Effects of Pediococcus acidilactici, mannan-oligosaccharide, butyric acid and their combination on growth performance and intestinal health in young broiler chickens challenged with Salmonella Typhimurium

Effects of Pediococcus acidilactici, mannan-oligosaccharide, butyric acid and their combination... ABSTRACT This study compared the efficacy of Pediococcus acidilactici, mannan-oligosaccharide, butyric acid, and their combination on growth performance and intestinal health in broiler chickens challenged with S. Typhimurium. Ross 308 male broilers (n = 420) were randomly assigned to one of the 6 treatments, resulting in 5 replicate pens of 14 chicks per treatment. The treatments included a negative control [(NC), no additive, not challenged]; positive control [(PC), no additive, but challenged with S. Typhimurium at d 3 posthatch], and 4 groups whereby birds were challenged with S. Typhimurium at d 3 posthatch and fed diets supplemented with either probiotic [0.1 g/kg Pediococcus acidilactici (PA)], prebiotic [2 g/kg mannan-oligosaccharides (MOS)], organic acid [0.5 g/kg butyric acid (BA)], or a combination of the 3 additives (MA). The S. Typhimurium challenge decreased feed intake, body weight gain and increased feed conversion ratio and reduced jejunum villus height (VH) and VH to crypt depth (CD) ratio (P < 0.05). Birds on the MA treatment exhibited similar performance to birds on the NC treatment (P > 0.05) and had a lower population of Salmonella in the ceca compared with birds on the PC treatment, at d 14 and 21 post-challenge (P < 0.05). The lowest heterophil to lymphocyte ratio was observed in birds on the MA and NC treatments (P < 0.05). Birds fed diets supplemented with MA or PA had greater VH and VH: CD ratio than birds on the PC treatment at d 7, 14 and 21 d post-challenge (P < 0.05). Suppressed amylase and protease activity was observed as a result of the S. Typhimurium challenge; the enzyme levels were restored in birds fed the additive-supplemented diets, when compared to the birds on the PC treatment, particularly at d 21 post-challenge (P < 0.05). These results indicate that dietary supplementation with a combination of PA, BA, and MOS in broiler chickens could be used as an effective tool for controlling S. Typhimurium and promoting growth performance. INTRODUCTION Salmonella enterica var. Typhimurium (S. Typhimurium) is one of the most prevalent serotypes of Salmonella, a key causative agent of salmonellosis, which is a disease that enters the human food chain through animal products, particularly raw poultry products (Chalghoumi et al., 2009; Thung et al., 2016). In-feed antibiotics have long been used at sub-therapeutic doses in poultry feed to control gut pathogenic bacteria load and hence to enhance feed efficiency, promote animal growth, and improve the quality of the animal products (Cheng et al., 2014). However, the increased risk of bacteria acquiring resistance to antibiotics and their residue in end-products such as meat and eggs led to a ban on the use of in-feed antibiotics as growth promotors (AGP) more than a decade ago in many countries in the European Union (Toghyani et al., 2010; Sugiharto, 2016). As a result of this growing pressure on livestock producers worldwide, various alternative strategies have been proposed and investigated as potential substitutes for AGPs in animal feeds. Particular attention has been paid to the use of probiotics (Menconi et al., 2011; Park and Kim, 2014), prebiotics (Pourabedin et al., 2016; Rajani et al., 2016), and organic acids (Fernandez-Rubio et al., 2009; Saleem et al., 2016), as AGPs alternatives in poultry feed. There is evidence to suggest that these aforementioned products exert beneficial effects on performance parameters, microbiota composition, and intestinal integrity, and potentially reduce the levels of pathogenic bacteria such as Salmonella in the tract. However, these positive effects have not always been consistent (Van der Aar et al., 2017). The main proposed modes of action of probiotics include 1) antagonistic action towards pathogenic bacteria, by secreting products that inhibit their development, such as bacteriocins, organic acids, and hydrogen peroxide, and 2) competitive exclusion, by competing with bacteria for locations in the intestinal mucous membrane to adhere to and nutrients (Patterson and Burkholder, 2003). Prebiotics serve as a substrate for endogenous beneficial bacteria, thus promoting competitive exclusion of pathogenic microbes and selective colonization by beneficial microbes (Biggs et al., 2007). Oligosaccharides display prebiotics properties, but they have also been reported to present immunomodulatory beneficial effects in the gut such as modifying clearance efficiency of pathogenic bacteria, activating T cell-dependent immune responses and repression of pro-inflammatory cytokines (Troy and Kasper, 2010; Bonos et al., 2011). Organic acids, such as lactic, acetic, butyric, tannic, fumaric and propionic acids, display antimicrobial properties and play a crucial role in controlling the population of pathogenic bacteria (Menconi et al., 2014), likely by lowering gastrointestinal tract pH (Partanen and Mroz, 1999) and stimulating protein digestion by converting pepsinogen to pepsin (Suiryanrayna and Ramana, 2015). The differing mechanisms and modes of action exhibited by probiotics, prebiotics, and organic acids suggest that there could be a complementary synergistic effect resulting from supplementing diets with a mixture of these additives, especially under the stressful conditions imposed by a S. Typhimurium challenge (Rajani et al., 2016). In view of an evident dearth of data in this field, the current study was designed to determine the efficacy of Pediococcus acidilactici (a species of gram-positive cocci that produces lactic acid and secrets a bacteriocins known as pediocins) as a probiotic supplement, mannan oligosaccharides as a prebiotic supplement, and micro-encapsulated butyric acid, singularly or in combination, on growth performance and gut health of young broiler chicks challenged with S. Typhimurium. MATRIALS AND METHODS Experimental Design, Dietary Treatments, and Bird Husbandry The experimental procedures in this study were reviewed and approved by the Animal Ethics committee of Islamic Azad University, Isfahan Branch, based on institutional and national guidelines for the care and use of animals. A total of 420 day-old Ross 308 male broiler chicks were obtained from a commercial hatchery (Navid Morgh Guilan Company., Guilan, Iran), were fed a common starter diet for the first 3 d. At d 3 posthatch, birds were weighed and randomly assigned to one of the 6 experimental treatments with 5 replicate pens of 14 birds each to have a starting body weight of 72 ± 1.5 g/bird. The treatments consisted of negative control [(NC), no additive and no challenge with S. Typhimurium]; positive control [(PC), no additive, but challenged with S. Typhimurium], and 4 groups whereby birds were challenged with S. Typhimurium and fed diets supplemented with either probiotic (Pediococcus acidilactici at 0.01% [Pedi Guard, Iran, concentration 1 × 1010 CFU/g]; PA), prebiotic (0.2% mannan-oligosaccharides, [ActiveMOS®, Biorigin, Brazil]; MOS), butyric acid (0.05% butyric acid provided in an encapsulated form consisting of 50% butyrate salt [ButiPEARL; Kemin Industries, Herentals, Belgium]; BA), or a combination of the 3 additives (MA). All the challenged birds were orally administered 105 CFU of S. Typhimurium at d 3 posthatch. Birds were allowed ad libitum access to the treatment diets and water for the duration of the trial (d 0 to 24). A basal corn-SBM diet was formulated to meet the nutrient requirements according to the Ross 308 (Aviagen, 2014) for starter (0- to 10-d) and grower (10- to 24-d) periods (Table 1). The temperature and lighting programs used followed the Ross 308 breed management recommendations (Aviagen, 2014). Table 1. Compositions of the experimental diets. % as-is Ingredient Starter (d 1–10) Grower (d 11–24) Corn 55.5 65.5 Soybean meal 37.7 28.1 Sunflower oil 2.37 2.24 Limestone 1.09 0.99 Dicalcium phosphate1 1.74 1.54 Sodium chloride 0.21 0.18 Sodium bicarbonate 0.16 0.19 Vitamin premix2 0.25 0.25 Mineral premix3 0.25 0.25 Choline chloride 60% 0.122 0.116 DL-methionine 0.296 0.254 L-lysine 0.188 0.269 L-threonine 0.091 0.079 Nutrient composition Metabolizable energy (Kcal/kg) 3000 3100 Crude protein (%) 22.94 19.41 Dig4 lysine (%) 1.28 1.11 Dig methionine (%) 0.63 0.55 Dig methionine + cysteine (%) 0.95 0.83 Dig threonine (%) 0.86 0.72 Calcium (%) 0.90 0.80 Available phosphorus (%) 0.45 0.40 Sodium (%) 0.16 0.17 Chloride (%) 0.21 0.21 Choline (mg/kg) 1700 1500 % as-is Ingredient Starter (d 1–10) Grower (d 11–24) Corn 55.5 65.5 Soybean meal 37.7 28.1 Sunflower oil 2.37 2.24 Limestone 1.09 0.99 Dicalcium phosphate1 1.74 1.54 Sodium chloride 0.21 0.18 Sodium bicarbonate 0.16 0.19 Vitamin premix2 0.25 0.25 Mineral premix3 0.25 0.25 Choline chloride 60% 0.122 0.116 DL-methionine 0.296 0.254 L-lysine 0.188 0.269 L-threonine 0.091 0.079 Nutrient composition Metabolizable energy (Kcal/kg) 3000 3100 Crude protein (%) 22.94 19.41 Dig4 lysine (%) 1.28 1.11 Dig methionine (%) 0.63 0.55 Dig methionine + cysteine (%) 0.95 0.83 Dig threonine (%) 0.86 0.72 Calcium (%) 0.90 0.80 Available phosphorus (%) 0.45 0.40 Sodium (%) 0.16 0.17 Chloride (%) 0.21 0.21 Choline (mg/kg) 1700 1500 1Dicalcium phosphate contained: phosphorus, 18%; calcium, 21%. 2Supplied per kg of diet: 1.8 mg all-trans-retinyl acetate, 0.02 mg cholecalciferol, 8.3 mg alphatocopheryl acetate, 2.2 mg menadione, 2 mg pyridoxine HCl, 8 mg cyanocobalamin,10 mg nicotine amid, 0.3 mg folic acid, 20 mg D-biotin and 160 mg choline chloride. 3Supplied per kg of diet: 32 mg Mn (MnSO4⋅H2O), 16 mg Fe (FeSO4⋅7H2O), 24 mg Zn (ZnO), 2 mg Cu (CuSO4⋅5H2O), 800 μg I (KI), 200 μg Co (CoSO4) and 60 μg Se. 4Dig, Digestible. View Large Table 1. Compositions of the experimental diets. % as-is Ingredient Starter (d 1–10) Grower (d 11–24) Corn 55.5 65.5 Soybean meal 37.7 28.1 Sunflower oil 2.37 2.24 Limestone 1.09 0.99 Dicalcium phosphate1 1.74 1.54 Sodium chloride 0.21 0.18 Sodium bicarbonate 0.16 0.19 Vitamin premix2 0.25 0.25 Mineral premix3 0.25 0.25 Choline chloride 60% 0.122 0.116 DL-methionine 0.296 0.254 L-lysine 0.188 0.269 L-threonine 0.091 0.079 Nutrient composition Metabolizable energy (Kcal/kg) 3000 3100 Crude protein (%) 22.94 19.41 Dig4 lysine (%) 1.28 1.11 Dig methionine (%) 0.63 0.55 Dig methionine + cysteine (%) 0.95 0.83 Dig threonine (%) 0.86 0.72 Calcium (%) 0.90 0.80 Available phosphorus (%) 0.45 0.40 Sodium (%) 0.16 0.17 Chloride (%) 0.21 0.21 Choline (mg/kg) 1700 1500 % as-is Ingredient Starter (d 1–10) Grower (d 11–24) Corn 55.5 65.5 Soybean meal 37.7 28.1 Sunflower oil 2.37 2.24 Limestone 1.09 0.99 Dicalcium phosphate1 1.74 1.54 Sodium chloride 0.21 0.18 Sodium bicarbonate 0.16 0.19 Vitamin premix2 0.25 0.25 Mineral premix3 0.25 0.25 Choline chloride 60% 0.122 0.116 DL-methionine 0.296 0.254 L-lysine 0.188 0.269 L-threonine 0.091 0.079 Nutrient composition Metabolizable energy (Kcal/kg) 3000 3100 Crude protein (%) 22.94 19.41 Dig4 lysine (%) 1.28 1.11 Dig methionine (%) 0.63 0.55 Dig methionine + cysteine (%) 0.95 0.83 Dig threonine (%) 0.86 0.72 Calcium (%) 0.90 0.80 Available phosphorus (%) 0.45 0.40 Sodium (%) 0.16 0.17 Chloride (%) 0.21 0.21 Choline (mg/kg) 1700 1500 1Dicalcium phosphate contained: phosphorus, 18%; calcium, 21%. 2Supplied per kg of diet: 1.8 mg all-trans-retinyl acetate, 0.02 mg cholecalciferol, 8.3 mg alphatocopheryl acetate, 2.2 mg menadione, 2 mg pyridoxine HCl, 8 mg cyanocobalamin,10 mg nicotine amid, 0.3 mg folic acid, 20 mg D-biotin and 160 mg choline chloride. 3Supplied per kg of diet: 32 mg Mn (MnSO4⋅H2O), 16 mg Fe (FeSO4⋅7H2O), 24 mg Zn (ZnO), 2 mg Cu (CuSO4⋅5H2O), 800 μg I (KI), 200 μg Co (CoSO4) and 60 μg Se. 4Dig, Digestible. View Large Bacterial Inoculum Preparation The S. Typhimurium strain (PTCC 1709) was obtained as a freeze-dried powder from the Iranian Research Organization for Science and Technology, Tehran. The frozen bacterial culture was thawed and cultured in a trypticase soy broth for 24 at 37°C. The number of CFU was determined by diluting the inoculum with sterile phosphate-buffered saline (PBS) and plating it on culture medium XLT4 agar for 24 h at 37°C. At d 3 posthatch, all birds (except those in the NC treatment group) were orally administered a one-off dose of 0.5 mL of 1 × 105 CFU S. Typhimurium suspension. The same dose per os of sterile PBS was orally administered to the birds in unchallenged group. Performance Measurements Growth performance parameters including feed intake (FI) and body weight gain (BWG) were measured during the starter (d 0 to 7 post challenge), grower (d 7 to 21 post challenge), and overall experimental period (d 0 to 21 post challenge), and used to calculate the feed conversion ratio (FCR), adjusted for mortality. Ten birds per treatment (2 birds from each replicate pen) were randomly selected for blood, digesta and tissue sample collection at d 7, 14, and 21 post-challenge. Hematological Parameters At d 7, 14, and 21 post-challenge, 3 mL of blood was obtained from the 2 birds per pen by puncturing the brachial vein and individually collecting the blood into non-heparinized tubes. Blood smears were prepared by the May-Grunwald-Giemsa coloring technique and heterophil to lymphocyte (H/L) ratio was calculated by counting 100 leukocytes per slide. Enumeration of Cecal Bacteria Following blood sample collection, the 2 birds were weighed and euthanized by CO2 asphyxiation. The contents of the ceca were collected by gently squeezing the digesta into plastic containers, and pooled per replicate. To determine S. Typhimurium counts, 1 g of the ceca digesta was diluted with 0.1% peptone water, homogenized for 1 min and serial dilutions were subsequently prepared in the same diluent. Samples at the optimum dilution level were then selected and 0.1 mL was plated in triplicate on XLT4 Agar (Difco) containing 100 mL of Nalidixic acid. The plates were then incubated for 24 h at 37°C under anaerobic conditions. To determine Lactobacillus, Bifidobacterium, and coliform counts in the ceca samples, again 10-fold serial dilutions were conducted with peptone water and then 0.1 mL of sample from the optimum dilution level was cultured on plates containing modified Rogosa SL agar, TOS Propionate agar, and MacConkey agar, respectively. Plates were incubated in anaerobic conditions for 24 to 48 h at 37°C. Following incubation, the number of colonies on each plate was counted and the obtained numbers were multiplied by reversed dilution, and reported as the number of CFU per 1 g sample. Intestinal Morphology The digesta content of the jejunum from the 2 birds was gently flushed out with ultra-pure water into a container, and then approximately 3 cm of the jejunum tissue (middle section) was transected and immersed in 10% buffered formalin. Tissues were serial dehydrated by passing the tissue through increasing concentrations of ethyl alcohol, before being embedded in paraffin wax. Histological examinations were carried out according to the method described by Xu et al. (2003). A total of 10 well-oriented, intact villi and crypts were randomly selected in duplicate from each tissue sample and the average of 20 values were obtained for each bird. All morphological parameters were measured using the ImageJ software package (http://rsb.info.nih.gov/ij/). Enzyme Activity The collected jejunum digesta samples were diluted 10 times with phosphate- saline buffer, based on weight, and were then centrifuged at 18,000 × g for 20 min at 4°C. The supernatant was stored at −80°C prior to enzyme analysis. Samples were processed to determine amylase (EC 3.2.1.1), lipase (EC 3.1.1.3), and neutral protease activities following the methods described by Xu et al. (2003). One unit of amylase activity was defined as the amount of amylase required to release 1 mg of glucose after 30 min incubation at 40°C, per mg of intestinal digesta protein. Lipase activity was equal to the volume (in mL) of 0.05 M NaOH required to neutralize the fatty acids liberated during a 6-h incubation with 3 mL of lipase substrate at 38°C, per mg of intestinal digesta protein. The protease activity unit was defined as milligrams of azocasein degraded after 2 h incubation at 37°C, per mg of intestinal digesta protein. Statistical Analysis Firstly, Kolmogorov-Smirnov testing was conducted to confirm normality of the data. Data were then subjected to one-way analysis of variance (ANOVA) using General Linear Model procedure of SAS 9.3 package (SAS Institute Inc., 2010) to determine the equality of the means. Each pen was considered as an experimental unit. The values presented in the tables represent the mean for each treatment, with a pooled standard error of mean (SEM). Tukey's HSD test was used to make pairwise comparisons between means, where appropriate. Statistical significance was declared at P < 0.05. RESULTS Bird's Performance and Mortality Rate The effects of the treatments on bird performance are reported in Table 2. The S. Typhimurium challenge decreased FI, BWG, and increased FCR in birds in the PC treatment group compared to the NC treatment group, at all time periods tested (P < 0.05). Dietary supplementation of PA, MOS, BA, and in particular their combination, improved FI, BWG, and FCR in the challenged birds, compared to birds in the PC treatment group (P < 0.05), at all measured time periods. However, only birds in the MA treatment group displayed a body weight gain (0 to 21 d post challenge) that was statistically similar to birds in the NC treatment group (P > 0.05); birds fed the diets containing just one of the dietary additives had a lower BWG and higher FCR compared to birds on the NC treatment (P < 0.05). No significant effect of challenge or dietary treatment was observed on bird mortality during the experimental period (data not shown). Table 2. Effects of dietary treatments on growth performance of broilers. Treatments1 Item2 NC PC PA MOS BA MA SEM P-value Day 3 BW (g/b) 72.5 73.2 72.9 73.4 73.0 72.7 0.48 0.88 0 to 7 d P-Ch BWG (g/b) 185.7a 144.5c 168.1b 166.8b 165.2b 170.5b 4.45 <.001 FI (g/b) 218.3a 186.9b 207.3a 205.5a 204.7a,b 203.6a,b 5.84 0.03 FCR (g/g) 1.17c 1.29a 1.23b 1.23b 1.24b 1.19b,c 0.01 0.001 7 to 21 d P-Ch BWG (g/b) 720.4a 604.2c 681.8b 682.9b 676.1b 712.2a 8.55 <.001 FI (g/b) 1004.3a 945.5b 983.9a,b 996.0a 998.4a 1011.4a 13.86 0.03 FCR (g/g) 1.39c 1.56a 1.44b,c 1.46b 1.47b 1.42b,c 0.02 0.001 0 to 21 d P-Ch BWG (g/b) 906.2a 748.7d 850.1b,c 849.8b,c 841.3c 882.8a,b 11.03 <.001 FI (g/b) 1222a 1132b 1191a 1201a 1203a 1215a 18.27 0.02 FCR(g/g) 1.35c 1.51a 1.40b,c 1.41b 1.43b 1.37b,c 0.02 <.001 Treatments1 Item2 NC PC PA MOS BA MA SEM P-value Day 3 BW (g/b) 72.5 73.2 72.9 73.4 73.0 72.7 0.48 0.88 0 to 7 d P-Ch BWG (g/b) 185.7a 144.5c 168.1b 166.8b 165.2b 170.5b 4.45 <.001 FI (g/b) 218.3a 186.9b 207.3a 205.5a 204.7a,b 203.6a,b 5.84 0.03 FCR (g/g) 1.17c 1.29a 1.23b 1.23b 1.24b 1.19b,c 0.01 0.001 7 to 21 d P-Ch BWG (g/b) 720.4a 604.2c 681.8b 682.9b 676.1b 712.2a 8.55 <.001 FI (g/b) 1004.3a 945.5b 983.9a,b 996.0a 998.4a 1011.4a 13.86 0.03 FCR (g/g) 1.39c 1.56a 1.44b,c 1.46b 1.47b 1.42b,c 0.02 0.001 0 to 21 d P-Ch BWG (g/b) 906.2a 748.7d 850.1b,c 849.8b,c 841.3c 882.8a,b 11.03 <.001 FI (g/b) 1222a 1132b 1191a 1201a 1203a 1215a 18.27 0.02 FCR(g/g) 1.35c 1.51a 1.40b,c 1.41b 1.43b 1.37b,c 0.02 <.001 a-cMeans with different superscripts in each row are significantly different (P < 0.05). 1NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. 2P-Hatch, Posthatch; P-Ch, Post-Challenge; BWG, body weight gain; FI, feed intake; FCR, feed conversion ratio. View Large Table 2. Effects of dietary treatments on growth performance of broilers. Treatments1 Item2 NC PC PA MOS BA MA SEM P-value Day 3 BW (g/b) 72.5 73.2 72.9 73.4 73.0 72.7 0.48 0.88 0 to 7 d P-Ch BWG (g/b) 185.7a 144.5c 168.1b 166.8b 165.2b 170.5b 4.45 <.001 FI (g/b) 218.3a 186.9b 207.3a 205.5a 204.7a,b 203.6a,b 5.84 0.03 FCR (g/g) 1.17c 1.29a 1.23b 1.23b 1.24b 1.19b,c 0.01 0.001 7 to 21 d P-Ch BWG (g/b) 720.4a 604.2c 681.8b 682.9b 676.1b 712.2a 8.55 <.001 FI (g/b) 1004.3a 945.5b 983.9a,b 996.0a 998.4a 1011.4a 13.86 0.03 FCR (g/g) 1.39c 1.56a 1.44b,c 1.46b 1.47b 1.42b,c 0.02 0.001 0 to 21 d P-Ch BWG (g/b) 906.2a 748.7d 850.1b,c 849.8b,c 841.3c 882.8a,b 11.03 <.001 FI (g/b) 1222a 1132b 1191a 1201a 1203a 1215a 18.27 0.02 FCR(g/g) 1.35c 1.51a 1.40b,c 1.41b 1.43b 1.37b,c 0.02 <.001 Treatments1 Item2 NC PC PA MOS BA MA SEM P-value Day 3 BW (g/b) 72.5 73.2 72.9 73.4 73.0 72.7 0.48 0.88 0 to 7 d P-Ch BWG (g/b) 185.7a 144.5c 168.1b 166.8b 165.2b 170.5b 4.45 <.001 FI (g/b) 218.3a 186.9b 207.3a 205.5a 204.7a,b 203.6a,b 5.84 0.03 FCR (g/g) 1.17c 1.29a 1.23b 1.23b 1.24b 1.19b,c 0.01 0.001 7 to 21 d P-Ch BWG (g/b) 720.4a 604.2c 681.8b 682.9b 676.1b 712.2a 8.55 <.001 FI (g/b) 1004.3a 945.5b 983.9a,b 996.0a 998.4a 1011.4a 13.86 0.03 FCR (g/g) 1.39c 1.56a 1.44b,c 1.46b 1.47b 1.42b,c 0.02 0.001 0 to 21 d P-Ch BWG (g/b) 906.2a 748.7d 850.1b,c 849.8b,c 841.3c 882.8a,b 11.03 <.001 FI (g/b) 1222a 1132b 1191a 1201a 1203a 1215a 18.27 0.02 FCR(g/g) 1.35c 1.51a 1.40b,c 1.41b 1.43b 1.37b,c 0.02 <.001 a-cMeans with different superscripts in each row are significantly different (P < 0.05). 1NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. 2P-Hatch, Posthatch; P-Ch, Post-Challenge; BWG, body weight gain; FI, feed intake; FCR, feed conversion ratio. View Large Characterization of Cecal Microbiota As illustrated in Figure 1, birds in the PC treatment group had the lowest ceca Lactobacillus population compared to all other treatments, at d 14 post-challenge (P < 0.05). Birds in the treatment groups with dietary supplementation of PA, MOS, BA and MA had a greater population of cecal lactobacilli compared to birds in the PC or NC treatment groups at d 14 and 21 post-challenge (P < 0.05), with the highest population observed in birds on the MA treatment. The cecal population of Bifidobacterium was not affected by the challenge (P > 0.05); however, dietary supplementation with MOS and MA significantly increased Bifidobacterium count compared to both the PC and NC treatment groups at d 21 post-challenge (P < 0.05; Figure 2). The S. Typhimurium challenge had no impact on the population of coliforms in the ceca (P > 0.05, Figure 3). Birds on the NC and PC treatments had the highest population of coliforms at d 7 and 21 post-challenge, whilst birds in the MA treatment group had the lowest population (P < 0.05; Figure 3). No Salmonella spp. were detected in the ceca of the unchallenged birds. Birds fed the dietary supplements had lower prevalence of Salmonella in the ceca compared to birds in the PC group at d 14 and 21 post-challenge (P < 0.05; Figure 4), and birds on the MA treatment had lower ceca Salmonella content compared to birds on any other treatment (P < 0.05; Figure 4). Figure 1. View largeDownload slide Effects of dietary treatments on cecal Lactobacillus population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. Figure 1. View largeDownload slide Effects of dietary treatments on cecal Lactobacillus population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. Figure 2. View largeDownload slide Effects of dietary treatments on cecal Bifidobacterium population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. Figure 2. View largeDownload slide Effects of dietary treatments on cecal Bifidobacterium population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. Figure 3. View largeDownload slide Effects of dietary treatments on cecal Coliforms population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. Figure 3. View largeDownload slide Effects of dietary treatments on cecal Coliforms population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. Figure 4. View largeDownload slide Effects of dietary treatments on cecal Salmonella population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. Figure 4. View largeDownload slide Effects of dietary treatments on cecal Salmonella population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. Hematological Parameters, Intestinal Enzyme Activities and Morphological Analysis Table 3 summarizes the effect of dietary treatments on heterophil and lymphocyte counts and the H/L ratio, measured at d 7, 14, and 21 post-challenge. An increased heterophil count at d 7 and 21 post-challenge and greater H/L ratios at all the periods tested were observed with the presence of the S. Typhimurium challenge (P < 0.05). The challenged birds fed the diets supplemented with PA, MOS, BA, and MA had significantly reduced heterophil and increased lymphocyte counts at d 7, 14, and 21 post-challenge compared to birds in the PC treatment group (P < 0.05). There were no significant differences between birds in the NC and MA treatment groups with regards to the H/L ratio at d 14 and 21 post-challenge. Heterophil counts at d 14 and lymphocyte counts at d 21 post-challenge were not influenced by the treatments (P > 0.05). Table 3. Effects of dietary treatments on heterophil and lymphocyte counts. Treatments1 Item Day P-Ch2 NC PC PA MOS BA MA SEM P-value Heterophil (H) 7 26.61c 39.85a 34.04b 34.95b 33.54b 30.19b,c 1.51 <.001 14 28.63 34.74 32.41 32.29 34.12 31.55 1.97 0.34 21 24.69c 33.37a 27.92b,c 29.56b 28.65b,c 25.28c 1.29 0.001 Lymphocyte (L) 7 64.3a 56.2c 60.1b 60.0b 60.5b 62.7a,b 1.22 0.002 14 61.9a 55.4b 57.2b 58.3a,b 58.0a,b 59.5a,b 1.34 0.04 21 64.1 59.5 58.8 63.1 61.7 64.3 1.69 0.13 H:L ratio 7 0.41d 0.71a 0.56b 0.58b 0.55b 0.48c 0.01 <.001 14 0.46c 0.62a 0.56a,b 0.55a,b 0.58a,b 0.52b,c 0.02 0.005 21 0.38c 0.58a 0.47b 0.46b 0.46b 0.39c 0.01 <.001 Treatments1 Item Day P-Ch2 NC PC PA MOS BA MA SEM P-value Heterophil (H) 7 26.61c 39.85a 34.04b 34.95b 33.54b 30.19b,c 1.51 <.001 14 28.63 34.74 32.41 32.29 34.12 31.55 1.97 0.34 21 24.69c 33.37a 27.92b,c 29.56b 28.65b,c 25.28c 1.29 0.001 Lymphocyte (L) 7 64.3a 56.2c 60.1b 60.0b 60.5b 62.7a,b 1.22 0.002 14 61.9a 55.4b 57.2b 58.3a,b 58.0a,b 59.5a,b 1.34 0.04 21 64.1 59.5 58.8 63.1 61.7 64.3 1.69 0.13 H:L ratio 7 0.41d 0.71a 0.56b 0.58b 0.55b 0.48c 0.01 <.001 14 0.46c 0.62a 0.56a,b 0.55a,b 0.58a,b 0.52b,c 0.02 0.005 21 0.38c 0.58a 0.47b 0.46b 0.46b 0.39c 0.01 <.001 a-cMeans with different superscripts in each row are significantly different (P < 0.05). Mean values are based on 2 observations per replicate (5 replicates per treatment) at each time point. 1NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. 2P-Ch, Post-Challenge. View Large Table 3. Effects of dietary treatments on heterophil and lymphocyte counts. Treatments1 Item Day P-Ch2 NC PC PA MOS BA MA SEM P-value Heterophil (H) 7 26.61c 39.85a 34.04b 34.95b 33.54b 30.19b,c 1.51 <.001 14 28.63 34.74 32.41 32.29 34.12 31.55 1.97 0.34 21 24.69c 33.37a 27.92b,c 29.56b 28.65b,c 25.28c 1.29 0.001 Lymphocyte (L) 7 64.3a 56.2c 60.1b 60.0b 60.5b 62.7a,b 1.22 0.002 14 61.9a 55.4b 57.2b 58.3a,b 58.0a,b 59.5a,b 1.34 0.04 21 64.1 59.5 58.8 63.1 61.7 64.3 1.69 0.13 H:L ratio 7 0.41d 0.71a 0.56b 0.58b 0.55b 0.48c 0.01 <.001 14 0.46c 0.62a 0.56a,b 0.55a,b 0.58a,b 0.52b,c 0.02 0.005 21 0.38c 0.58a 0.47b 0.46b 0.46b 0.39c 0.01 <.001 Treatments1 Item Day P-Ch2 NC PC PA MOS BA MA SEM P-value Heterophil (H) 7 26.61c 39.85a 34.04b 34.95b 33.54b 30.19b,c 1.51 <.001 14 28.63 34.74 32.41 32.29 34.12 31.55 1.97 0.34 21 24.69c 33.37a 27.92b,c 29.56b 28.65b,c 25.28c 1.29 0.001 Lymphocyte (L) 7 64.3a 56.2c 60.1b 60.0b 60.5b 62.7a,b 1.22 0.002 14 61.9a 55.4b 57.2b 58.3a,b 58.0a,b 59.5a,b 1.34 0.04 21 64.1 59.5 58.8 63.1 61.7 64.3 1.69 0.13 H:L ratio 7 0.41d 0.71a 0.56b 0.58b 0.55b 0.48c 0.01 <.001 14 0.46c 0.62a 0.56a,b 0.55a,b 0.58a,b 0.52b,c 0.02 0.005 21 0.38c 0.58a 0.47b 0.46b 0.46b 0.39c 0.01 <.001 a-cMeans with different superscripts in each row are significantly different (P < 0.05). Mean values are based on 2 observations per replicate (5 replicates per treatment) at each time point. 1NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. 2P-Ch, Post-Challenge. View Large As illustrated in Table 4, S. Typhimurium challenge significantly decreased amylase and protease activity measured in the jejunum at d 7, 14, and 21 post-challenge, and lipase activity at d 7 post-challenge (P < 0.05). Amylase and protease activity in birds fed the diets supplemented with PA, MOS, BA, or MA was either higher or equal to that measured in birds on the PC treatment group at all ages studied, and was consistently higher in birds fed the supplemented diets compared to birds on the NC treatment at d 21 post-challenge (P < 0.05). Additionally, at d 7 post-challenge there was no significant difference in lipase activity observed between birds on the NC treatment and those fed the supplemented diets (P > 0.05). Table 4. Effects of dietary treatments on intestinal digestive enzyme activities (U/mg of digesta protein). Treatments1 Enzymes Day P-Ch2 NC PC PA MOS BA MA SEM P-value Amylase 7 9.21a 3.64c 6.45b 6.84b 6.26b 7.12b 0.31 <.001 14 9.74a 5.58c 8.29b,c 7.78c 8.34b,c 9.57a,b 0.50 <.001 21 12.09a 8.92b 11.56a 11.32a 10.95a 11.68a 0.47 0.008 Protease 7 86.0a 57.8c 69.7b 72.4b 64.2b,c 74.6b 3.64 0.002 14 94.6a 73.1c 86.3a,b 84.6a,b 79.6b,c 89.7a,b 3.78 0.006 21 116.3a 96.1b 112.5a 111.3a 111.3a 114.6a 3.99 0.01 Lipase 7 24.31a 15.25b 21.57a 19.40a,b 20.85a 21.51a 1.82 0.01 14 24.47 19.54 19.90 21.55 19.62 22.35 1.55 0.19 21 28.76 24.05 25.61 24.62 26.40 29.60 1.70 0.16 Treatments1 Enzymes Day P-Ch2 NC PC PA MOS BA MA SEM P-value Amylase 7 9.21a 3.64c 6.45b 6.84b 6.26b 7.12b 0.31 <.001 14 9.74a 5.58c 8.29b,c 7.78c 8.34b,c 9.57a,b 0.50 <.001 21 12.09a 8.92b 11.56a 11.32a 10.95a 11.68a 0.47 0.008 Protease 7 86.0a 57.8c 69.7b 72.4b 64.2b,c 74.6b 3.64 0.002 14 94.6a 73.1c 86.3a,b 84.6a,b 79.6b,c 89.7a,b 3.78 0.006 21 116.3a 96.1b 112.5a 111.3a 111.3a 114.6a 3.99 0.01 Lipase 7 24.31a 15.25b 21.57a 19.40a,b 20.85a 21.51a 1.82 0.01 14 24.47 19.54 19.90 21.55 19.62 22.35 1.55 0.19 21 28.76 24.05 25.61 24.62 26.40 29.60 1.70 0.16 a-cMeans with different superscripts in each row are significantly different (P < 0.05). Mean values are based on 2 observations per replicate (5 replicates per treatment) at each time point 1NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. 2P-Ch, Post-Challenge View Large Table 4. Effects of dietary treatments on intestinal digestive enzyme activities (U/mg of digesta protein). Treatments1 Enzymes Day P-Ch2 NC PC PA MOS BA MA SEM P-value Amylase 7 9.21a 3.64c 6.45b 6.84b 6.26b 7.12b 0.31 <.001 14 9.74a 5.58c 8.29b,c 7.78c 8.34b,c 9.57a,b 0.50 <.001 21 12.09a 8.92b 11.56a 11.32a 10.95a 11.68a 0.47 0.008 Protease 7 86.0a 57.8c 69.7b 72.4b 64.2b,c 74.6b 3.64 0.002 14 94.6a 73.1c 86.3a,b 84.6a,b 79.6b,c 89.7a,b 3.78 0.006 21 116.3a 96.1b 112.5a 111.3a 111.3a 114.6a 3.99 0.01 Lipase 7 24.31a 15.25b 21.57a 19.40a,b 20.85a 21.51a 1.82 0.01 14 24.47 19.54 19.90 21.55 19.62 22.35 1.55 0.19 21 28.76 24.05 25.61 24.62 26.40 29.60 1.70 0.16 Treatments1 Enzymes Day P-Ch2 NC PC PA MOS BA MA SEM P-value Amylase 7 9.21a 3.64c 6.45b 6.84b 6.26b 7.12b 0.31 <.001 14 9.74a 5.58c 8.29b,c 7.78c 8.34b,c 9.57a,b 0.50 <.001 21 12.09a 8.92b 11.56a 11.32a 10.95a 11.68a 0.47 0.008 Protease 7 86.0a 57.8c 69.7b 72.4b 64.2b,c 74.6b 3.64 0.002 14 94.6a 73.1c 86.3a,b 84.6a,b 79.6b,c 89.7a,b 3.78 0.006 21 116.3a 96.1b 112.5a 111.3a 111.3a 114.6a 3.99 0.01 Lipase 7 24.31a 15.25b 21.57a 19.40a,b 20.85a 21.51a 1.82 0.01 14 24.47 19.54 19.90 21.55 19.62 22.35 1.55 0.19 21 28.76 24.05 25.61 24.62 26.40 29.60 1.70 0.16 a-cMeans with different superscripts in each row are significantly different (P < 0.05). Mean values are based on 2 observations per replicate (5 replicates per treatment) at each time point 1NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. 2P-Ch, Post-Challenge View Large As demonstrated in Table 5, the S. Typhimurium challenge decreased jejunum villus height (VH) in birds on the PC treatment at d 14 and 21 post-challenge (P < 0.05). Birds fed the supplemented diets had significantly greater VH compared to birds on the PC treatment (P < 0.05), and birds fed the diets supplemented with PA or MA presented VH that was similar to that observed in birds on the NC treatment (P > 0.05). The dietary treatments had no significant impact on crypt depth (CD; P > 0.05), but at d 7 lower VH: CD ratios were observed in birds fed the supplemented diets compared to those on the PC treatment. At d 14 and 21 there was no significant difference between birds on the NC treatment and those fed the supplemented diets (P > 0.05), except for birds fed the diets supplemented with BA. Table 5. Effects of dietary treatments on the morphology of jejunum (μm) of broilers. Treatments1 Items Day P-Ch2 NC PC PA MOS BA MA SEM P-value Villus height 7 438.4 403.6 421.5 423.9 416.3 429.5 11.14 0.36 14 654.6a 572.7c 626.0a,b 618.4b 611.7b 632.4a,b 9.46 <.001 21 922.8a 841.3c 894.6a,b 882.1b 879.4b 919.6a 10.15 <.001 Crypt depth 7 91.5 98.2 90.7 95.8 93.5 88.3 4.83 0.73 14 134.1 152.9 138.8 140.6 150.7 135.4 7.78 0.41 21 167.2 180.0 169.6 170.2 177.7 170.2 6.90 0.74 VH: CD 7 4.90a 4.13b 4.66a 4.45a,b 4.46a,b 4.89a 0.16 0.01 14 4.98a 3.77c 4.56a,b 4.44a,b 4.09b,c 4.71a,b 0.20 0.002 21 5.53a 4.72c 5.30a,b 5.20a,b 4.98b,c 5.42a,b 0.15 0.008 Treatments1 Items Day P-Ch2 NC PC PA MOS BA MA SEM P-value Villus height 7 438.4 403.6 421.5 423.9 416.3 429.5 11.14 0.36 14 654.6a 572.7c 626.0a,b 618.4b 611.7b 632.4a,b 9.46 <.001 21 922.8a 841.3c 894.6a,b 882.1b 879.4b 919.6a 10.15 <.001 Crypt depth 7 91.5 98.2 90.7 95.8 93.5 88.3 4.83 0.73 14 134.1 152.9 138.8 140.6 150.7 135.4 7.78 0.41 21 167.2 180.0 169.6 170.2 177.7 170.2 6.90 0.74 VH: CD 7 4.90a 4.13b 4.66a 4.45a,b 4.46a,b 4.89a 0.16 0.01 14 4.98a 3.77c 4.56a,b 4.44a,b 4.09b,c 4.71a,b 0.20 0.002 21 5.53a 4.72c 5.30a,b 5.20a,b 4.98b,c 5.42a,b 0.15 0.008 a-cMeans with different superscripts in each row are significantly different (P < 0.05). Mean values are based on 2 observations per replicate (5 replicates per treatment) at each time point 1NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. 2P-Ch, Post-Challenge. View Large Table 5. Effects of dietary treatments on the morphology of jejunum (μm) of broilers. Treatments1 Items Day P-Ch2 NC PC PA MOS BA MA SEM P-value Villus height 7 438.4 403.6 421.5 423.9 416.3 429.5 11.14 0.36 14 654.6a 572.7c 626.0a,b 618.4b 611.7b 632.4a,b 9.46 <.001 21 922.8a 841.3c 894.6a,b 882.1b 879.4b 919.6a 10.15 <.001 Crypt depth 7 91.5 98.2 90.7 95.8 93.5 88.3 4.83 0.73 14 134.1 152.9 138.8 140.6 150.7 135.4 7.78 0.41 21 167.2 180.0 169.6 170.2 177.7 170.2 6.90 0.74 VH: CD 7 4.90a 4.13b 4.66a 4.45a,b 4.46a,b 4.89a 0.16 0.01 14 4.98a 3.77c 4.56a,b 4.44a,b 4.09b,c 4.71a,b 0.20 0.002 21 5.53a 4.72c 5.30a,b 5.20a,b 4.98b,c 5.42a,b 0.15 0.008 Treatments1 Items Day P-Ch2 NC PC PA MOS BA MA SEM P-value Villus height 7 438.4 403.6 421.5 423.9 416.3 429.5 11.14 0.36 14 654.6a 572.7c 626.0a,b 618.4b 611.7b 632.4a,b 9.46 <.001 21 922.8a 841.3c 894.6a,b 882.1b 879.4b 919.6a 10.15 <.001 Crypt depth 7 91.5 98.2 90.7 95.8 93.5 88.3 4.83 0.73 14 134.1 152.9 138.8 140.6 150.7 135.4 7.78 0.41 21 167.2 180.0 169.6 170.2 177.7 170.2 6.90 0.74 VH: CD 7 4.90a 4.13b 4.66a 4.45a,b 4.46a,b 4.89a 0.16 0.01 14 4.98a 3.77c 4.56a,b 4.44a,b 4.09b,c 4.71a,b 0.20 0.002 21 5.53a 4.72c 5.30a,b 5.20a,b 4.98b,c 5.42a,b 0.15 0.008 a-cMeans with different superscripts in each row are significantly different (P < 0.05). Mean values are based on 2 observations per replicate (5 replicates per treatment) at each time point 1NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. 2P-Ch, Post-Challenge. View Large DISCUSSION Enteric microbiota play an important role in dictating health and consequently growth responses in broiler chickens (Van der Aar et al., 2017), but there is a concern that pathogenic bacteria in this microbiota may contaminate poultry meat during processing, resulting in foodborne diseases such as salmonellosis. Reducing Salmonella load in live animals is considered to be the most effective strategy for reducing this contamination and thus the number of human salmonellosis cases (EFSA, 2004). The current study investigated the efficacy of probiotic, prebiotic, butyric acid and their mixture on diminishing Salmonella load, by evaluating their impact on growth performance, intestinal parameters, and cecal digesta microbiota compositions in broiler chickens orally challenged with S. Typhimurium. Challenging the birds with S. Typhimurium decreased FI, BWG, and feed efficiency, which is in agreement with a number of former studies (Vandeplas et al., 2009; Marcq et al., 2011). The reduced performance observed in the challenged birds is probably due to intestinal mucosal damage induced by the S. Typhimurium (Vandeplas et al., 2009). In contrast, Mountzouris et al. (2009) and Amerah et al. (2012) found that challenging birds with Salmonella had no effect on performance response; this may be due to discrepancies between the species, strains or dose of Salmonella administered leading to different extents of intestinal damage (Malago et al., 2003), or because of differences in dietary cereal type fed to the birds (Teirlynck et al., 2009). In the present study, dietary supplementation with PA, BA, and MOS improved bird performance and reduced the presence of Salmonella in the ceca. Interestingly, only birds fed the mixture of additives (MA) showed complete recovery following the challenge, exhibiting similar BWG and FCR as the unchallenged birds, highlighting a synergistic rather than being overlapping effect of the additives. Similarly, previous studies have reported that dietary supplementation with prebiotic-based mannan-oligosaccharides (Lourenco et al., 2016; Rajani et al., 2016), B. subtilis (Park and Kim, 2014), and butyric acid (Saleem et al., 2016) resulted in improved growth performance in broilers challenged with Salmonella. The positive effect of MOS on bird's performance is likely attributed to their ability to bind to pathogens, stimulating the immune system and improving intestinal function by enhancing villus uniformity (Baurhoo et al., 2007; Lourenco et al., 2016; Pourabedin et al., 2016). Additionally, MOS-based prebiotics serve as a substrate for endogenous beneficial bacteria, thus promoting competitive exclusion of pathogenic microbes and selective colonization of beneficial microbes (Spring et al., 2000; Biggs et al., 2007). Fermentation of oligosaccharides also results in production of short-chain fatty acids, which display antimicrobial effects by penetrating the cell membrane of gram negative bacteria, such as Salmonella and coliforms, and altering the environmental pH, leading to death of the bacteria (Faber et al., 2012; Suiryanrayna and Ramana, 2015). The beneficial effects of probiotic supplements on broiler performance is associated with their role in maintaining healthy balance of bacteria in the digestive tract and improving metabolism and digestion by increasing digestive enzyme activity (Sugiharto, 2016). Organic acids potentially exhibit antimicrobial activities, by reducing gastrointestinal tract pH and stimulating protein digestion by converting pepsinogen to pepsin (Suiryanrayna and Ramana, 2015). Thus, the observed synergistic and complementary effect of the additives used in this study is likely due to the dissimilar beneficial mechanisms exhibited by each supplement. The cecal samples of the unchallenged birds were detected negative for the presence of Salmonella, indicating that there was no cross contamination between the challenged and unchallenged birds. The dietary supplements used in the current study increased Lactobacillus and Bifidobacterium concentration and decreased the population of coliforms and Salmonella in the ceca. In agreement with this study, Prado-Rebolledo et al. (2017) and Pourabedin et al. (2016) reported that supplementing LAB-based probiotics and MOS-based prebiotics to the diets of broilers challenged with Salmonella enterica decreased cecal colonization of Salmonella. Similar effects on controlling and reducing cecal Salmonella colonization have also been described as a result of dietary application of organic acids (Fernandez-Rubio et al., 2009; Saleem et al., 2016). The metabolic activity of P. acidilactici bacteria results in production of lactic acid and secretion of bacteriocins, which create unfavorable conditions for the growth of pathogenic bacteria such as coliforms and Salmonella (Taheri et al., 2010). This suggests that the success of the MA diet may be largely attributed to decreased acid-binding capacity and consequential reduction in digesta pH in birds fed this diet (Emami et al., 2017), resulting in selective stimulation of beneficial bacteria, increased production of short-chain fatty acids and thus reduced colonization of pathogenic bacteria (Sugiharto, 2016). The reliability of the H/L ratio as a biological index of stress in avian species is well documented (Maxwell, 1993; Toghyani et al., 2011); heterophils display phagocytic, chemotactic, and adhesion activities and are responsible for protecting the body against pathogens (Munyaka et al., 2012). The S. Typhimurium challenged birds had significantly increased heterophil and reduced lymphocyte numbers, causing the H/L ratio to increase from 0.38 in the NC birds to 0.58 in the PC birds at d 21 post-challenge. The ratio decreased to approximately 0.46 in the challenged birds fed the diets supplemented with PA, MOS, and BA. A complementary effect of the additives was observed for the H/L ratio, with birds on the MA treatment displaying similar ratios to those on the NC treatment at d 14 and 21 post-challenge. These findings are in agreement with previous reports highlighting that probiotics (Prado-Rebolledo et al., 2017), prebiotics (Sadeghi et al., 2013) and short-chain organic acids (Rath et al., 2006) possess immunomodulatory effects. LAB-based probiotics exert immunomodulatory activities by interacting with the host immune system, leading to changes in gene expression of cytokines and thus altered intestinal microbiota composition (Menconi et al., 2011; Pourabedin et al., 2016). Janardhana et al. (2009) demonstrated that, in addition to influencing the proliferative function and phenotypic expression of immune cells, prebiotics can also influence systemic antibody levels such as IgA, IgM, and IgG in broiler chickens. Thus, it is probable that the systemic effect of the supplements on immune response stimulation could have alleviated the impact of the S. Typhimurium challenge on increasing the H/L ratio in the current study. The S. Typhimurium challenge suppressed the activity of amylase, lipase and protease in this study. Some species of pathogenic bacteria, such as Escherichia coli (Zhang et al., 2016) and Clostridium (Mitsch et al., 2004), have been shown to inhibit secretion of digestive enzyme by damaging the villus and microvillus of the intestinal mucosa. Dietary supplementation with PA, MOS, BA, and MA ameliorated the negative impact of the challenge on digestive enzyme activity, particularly in the older birds. This is in agreement with a number of previous studies that have observed improved digestive enzyme activity in birds fed probiotics (Wang and Gu, 2010; Zhang et al., 2016), and prebiotics (Xu et al., 2003). The positive effect of these additives on enzymes activity is likely attributed to their impact on improving integrity of intestinal lining (Mitsch et al., 2004) and modifying the microbial ecosystem; for example, lactic-acid producing bacteria stimulate the secretion of digestive enzymes and reduce gastrointestinal pH (Jin et al., 2000; Xu et al., 2003; Suiryanrayna and Ramana, 2015). As stated by Choct (2009), the presence of stressors throughout the intestinal tract can quickly alter the structure of the intestine. Outcomes of the VH and CD measurements in this study, as indices of intestinal integrity and development, suggest that although intestinal development was not immediately affected by the challenge (determined at d 7 post-challenge), the S. Typhimurium challenge resulted in a noticeable reduction in VH at d 14 and 21 post-challenge, leading to a decreased VH: CD ratios. In agreement with the current findings, Rajani et al. (2016) also observed reduced VH and VH: CD ratio in the small intestine of broilers challenged with S. Typhimurium. Dietary supplementation with PA, MOS, BA, and MA improved VH and VH: CD ratio, which is reflected by the growth performance responses, in that shorter villi have a smaller surface area for nutrient absorption. Previous studies have also reported the beneficial effects of P. acidilacticis, MOS and organic acids on intestinal morphology (Baurhoo et al., 2007; Panda et al., 2009; Taheri et al., 2010). According to the literature and as observed in current study, evidently the improved intestinal microbiota balance in favor of beneficial bacteria by using the aforementioned additives can be responsible for the rectified jejunum morphological parameters (Antongiovanni et al., 2007; Panda et al., 2009; Mallo et al., 2012). CONCLUSION The results obtained in the present study indicate that S. Typhimurium challenge does not directly influence bird mortality, but it negatively affects growth performance by impairing gut morphology, digestive enzyme secretion and microbiota composition, resulting in reduced feed efficiency and bird health. The probiotic, prebiotic and butyric acid supplements fed in this study were, to some extent, efficacious at combatting the negative effects of the challenge on broiler performance and gut health. However, a complete recovery from the challenge effects was observed only when a mixture of the additives was administered, suggesting there was a synergistic effect between the additives. Therefore, it can be concluded that dietary supplementation with a combination of MOS, PA, and BA has the potential to be considered as a practical and effective strategy for controlling the incidence of Salmonella in broiler chickens in a post-antibiotic era. ACKNOWLEDGMENTS The authors greatly acknowledge and appreciate the technical support from the staff of the microbiology laboratory, College of Medical Sciences, Islamic Azad University, Isfahan (Najafabad), Iran. We are also grateful to Tak Genezist Company and Dr. Alireza Bidram for supplying the probiotic and butyric acid. REFERENCES Amerah A. M. , Mathis G. , Hofacre C. L. . 2012 . Effect of xylanase and a blend of essential oils on performance and Salmonella colonization of broiler chickens challenged with Salmonella Heidelberg . Poult. Sci. 91 : 943 – 947 . Google Scholar CrossRef Search ADS PubMed Antongiovanni M. , Buccioni A. , Petacchi F. , Leeson S. , Minieri S. , Martini A. , Cecchi R. . 2007 . Butyric acid glycerides in the diet of broiler chickens: effects on gut histology and carcass composition . Ital. J. Anim. Sci . 6 : 19 – 25 . Google Scholar CrossRef Search ADS Aviagen . 2014 . Broiler Nutrition Specification Ross 308 . Aviagen , Huntsville, Alabama . Baurhoo B. , Phillip L. , Ruiz-Feria C. A. . 2007 . Effects of purified lignin and mannan oligosaccharides on intestinal integrity and microbial populations in the ceca and litter of broiler chickens . Poult. Sci . 86 : 1070 – 1078 . Google Scholar CrossRef Search ADS PubMed Biggs P. , Parsons C. M. , Fahey G. C. . 2007 . The effects of several oligosaccharides on growth performance, nutrient digestibilities, and cecal microbial populations in young chicks . Poult. Sci . 86 : 2327 – 2336 . Google Scholar CrossRef Search ADS PubMed Bonos E. , Christaki E. , Abrahim A. , Soultos N. , Florou-Paneri P . 2011 . The influence of mannan oligosaccharides, acidifiers and their combination on cecal microflora of Japanese quail (Coturnix japonica) . Anaerobe . 17 : 436 – 439 . Google Scholar CrossRef Search ADS PubMed Chalghoumi R. , Marcq C. , Thewis A. , Portetelle D. , Beckers Y. . 2009 . Effects of feed supplementation with specific hen egg yolk antibody (immunoglobin Y) on Salmonella species cecal colonization and growth performances of challenged broiler chickens . Poult. Sci. 88 : 2081 – 2092 . Google Scholar CrossRef Search ADS PubMed Cheng G. , Hao H. , Xie S. , Wang X. , Dai M. , Huang L. , Yuan Z. . 2014 . Antibiotic alternatives: the substitution of antibiotics in animal husbandry? Front. Microbiol . 4 : 137 – 156 . Choct M. 2009 . Managing gut health through nutrition . Br. Poult. Sci. 50 : 9 – 15 . Google Scholar CrossRef Search ADS PubMed EFSA . 2004 . Opinion of the scientific panel on biological hazards on a request from the Commission related to the use of vaccines for the control of Salmonella in poultry . EFSA J. 114 : 1 – 74 . Emami N. K. , Daneshmand A. , Naeini S. Z. , Graystone E. N. , Broom L. J. . 2017 . Effects of commercial organic acid blends on male broilers challenged with E. coli K88: Performance, microbiology, intestinal morphology, and immune response . Poult. Sci. 96 : 3254 – 3263 . Google Scholar CrossRef Search ADS PubMed Faber T. A. , Dilger R. N. , Hopkins A. C. , Price N. P. , Fahey G. C. . 2012 . Effects of oligosaccharides in a soybean meal-based diet on fermentative and immune responses in broiler chicks challenged with Eimeria acervulina . Poult. Sci . 91 : 3132 – 3140 . Google Scholar CrossRef Search ADS PubMed Fernandez-Rubio C. , Ordonez C. , Abad-Gonzalez J. , Garcia-Gallego A. , Pilar M. , Honrubia J. , Mallo J. , Balana-Fouce R . 2009 . Butyric acid-based feed additives help protect broiler chickens from Salmonella enteritidis infection . Poult. Sci. 88 : 943 – 948 . Google Scholar CrossRef Search ADS PubMed Janardhana V. , Broadway M. M. , Bruce M. P. , Lowenthal J. W. , Geier M. S. , Hughes R. H. , Bean A. G. D. . 2009 . Prebiotics modulate immune responses in gut-associated lymphoid tissue of chickens . J. Nutr . 139 : 1404 – 1409 . Google Scholar CrossRef Search ADS PubMed Jin L. Z. , Ho Y. W. , Abdullah N. , Jalaludin S. . 2000 . Digestive and bacterial enzyme activities in broilers fed diets supplemented with Lactobacillus cultures . Poult. Sci. 79 : 886 – 891 . Google Scholar CrossRef Search ADS PubMed Lourenco M. C. , de Souza A. M. , Hayashi R. M. , da Silva A. B. , Santin E. . 2016 . Immune response of broiler chickens supplemented with prebiotic from Saccharomyces cerevisiae challenged with Salmonella enteritidis or Minnesota . J. Appl. Poult. Res. 25 : 165 – 172 . Google Scholar CrossRef Search ADS Malago J. J. , Koninkx J. F. J. G. , Ovelgonne H. H. , van Asten F .J .A. M. , Swennenhuis J. F. , van Dijk J. E. . 2003 . Expression levels of heat shock proteins in enterocyte-like Caco-2 cells after exposure to Salmonella enteritidis . Cell Stress Chaperones . 8 : 194 – 203 . Google Scholar CrossRef Search ADS PubMed Mallo J. J. , Puyalto M. , Rama Rao S. V. . 2012 . Evaluation of the effect of sodium butyrate addition to broilers diet on energy and protein digestibility, productive parameters and size of intestinal villi of animals . Feed. Livest . 8 : 26 – 30 . Marcq C. , Cox E. , Szalo I. M. , Thewis A. , Beckers Y. . 2011 . Salmonella Typhimurium oral challenge model in mature broilers: Bacteriological, immunological, and growth performance aspects . Poult. Sci. 90 : 59 – 67 . Google Scholar CrossRef Search ADS PubMed Maxwell M. H. 1993 . Avian blood leucocyte responses to stress . W. Poult. Sci. J. 49 : 34 – 43 . Google Scholar CrossRef Search ADS Menconi A. , Wolfenden A. D. , Shivaramaiah S. , Terraes J. C. , Urbano T. , Kuttel J. , Kremer C. , Hargis B. M. , Tellez G. . 2011 . Effect of lactic acid bacteria probiotic culture for the treatment of Salmonella enterica serovar Heidelberg in neonatal broiler chickens and turkey poults . Poult. Sci. 90 : 561 – 565 . Google Scholar CrossRef Search ADS PubMed Menconi A. , Kuttappan V. A. , Hernandez-Velasco X. , Urbano T. , Matte F. , Layton S. , Kallapura G. , Latorre J. , Morales B. E. , Prado O. , Vicente J. L. , Barton J. , Filho R. L. A. , Lovato M. , Hargis B. M. , Tellez G. . 2014 . Evaluation of a commercially available organic acid product on body weight loss, carcass yield, and meat quality during preslaughter feed withdrawal in broiler chickens: a poultry welfare and economic perspective . Poult. Sci . 93 : 448 – 455 . Google Scholar CrossRef Search ADS PubMed Mitsch P. , Zitterl-Eglseer K. , Kohler B. , Gabler C. , Losa R. , Zimpernik I. . 2004 . The effect of two different blends of essential oil components on the proliferation of Clostridium perfringens in the intestines of broiler chickens . Poult. Sci. 83 : 669 – 675 . Google Scholar CrossRef Search ADS PubMed Mountzouris K. C. , Balaskas C. , Xanthakos I. , Tzivinikou A. , Fegeros K. . 2009 . Effects of a multi-species probiotic on biomarkers of competitive exclusion efficacy in broilers challenged with Salmonella Enteritidis . Br. Poult. Sci. 50 : 467 – 478 . Google Scholar CrossRef Search ADS PubMed Munyaka P. M. , Echeverry H. , Yitbarek A. , Camelo-Jaimes G. , Sharif S. , Guenter W. , House J. D. , Rodriguez-Lecompte J. C. . 2012 . Local and systemic innate immunity in broiler chickens supplemented with yeast-derived carbohydrates . Poult. Sci . 91 : 2164 – 2172 . Google Scholar CrossRef Search ADS PubMed Panda A. K. , Rao S. V. R. , Raju M. V. L. N. , Sunder G. S. . 2009 . Effect of butyric acid on performance, gastrointestinal tract health and carcass characteristics in broiler chickens. Asian-Australas . J. Anim. Sci . 22 : 1026 – 1031 . Park J. H. , Kim I. H. . 2014 . Supplemental effect of probiotic Bacillus subtilis B2A on productivity, organ weight, intestinal Salmonella microflora, and breast meat quality of growing broiler chicks . Poult. Sci. 93 : 2054 – 2059 . Google Scholar CrossRef Search ADS PubMed Partanen K. H. , Mroz Z. . 1999 . Organic acids for performance enhancement in pig diets . Nutr. Res. Rev . 12 : 117 – 145 . Google Scholar CrossRef Search ADS PubMed Patterson J. A. , Burkholder K. M. . 2003 . Application of prebiotics and probiotics in poultry production . Poult. Sci . 82 : 627 – 631 . Google Scholar CrossRef Search ADS PubMed Pourabedin M. , Chen Q. , Yang M. , Zhao X. . 2016 . Mannan-and xylooligosaccharides modulate cecal microbiota and expression of inflammatory-related cytokines and reduce cecal Salmonella Enteritidis colonisation in young chickens . FEMS. Microbiol. Ecol. 93 : 226 . Google Scholar CrossRef Search ADS Prado-Rebolledo O. F. , Delgado-Machuca J. D. J. , Macedo-Barragan R. J. , Garcia-Marquez L. J. , Barrera M. J. E. , Latorre J. D. , Hernandez-Velasco X. , Tellez G. . 2017 . Evaluation of a selected lactic acid bacteria-based probiotic on Salmonella enterica serovar Enteritidis colonization and intestinal permeability in broiler chickens . Avian Pathol . 46 : 90 – 94 . Google Scholar CrossRef Search ADS PubMed Rajani J. , Dastar B. , Samadi F. , Karimi Torshizi M. A. , Abdulkhani A. , Esfandyarpour S. . 2016 . Effect of extracted galactoglucomannan oligosaccharides from pine wood (Pinus brutia) on Salmonella Typhimurium colonisation, growth performance and intestinal morphology in broiler chicks . Br. Poult. Sci. 57 : 682 – 692 . Google Scholar PubMed Rath N. C. , Huff W. E. , Huff G. R. . 2006 . Effects of humic acid on broiler chickens . Poult. Sci . 85 : 410 – 414 . Google Scholar CrossRef Search ADS PubMed Sadeghi A. A. , Mohammadi A. , Shawrang P. , Aminafshar M. . 2013 . Immune responses to dietary inclusion of prebiotic-based mannan-oligosaccharide and β-glucan in broiler chicks challenged with Salmonella enteritidis . Turk. J. Vet. Anim. Sci. 37 : 206 – 213 . SAS Institute Inc . 2010 . SAS User's Guide. Statistics. Version 9.3ed . SAS Inst. Inc. , Cary, NC . Saleem G. , Ramzaan R. , Khattak F. M. , Akhtar R. . 2016 . Effects of acetic acid supplementation in broiler chickens orally challenged with Salmonella Pullorum . Turk. J. Vet. Anim. Sci. 40 : 434 – 443 . Google Scholar CrossRef Search ADS Spring P. , Wenk C. , Dawson K. A. , Newman K. E. . 2000 . The effects of dietary mannaoligosaccharides on cecal parameters and the concentrations of enteric bacteria in the ceca of Salmonella-challenged broiler chicks . Poult. Sci. 79 : 205 – 211 . Google Scholar CrossRef Search ADS PubMed Sugiharto S. 2016 . Role of nutraceuticals in gut health and growth performance of poultry . J. Sau. Soc. Agric. Sci. 15 : 99 – 111 . Suiryanrayna M. V. , Ramana J. V. . 2015 . A review of the effects of dietary organic acids fed to swine . J. Anim. Sci. Biotechnol. 6 : 45 . Google Scholar CrossRef Search ADS PubMed Taheri H. R. , Moravej H. , Malakzadegan A. , Tabandeh F. , Zaghari M. , Shivazad M. , Adibmoradi M. . 2010 . Efficacy of Pediococcus acidlactici-based probiotic on intestinal Coliforms and villus height, serum cholesterol level and performance of broiler chickens . Afr. J. Biotechnol. 9 : 7564 – 7567 . Google Scholar CrossRef Search ADS Teirlynck E. , Haesebrouck F. , Pasmans F. , Dewulf J. , Ducatelle R. , Van Immerseel F. . 2009 . The cereal type in feed influences Salmonella Enteritidis colonization in broilers . Poult. Sci. 88 : 2108 – 2112 . Google Scholar CrossRef Search ADS PubMed Thung T. Y. , Mahyudin N. A. , Basri D. F. , Wan Mohamed Radzi C. W. J. , Nakaguchi Y. , Nishibuchi M. , Radu S. . 2016 . Prevalence and antibiotic resistance of Salmonella Enteritidis and Salmonella Typhimurium in raw chicken meat at retail markets in Malaysia . Poult. Sci. 95 : 1888 – 1893 . Google Scholar CrossRef Search ADS PubMed Toghyani M. , Toghyani M. , Gheisari A. , Ghalamkari G. , Mohammadrezaei M. . 2010 . Growth performance, serum biochemistry and blood hematology of broiler chicks fed different levels of black seed (Nigellasativa) and peppermint (Mentha piperita) . Livest. Sci . 129 : 173 – 178 . Google Scholar CrossRef Search ADS Toghyani M. , Toghyani M. , Gheisari A. , Ghalamkari G. , Eghbalsaied S. . 2011 . Evaluation of cinnamon and garlic as antibiotic growth promoter substitutions on performance, immune responses, serum biochemical and haematological parameters in broiler chicks . Livest. Sci . 138 : 167 – 173 . Google Scholar CrossRef Search ADS Troy E. B. , Kasper D. L. . 2010 . Beneficial effects of Bacteroides fragilis polysaccharides on the immune system . Front. Biosci. 15 : 25 – 34 . Google Scholar CrossRef Search ADS Van der Aar P. J. , Molist F. , van der Klis J. D. . 2017 . The central role of intestinal health on the effect of feed additives on feed intake in swine and poultry . Anim. Feed Sci. Technol . 233 : 64 – 75 . Google Scholar CrossRef Search ADS Vandeplas S. , Dauphin R. D. , Thiry C. , Beckers Y. , Welling G. W. , Thonart P. , Thewis A. . 2009 . Efficiency of a Lactobacillus plantarum-xylanase combination on growth performances, microflora populations, and nutrient digestibilities of broilers infected with Salmonella Typhimurium . Poult. Sci. 88 : 1643 – 1654 . Google Scholar CrossRef Search ADS PubMed Wang Y. , Gu Q. . 2010 . Effect of probiotic on growth performance and digestive enzyme activity of Arbor Acres broilers . Res. Vet. Sci. 89 : 163 – 170 . Google Scholar CrossRef Search ADS PubMed Xu Z. R. , Hu C. H. , Xia M. S. , Zhan X. A. , Wang M. Q. . 2003 . Effects of dietary fructooligosaccharide on digestive enzyme activities, intestinal microflora and morphology of male broilers . Poult. Sci . 82 : 1030 – 1036 . Google Scholar CrossRef Search ADS PubMed Zhang L. , Zhang L. , Zeng X. , Zhou L. , Cao G. , Yang C. . 2016 . Effects of dietary supplementation of probiotic, Clostridium butyricum, on growth performance, immune response, intestinal barrier function, and digestive enzyme activity in broiler chickens challenged with Escherichia coli K88 . J. Anim. Sci. Biotechnol. 7 : 3 – 12 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Effects of Pediococcus acidilactici, mannan-oligosaccharide, butyric acid and their combination on growth performance and intestinal health in young broiler chickens challenged with Salmonella Typhimurium

Loading next page...
 
/lp/ou_press/effects-of-pediococcus-acidilactici-mannan-oligosaccharide-butyric-E99MR76wNq
Publisher
Oxford University Press
Copyright
© 2018 Poultry Science Association Inc.
ISSN
0032-5791
eISSN
1525-3171
D.O.I.
10.3382/ps/pey035
Publisher site
See Article on Publisher Site

Abstract

ABSTRACT This study compared the efficacy of Pediococcus acidilactici, mannan-oligosaccharide, butyric acid, and their combination on growth performance and intestinal health in broiler chickens challenged with S. Typhimurium. Ross 308 male broilers (n = 420) were randomly assigned to one of the 6 treatments, resulting in 5 replicate pens of 14 chicks per treatment. The treatments included a negative control [(NC), no additive, not challenged]; positive control [(PC), no additive, but challenged with S. Typhimurium at d 3 posthatch], and 4 groups whereby birds were challenged with S. Typhimurium at d 3 posthatch and fed diets supplemented with either probiotic [0.1 g/kg Pediococcus acidilactici (PA)], prebiotic [2 g/kg mannan-oligosaccharides (MOS)], organic acid [0.5 g/kg butyric acid (BA)], or a combination of the 3 additives (MA). The S. Typhimurium challenge decreased feed intake, body weight gain and increased feed conversion ratio and reduced jejunum villus height (VH) and VH to crypt depth (CD) ratio (P < 0.05). Birds on the MA treatment exhibited similar performance to birds on the NC treatment (P > 0.05) and had a lower population of Salmonella in the ceca compared with birds on the PC treatment, at d 14 and 21 post-challenge (P < 0.05). The lowest heterophil to lymphocyte ratio was observed in birds on the MA and NC treatments (P < 0.05). Birds fed diets supplemented with MA or PA had greater VH and VH: CD ratio than birds on the PC treatment at d 7, 14 and 21 d post-challenge (P < 0.05). Suppressed amylase and protease activity was observed as a result of the S. Typhimurium challenge; the enzyme levels were restored in birds fed the additive-supplemented diets, when compared to the birds on the PC treatment, particularly at d 21 post-challenge (P < 0.05). These results indicate that dietary supplementation with a combination of PA, BA, and MOS in broiler chickens could be used as an effective tool for controlling S. Typhimurium and promoting growth performance. INTRODUCTION Salmonella enterica var. Typhimurium (S. Typhimurium) is one of the most prevalent serotypes of Salmonella, a key causative agent of salmonellosis, which is a disease that enters the human food chain through animal products, particularly raw poultry products (Chalghoumi et al., 2009; Thung et al., 2016). In-feed antibiotics have long been used at sub-therapeutic doses in poultry feed to control gut pathogenic bacteria load and hence to enhance feed efficiency, promote animal growth, and improve the quality of the animal products (Cheng et al., 2014). However, the increased risk of bacteria acquiring resistance to antibiotics and their residue in end-products such as meat and eggs led to a ban on the use of in-feed antibiotics as growth promotors (AGP) more than a decade ago in many countries in the European Union (Toghyani et al., 2010; Sugiharto, 2016). As a result of this growing pressure on livestock producers worldwide, various alternative strategies have been proposed and investigated as potential substitutes for AGPs in animal feeds. Particular attention has been paid to the use of probiotics (Menconi et al., 2011; Park and Kim, 2014), prebiotics (Pourabedin et al., 2016; Rajani et al., 2016), and organic acids (Fernandez-Rubio et al., 2009; Saleem et al., 2016), as AGPs alternatives in poultry feed. There is evidence to suggest that these aforementioned products exert beneficial effects on performance parameters, microbiota composition, and intestinal integrity, and potentially reduce the levels of pathogenic bacteria such as Salmonella in the tract. However, these positive effects have not always been consistent (Van der Aar et al., 2017). The main proposed modes of action of probiotics include 1) antagonistic action towards pathogenic bacteria, by secreting products that inhibit their development, such as bacteriocins, organic acids, and hydrogen peroxide, and 2) competitive exclusion, by competing with bacteria for locations in the intestinal mucous membrane to adhere to and nutrients (Patterson and Burkholder, 2003). Prebiotics serve as a substrate for endogenous beneficial bacteria, thus promoting competitive exclusion of pathogenic microbes and selective colonization by beneficial microbes (Biggs et al., 2007). Oligosaccharides display prebiotics properties, but they have also been reported to present immunomodulatory beneficial effects in the gut such as modifying clearance efficiency of pathogenic bacteria, activating T cell-dependent immune responses and repression of pro-inflammatory cytokines (Troy and Kasper, 2010; Bonos et al., 2011). Organic acids, such as lactic, acetic, butyric, tannic, fumaric and propionic acids, display antimicrobial properties and play a crucial role in controlling the population of pathogenic bacteria (Menconi et al., 2014), likely by lowering gastrointestinal tract pH (Partanen and Mroz, 1999) and stimulating protein digestion by converting pepsinogen to pepsin (Suiryanrayna and Ramana, 2015). The differing mechanisms and modes of action exhibited by probiotics, prebiotics, and organic acids suggest that there could be a complementary synergistic effect resulting from supplementing diets with a mixture of these additives, especially under the stressful conditions imposed by a S. Typhimurium challenge (Rajani et al., 2016). In view of an evident dearth of data in this field, the current study was designed to determine the efficacy of Pediococcus acidilactici (a species of gram-positive cocci that produces lactic acid and secrets a bacteriocins known as pediocins) as a probiotic supplement, mannan oligosaccharides as a prebiotic supplement, and micro-encapsulated butyric acid, singularly or in combination, on growth performance and gut health of young broiler chicks challenged with S. Typhimurium. MATRIALS AND METHODS Experimental Design, Dietary Treatments, and Bird Husbandry The experimental procedures in this study were reviewed and approved by the Animal Ethics committee of Islamic Azad University, Isfahan Branch, based on institutional and national guidelines for the care and use of animals. A total of 420 day-old Ross 308 male broiler chicks were obtained from a commercial hatchery (Navid Morgh Guilan Company., Guilan, Iran), were fed a common starter diet for the first 3 d. At d 3 posthatch, birds were weighed and randomly assigned to one of the 6 experimental treatments with 5 replicate pens of 14 birds each to have a starting body weight of 72 ± 1.5 g/bird. The treatments consisted of negative control [(NC), no additive and no challenge with S. Typhimurium]; positive control [(PC), no additive, but challenged with S. Typhimurium], and 4 groups whereby birds were challenged with S. Typhimurium and fed diets supplemented with either probiotic (Pediococcus acidilactici at 0.01% [Pedi Guard, Iran, concentration 1 × 1010 CFU/g]; PA), prebiotic (0.2% mannan-oligosaccharides, [ActiveMOS®, Biorigin, Brazil]; MOS), butyric acid (0.05% butyric acid provided in an encapsulated form consisting of 50% butyrate salt [ButiPEARL; Kemin Industries, Herentals, Belgium]; BA), or a combination of the 3 additives (MA). All the challenged birds were orally administered 105 CFU of S. Typhimurium at d 3 posthatch. Birds were allowed ad libitum access to the treatment diets and water for the duration of the trial (d 0 to 24). A basal corn-SBM diet was formulated to meet the nutrient requirements according to the Ross 308 (Aviagen, 2014) for starter (0- to 10-d) and grower (10- to 24-d) periods (Table 1). The temperature and lighting programs used followed the Ross 308 breed management recommendations (Aviagen, 2014). Table 1. Compositions of the experimental diets. % as-is Ingredient Starter (d 1–10) Grower (d 11–24) Corn 55.5 65.5 Soybean meal 37.7 28.1 Sunflower oil 2.37 2.24 Limestone 1.09 0.99 Dicalcium phosphate1 1.74 1.54 Sodium chloride 0.21 0.18 Sodium bicarbonate 0.16 0.19 Vitamin premix2 0.25 0.25 Mineral premix3 0.25 0.25 Choline chloride 60% 0.122 0.116 DL-methionine 0.296 0.254 L-lysine 0.188 0.269 L-threonine 0.091 0.079 Nutrient composition Metabolizable energy (Kcal/kg) 3000 3100 Crude protein (%) 22.94 19.41 Dig4 lysine (%) 1.28 1.11 Dig methionine (%) 0.63 0.55 Dig methionine + cysteine (%) 0.95 0.83 Dig threonine (%) 0.86 0.72 Calcium (%) 0.90 0.80 Available phosphorus (%) 0.45 0.40 Sodium (%) 0.16 0.17 Chloride (%) 0.21 0.21 Choline (mg/kg) 1700 1500 % as-is Ingredient Starter (d 1–10) Grower (d 11–24) Corn 55.5 65.5 Soybean meal 37.7 28.1 Sunflower oil 2.37 2.24 Limestone 1.09 0.99 Dicalcium phosphate1 1.74 1.54 Sodium chloride 0.21 0.18 Sodium bicarbonate 0.16 0.19 Vitamin premix2 0.25 0.25 Mineral premix3 0.25 0.25 Choline chloride 60% 0.122 0.116 DL-methionine 0.296 0.254 L-lysine 0.188 0.269 L-threonine 0.091 0.079 Nutrient composition Metabolizable energy (Kcal/kg) 3000 3100 Crude protein (%) 22.94 19.41 Dig4 lysine (%) 1.28 1.11 Dig methionine (%) 0.63 0.55 Dig methionine + cysteine (%) 0.95 0.83 Dig threonine (%) 0.86 0.72 Calcium (%) 0.90 0.80 Available phosphorus (%) 0.45 0.40 Sodium (%) 0.16 0.17 Chloride (%) 0.21 0.21 Choline (mg/kg) 1700 1500 1Dicalcium phosphate contained: phosphorus, 18%; calcium, 21%. 2Supplied per kg of diet: 1.8 mg all-trans-retinyl acetate, 0.02 mg cholecalciferol, 8.3 mg alphatocopheryl acetate, 2.2 mg menadione, 2 mg pyridoxine HCl, 8 mg cyanocobalamin,10 mg nicotine amid, 0.3 mg folic acid, 20 mg D-biotin and 160 mg choline chloride. 3Supplied per kg of diet: 32 mg Mn (MnSO4⋅H2O), 16 mg Fe (FeSO4⋅7H2O), 24 mg Zn (ZnO), 2 mg Cu (CuSO4⋅5H2O), 800 μg I (KI), 200 μg Co (CoSO4) and 60 μg Se. 4Dig, Digestible. View Large Table 1. Compositions of the experimental diets. % as-is Ingredient Starter (d 1–10) Grower (d 11–24) Corn 55.5 65.5 Soybean meal 37.7 28.1 Sunflower oil 2.37 2.24 Limestone 1.09 0.99 Dicalcium phosphate1 1.74 1.54 Sodium chloride 0.21 0.18 Sodium bicarbonate 0.16 0.19 Vitamin premix2 0.25 0.25 Mineral premix3 0.25 0.25 Choline chloride 60% 0.122 0.116 DL-methionine 0.296 0.254 L-lysine 0.188 0.269 L-threonine 0.091 0.079 Nutrient composition Metabolizable energy (Kcal/kg) 3000 3100 Crude protein (%) 22.94 19.41 Dig4 lysine (%) 1.28 1.11 Dig methionine (%) 0.63 0.55 Dig methionine + cysteine (%) 0.95 0.83 Dig threonine (%) 0.86 0.72 Calcium (%) 0.90 0.80 Available phosphorus (%) 0.45 0.40 Sodium (%) 0.16 0.17 Chloride (%) 0.21 0.21 Choline (mg/kg) 1700 1500 % as-is Ingredient Starter (d 1–10) Grower (d 11–24) Corn 55.5 65.5 Soybean meal 37.7 28.1 Sunflower oil 2.37 2.24 Limestone 1.09 0.99 Dicalcium phosphate1 1.74 1.54 Sodium chloride 0.21 0.18 Sodium bicarbonate 0.16 0.19 Vitamin premix2 0.25 0.25 Mineral premix3 0.25 0.25 Choline chloride 60% 0.122 0.116 DL-methionine 0.296 0.254 L-lysine 0.188 0.269 L-threonine 0.091 0.079 Nutrient composition Metabolizable energy (Kcal/kg) 3000 3100 Crude protein (%) 22.94 19.41 Dig4 lysine (%) 1.28 1.11 Dig methionine (%) 0.63 0.55 Dig methionine + cysteine (%) 0.95 0.83 Dig threonine (%) 0.86 0.72 Calcium (%) 0.90 0.80 Available phosphorus (%) 0.45 0.40 Sodium (%) 0.16 0.17 Chloride (%) 0.21 0.21 Choline (mg/kg) 1700 1500 1Dicalcium phosphate contained: phosphorus, 18%; calcium, 21%. 2Supplied per kg of diet: 1.8 mg all-trans-retinyl acetate, 0.02 mg cholecalciferol, 8.3 mg alphatocopheryl acetate, 2.2 mg menadione, 2 mg pyridoxine HCl, 8 mg cyanocobalamin,10 mg nicotine amid, 0.3 mg folic acid, 20 mg D-biotin and 160 mg choline chloride. 3Supplied per kg of diet: 32 mg Mn (MnSO4⋅H2O), 16 mg Fe (FeSO4⋅7H2O), 24 mg Zn (ZnO), 2 mg Cu (CuSO4⋅5H2O), 800 μg I (KI), 200 μg Co (CoSO4) and 60 μg Se. 4Dig, Digestible. View Large Bacterial Inoculum Preparation The S. Typhimurium strain (PTCC 1709) was obtained as a freeze-dried powder from the Iranian Research Organization for Science and Technology, Tehran. The frozen bacterial culture was thawed and cultured in a trypticase soy broth for 24 at 37°C. The number of CFU was determined by diluting the inoculum with sterile phosphate-buffered saline (PBS) and plating it on culture medium XLT4 agar for 24 h at 37°C. At d 3 posthatch, all birds (except those in the NC treatment group) were orally administered a one-off dose of 0.5 mL of 1 × 105 CFU S. Typhimurium suspension. The same dose per os of sterile PBS was orally administered to the birds in unchallenged group. Performance Measurements Growth performance parameters including feed intake (FI) and body weight gain (BWG) were measured during the starter (d 0 to 7 post challenge), grower (d 7 to 21 post challenge), and overall experimental period (d 0 to 21 post challenge), and used to calculate the feed conversion ratio (FCR), adjusted for mortality. Ten birds per treatment (2 birds from each replicate pen) were randomly selected for blood, digesta and tissue sample collection at d 7, 14, and 21 post-challenge. Hematological Parameters At d 7, 14, and 21 post-challenge, 3 mL of blood was obtained from the 2 birds per pen by puncturing the brachial vein and individually collecting the blood into non-heparinized tubes. Blood smears were prepared by the May-Grunwald-Giemsa coloring technique and heterophil to lymphocyte (H/L) ratio was calculated by counting 100 leukocytes per slide. Enumeration of Cecal Bacteria Following blood sample collection, the 2 birds were weighed and euthanized by CO2 asphyxiation. The contents of the ceca were collected by gently squeezing the digesta into plastic containers, and pooled per replicate. To determine S. Typhimurium counts, 1 g of the ceca digesta was diluted with 0.1% peptone water, homogenized for 1 min and serial dilutions were subsequently prepared in the same diluent. Samples at the optimum dilution level were then selected and 0.1 mL was plated in triplicate on XLT4 Agar (Difco) containing 100 mL of Nalidixic acid. The plates were then incubated for 24 h at 37°C under anaerobic conditions. To determine Lactobacillus, Bifidobacterium, and coliform counts in the ceca samples, again 10-fold serial dilutions were conducted with peptone water and then 0.1 mL of sample from the optimum dilution level was cultured on plates containing modified Rogosa SL agar, TOS Propionate agar, and MacConkey agar, respectively. Plates were incubated in anaerobic conditions for 24 to 48 h at 37°C. Following incubation, the number of colonies on each plate was counted and the obtained numbers were multiplied by reversed dilution, and reported as the number of CFU per 1 g sample. Intestinal Morphology The digesta content of the jejunum from the 2 birds was gently flushed out with ultra-pure water into a container, and then approximately 3 cm of the jejunum tissue (middle section) was transected and immersed in 10% buffered formalin. Tissues were serial dehydrated by passing the tissue through increasing concentrations of ethyl alcohol, before being embedded in paraffin wax. Histological examinations were carried out according to the method described by Xu et al. (2003). A total of 10 well-oriented, intact villi and crypts were randomly selected in duplicate from each tissue sample and the average of 20 values were obtained for each bird. All morphological parameters were measured using the ImageJ software package (http://rsb.info.nih.gov/ij/). Enzyme Activity The collected jejunum digesta samples were diluted 10 times with phosphate- saline buffer, based on weight, and were then centrifuged at 18,000 × g for 20 min at 4°C. The supernatant was stored at −80°C prior to enzyme analysis. Samples were processed to determine amylase (EC 3.2.1.1), lipase (EC 3.1.1.3), and neutral protease activities following the methods described by Xu et al. (2003). One unit of amylase activity was defined as the amount of amylase required to release 1 mg of glucose after 30 min incubation at 40°C, per mg of intestinal digesta protein. Lipase activity was equal to the volume (in mL) of 0.05 M NaOH required to neutralize the fatty acids liberated during a 6-h incubation with 3 mL of lipase substrate at 38°C, per mg of intestinal digesta protein. The protease activity unit was defined as milligrams of azocasein degraded after 2 h incubation at 37°C, per mg of intestinal digesta protein. Statistical Analysis Firstly, Kolmogorov-Smirnov testing was conducted to confirm normality of the data. Data were then subjected to one-way analysis of variance (ANOVA) using General Linear Model procedure of SAS 9.3 package (SAS Institute Inc., 2010) to determine the equality of the means. Each pen was considered as an experimental unit. The values presented in the tables represent the mean for each treatment, with a pooled standard error of mean (SEM). Tukey's HSD test was used to make pairwise comparisons between means, where appropriate. Statistical significance was declared at P < 0.05. RESULTS Bird's Performance and Mortality Rate The effects of the treatments on bird performance are reported in Table 2. The S. Typhimurium challenge decreased FI, BWG, and increased FCR in birds in the PC treatment group compared to the NC treatment group, at all time periods tested (P < 0.05). Dietary supplementation of PA, MOS, BA, and in particular their combination, improved FI, BWG, and FCR in the challenged birds, compared to birds in the PC treatment group (P < 0.05), at all measured time periods. However, only birds in the MA treatment group displayed a body weight gain (0 to 21 d post challenge) that was statistically similar to birds in the NC treatment group (P > 0.05); birds fed the diets containing just one of the dietary additives had a lower BWG and higher FCR compared to birds on the NC treatment (P < 0.05). No significant effect of challenge or dietary treatment was observed on bird mortality during the experimental period (data not shown). Table 2. Effects of dietary treatments on growth performance of broilers. Treatments1 Item2 NC PC PA MOS BA MA SEM P-value Day 3 BW (g/b) 72.5 73.2 72.9 73.4 73.0 72.7 0.48 0.88 0 to 7 d P-Ch BWG (g/b) 185.7a 144.5c 168.1b 166.8b 165.2b 170.5b 4.45 <.001 FI (g/b) 218.3a 186.9b 207.3a 205.5a 204.7a,b 203.6a,b 5.84 0.03 FCR (g/g) 1.17c 1.29a 1.23b 1.23b 1.24b 1.19b,c 0.01 0.001 7 to 21 d P-Ch BWG (g/b) 720.4a 604.2c 681.8b 682.9b 676.1b 712.2a 8.55 <.001 FI (g/b) 1004.3a 945.5b 983.9a,b 996.0a 998.4a 1011.4a 13.86 0.03 FCR (g/g) 1.39c 1.56a 1.44b,c 1.46b 1.47b 1.42b,c 0.02 0.001 0 to 21 d P-Ch BWG (g/b) 906.2a 748.7d 850.1b,c 849.8b,c 841.3c 882.8a,b 11.03 <.001 FI (g/b) 1222a 1132b 1191a 1201a 1203a 1215a 18.27 0.02 FCR(g/g) 1.35c 1.51a 1.40b,c 1.41b 1.43b 1.37b,c 0.02 <.001 Treatments1 Item2 NC PC PA MOS BA MA SEM P-value Day 3 BW (g/b) 72.5 73.2 72.9 73.4 73.0 72.7 0.48 0.88 0 to 7 d P-Ch BWG (g/b) 185.7a 144.5c 168.1b 166.8b 165.2b 170.5b 4.45 <.001 FI (g/b) 218.3a 186.9b 207.3a 205.5a 204.7a,b 203.6a,b 5.84 0.03 FCR (g/g) 1.17c 1.29a 1.23b 1.23b 1.24b 1.19b,c 0.01 0.001 7 to 21 d P-Ch BWG (g/b) 720.4a 604.2c 681.8b 682.9b 676.1b 712.2a 8.55 <.001 FI (g/b) 1004.3a 945.5b 983.9a,b 996.0a 998.4a 1011.4a 13.86 0.03 FCR (g/g) 1.39c 1.56a 1.44b,c 1.46b 1.47b 1.42b,c 0.02 0.001 0 to 21 d P-Ch BWG (g/b) 906.2a 748.7d 850.1b,c 849.8b,c 841.3c 882.8a,b 11.03 <.001 FI (g/b) 1222a 1132b 1191a 1201a 1203a 1215a 18.27 0.02 FCR(g/g) 1.35c 1.51a 1.40b,c 1.41b 1.43b 1.37b,c 0.02 <.001 a-cMeans with different superscripts in each row are significantly different (P < 0.05). 1NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. 2P-Hatch, Posthatch; P-Ch, Post-Challenge; BWG, body weight gain; FI, feed intake; FCR, feed conversion ratio. View Large Table 2. Effects of dietary treatments on growth performance of broilers. Treatments1 Item2 NC PC PA MOS BA MA SEM P-value Day 3 BW (g/b) 72.5 73.2 72.9 73.4 73.0 72.7 0.48 0.88 0 to 7 d P-Ch BWG (g/b) 185.7a 144.5c 168.1b 166.8b 165.2b 170.5b 4.45 <.001 FI (g/b) 218.3a 186.9b 207.3a 205.5a 204.7a,b 203.6a,b 5.84 0.03 FCR (g/g) 1.17c 1.29a 1.23b 1.23b 1.24b 1.19b,c 0.01 0.001 7 to 21 d P-Ch BWG (g/b) 720.4a 604.2c 681.8b 682.9b 676.1b 712.2a 8.55 <.001 FI (g/b) 1004.3a 945.5b 983.9a,b 996.0a 998.4a 1011.4a 13.86 0.03 FCR (g/g) 1.39c 1.56a 1.44b,c 1.46b 1.47b 1.42b,c 0.02 0.001 0 to 21 d P-Ch BWG (g/b) 906.2a 748.7d 850.1b,c 849.8b,c 841.3c 882.8a,b 11.03 <.001 FI (g/b) 1222a 1132b 1191a 1201a 1203a 1215a 18.27 0.02 FCR(g/g) 1.35c 1.51a 1.40b,c 1.41b 1.43b 1.37b,c 0.02 <.001 Treatments1 Item2 NC PC PA MOS BA MA SEM P-value Day 3 BW (g/b) 72.5 73.2 72.9 73.4 73.0 72.7 0.48 0.88 0 to 7 d P-Ch BWG (g/b) 185.7a 144.5c 168.1b 166.8b 165.2b 170.5b 4.45 <.001 FI (g/b) 218.3a 186.9b 207.3a 205.5a 204.7a,b 203.6a,b 5.84 0.03 FCR (g/g) 1.17c 1.29a 1.23b 1.23b 1.24b 1.19b,c 0.01 0.001 7 to 21 d P-Ch BWG (g/b) 720.4a 604.2c 681.8b 682.9b 676.1b 712.2a 8.55 <.001 FI (g/b) 1004.3a 945.5b 983.9a,b 996.0a 998.4a 1011.4a 13.86 0.03 FCR (g/g) 1.39c 1.56a 1.44b,c 1.46b 1.47b 1.42b,c 0.02 0.001 0 to 21 d P-Ch BWG (g/b) 906.2a 748.7d 850.1b,c 849.8b,c 841.3c 882.8a,b 11.03 <.001 FI (g/b) 1222a 1132b 1191a 1201a 1203a 1215a 18.27 0.02 FCR(g/g) 1.35c 1.51a 1.40b,c 1.41b 1.43b 1.37b,c 0.02 <.001 a-cMeans with different superscripts in each row are significantly different (P < 0.05). 1NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. 2P-Hatch, Posthatch; P-Ch, Post-Challenge; BWG, body weight gain; FI, feed intake; FCR, feed conversion ratio. View Large Characterization of Cecal Microbiota As illustrated in Figure 1, birds in the PC treatment group had the lowest ceca Lactobacillus population compared to all other treatments, at d 14 post-challenge (P < 0.05). Birds in the treatment groups with dietary supplementation of PA, MOS, BA and MA had a greater population of cecal lactobacilli compared to birds in the PC or NC treatment groups at d 14 and 21 post-challenge (P < 0.05), with the highest population observed in birds on the MA treatment. The cecal population of Bifidobacterium was not affected by the challenge (P > 0.05); however, dietary supplementation with MOS and MA significantly increased Bifidobacterium count compared to both the PC and NC treatment groups at d 21 post-challenge (P < 0.05; Figure 2). The S. Typhimurium challenge had no impact on the population of coliforms in the ceca (P > 0.05, Figure 3). Birds on the NC and PC treatments had the highest population of coliforms at d 7 and 21 post-challenge, whilst birds in the MA treatment group had the lowest population (P < 0.05; Figure 3). No Salmonella spp. were detected in the ceca of the unchallenged birds. Birds fed the dietary supplements had lower prevalence of Salmonella in the ceca compared to birds in the PC group at d 14 and 21 post-challenge (P < 0.05; Figure 4), and birds on the MA treatment had lower ceca Salmonella content compared to birds on any other treatment (P < 0.05; Figure 4). Figure 1. View largeDownload slide Effects of dietary treatments on cecal Lactobacillus population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. Figure 1. View largeDownload slide Effects of dietary treatments on cecal Lactobacillus population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. Figure 2. View largeDownload slide Effects of dietary treatments on cecal Bifidobacterium population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. Figure 2. View largeDownload slide Effects of dietary treatments on cecal Bifidobacterium population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. Figure 3. View largeDownload slide Effects of dietary treatments on cecal Coliforms population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. Figure 3. View largeDownload slide Effects of dietary treatments on cecal Coliforms population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. Figure 4. View largeDownload slide Effects of dietary treatments on cecal Salmonella population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. Figure 4. View largeDownload slide Effects of dietary treatments on cecal Salmonella population in broiler chicks at 7 to 14 and 21 d after challenge with S. Typhimurium. Each mean represents 10 chicks. Bars with different letters (a, c) differ significantly (P < 0.05) within the same day. NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. Hematological Parameters, Intestinal Enzyme Activities and Morphological Analysis Table 3 summarizes the effect of dietary treatments on heterophil and lymphocyte counts and the H/L ratio, measured at d 7, 14, and 21 post-challenge. An increased heterophil count at d 7 and 21 post-challenge and greater H/L ratios at all the periods tested were observed with the presence of the S. Typhimurium challenge (P < 0.05). The challenged birds fed the diets supplemented with PA, MOS, BA, and MA had significantly reduced heterophil and increased lymphocyte counts at d 7, 14, and 21 post-challenge compared to birds in the PC treatment group (P < 0.05). There were no significant differences between birds in the NC and MA treatment groups with regards to the H/L ratio at d 14 and 21 post-challenge. Heterophil counts at d 14 and lymphocyte counts at d 21 post-challenge were not influenced by the treatments (P > 0.05). Table 3. Effects of dietary treatments on heterophil and lymphocyte counts. Treatments1 Item Day P-Ch2 NC PC PA MOS BA MA SEM P-value Heterophil (H) 7 26.61c 39.85a 34.04b 34.95b 33.54b 30.19b,c 1.51 <.001 14 28.63 34.74 32.41 32.29 34.12 31.55 1.97 0.34 21 24.69c 33.37a 27.92b,c 29.56b 28.65b,c 25.28c 1.29 0.001 Lymphocyte (L) 7 64.3a 56.2c 60.1b 60.0b 60.5b 62.7a,b 1.22 0.002 14 61.9a 55.4b 57.2b 58.3a,b 58.0a,b 59.5a,b 1.34 0.04 21 64.1 59.5 58.8 63.1 61.7 64.3 1.69 0.13 H:L ratio 7 0.41d 0.71a 0.56b 0.58b 0.55b 0.48c 0.01 <.001 14 0.46c 0.62a 0.56a,b 0.55a,b 0.58a,b 0.52b,c 0.02 0.005 21 0.38c 0.58a 0.47b 0.46b 0.46b 0.39c 0.01 <.001 Treatments1 Item Day P-Ch2 NC PC PA MOS BA MA SEM P-value Heterophil (H) 7 26.61c 39.85a 34.04b 34.95b 33.54b 30.19b,c 1.51 <.001 14 28.63 34.74 32.41 32.29 34.12 31.55 1.97 0.34 21 24.69c 33.37a 27.92b,c 29.56b 28.65b,c 25.28c 1.29 0.001 Lymphocyte (L) 7 64.3a 56.2c 60.1b 60.0b 60.5b 62.7a,b 1.22 0.002 14 61.9a 55.4b 57.2b 58.3a,b 58.0a,b 59.5a,b 1.34 0.04 21 64.1 59.5 58.8 63.1 61.7 64.3 1.69 0.13 H:L ratio 7 0.41d 0.71a 0.56b 0.58b 0.55b 0.48c 0.01 <.001 14 0.46c 0.62a 0.56a,b 0.55a,b 0.58a,b 0.52b,c 0.02 0.005 21 0.38c 0.58a 0.47b 0.46b 0.46b 0.39c 0.01 <.001 a-cMeans with different superscripts in each row are significantly different (P < 0.05). Mean values are based on 2 observations per replicate (5 replicates per treatment) at each time point. 1NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. 2P-Ch, Post-Challenge. View Large Table 3. Effects of dietary treatments on heterophil and lymphocyte counts. Treatments1 Item Day P-Ch2 NC PC PA MOS BA MA SEM P-value Heterophil (H) 7 26.61c 39.85a 34.04b 34.95b 33.54b 30.19b,c 1.51 <.001 14 28.63 34.74 32.41 32.29 34.12 31.55 1.97 0.34 21 24.69c 33.37a 27.92b,c 29.56b 28.65b,c 25.28c 1.29 0.001 Lymphocyte (L) 7 64.3a 56.2c 60.1b 60.0b 60.5b 62.7a,b 1.22 0.002 14 61.9a 55.4b 57.2b 58.3a,b 58.0a,b 59.5a,b 1.34 0.04 21 64.1 59.5 58.8 63.1 61.7 64.3 1.69 0.13 H:L ratio 7 0.41d 0.71a 0.56b 0.58b 0.55b 0.48c 0.01 <.001 14 0.46c 0.62a 0.56a,b 0.55a,b 0.58a,b 0.52b,c 0.02 0.005 21 0.38c 0.58a 0.47b 0.46b 0.46b 0.39c 0.01 <.001 Treatments1 Item Day P-Ch2 NC PC PA MOS BA MA SEM P-value Heterophil (H) 7 26.61c 39.85a 34.04b 34.95b 33.54b 30.19b,c 1.51 <.001 14 28.63 34.74 32.41 32.29 34.12 31.55 1.97 0.34 21 24.69c 33.37a 27.92b,c 29.56b 28.65b,c 25.28c 1.29 0.001 Lymphocyte (L) 7 64.3a 56.2c 60.1b 60.0b 60.5b 62.7a,b 1.22 0.002 14 61.9a 55.4b 57.2b 58.3a,b 58.0a,b 59.5a,b 1.34 0.04 21 64.1 59.5 58.8 63.1 61.7 64.3 1.69 0.13 H:L ratio 7 0.41d 0.71a 0.56b 0.58b 0.55b 0.48c 0.01 <.001 14 0.46c 0.62a 0.56a,b 0.55a,b 0.58a,b 0.52b,c 0.02 0.005 21 0.38c 0.58a 0.47b 0.46b 0.46b 0.39c 0.01 <.001 a-cMeans with different superscripts in each row are significantly different (P < 0.05). Mean values are based on 2 observations per replicate (5 replicates per treatment) at each time point. 1NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. 2P-Ch, Post-Challenge. View Large As illustrated in Table 4, S. Typhimurium challenge significantly decreased amylase and protease activity measured in the jejunum at d 7, 14, and 21 post-challenge, and lipase activity at d 7 post-challenge (P < 0.05). Amylase and protease activity in birds fed the diets supplemented with PA, MOS, BA, or MA was either higher or equal to that measured in birds on the PC treatment group at all ages studied, and was consistently higher in birds fed the supplemented diets compared to birds on the NC treatment at d 21 post-challenge (P < 0.05). Additionally, at d 7 post-challenge there was no significant difference in lipase activity observed between birds on the NC treatment and those fed the supplemented diets (P > 0.05). Table 4. Effects of dietary treatments on intestinal digestive enzyme activities (U/mg of digesta protein). Treatments1 Enzymes Day P-Ch2 NC PC PA MOS BA MA SEM P-value Amylase 7 9.21a 3.64c 6.45b 6.84b 6.26b 7.12b 0.31 <.001 14 9.74a 5.58c 8.29b,c 7.78c 8.34b,c 9.57a,b 0.50 <.001 21 12.09a 8.92b 11.56a 11.32a 10.95a 11.68a 0.47 0.008 Protease 7 86.0a 57.8c 69.7b 72.4b 64.2b,c 74.6b 3.64 0.002 14 94.6a 73.1c 86.3a,b 84.6a,b 79.6b,c 89.7a,b 3.78 0.006 21 116.3a 96.1b 112.5a 111.3a 111.3a 114.6a 3.99 0.01 Lipase 7 24.31a 15.25b 21.57a 19.40a,b 20.85a 21.51a 1.82 0.01 14 24.47 19.54 19.90 21.55 19.62 22.35 1.55 0.19 21 28.76 24.05 25.61 24.62 26.40 29.60 1.70 0.16 Treatments1 Enzymes Day P-Ch2 NC PC PA MOS BA MA SEM P-value Amylase 7 9.21a 3.64c 6.45b 6.84b 6.26b 7.12b 0.31 <.001 14 9.74a 5.58c 8.29b,c 7.78c 8.34b,c 9.57a,b 0.50 <.001 21 12.09a 8.92b 11.56a 11.32a 10.95a 11.68a 0.47 0.008 Protease 7 86.0a 57.8c 69.7b 72.4b 64.2b,c 74.6b 3.64 0.002 14 94.6a 73.1c 86.3a,b 84.6a,b 79.6b,c 89.7a,b 3.78 0.006 21 116.3a 96.1b 112.5a 111.3a 111.3a 114.6a 3.99 0.01 Lipase 7 24.31a 15.25b 21.57a 19.40a,b 20.85a 21.51a 1.82 0.01 14 24.47 19.54 19.90 21.55 19.62 22.35 1.55 0.19 21 28.76 24.05 25.61 24.62 26.40 29.60 1.70 0.16 a-cMeans with different superscripts in each row are significantly different (P < 0.05). Mean values are based on 2 observations per replicate (5 replicates per treatment) at each time point 1NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. 2P-Ch, Post-Challenge View Large Table 4. Effects of dietary treatments on intestinal digestive enzyme activities (U/mg of digesta protein). Treatments1 Enzymes Day P-Ch2 NC PC PA MOS BA MA SEM P-value Amylase 7 9.21a 3.64c 6.45b 6.84b 6.26b 7.12b 0.31 <.001 14 9.74a 5.58c 8.29b,c 7.78c 8.34b,c 9.57a,b 0.50 <.001 21 12.09a 8.92b 11.56a 11.32a 10.95a 11.68a 0.47 0.008 Protease 7 86.0a 57.8c 69.7b 72.4b 64.2b,c 74.6b 3.64 0.002 14 94.6a 73.1c 86.3a,b 84.6a,b 79.6b,c 89.7a,b 3.78 0.006 21 116.3a 96.1b 112.5a 111.3a 111.3a 114.6a 3.99 0.01 Lipase 7 24.31a 15.25b 21.57a 19.40a,b 20.85a 21.51a 1.82 0.01 14 24.47 19.54 19.90 21.55 19.62 22.35 1.55 0.19 21 28.76 24.05 25.61 24.62 26.40 29.60 1.70 0.16 Treatments1 Enzymes Day P-Ch2 NC PC PA MOS BA MA SEM P-value Amylase 7 9.21a 3.64c 6.45b 6.84b 6.26b 7.12b 0.31 <.001 14 9.74a 5.58c 8.29b,c 7.78c 8.34b,c 9.57a,b 0.50 <.001 21 12.09a 8.92b 11.56a 11.32a 10.95a 11.68a 0.47 0.008 Protease 7 86.0a 57.8c 69.7b 72.4b 64.2b,c 74.6b 3.64 0.002 14 94.6a 73.1c 86.3a,b 84.6a,b 79.6b,c 89.7a,b 3.78 0.006 21 116.3a 96.1b 112.5a 111.3a 111.3a 114.6a 3.99 0.01 Lipase 7 24.31a 15.25b 21.57a 19.40a,b 20.85a 21.51a 1.82 0.01 14 24.47 19.54 19.90 21.55 19.62 22.35 1.55 0.19 21 28.76 24.05 25.61 24.62 26.40 29.60 1.70 0.16 a-cMeans with different superscripts in each row are significantly different (P < 0.05). Mean values are based on 2 observations per replicate (5 replicates per treatment) at each time point 1NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. 2P-Ch, Post-Challenge View Large As demonstrated in Table 5, the S. Typhimurium challenge decreased jejunum villus height (VH) in birds on the PC treatment at d 14 and 21 post-challenge (P < 0.05). Birds fed the supplemented diets had significantly greater VH compared to birds on the PC treatment (P < 0.05), and birds fed the diets supplemented with PA or MA presented VH that was similar to that observed in birds on the NC treatment (P > 0.05). The dietary treatments had no significant impact on crypt depth (CD; P > 0.05), but at d 7 lower VH: CD ratios were observed in birds fed the supplemented diets compared to those on the PC treatment. At d 14 and 21 there was no significant difference between birds on the NC treatment and those fed the supplemented diets (P > 0.05), except for birds fed the diets supplemented with BA. Table 5. Effects of dietary treatments on the morphology of jejunum (μm) of broilers. Treatments1 Items Day P-Ch2 NC PC PA MOS BA MA SEM P-value Villus height 7 438.4 403.6 421.5 423.9 416.3 429.5 11.14 0.36 14 654.6a 572.7c 626.0a,b 618.4b 611.7b 632.4a,b 9.46 <.001 21 922.8a 841.3c 894.6a,b 882.1b 879.4b 919.6a 10.15 <.001 Crypt depth 7 91.5 98.2 90.7 95.8 93.5 88.3 4.83 0.73 14 134.1 152.9 138.8 140.6 150.7 135.4 7.78 0.41 21 167.2 180.0 169.6 170.2 177.7 170.2 6.90 0.74 VH: CD 7 4.90a 4.13b 4.66a 4.45a,b 4.46a,b 4.89a 0.16 0.01 14 4.98a 3.77c 4.56a,b 4.44a,b 4.09b,c 4.71a,b 0.20 0.002 21 5.53a 4.72c 5.30a,b 5.20a,b 4.98b,c 5.42a,b 0.15 0.008 Treatments1 Items Day P-Ch2 NC PC PA MOS BA MA SEM P-value Villus height 7 438.4 403.6 421.5 423.9 416.3 429.5 11.14 0.36 14 654.6a 572.7c 626.0a,b 618.4b 611.7b 632.4a,b 9.46 <.001 21 922.8a 841.3c 894.6a,b 882.1b 879.4b 919.6a 10.15 <.001 Crypt depth 7 91.5 98.2 90.7 95.8 93.5 88.3 4.83 0.73 14 134.1 152.9 138.8 140.6 150.7 135.4 7.78 0.41 21 167.2 180.0 169.6 170.2 177.7 170.2 6.90 0.74 VH: CD 7 4.90a 4.13b 4.66a 4.45a,b 4.46a,b 4.89a 0.16 0.01 14 4.98a 3.77c 4.56a,b 4.44a,b 4.09b,c 4.71a,b 0.20 0.002 21 5.53a 4.72c 5.30a,b 5.20a,b 4.98b,c 5.42a,b 0.15 0.008 a-cMeans with different superscripts in each row are significantly different (P < 0.05). Mean values are based on 2 observations per replicate (5 replicates per treatment) at each time point 1NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. 2P-Ch, Post-Challenge. View Large Table 5. Effects of dietary treatments on the morphology of jejunum (μm) of broilers. Treatments1 Items Day P-Ch2 NC PC PA MOS BA MA SEM P-value Villus height 7 438.4 403.6 421.5 423.9 416.3 429.5 11.14 0.36 14 654.6a 572.7c 626.0a,b 618.4b 611.7b 632.4a,b 9.46 <.001 21 922.8a 841.3c 894.6a,b 882.1b 879.4b 919.6a 10.15 <.001 Crypt depth 7 91.5 98.2 90.7 95.8 93.5 88.3 4.83 0.73 14 134.1 152.9 138.8 140.6 150.7 135.4 7.78 0.41 21 167.2 180.0 169.6 170.2 177.7 170.2 6.90 0.74 VH: CD 7 4.90a 4.13b 4.66a 4.45a,b 4.46a,b 4.89a 0.16 0.01 14 4.98a 3.77c 4.56a,b 4.44a,b 4.09b,c 4.71a,b 0.20 0.002 21 5.53a 4.72c 5.30a,b 5.20a,b 4.98b,c 5.42a,b 0.15 0.008 Treatments1 Items Day P-Ch2 NC PC PA MOS BA MA SEM P-value Villus height 7 438.4 403.6 421.5 423.9 416.3 429.5 11.14 0.36 14 654.6a 572.7c 626.0a,b 618.4b 611.7b 632.4a,b 9.46 <.001 21 922.8a 841.3c 894.6a,b 882.1b 879.4b 919.6a 10.15 <.001 Crypt depth 7 91.5 98.2 90.7 95.8 93.5 88.3 4.83 0.73 14 134.1 152.9 138.8 140.6 150.7 135.4 7.78 0.41 21 167.2 180.0 169.6 170.2 177.7 170.2 6.90 0.74 VH: CD 7 4.90a 4.13b 4.66a 4.45a,b 4.46a,b 4.89a 0.16 0.01 14 4.98a 3.77c 4.56a,b 4.44a,b 4.09b,c 4.71a,b 0.20 0.002 21 5.53a 4.72c 5.30a,b 5.20a,b 4.98b,c 5.42a,b 0.15 0.008 a-cMeans with different superscripts in each row are significantly different (P < 0.05). Mean values are based on 2 observations per replicate (5 replicates per treatment) at each time point 1NC, negative control (not challenged with S. Typhimurium); PC, positive control (challenged with S. Typhimurium); PA, P. acidilactici group + challenge with S. Typhimurium; MOS, mannan-oligosaccharide group + challenge with S. Typhimurium; BA, butyric acid group + challenge with S. Typhimurium; MA, a mixture of additives + challenge with S. Typhimurium. 2P-Ch, Post-Challenge. View Large DISCUSSION Enteric microbiota play an important role in dictating health and consequently growth responses in broiler chickens (Van der Aar et al., 2017), but there is a concern that pathogenic bacteria in this microbiota may contaminate poultry meat during processing, resulting in foodborne diseases such as salmonellosis. Reducing Salmonella load in live animals is considered to be the most effective strategy for reducing this contamination and thus the number of human salmonellosis cases (EFSA, 2004). The current study investigated the efficacy of probiotic, prebiotic, butyric acid and their mixture on diminishing Salmonella load, by evaluating their impact on growth performance, intestinal parameters, and cecal digesta microbiota compositions in broiler chickens orally challenged with S. Typhimurium. Challenging the birds with S. Typhimurium decreased FI, BWG, and feed efficiency, which is in agreement with a number of former studies (Vandeplas et al., 2009; Marcq et al., 2011). The reduced performance observed in the challenged birds is probably due to intestinal mucosal damage induced by the S. Typhimurium (Vandeplas et al., 2009). In contrast, Mountzouris et al. (2009) and Amerah et al. (2012) found that challenging birds with Salmonella had no effect on performance response; this may be due to discrepancies between the species, strains or dose of Salmonella administered leading to different extents of intestinal damage (Malago et al., 2003), or because of differences in dietary cereal type fed to the birds (Teirlynck et al., 2009). In the present study, dietary supplementation with PA, BA, and MOS improved bird performance and reduced the presence of Salmonella in the ceca. Interestingly, only birds fed the mixture of additives (MA) showed complete recovery following the challenge, exhibiting similar BWG and FCR as the unchallenged birds, highlighting a synergistic rather than being overlapping effect of the additives. Similarly, previous studies have reported that dietary supplementation with prebiotic-based mannan-oligosaccharides (Lourenco et al., 2016; Rajani et al., 2016), B. subtilis (Park and Kim, 2014), and butyric acid (Saleem et al., 2016) resulted in improved growth performance in broilers challenged with Salmonella. The positive effect of MOS on bird's performance is likely attributed to their ability to bind to pathogens, stimulating the immune system and improving intestinal function by enhancing villus uniformity (Baurhoo et al., 2007; Lourenco et al., 2016; Pourabedin et al., 2016). Additionally, MOS-based prebiotics serve as a substrate for endogenous beneficial bacteria, thus promoting competitive exclusion of pathogenic microbes and selective colonization of beneficial microbes (Spring et al., 2000; Biggs et al., 2007). Fermentation of oligosaccharides also results in production of short-chain fatty acids, which display antimicrobial effects by penetrating the cell membrane of gram negative bacteria, such as Salmonella and coliforms, and altering the environmental pH, leading to death of the bacteria (Faber et al., 2012; Suiryanrayna and Ramana, 2015). The beneficial effects of probiotic supplements on broiler performance is associated with their role in maintaining healthy balance of bacteria in the digestive tract and improving metabolism and digestion by increasing digestive enzyme activity (Sugiharto, 2016). Organic acids potentially exhibit antimicrobial activities, by reducing gastrointestinal tract pH and stimulating protein digestion by converting pepsinogen to pepsin (Suiryanrayna and Ramana, 2015). Thus, the observed synergistic and complementary effect of the additives used in this study is likely due to the dissimilar beneficial mechanisms exhibited by each supplement. The cecal samples of the unchallenged birds were detected negative for the presence of Salmonella, indicating that there was no cross contamination between the challenged and unchallenged birds. The dietary supplements used in the current study increased Lactobacillus and Bifidobacterium concentration and decreased the population of coliforms and Salmonella in the ceca. In agreement with this study, Prado-Rebolledo et al. (2017) and Pourabedin et al. (2016) reported that supplementing LAB-based probiotics and MOS-based prebiotics to the diets of broilers challenged with Salmonella enterica decreased cecal colonization of Salmonella. Similar effects on controlling and reducing cecal Salmonella colonization have also been described as a result of dietary application of organic acids (Fernandez-Rubio et al., 2009; Saleem et al., 2016). The metabolic activity of P. acidilactici bacteria results in production of lactic acid and secretion of bacteriocins, which create unfavorable conditions for the growth of pathogenic bacteria such as coliforms and Salmonella (Taheri et al., 2010). This suggests that the success of the MA diet may be largely attributed to decreased acid-binding capacity and consequential reduction in digesta pH in birds fed this diet (Emami et al., 2017), resulting in selective stimulation of beneficial bacteria, increased production of short-chain fatty acids and thus reduced colonization of pathogenic bacteria (Sugiharto, 2016). The reliability of the H/L ratio as a biological index of stress in avian species is well documented (Maxwell, 1993; Toghyani et al., 2011); heterophils display phagocytic, chemotactic, and adhesion activities and are responsible for protecting the body against pathogens (Munyaka et al., 2012). The S. Typhimurium challenged birds had significantly increased heterophil and reduced lymphocyte numbers, causing the H/L ratio to increase from 0.38 in the NC birds to 0.58 in the PC birds at d 21 post-challenge. The ratio decreased to approximately 0.46 in the challenged birds fed the diets supplemented with PA, MOS, and BA. A complementary effect of the additives was observed for the H/L ratio, with birds on the MA treatment displaying similar ratios to those on the NC treatment at d 14 and 21 post-challenge. These findings are in agreement with previous reports highlighting that probiotics (Prado-Rebolledo et al., 2017), prebiotics (Sadeghi et al., 2013) and short-chain organic acids (Rath et al., 2006) possess immunomodulatory effects. LAB-based probiotics exert immunomodulatory activities by interacting with the host immune system, leading to changes in gene expression of cytokines and thus altered intestinal microbiota composition (Menconi et al., 2011; Pourabedin et al., 2016). Janardhana et al. (2009) demonstrated that, in addition to influencing the proliferative function and phenotypic expression of immune cells, prebiotics can also influence systemic antibody levels such as IgA, IgM, and IgG in broiler chickens. Thus, it is probable that the systemic effect of the supplements on immune response stimulation could have alleviated the impact of the S. Typhimurium challenge on increasing the H/L ratio in the current study. The S. Typhimurium challenge suppressed the activity of amylase, lipase and protease in this study. Some species of pathogenic bacteria, such as Escherichia coli (Zhang et al., 2016) and Clostridium (Mitsch et al., 2004), have been shown to inhibit secretion of digestive enzyme by damaging the villus and microvillus of the intestinal mucosa. Dietary supplementation with PA, MOS, BA, and MA ameliorated the negative impact of the challenge on digestive enzyme activity, particularly in the older birds. This is in agreement with a number of previous studies that have observed improved digestive enzyme activity in birds fed probiotics (Wang and Gu, 2010; Zhang et al., 2016), and prebiotics (Xu et al., 2003). The positive effect of these additives on enzymes activity is likely attributed to their impact on improving integrity of intestinal lining (Mitsch et al., 2004) and modifying the microbial ecosystem; for example, lactic-acid producing bacteria stimulate the secretion of digestive enzymes and reduce gastrointestinal pH (Jin et al., 2000; Xu et al., 2003; Suiryanrayna and Ramana, 2015). As stated by Choct (2009), the presence of stressors throughout the intestinal tract can quickly alter the structure of the intestine. Outcomes of the VH and CD measurements in this study, as indices of intestinal integrity and development, suggest that although intestinal development was not immediately affected by the challenge (determined at d 7 post-challenge), the S. Typhimurium challenge resulted in a noticeable reduction in VH at d 14 and 21 post-challenge, leading to a decreased VH: CD ratios. In agreement with the current findings, Rajani et al. (2016) also observed reduced VH and VH: CD ratio in the small intestine of broilers challenged with S. Typhimurium. Dietary supplementation with PA, MOS, BA, and MA improved VH and VH: CD ratio, which is reflected by the growth performance responses, in that shorter villi have a smaller surface area for nutrient absorption. Previous studies have also reported the beneficial effects of P. acidilacticis, MOS and organic acids on intestinal morphology (Baurhoo et al., 2007; Panda et al., 2009; Taheri et al., 2010). According to the literature and as observed in current study, evidently the improved intestinal microbiota balance in favor of beneficial bacteria by using the aforementioned additives can be responsible for the rectified jejunum morphological parameters (Antongiovanni et al., 2007; Panda et al., 2009; Mallo et al., 2012). CONCLUSION The results obtained in the present study indicate that S. Typhimurium challenge does not directly influence bird mortality, but it negatively affects growth performance by impairing gut morphology, digestive enzyme secretion and microbiota composition, resulting in reduced feed efficiency and bird health. The probiotic, prebiotic and butyric acid supplements fed in this study were, to some extent, efficacious at combatting the negative effects of the challenge on broiler performance and gut health. However, a complete recovery from the challenge effects was observed only when a mixture of the additives was administered, suggesting there was a synergistic effect between the additives. Therefore, it can be concluded that dietary supplementation with a combination of MOS, PA, and BA has the potential to be considered as a practical and effective strategy for controlling the incidence of Salmonella in broiler chickens in a post-antibiotic era. ACKNOWLEDGMENTS The authors greatly acknowledge and appreciate the technical support from the staff of the microbiology laboratory, College of Medical Sciences, Islamic Azad University, Isfahan (Najafabad), Iran. We are also grateful to Tak Genezist Company and Dr. Alireza Bidram for supplying the probiotic and butyric acid. REFERENCES Amerah A. M. , Mathis G. , Hofacre C. L. . 2012 . Effect of xylanase and a blend of essential oils on performance and Salmonella colonization of broiler chickens challenged with Salmonella Heidelberg . Poult. Sci. 91 : 943 – 947 . Google Scholar CrossRef Search ADS PubMed Antongiovanni M. , Buccioni A. , Petacchi F. , Leeson S. , Minieri S. , Martini A. , Cecchi R. . 2007 . Butyric acid glycerides in the diet of broiler chickens: effects on gut histology and carcass composition . Ital. J. Anim. Sci . 6 : 19 – 25 . Google Scholar CrossRef Search ADS Aviagen . 2014 . Broiler Nutrition Specification Ross 308 . Aviagen , Huntsville, Alabama . Baurhoo B. , Phillip L. , Ruiz-Feria C. A. . 2007 . Effects of purified lignin and mannan oligosaccharides on intestinal integrity and microbial populations in the ceca and litter of broiler chickens . Poult. Sci . 86 : 1070 – 1078 . Google Scholar CrossRef Search ADS PubMed Biggs P. , Parsons C. M. , Fahey G. C. . 2007 . The effects of several oligosaccharides on growth performance, nutrient digestibilities, and cecal microbial populations in young chicks . Poult. Sci . 86 : 2327 – 2336 . Google Scholar CrossRef Search ADS PubMed Bonos E. , Christaki E. , Abrahim A. , Soultos N. , Florou-Paneri P . 2011 . The influence of mannan oligosaccharides, acidifiers and their combination on cecal microflora of Japanese quail (Coturnix japonica) . Anaerobe . 17 : 436 – 439 . Google Scholar CrossRef Search ADS PubMed Chalghoumi R. , Marcq C. , Thewis A. , Portetelle D. , Beckers Y. . 2009 . Effects of feed supplementation with specific hen egg yolk antibody (immunoglobin Y) on Salmonella species cecal colonization and growth performances of challenged broiler chickens . Poult. Sci. 88 : 2081 – 2092 . Google Scholar CrossRef Search ADS PubMed Cheng G. , Hao H. , Xie S. , Wang X. , Dai M. , Huang L. , Yuan Z. . 2014 . Antibiotic alternatives: the substitution of antibiotics in animal husbandry? Front. Microbiol . 4 : 137 – 156 . Choct M. 2009 . Managing gut health through nutrition . Br. Poult. Sci. 50 : 9 – 15 . Google Scholar CrossRef Search ADS PubMed EFSA . 2004 . Opinion of the scientific panel on biological hazards on a request from the Commission related to the use of vaccines for the control of Salmonella in poultry . EFSA J. 114 : 1 – 74 . Emami N. K. , Daneshmand A. , Naeini S. Z. , Graystone E. N. , Broom L. J. . 2017 . Effects of commercial organic acid blends on male broilers challenged with E. coli K88: Performance, microbiology, intestinal morphology, and immune response . Poult. Sci. 96 : 3254 – 3263 . Google Scholar CrossRef Search ADS PubMed Faber T. A. , Dilger R. N. , Hopkins A. C. , Price N. P. , Fahey G. C. . 2012 . Effects of oligosaccharides in a soybean meal-based diet on fermentative and immune responses in broiler chicks challenged with Eimeria acervulina . Poult. Sci . 91 : 3132 – 3140 . Google Scholar CrossRef Search ADS PubMed Fernandez-Rubio C. , Ordonez C. , Abad-Gonzalez J. , Garcia-Gallego A. , Pilar M. , Honrubia J. , Mallo J. , Balana-Fouce R . 2009 . Butyric acid-based feed additives help protect broiler chickens from Salmonella enteritidis infection . Poult. Sci. 88 : 943 – 948 . Google Scholar CrossRef Search ADS PubMed Janardhana V. , Broadway M. M. , Bruce M. P. , Lowenthal J. W. , Geier M. S. , Hughes R. H. , Bean A. G. D. . 2009 . Prebiotics modulate immune responses in gut-associated lymphoid tissue of chickens . J. Nutr . 139 : 1404 – 1409 . Google Scholar CrossRef Search ADS PubMed Jin L. Z. , Ho Y. W. , Abdullah N. , Jalaludin S. . 2000 . Digestive and bacterial enzyme activities in broilers fed diets supplemented with Lactobacillus cultures . Poult. Sci. 79 : 886 – 891 . Google Scholar CrossRef Search ADS PubMed Lourenco M. C. , de Souza A. M. , Hayashi R. M. , da Silva A. B. , Santin E. . 2016 . Immune response of broiler chickens supplemented with prebiotic from Saccharomyces cerevisiae challenged with Salmonella enteritidis or Minnesota . J. Appl. Poult. Res. 25 : 165 – 172 . Google Scholar CrossRef Search ADS Malago J. J. , Koninkx J. F. J. G. , Ovelgonne H. H. , van Asten F .J .A. M. , Swennenhuis J. F. , van Dijk J. E. . 2003 . Expression levels of heat shock proteins in enterocyte-like Caco-2 cells after exposure to Salmonella enteritidis . Cell Stress Chaperones . 8 : 194 – 203 . Google Scholar CrossRef Search ADS PubMed Mallo J. J. , Puyalto M. , Rama Rao S. V. . 2012 . Evaluation of the effect of sodium butyrate addition to broilers diet on energy and protein digestibility, productive parameters and size of intestinal villi of animals . Feed. Livest . 8 : 26 – 30 . Marcq C. , Cox E. , Szalo I. M. , Thewis A. , Beckers Y. . 2011 . Salmonella Typhimurium oral challenge model in mature broilers: Bacteriological, immunological, and growth performance aspects . Poult. Sci. 90 : 59 – 67 . Google Scholar CrossRef Search ADS PubMed Maxwell M. H. 1993 . Avian blood leucocyte responses to stress . W. Poult. Sci. J. 49 : 34 – 43 . Google Scholar CrossRef Search ADS Menconi A. , Wolfenden A. D. , Shivaramaiah S. , Terraes J. C. , Urbano T. , Kuttel J. , Kremer C. , Hargis B. M. , Tellez G. . 2011 . Effect of lactic acid bacteria probiotic culture for the treatment of Salmonella enterica serovar Heidelberg in neonatal broiler chickens and turkey poults . Poult. Sci. 90 : 561 – 565 . Google Scholar CrossRef Search ADS PubMed Menconi A. , Kuttappan V. A. , Hernandez-Velasco X. , Urbano T. , Matte F. , Layton S. , Kallapura G. , Latorre J. , Morales B. E. , Prado O. , Vicente J. L. , Barton J. , Filho R. L. A. , Lovato M. , Hargis B. M. , Tellez G. . 2014 . Evaluation of a commercially available organic acid product on body weight loss, carcass yield, and meat quality during preslaughter feed withdrawal in broiler chickens: a poultry welfare and economic perspective . Poult. Sci . 93 : 448 – 455 . Google Scholar CrossRef Search ADS PubMed Mitsch P. , Zitterl-Eglseer K. , Kohler B. , Gabler C. , Losa R. , Zimpernik I. . 2004 . The effect of two different blends of essential oil components on the proliferation of Clostridium perfringens in the intestines of broiler chickens . Poult. Sci. 83 : 669 – 675 . Google Scholar CrossRef Search ADS PubMed Mountzouris K. C. , Balaskas C. , Xanthakos I. , Tzivinikou A. , Fegeros K. . 2009 . Effects of a multi-species probiotic on biomarkers of competitive exclusion efficacy in broilers challenged with Salmonella Enteritidis . Br. Poult. Sci. 50 : 467 – 478 . Google Scholar CrossRef Search ADS PubMed Munyaka P. M. , Echeverry H. , Yitbarek A. , Camelo-Jaimes G. , Sharif S. , Guenter W. , House J. D. , Rodriguez-Lecompte J. C. . 2012 . Local and systemic innate immunity in broiler chickens supplemented with yeast-derived carbohydrates . Poult. Sci . 91 : 2164 – 2172 . Google Scholar CrossRef Search ADS PubMed Panda A. K. , Rao S. V. R. , Raju M. V. L. N. , Sunder G. S. . 2009 . Effect of butyric acid on performance, gastrointestinal tract health and carcass characteristics in broiler chickens. Asian-Australas . J. Anim. Sci . 22 : 1026 – 1031 . Park J. H. , Kim I. H. . 2014 . Supplemental effect of probiotic Bacillus subtilis B2A on productivity, organ weight, intestinal Salmonella microflora, and breast meat quality of growing broiler chicks . Poult. Sci. 93 : 2054 – 2059 . Google Scholar CrossRef Search ADS PubMed Partanen K. H. , Mroz Z. . 1999 . Organic acids for performance enhancement in pig diets . Nutr. Res. Rev . 12 : 117 – 145 . Google Scholar CrossRef Search ADS PubMed Patterson J. A. , Burkholder K. M. . 2003 . Application of prebiotics and probiotics in poultry production . Poult. Sci . 82 : 627 – 631 . Google Scholar CrossRef Search ADS PubMed Pourabedin M. , Chen Q. , Yang M. , Zhao X. . 2016 . Mannan-and xylooligosaccharides modulate cecal microbiota and expression of inflammatory-related cytokines and reduce cecal Salmonella Enteritidis colonisation in young chickens . FEMS. Microbiol. Ecol. 93 : 226 . Google Scholar CrossRef Search ADS Prado-Rebolledo O. F. , Delgado-Machuca J. D. J. , Macedo-Barragan R. J. , Garcia-Marquez L. J. , Barrera M. J. E. , Latorre J. D. , Hernandez-Velasco X. , Tellez G. . 2017 . Evaluation of a selected lactic acid bacteria-based probiotic on Salmonella enterica serovar Enteritidis colonization and intestinal permeability in broiler chickens . Avian Pathol . 46 : 90 – 94 . Google Scholar CrossRef Search ADS PubMed Rajani J. , Dastar B. , Samadi F. , Karimi Torshizi M. A. , Abdulkhani A. , Esfandyarpour S. . 2016 . Effect of extracted galactoglucomannan oligosaccharides from pine wood (Pinus brutia) on Salmonella Typhimurium colonisation, growth performance and intestinal morphology in broiler chicks . Br. Poult. Sci. 57 : 682 – 692 . Google Scholar PubMed Rath N. C. , Huff W. E. , Huff G. R. . 2006 . Effects of humic acid on broiler chickens . Poult. Sci . 85 : 410 – 414 . Google Scholar CrossRef Search ADS PubMed Sadeghi A. A. , Mohammadi A. , Shawrang P. , Aminafshar M. . 2013 . Immune responses to dietary inclusion of prebiotic-based mannan-oligosaccharide and β-glucan in broiler chicks challenged with Salmonella enteritidis . Turk. J. Vet. Anim. Sci. 37 : 206 – 213 . SAS Institute Inc . 2010 . SAS User's Guide. Statistics. Version 9.3ed . SAS Inst. Inc. , Cary, NC . Saleem G. , Ramzaan R. , Khattak F. M. , Akhtar R. . 2016 . Effects of acetic acid supplementation in broiler chickens orally challenged with Salmonella Pullorum . Turk. J. Vet. Anim. Sci. 40 : 434 – 443 . Google Scholar CrossRef Search ADS Spring P. , Wenk C. , Dawson K. A. , Newman K. E. . 2000 . The effects of dietary mannaoligosaccharides on cecal parameters and the concentrations of enteric bacteria in the ceca of Salmonella-challenged broiler chicks . Poult. Sci. 79 : 205 – 211 . Google Scholar CrossRef Search ADS PubMed Sugiharto S. 2016 . Role of nutraceuticals in gut health and growth performance of poultry . J. Sau. Soc. Agric. Sci. 15 : 99 – 111 . Suiryanrayna M. V. , Ramana J. V. . 2015 . A review of the effects of dietary organic acids fed to swine . J. Anim. Sci. Biotechnol. 6 : 45 . Google Scholar CrossRef Search ADS PubMed Taheri H. R. , Moravej H. , Malakzadegan A. , Tabandeh F. , Zaghari M. , Shivazad M. , Adibmoradi M. . 2010 . Efficacy of Pediococcus acidlactici-based probiotic on intestinal Coliforms and villus height, serum cholesterol level and performance of broiler chickens . Afr. J. Biotechnol. 9 : 7564 – 7567 . Google Scholar CrossRef Search ADS Teirlynck E. , Haesebrouck F. , Pasmans F. , Dewulf J. , Ducatelle R. , Van Immerseel F. . 2009 . The cereal type in feed influences Salmonella Enteritidis colonization in broilers . Poult. Sci. 88 : 2108 – 2112 . Google Scholar CrossRef Search ADS PubMed Thung T. Y. , Mahyudin N. A. , Basri D. F. , Wan Mohamed Radzi C. W. J. , Nakaguchi Y. , Nishibuchi M. , Radu S. . 2016 . Prevalence and antibiotic resistance of Salmonella Enteritidis and Salmonella Typhimurium in raw chicken meat at retail markets in Malaysia . Poult. Sci. 95 : 1888 – 1893 . Google Scholar CrossRef Search ADS PubMed Toghyani M. , Toghyani M. , Gheisari A. , Ghalamkari G. , Mohammadrezaei M. . 2010 . Growth performance, serum biochemistry and blood hematology of broiler chicks fed different levels of black seed (Nigellasativa) and peppermint (Mentha piperita) . Livest. Sci . 129 : 173 – 178 . Google Scholar CrossRef Search ADS Toghyani M. , Toghyani M. , Gheisari A. , Ghalamkari G. , Eghbalsaied S. . 2011 . Evaluation of cinnamon and garlic as antibiotic growth promoter substitutions on performance, immune responses, serum biochemical and haematological parameters in broiler chicks . Livest. Sci . 138 : 167 – 173 . Google Scholar CrossRef Search ADS Troy E. B. , Kasper D. L. . 2010 . Beneficial effects of Bacteroides fragilis polysaccharides on the immune system . Front. Biosci. 15 : 25 – 34 . Google Scholar CrossRef Search ADS Van der Aar P. J. , Molist F. , van der Klis J. D. . 2017 . The central role of intestinal health on the effect of feed additives on feed intake in swine and poultry . Anim. Feed Sci. Technol . 233 : 64 – 75 . Google Scholar CrossRef Search ADS Vandeplas S. , Dauphin R. D. , Thiry C. , Beckers Y. , Welling G. W. , Thonart P. , Thewis A. . 2009 . Efficiency of a Lactobacillus plantarum-xylanase combination on growth performances, microflora populations, and nutrient digestibilities of broilers infected with Salmonella Typhimurium . Poult. Sci. 88 : 1643 – 1654 . Google Scholar CrossRef Search ADS PubMed Wang Y. , Gu Q. . 2010 . Effect of probiotic on growth performance and digestive enzyme activity of Arbor Acres broilers . Res. Vet. Sci. 89 : 163 – 170 . Google Scholar CrossRef Search ADS PubMed Xu Z. R. , Hu C. H. , Xia M. S. , Zhan X. A. , Wang M. Q. . 2003 . Effects of dietary fructooligosaccharide on digestive enzyme activities, intestinal microflora and morphology of male broilers . Poult. Sci . 82 : 1030 – 1036 . Google Scholar CrossRef Search ADS PubMed Zhang L. , Zhang L. , Zeng X. , Zhou L. , Cao G. , Yang C. . 2016 . Effects of dietary supplementation of probiotic, Clostridium butyricum, on growth performance, immune response, intestinal barrier function, and digestive enzyme activity in broiler chickens challenged with Escherichia coli K88 . J. Anim. Sci. Biotechnol. 7 : 3 – 12 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Poultry ScienceOxford University Press

Published: Mar 5, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off