challenged at 3 d of age, and the effects of the concurrent supplementation of Arg and VE on immune response were more consistent, and in agreement with our previous findings (Abdukalykova et al., 2008; Ruiz-Feria and Abdukalykova, 2009; Perez-Carbajal et al., 2010; Chan-Díaz et al., 2012). Seven days after challenge, MOB was not different among treatments, whereas at this time birds fed the AVE diet had significantly higher HOB than birds fed the CTL+ or the AVM diet, and higher (P < 0.05) LPR than birds fed the other diets. Furthermore, 14 d after challenge birds fed the AVE diet had consistently
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LIU ET AL. Table 2. Effects of Arg, vitamin E, and mannanoligosaccharides on cecal Salmonella Typhimurium (cfu, log10), and on number of Salmonella-positive samples (out of 10 samples, in parentheses) after an experimental challenge with 106 cfu of Salmonella Typhimurium in broiler chickens1 Treatment2 Days after challenge3 Experiment 1 3 10 17 Experiment 2 7 14 21
CTL−
CTL+
AVE
AVM
3.0 ± 0.5a (8) 0.8 ± 0.4 (1) 0.5 ± 0.4 (1)
2.1 ± 0.5ab (5) 1.5 ± 0.4 (1) 0.7 ± 0.3 (0)
1.6 ± 0.5ab (3) 0.2 ± 0.2 (0) 1.0 ± 0.6 (2)
0.8 ± 0.4b (1) 0.7 ± 0.4 (0) 0.6 ± 0.4 (1)
4.4 ± 0.6 (10) 1.5 ± 0.5 (7) 0.3 ± 0.3 (1)
4.0 ± 1.1 (10) 1.9 ± 0.6 (2) 0 ± 0 (0)
4.4 ± 1.1 (10) 1.9 ± 0.6 (7) 0.8 ± 0.4 (0)
4.4 ± 1.14 (9) 0.9 ± 0.6 (7) 0.4 ± 0.4 (0)
a,bValues
(Bunout et al., 2002) or leukocyte proliferation (Janardhana et al., 2009). In experiment 2, the dietary treatments did not affect antibody titers of IgG, but the antibody titers of IgM were significantly higher in birds fed the AVM diet compared with birds fed the CTL+ diet, and IgA titers were highest (P < 0.05) in birds fed the AVM diet. These results are in contrast with the effects of MOS on the innate immune response and LPR discussed above. It is well documented that infection with Salmonella leads to increased levels of IgG, IgM, and IgA antibodies (Withanage et al., 2005). However, the role of humoral immunity on Salmonella clearance is not well understood. Beal et al. (2006) demonstrated that although Salmonella infection in chickens elicited high concentrations of antibodies, B cells did not play an essential role in clearance of the primary or secondary infection. More recently, Nanton et al. (2012) suggested that B cells play an indirect role in protection against Salmonella through the development of T cell immunity after secondary infection. Thus, the higher concentrations of IgA in birds fed the AVM diet may be beneficial in a secondary challenge, or may reduce horizontal transmission of Salmonella during growing. However, in these experiments the dietary treatments did not affect Salmonella recovery in the ceca after challenge. Only in experiment 1, when birds were challenged at d 7, birds fed the AVM diet had lower (P < 0.05) concentrations of Salmonella than birds fed the CTL− diet, but not different from the ceca Salmonella levels of birds fed the AVE or the CTL+ diet 3 d after challenge (Table 2). At 10 and 17 d after challenge, the recovery of Salmonella was very low and not different among treatments. In experiment 2, when the challenge was performed at an earlier age (d 3) Salmonella recovery was high in all treatments 7 d after challenge (4–4.4 logs), was reduced by more than half by 14 d after challenge (0.9–1.9 logs), and was very low by 21 d after challenge (0–0.8 logs), but there were
no significant differences among treatments. Therefore, the higher immune response elicited by Arg and VE in experiment 2 was not correlated with a lower Salmonella recovery in the ceca compared with birds fed the control diets; this could be attributed to a low dose of Salmonella in the challenge, or to the lack of other stresses in otherwise healthy birds. Also, it has been suggested that the expression of cytokines and chemokines during primary Salmonella infection will result in the modulation of CD4 T cell response to improve resistance against secondary infection (Withanage et al., 2005; Nanton et al., 2012); accordingly, the higher HOB and MOB found in this experiment, along with our previous findings on the effects of Arg and VE on T cell differentiation (Abdukalykova et al., 2008) may be important in the development of acquired immunity after a secondary challenge. Further research is warranted on this subject. In the same way, the higher concentrations of IgA in birds fed the AVM diet did not correlate with lower concentrations of Salmonella in birds fed the AVM diet. The effects of prebiotics on Salmonella prevention have been variable. Agunos et al. (2007) reported reductions in fecal Salmonella 16 and 19 d after a challenge with 2 × 107 cfu of Salmonella when birds were fed β1–4-mannobiose, whereas Revolledo et al. (2009) did not find any effect on ceca Salmonella in birds fed β-glucans. In the same way, Burkey et al. (2004) and Calveyra et al. (2011) reported no effects of MOS on Salmonella shedding in pigs experimentally infected with Salmonella Typhimurium. In summary, our results show that supplementing diets with Arg and VE improve the innate and acquired immune response in chicks challenged with Salmonella, but only when the challenge occurred early in their life (3 d old). We also found that further supplementing the diet with MOS did not improve the effects of Arg and VE, except in the production of IgA. Although the dietary treatments did not reduce Salmonella con-
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with different superscripts within the same row are different (P < 0.05). of 10 observations ± SEM (number of birds tested positive, out of 10, after Rappaport-Vassiliadis broth media enrichment for 24 h). 2CTL−, antibiotic-free diet; CTL+, diet with 40 mg of bacitracin/kg of feed; AVE, antibiotic-free diet plus 0.8% l-Arg, and 40 IU of vitamin E/kg of feed; AVM, AVE diet supplemented with 0.2% mannanoligosaccharides (Biomos, Alltech Inc., Nicholasville, KY). 3Birds were orally challenged at d 7 (experiment 1) or d 3 (experiment 2). 1Mean
IMMUNE FUNCTION AFTER SALMONELLA CHALLENGE
centrations in the ceca, the better immune response of birds fed Arg and VE may benefit birds grown under commercial conditions, where they face more stressful conditions and are exposed to several microbial challenges. Further studies are needed to evaluate the effects of Arg and VE on Salmonella clearance after a secondary Salmonella challenge.
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