Fermented soybean meal ameliorates Salmonella Typhimurium infection in young broiler chickens

Fermented soybean meal ameliorates Salmonella Typhimurium infection in young broiler chickens ,∗,1 H. Mohebodini,† A. Ashayerizadeh,∗ A. Shabani,∗ and R. Barekatain

‡,1

∗

Department of Animal and Poultry Nutrition, Faculty of Animal Science, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan 49189-4364, Iran; † Department of Animal Sciences, University of Mohaghegh Ardabili, Ardabil 56199-11367, Iran; and ‡ South Australian Research and Development Institute, Roseworthy Campus, University of Adelaide, Roseworthy, SA 5371, Australia sion ratio was alleviated by the addition of XOS, LAC, and FSBM in broiler diets compared with IC birds (P < 0.05). The ST infection reduced duodenum and jejunum villus height and increased Salmonella colonization throughout the gut as well as internal organ invasion compared with NC birds (P < 0.05). However, ST-infected broilers fed the XOS, LAC, and FSBMcontaining diets showed a significant decrease in gut Salmonella colonization, and internal organ invasion, an increase in LAB counts, and improvement in intestinal mucosa morphology (P < 0.05). The tested feed additives or FSBM reduced heterophil to lymphocyte ratio compared with the IC group (P < 0.05). The results suggest that XOS, LAC, and FSBM improve growth performance, lower Salmonella colonization, and improve intestinal characteristics and immune response in ST-challenged broiler chicks. Therefore, fermented feeds due to having functional ingredients can be considered as an effective strategy to lessen the colonization of gut pathogens in broiler chickens.

ABSTRACT The present study was designed to investigate the effectiveness of dietary fermented soybean meal (FSBM) in comparison with prebiotic (Xylooligosaccharide; XOS) and probiotic (Lactic acid bacteria-based probiotic; LAC) for prevention of Salmonella Typhimurium (ST) infection in young broiler chickens from 1 to 24 d. The in vitro study revealed that soybean meal (SBM) fermentation increased the number of lactic acid bacteria (LAB) and lactic acid content and inhibited the growth of enterobacteria such as coliforms in SBM. A total of 450 dayold Ross 308 broiler chicks were placed in 30 pens (15 birds/pen) and allocated to 5 experimental treatments that consisted an un-supplemented basal diet and not infected (NC) or infected with ST (IC); IC plus 2 g XOS/kg; IC plus 0.2 g LAC/kg; and IC containing a complete replacement of SBM with FSBM. All birds (except NC) were orally administered with 0.5 mL of the ST solution (1 × 106 CFU/mL) at d 3 post-hatch. The ST challenge decreased body weight gain and feed intake (P < 0.05). The impairment of feed conver-

Key words: fermented soybean meal, gut microbiota, Salmonella, intestinal morphology 2019 Poultry Science 0:1–13 http://dx.doi.org/10.3382/ps/pez338

INTRODUCTION

Salmonella to humans (Chen and Jaing, 2014). On the other hand, Salmonella infection in poultry (especially in young birds) is associated with severe morbidity and mortality, malabsorption, impaired growth rate, and inefficient feed utilization which can lead to substantial economic losses in the poultry production systems (Yan et al., 2011; Shao et al., 2016). Therefore, the development of effective and applied strategies to control and deal with bacterial infections in the poultry industry is essential. Possible strategies include the development of vaccines against diseases and/or administration of probiotics, prebiotics, and fermented feeds (FF). The beneficial effects of lactic acid bacteria (LAB)based probiotics on suppressing bacterial infections are probably through creating a hostile microecology with the production of lactic acid and decrease in pH, inhibiting bacterial adherence and competing for nutrients with undesired microorganisms (Gadde et al.,

The limitation of the application of antibiotics (AB) in the poultry industry due to their negative aspects, such as antimicrobial resistance, has increased the prevalence of avian pathogens such as Salmonella. Among the different subspecies of Salmonella, Salmonella enterica serovar Typhimurium (ST) is one of the most common serotypes causing foodborne infections in humans, around the world (Rodpai et al., 2013). Poultry products have been recognized as an important reservoir for the transmission of

 C 2019 Poultry Science Association Inc. Received February 25, 2019. Accepted May 24, 2019. 1 Corresponding authors: [email protected] (RB); Jazi. [email protected] (VJ)

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V. Jazi

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MATERIALS AND METHODS All experimental procedures used in this study were approved by the Animal Care and Use Committee of the University of Mohaghegh Ardabili, Ardabil, Iran.

Fermentation of Soybean Meal The FSBM was produced through fermentation of the soybean meal (SBM) with Lactobacillus acidophilus (PTCC1643), Lactobacillus plantarum

(PTCC1058), Bacillus subtilis (PTCC1156) bacteria, and Aspergillus oryzae (PTCC5163) fungus (Iranian Research Organization for Science and Technology; IROST, Tehran, Iran) according to a previous study (Jazi et al., 2018b). Briefly, each kilogram of SBM as substrate was mixed and inoculated with 1 L of distilled water containing approximately 108 CFU/mL of Lactobacillus acidophilus, Lactobacillus plantarum, Bacillus subtilis, and 106 spores/mL of Aspergillus oryzae and incubated at 30◦ C for 7 d in three fermentation tanks equipped with a one-way valve. Fermented products were then dried for 2 d at 50◦ C and ultimately ground to 0.5 mm in particle size and kept at room temperature until mixed in the experimental diets. All the chemicals used in this experiment were of analytical grade (Merck, Darmstadt, Germany). Three replicates (one sample from each tanks) from SBM were collected before and after fermentation for the determination of microbial population, pH, lactic acid content, and chemical composition.

Physiological Characteristics of SBM and FSBM In order to enumerate the bacterial counts, 1 g of each sample (SBM or FSBM) was used to make 10fold serial dilutions using buffered peptone water. Then 0.1 mL of appropriate dilutions was spread on the plates. The enumeration of LAB and coliform bacteria was determined on de Man Rogosa and Sharpe agar and violet red bile agar following anaerobic incubation at 37◦ C for 24 to 48 h. The lactic acid content was measured by HPLC according to the method described by Marsili et al. (1983). The pH value was measured using a portable pH meter (pH Meter CG 804, Schott Gerate).

Proximate Analysis of SBM and FSBM Unfermented and fermented samples were analyzed for dry matter, crude protein, ether extract, crude fiber, and ash using standard methods of the Association of Official Analytical Chemists (AOAC, 2005, Table 1). The amino acid profile of feedstuffs was analyzed using an automatic amino acid analyzer (Hitachi L-8800, Japan). Before analysis, samples were hydrolyzed with 6 M HCl at 110◦ C for 24 h. The sulfur-containing amino acids, cysteine, and methionine were oxidized using performic acid before the acid hydrolysis. Phytic acid concentration was analyzed after the extraction of the samples with HCl and Na2 SO4 at 660 nm absorbance (Vaintraub and Lapteva, 1988). Trypsin inhibitor activity in the feedstuffs was assessed by the method of Smith et al. (1980), and their results were presented as milligram trypsin inhibited per gram of dry sample. Amounts of β -conglycinin and glycinin in unfermented and fermented samples were determined following the methods described by Wang et al. (2014).

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2017). Previous studies have shown that LAB can reduce the Salmonella colonization, invasion, and inflammation caused by Salmonella infection in broilers (Chen et al., 2012; Jazi et al., 2018a). Xylooligosaccharides (XOS) are xylose-based oligomers, which are considered as one of the functional oligosaccharides. It was shown that dietary XOS increased the relative abundance of genus Lactobacillus and stimulated the immune response against infection by reducing Salmonella colonization (Pourabedin et al., 2016). In recent years, the fermentation of feed has been regarded as a novel and effective biosafe nutritional strategy for substituting AB in animal feed industry (Shi et al., 2017; Jazi et al., 2018b). One of the beneficial and key properties of FF is associated with the presence of antibacterial compounds in such feeds. FF are usually rich in probiotics (e.g., LAB) and prebiotics and are characterized by high concentrations of lactic acid and acetic acid and an acidic pH of about 4 (Wang et al., 2018; Jazi et al., 2018b). Apparently, LAB and organic acids present in FF by acidifying the various sections of the gut and promoting beneficial microbial species prevent the establishment, growth, and proliferation of pathogenic bacteria and thereby improve animal sanitary status and growth performance (Shabani et al., 2019). Furthermore, the FF could affect the gut microbiota by providing nutrients and energy to useful bacterial species in the microbial community (Wang et al., 2018). It is also accepted that the fermentation process can improve the nutritional value of raw plant protein meals by reducing the anti-nutritional factors and improving nutrient bioavailability (Chen et al., 2013; Jazi et al., 2017; Shi et al., 2017), which may result in better usage of nutrients in birds. Thus, it was hypothesized that fermented soybean meal (FSBM) due to possessing probiotic and/or prebiotic effects may provide protection to broilers by maintaining growth performance and gut health during a bacterial challenge. Moreover, in view of the scarcity of data in this field, the current study aimed to compare the efficacy of dietary FSBM, probiotic and prebiotic supplements on growth performance, gut microbiota, intestinal morphology, and hematology parameters in ST-infected broiler chickens.

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FERMENTED SOYBEAN MEAL AND SALMONELLA Table 1. Composition of SBM and FSBM (% of dry matter basis unless otherwise specified). Ingredients1 SBM

FSBM

SEM

P-value

pH Lactic acid (mmol/kg) LAB2 (Log10 CFU/g) Coliform (Log10 CFU/g) Dry matter Crude protein Ether extract Crude fiber Ash Phytic acid (g/100 g) Trypsin inhibitor (mg/g) β -conglycinin (mg/g) Glycinin (mg/g) Indispensable amino acids Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine Dispensable amino acids Alanine Asparagine Cysteine Glutamine Glycine Proline Serine Tyrosine

6.14a 25.57b 3.49b 4.90a 91.87 44.29b 1.96a 7.02a 6.15 0.51a 2.68a 34.02a 61.54a

4.01b 176.32a 10.08a 0.00b 90.39 46.75a 1.04b 5.85b 6.37 0.14b 0.73b 18.61b 15.20b

0.09 1.28 0.25 0.34 1.17 0.26 0.13 0.20 0.13 0.04 0.16 1.98 1.77

0.001 < 0.001 < 0.001 0.001 0.425 0.002 0.016 0.013 0.310 0.002 0.001 0.005 < 0.001

2.91 1.27 2.01 3.18 2.60 0.58 2.09 1.68 1.85

3.02 1.23 2.04 3.09 2.82 0.50 2.15 1.70 1.81

0.12 0.04 0.05 0.13 0.08 0.07 0.05 0.07 0.07

0.585 0.870 0.691 0.605 0.138 0.152 0.474 0.862 0.730

1.73 4.92 0.61 7.82 1.75 2.20 1.98 1.57

1.68 5.01 0.60 8.58 1.64 2.07 2.25 1.61

0.08 0.10 0.07 0.20 0.05 0.05 0.11 0.05

0.687 0.545 0.224 0.059 0.902 0.688 0.160 0.642

1

SBM = soybean meal; FSBM = fermented soybean meal. LAB = lactic acid bacteria. a,b Means with no common superscript within each column are significantly (P < 0.05) different. 2

Birds, Experimental Diets, and ST Challenge Four hundred and fifty newly hatched male broiler chickens (Ross 308) were obtained from a commercial hatchery (Sepid Makian Company, Guilan, Iran). Birds were randomly housed in 30 pens and each pen was randomly assigned to one of the 5 experimental treatments in a 24-d study. Each treatment was replicated 6 times with each pen accommodating 15 birds. The experimental treatments were as follows: (1) non-infected control (NC), birds fed the unsupplemented basal diet and not challenged with Salmonella Typhimurium (ST); (2) infected control (IC), birds fed the unsupplemented basal diet and orally challenged with ST; (3) XOS, birds fed the basal diet supplemented with 2 g/kg XOS (Longlive 95p; Shandong Longlive Bio-Technology Company, China) and orally challenged with ST; (4) LAC, birds fed the basal diet supplemented with 0.2 g/kg LAB-based probiotic (Lactofeed; Tak Genezist Company, Iran, contains 5.2 × 1010 cfu/g of 4 different LAB including Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium thermophilum, and Enterococcus faecium) and orally challenged with ST; (5) FSBM, birds fed the diet in which

SBM was replaced with FSBM and orally challenged with ST. All birds had free access to clean water and feed throughout the study and ST challenge was performed on d 3 post-hatch. The experimental diets were AB-free and coccidiostat-free and were formulated to meet or exceed minimum nutrient requirements of Ross 308 broiler chickens (Aviagen, 2014) for starter (1 to 10 d) and grower (11 to 24 d) phases (Table 2). Diets were fed in mash form. The lighting and temperature program followed the industry standards described in the Ross 308 breed management manual (Aviagen, 2014). A primary poultry isolate of ST strain (ATCC 14028) was sourced from IROST (Tehran, Iran) and was used in this study to challenge the birds. After cultivating ST on special culture medium Xylose lysine tergitol 4 (XLT4) agar, single colony with a black center was picked up and transferred into a tube contained 10 mL trypticase soy broth and then incubated at 37◦ C for 24 h. The concentration of ST, after diluting the inoculum with sterile phosphate-buffered, was determined as 1 × 106 cfu/mL. On d 3 post-hatch, each bird in the ST-challenge group was orally administrated a one-off dose of 0.5 mL ST suspension. All the unchallenged

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Item

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JAZI ET AL. Table 2. Ingredients and nutrient compositions of experimental diets. Experimental diets1 Starter (0 to 10 D) Ingredients (%)

FSBM

Grower (11 to 24 D) SBM

FSBM

50.58 38.11 – 3.0 3.74 2.12 0.81 0.3 0.07 0.25 0.25 0.32 0.32 0.13

53.66 – 35.62 3.0 3.13 2.13 0.83 0.3 0.07 0.25 0.25 0.32 0.29 0.15

51.07 39.20 – – 5.74 1.92 0.68 0.3 0.09 0.25 0.25 0.31 0.12 0.07

54.24 – 36.64 – 5.11 1.92 0.70 0.3 0.09 0.25 0.25 0.31 0.09 0.10

3,000 23.08 0.96 0.48 0.16 1.30 0.64 0.94 0.86

3,000 23.08 0.96 0.48 0.16 1.30 0.64 0.94 0.86

3,100 21.53 0.87 0.43 0.16 1.15 0.59 0.87 0.77

3,100 21.53 0.87 0.43 0.16 1.15 0.59 0.87 0.77

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SBM = soybean meal; FSBM = fermented soybean meal. Vitamin premix provided the following per kilogram of diet: vitamin A, 11,500 IU; cholecalciferol, 2,100 IU; vitamin E, 22 IU; vitamin K3, 1.50 mg; thiamine, 3 mg; riboflavin, 4.4 mg; pantothenic acid, 25 mg; niacin, 40 mg; choline chloride, 560 mg; biotin, 0.1 mg; folic acid, 0.8 mg; pyridoxine 10 mg; vitamin B12, 0.060 mg. 3 Trace mineral premix provided the following in milligrams per kilogram of diet: iron, 50 mg; zinc, 55 mg; manganese, 75 mg; iodine, 1.8 mg; copper, 8 mg; selenium, 0.3 mg; cobalt, 0.2 mg. 4 SID = Standardized ileal digestible. 2

birds, received the same dose per os of sterile PBS. To avoid cross contamination, birds in the unchallenged and challenged groups were placed in separate rooms and reared under the same management conditions.

Growth Performance and Sampling The feed intake (FI) and body weight (BW) were determined for the starter and grower phases; BW gain, FI, and mortality-corrected feed conversion ratio (FCR) calculated for d 1 to 10, d 11 to 24, and d 1 to 24. Two birds per pen with BW close to the pen mean weight (12 chicks/treatment) were selected for blood, digesta, and tissue sample collection at d 10 and 24 post-hatch.

Gut Colonization and Organ Invasion by Salmonella On d 10 and 24 post-hatch, the contents of crop, ileum, and ceca were collected by gently squeezing the digesta into plastic containers, and pooled per replicate. To measure ST counts in the crop, ileum, and ceca, approximately 1 g of the digesta contents of each sample was diluted with 0.1% peptone water and prepared for serial dilutions. Thereafter, the samples were

homogenized for 1 min. From each dilution, 0.1 mL of dilution suspension was plated in triplicate on XLT4 agar (Difco) containing 100 mg/mL of nalidixic acid, and plates were then incubated for 24 h at 37◦ C under anaerobic conditions. In order to determine the number of total LAB, 1 g of each sample (crop, ileum, and ceca) was diluted to 1:4 wt/vol with sterile 0.9% saline. Ten-fold dilutions of each sample was made in a sterile 96-well Bacti flat bottom plate, and the diluted samples were plated on 3 different plates of medium to evaluate the total number of LAB in modified de Man, Rogosa, Sharpe agar. All plates were incubated at 37◦ C for 48 h and bacterial numbers were expressed as log10 CFU per gram sample. After recording the weight of liver, spleen, and bursa of Fabricius, the organ invasion was determined by counting ST colonies on XLT4 agar. Briefly, samples of each organ were homogenized separately. These homogenized solutions were diluted 1:10 with a sterile solution of 0.1% peptone water and then 100 μL of suspension for each sample was spread on XLT4 agar. To evaluate the pH value of crop, ileum, and cecal contents, 1 g of each digesta from sampled birds was collected and placed into 2 mL distilled water. The pH value was determined using a portable pH meter (pH meter CG 804, Schott Gerate).

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Corn Soybean meal Fermented soybean meal Corn gluten meal Soybean oil Dicalcium phosphate Calcium carbonate Salt Sodium bicarbonate Vitamin premix2 Mineral premix3 DL-Methionine L-Lysine HCL L-Threonine Nutrient composition ME (Kcal/kg) Crude protein (%) Ca (%) Av. P (%) Na (%) SID4 Lys (%) SID Met (%) SID Met + Cys (%) SID Thr (%)

SBM

FERMENTED SOYBEAN MEAL AND SALMONELLA

Intestinal Morphology and Hematological Parameters

Statistical Analysis Microbial characteristics and chemical composition of SBM samples before and after fermentation process were compared using t-test. The incidence of ST recovery within experiments was compared, testing all possibilities, using the chi-squared test of independence to determine significant (P < 0.05) differences between groups within experiments. Data obtained during the feeding trial of broiler chickens were analyzed in a completely randomized design using the GLM procedures of SAS software (SAS Institute Inc., 2010). Results were expressed as treatment means with their pooled standard error of the mean. When a significant effect of treatment was detected, means were compared using Tukey’s HSD test at P < 0.05.

RESULTS Physiological and Chemical Characteristics of SBM and FSBM The analyzed chemical composition and physiological characteristics of SBM and FSBM are listed in Table 1. Results demonstrated that microbial fermentation of SBM significantly increased crude protein, lactic acid concentrations, and number of LAB (P < 0.05). The pH value, counts of coliform, crude fiber, ether extract, phytic acid, trypsin inhibitor, β -conglycinin, and glycinin in FSBM were significantly lower compared with SBM (P < 0.05). However, the process of microbial fermentation had no significant effect on SBM dry matter, ash, and amino acid concentration.

Growth Performance Effects of experimental treatments on the growth performance of birds from d 1 to 24 are shown in Table 3. Birds in the IC group (infected control) demonstrated a depressed performance. In IC group, FI and BW gain significantly reduced in periods of d 1 to 10, d 11 to 24, and d 1 to 24, compared with the NC (noninfected control) group (P < 0.05). During the entire study, ST challenge in IC group significantly increased FCR compared with the NC birds (P < 0.05). In the starter phase, feed additives or FSBM had no effect on BW gain and FI of birds infected with ST when compared with IC birds. However, challenged birds fed the diets containing XOS, LAC, and FSBM had significantly lower FCR compared with the IC birds (P < 0.05). Broilers fed the diets containing XOS, LAC, and FSBM had greater BW gain, FI, and lower FCR than birds in the IC group during d 11 to 24 and d 1 to 24 (P < 0.05). For the FCR during the starter and overall period, there was no difference between birds fed the FSBM diet compared with the NC broilers. During the entire trial (d 1 to 24), the mortality rate was lower than 3.5% and deaths were not related to any of the dietary treatments or challenge group.

The Relative Organ Weights The relative weights of organs are given in Table 4. On d 10, all birds challenged with ST had heavier liver and bursa of Fabricius than birds in the NC group (P < 0.05). Compared to IC birds, dietary supplementation of XOS decreased the liver weight of broilers challenged with ST (P < 0.05). The bursa of Fabricius weight was not affected by the experimental treatments. On d 24, no effect of challenge was observed on liver weight, while birds fed the diets containing LAC and FSBM had heavier liver (P < 0.05). The spleen weight at d 10 and 24 and bursa of Fabricius weight at d 24 were not affected by the challenge or dietary treatments.

Organ Invasion by Salmonella Table 5 outlines internal organ invasion by Salmonella at d 10 and 24. The internal organ samples (liver, spleen, and bursa of Fabricius) taken from the uninfected birds were free of ST on d 10 and 24. In birds infected with ST, the levels of Salmonella recovered from liver, spleen, and bursa of Fabricius were significantly higher than birds in the NC group at d 10 (P < 0.05). In contrast, the number of Salmonella in the liver, spleen, and bursa of Fabricius of birds fed the XOS, LAC, and FSBM containing diets at d 10 were significantly lower compared with the IC birds (P < 0.05). Nevertheless, on d 24, the prevalence of Salmonella in the most samples that were taken from internal organs of challenged birds were detected negative.

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For morphometric analysis, the intestinal contents were flushed away with cold PBS and approximately 3 cm pieces from the midpoint of duodenum, jejunum, and ileum were cut and immersed in 10% formaldehyde solution before embedded into a fixation Bouin’s solution and paraffin embedding. Intestinal morphological examinations were carried out similar to the procedures described by Jazi et al. (2018b). All morphological parameters were measured using the ImageJ Software Package (http://rsb.info.nih.gov/ij/). To acquire heterophil to lymphocyte (H:L) ratio, blood samples were collected in heparinized tubes by puncturing the brachial vein from the same selected birds, 1 drop was smeared on each of 2 glass slides. The smears of blood were prepared by MayGrunwald-Giemsa coloring, and H:L ratio was calculated by counting 100 leukocytes per slide for each bird.

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Table 3. Influence of the experimental treatments on growth performance of Salmonella Typhimurium (ST)-infected broiler chicks. Treatments1 Items2

IC

204a 252a 1.23c

176b 230b 1.31a

767a 1132a 1.47c

659c 1049c 1.59a

972a 1384a 1.42c 1.10

835c 1279c 1.53a 3.33

XOS

LAC

181b 234b 1.29a,b

FSBM

SEM

P-value

183b 233b 1.27b

189b 241a,b 1.27b

4.16 4.57 0.01

0.005 0.023 0.001

725b 1099b 1.51b

736b 1105a,b 1.50b,c

749a,b 1115a,b 1.49b,c

7.88 10.33 0.01

0.001 0.002 0.001

906b 1333b 1.47b 2.22

919b 1338b 1.45b 3.33

938b 1356a,b 1.44b,c 2.22

11.01 13.24 0.01 1.09

0.001 0.004 0.001 0.768

1 NC = birds fed a basal diet; IC = birds fed a basal diet and infected with ST; XOS = birds fed a basal diet containing xylooligosaccharide (2 g/kg) and infected with ST; LAC = birds fed a basal diet containing lactic acid bacteria (0.2 g/kg) and infected with ST; FSBM = birds fed diet containing fermented soybean meal (complete replacement of SBM with fermented SBM) and infected with ST. 2 BW gain = body weight gain; FI = feed intake; FCR = feed conversion ratio. a–c Means with no common superscript within each row are significantly (P < 0.05) different.

Table 4. Influence of the experimental treatments on internal organ weight (% of live body weight) of Salmonella Typhimurium (ST)-infected broiler chicks. Treatments1 Items d 10 Liver Spleen Bursa of Fabricius d 24 Liver Spleen Bursa of Fabricius

NC

IC

XOS

LAC

FSBM

SEM

P-value

2.49c 0.18 0.26b

3.24a 0.23 0.38a

2.77b 0.20 0.36a

2.95a,b 0.22 0.34a

3.04a,b 0.23 0.33a

0.09 0.01 0.02

0.002 0.183 0.001

2.55b 0.15 0.29

2.68b 0.16 0.35

2.60b 0.21 0.29

2.77a,b 0.19 0.33

2.96a 0.19 0.30

0.07 0.02 0.02

0.005 0.347 0.219

1 NC = birds fed a basal diet; IC = birds fed a basal diet and infected with ST; XOS = birds fed a basal diet containing xylooligosaccharide (2 g/kg) and infected with ST; LAC = birds fed a basal diet containing lactic acid bacteria (0.2 g/kg) and infected with ST; FSBM = birds fed diet containing fermented soybean meal (complete replacement of SBM with fermented SBM) and infected with ST. a–c Means with no common superscript within each row are significantly (P < 0.05) different.

Gut Microbes and pH Value Aa illustrated in Table 6, feeding LAC and FBSM diets significantly decreased pH value in the crop, ileum, and ceca of ST-challenged birds compared with both the IC and NC groups, at d 10 and 24 (P < 0.05). In addition, diets supplemented with XOS reduced pH in the ileal and cecal digesta contents at d 10 and 24 (P < 0.05). An increased level of Salmonella in the contents of crop, ileum, and ceca was observed in the IC group on d 10 compared with NC group (Figure 1; P < 0.05). Broilers fed the diets containing feed additives or FSBM had lower counts of Salmonella in the crop, ileal, and cecal digesta compared to the birds in the IC group (P < 0.05). Birds on the FSBM treatment group had fewer Salmonella counts compared to other experimental treatments (P < 0.05). On d 10, counts of LAB in crop, ileum, and ceca were increased (Figure 2; P < 0.05) in response to feeding the FSBM and LAC diets. In addition, on d 24, counts of LAB in crop, ileum, and ceca of the birds fed FBSM, LAC, and

XOS (except crop) diets were greater (Figure 3; P < 0.05) than those fed the control diet. At the same time, birds on the FSBM treatment group had the highest LAB populations in the ileal, and cecal digesta compared with other experimental treatments (P < 0.05). The crop, ileum, and ceca digesta samples recovered from the NC broiler chicks were free from ST contamination. at both times. No ST colony was observed in the crop, ileal, and cecal contents of birds challenged with ST compared with the unchallenged birds on d 24.

Hematological Parameters As demonstrated in Table 7, although ST challenge had no influence on heterophil and lymphocyte numbers, but significantly increased the H:L ratio on d 10. Heterophil counts as well as H:L ratio were significantly higher in birds challenged with ST than unchallenged birds on d 24 (P < 0.05). The lowest H:L ratio among ST-infected birds belonged to XOS, LAC, and FSBM

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d 1 to 10 BW gain, g FI, g FCR d 11 to 24 BW gain, g FI, g FCR d 1 to 24 BW gain, g FI, g FCR Mortality (%)

NC

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FERMENTED SOYBEAN MEAL AND SALMONELLA

Table 5. Influence of the experimental treatments on organ Salmonella invasion in Salmonella Typhimurium (ST)-infected broiler chicks. Treatments1 Items

Spleen Bursa of Fabricius d 24 Liver Spleen Bursa of Fabricius

IC

XOS

LAC

FSBM

SEM

P-value

(0/12)2 0d (0/12) 0c (0/12) 0c

(12/12) 100a (11/12) 92a (12/12) 100a

(9/12) 75a,b (7/12) 58a,b (9/12) 75a,b

(5/12) 41c (6/12) 50b (6/12) 50b

(6/12) 50c (6/12) 50b (6/12) 50b

11.12

< 0.001

12.20

0.001

11.18

< 0.001

(0/12) 0 (0/12) 0 (0/12) 0

(1/12) 8.3 (1/12) 8.3 (0/12) 0

(0/12) 0 (1/12) 8.3 (0/12) 0

(0/12) 0 (0/12) 0 (0/12) 0

(0/12) 0 (0/12) 0 (0/12) 0

3.72

0.41

5.27

0.56

0.00

–

1 NC = birds fed a basal diet; IC = birds fed a basal diet and infected with ST; XOS = birds fed a basal diet containing xylooligosaccharide (2 g/kg) and infected with ST; LAC = birds fed a basal diet containing lactic acid bacteria (0.2 g/kg) and infected with ST; FSBM = birds fed diet containing fermented soybean meal (complete replacement of SBM with fermented SBM) and infected with ST. 2 Data expressed as positive test of ST contamination in birds (%). a-c Means with no common superscript within each row are significantly (P < 0.05) different.

Table 6. Influence of the experimental treatments on pH value of gastrointestinal tract of Salmonella Typhimurium (ST)-infected broiler chicks. Treatments1 Items d 10 Crop Ileum Ceca d 24 Crop Ileum Ceca

NC

IC

XOS

LAC

FSBM

SEM

P-value

4.86a 6.74a 6.12a

4.89a 6.76a 6.20a

4.82a 6.64a,b 5.81b

4.65b 6.54b 5.79b

4.51b 6.51b 5.64b

0.05 0.07 0.05

0.005 0.040 0.001

4.79a 6.43a 6.25a

4.75a 6.48a 6.17a

4.71a,b 6.18b 5.92b

4.58b 6.11b,c 5.87b

4.42c 6.01c 5.85b

0.04 0.03 0.03

0.001 < 0.001 0.001

1 NC = birds fed a basal diet; IC = birds fed a basal diet and infected with ST; XOS = birds fed a basal diet containing xylooligosaccharide (2 g/kg) and infected with ST; LAC = birds fed a basal diet containing lactic acid bacteria (0.2 g/kg) and infected with ST; FSBM = birds fed diet containing fermented soybean meal (complete replacement of SBM with fermented SBM) and infected with ST. a-c Means with no common superscript within each row are significantly (P < 0.05) different.

Figure 1. Influence of the experimental treatments on Salmonella population in crop, ileum, and ceca of broiler chicks challenged with ST at d 10. IC = birds fed a basal diet and infected with ST; XOS = birds fed a basal diet containing xylooligosaccharide (2 g/kg) and infected with ST; LAC = birds fed a basal diet containing lactic acid bacteria (0.2 g/kg) and infected with ST; FSBM = birds fed diet containing fermented soybean meal (complete replacement of SBM with fermented SBM) and infected with ST. a-c Means within the same intestinal segment not sharing a same letter are significantly different (P < 0.05).

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d 10 Liver

NC

8

JAZI ET AL. 8.4 a a

b

a

8

ab b

7.8

b

b

ab

a b

b

7.6

b

7.4 7.2 7 Crop

Ileum NC

IC

XOS

LAC

Ceca FSBM

Figure 2. Influence of the experimental treatments on lactic acid bacteria population in crop, ileum, and ceca of broiler chicks challenged with ST at d 10. NC = birds fed a basal diet; IC = birds fed a basal diet and infected with ST; XOS = birds fed a basal diet containing xylooligosaccharide (2 g/kg) and infected with ST; LAC = birds fed a basal diet containing lactic acid bacteria (0.2 g/kg) and infected with ST; FSBM = birds fed diet containing fermented soybean meal (complete replacement of SBM with fermented SBM) and infected with ST. a-c Means within the same intestinal segment not sharing a same letter are significantly different (P < 0.05).

Figure 3. Influence of the experimental treatments on lactic acid bacteria population in crop, ileum, and ceca of broiler chicks challenged with ST at d 24. NC = birds fed a basal diet; IC = birds fed a basal diet and infected with ST; XOS = birds fed a basal diet containing xylooligosaccharide (2 g/kg) and infected with ST; LAC = birds fed a basal diet containing lactic acid bacteria (0.2 g/kg) and infected with ST; FSBM = birds fed diet containing fermented soybean meal (complete replacement of SBM with fermented SBM) and infected with ST. a-c Means within the same intestinal segment not sharing a same letter are significantly different (P < 0.05).

treatment groups at d 10 and 24 (P < 0.05). There was no significant difference between birds on the NC, LAC, and FSBM groups in terms of H:L ratio at d 21. Heterophil counts at d 10 and lymphocyte counts at d 10 and 24 were not affected by the challenge or experimental treatments.

Small Intestinal Morphology The effect of experimental treatments on intestinal morphology of broiler chicks challenged by ST are presented in Tables 8 and 9. To investigate whether Salmonella colonization and LAB affect intestinal morphology, we evaluated the morphological characteristics (such as villi height [VH], crypt depth [CD], and VH:CD ratio) of all 3 parts of the small intestine. VH

and VH:CD ratio in duodenum, and jejunum significantly decreased in the ST-challenged birds compared with the unchallenged birds at d 10 (P < 0.05). On d 24, ST challenge also reduced duodenal VH and VH:CD ratio in broilers fed the control diet (P < 0.05). However, the XOS, LAC, and FSBM treatment groups had significantly greater VH and VH:CD ratio in the duodenum (at d 10 and 24) and jejunum (at d 10), compared to the IC group (P < 0.05). On d 24, no significant difference was observed in the morphological parameters between NC and FSBM treatment groups (Table 9). The VH, CD, and VH:CD ratio in the ileum at d 10 and 24 and the CD in the duodenum and jejunum at d 10 and 24 as well as the VH, and VH:CD ratio in the jejunum at d 24 were not influenced by the dietary treatments or challenge.

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Lactic acid bacteria [log(CFU/g)]

a ab

8.2

9

FERMENTED SOYBEAN MEAL AND SALMONELLA Table 7. Influence of the experimental treatments on differential blood leucocytes counts (%) of Salmonella Typhimurium (ST)-infected broiler chicks. Treatments1 Items

IC

XOS

LAC

FSBM

SEM

P-value

25.79 57.64 0.44c

31.45 52.35 0.60a

30.56 54.75 0.50b

27.87 54.89 0.50b

28.50 55.13 0.51b

1.85 2.36 0.01

0.252 0.647 0.001

20.12b 53.75 0.37c

28.87a 50.31 0.57a

24.50a,b 51.37 0.47b

22.25b 53.62 0.41b,c

21.75b 54.25 0.40b,c

1.65 1.45 0.03

0.012 0.275 0.011

1 NC = birds fed a basal diet; IC = birds fed a basal diet and infected with ST; XOS = birds fed a basal diet containing xylooligosaccharide (2 g/kg) and infected with ST; LAC = birds fed a basal diet containing lactic acid bacteria (0.2 g/kg) and infected with ST; FSBM = birds fed diet containing fermented soybean meal (complete replacement of SBM with fermented SBM) and infected with ST. a–c Means with no common superscript within each row are significantly (P < 0.05) different.

Table 8. Influence of the experimental treatments on small intestinal morphology (μm) of Salmonella Typhimurium (ST)-infected broiler chicks at d 10. Treatments1 Items Duodenum Villus height (VH) Crypt depth (CD) VH:CD ratio Jejunum Villus height (VH) Crypt depth (CD) VH:CD ratio Ileum Villus height (VH) Crypt depth (CD) VH:CD ratio

NC

IC

XOS

LAC

FSBM

SEM

P-value

895a 182 4.91a

784c 191 4.10c

836b 183 4.58b

840b 181 4.63b

846b 185 4.57b

9.46 4.06 0.07

< .001 0.475 0.001

598a 141 4.24a

464c 136 3.40c

541b 140 3.85b

549b 143 3.84b

553b 139 3.96b

11.23 2.58 0.05

< 0.001 0.490 0.001

415 120 3.48

381 117 3.24

387 123 3.14

404 120 3.38

393 118 3.32

11.12 4.10 0.10

0.271 0.895 0.283

1 NC = birds fed a basal diet; IC = birds fed a basal diet and infected with ST; XOS = birds fed a basal diet containing xylooligosaccharide (2 g/kg) and infected with ST; LAC = birds fed a basal diet containing lactic acid bacteria (0.2 g/kg) and infected with ST; FSBM = birds fed diet containing fermented soybean meal (complete replacement of SBM with fermented SBM) and infected with ST. a–c Means with no common superscript within each row are significantly (P < 0.05) different.

DISCUSSION In the present study, the fermentation process was successfully established evidenced by the decrease in pH value (from 6.14 to 4.01), and coliforms counts (from 4.90 to 0.00 Log10 cfu/g), and in contrast the increase in LAB numbers (from 3.49 to 10.08 Log10 cfu/g) and concentration of lactic acid (from 25.57 to 176.32 mmol/kg) observed in the FSBM. The increase in the amount of lactic acid of FSBM can be mainly attributed to the presence of LAB and their microbial metabolites such as organic acids during the fermentation process, which results in a reduced pH value and inhibited growth of acid-sensitive pathogenic bacteria e.g., coliform (Shi et al., 2017; Jazi et al., 2018b). In addition, the LAB is capable of producing antimicrobial compounds such as bacteriocins with a bacteriostatic or bactericidal operation, which control the growth of undesirable bacteria (Parente and Ricciardi, 1999). The increase in LAB is probably due to the following events. Aspergillus oryzae consume the oxygen present in the fermentation envi-

ronment and supply a favorable environment for the growth and viability of Lactobacillus acidophilus, Lactobacillus plantarum, and Bacillus subtilis. Likewise, it has been shown that Bacillus subtilis by producing subtilisin and catalase is able to stimulate the growth of Lactobacillus spp. (Hosoi et al., 2000). Similarly, other studies showed that the use of probiotic species for fermentation of plant protein by-products significantly decreased pH value and increased LAB (Ashayerizadeh et al., 2017; Jazi et al., 2017). Therefore, feeding FF with low pH, high amount of lactic acid and acetic acid, and high numbers of LAB can be effective in improving the gut health through intestinal microflora balance and development of intestine. Microbial fermentation is also able to change the chemical composition of SBM. Similar to the results of this study, Kook et al. (2014) and De Oliveira Silva et al. (2018) showed that the microbial fermentation of SBM increased the crude protein content, while it decreased the crude fiber, ether extract, and phytic acid. The increase in crude protein may be due to the

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d 10 Heterophil (H) Lymphocyte (L) H:L ratio d 24 Heterophil (H) Lymphocyte (L) H:L ratio

NC

10

JAZI ET AL. Table 9. Influence of the experimental treatments on small intestinal morphology (μm) of Salmonella Typhimurium (ST)-infected broiler chicks at d 24. Treatments1 Items

IC

XOS

LAC

FSBM

SEM

P-value

1103a 167 6.60a

1001c 168 5.96c

1045b 170 6.18b,c

1051b 165 6.32a,b

1087a 164 6.63a

11.31 3.51 0.11

< 0.001 0.776 0.001

835 139 5.99

799 144 5.56

817 138 5.92

821 136 6.03

832 141 5.90

11.16 2.85 0.09

0.201 0.391 0.212

585 112 5.25

552 118 4.73

560 115 4.84

563 110 5.13

578 112 5.18

11.26 5.38 0.19

0.250 0.813 0.276

1 NC = birds fed a basal diet; IC = birds fed a basal diet and infected with ST; XOS = birds fed a basal diet containing xylooligosaccharide (2 g/kg) and infected with ST; LAC = birds fed a basal diet containing lactic acid bacteria (0.2 g/kg) and infected with ST; FSBM = birds fed diet containing fermented soybean meal (complete replacement of SBM with fermented SBM) and infected with ST. a–c Means with no common superscript within each row are significantly (P < 0.05) different.

hydrolysis of macro-molecular proteins and increased small-size peptides through proteases produced by microorganisms (Hong et al., 2004; Kook et al., 2014). In addition, the formation of microbial protein and an increase in the number of microflora (above 60% microbial biomass is composed of the protein), as well as a reduction in carbohydrates during fermentation, can explain the increase in the crude protein content of the fermented products (Terlabie et al., 2006; Jazi et al., 2017). The reduction in crude fiber and phytic acid contents of FSBM compared with SBM is likely due to the synthesis of enzymes such as cellulase, xylanase, glucanase, and phytase by fungal and bacterial species used in this study, as previously documented by Ashayerizadeh et al. (2017). The destruction of fiber cells also can lead to an increase in the amount of nonprotein nitrogen and nitrogenous compounds (Kanyinji and Sichangwa, 2014). The microbial lipase activity of Bacillus subtilis could possibly be responsible for the lower amount of ether extract in FSBM compared with SBM (Terlabie et al., 2006). Some of the SBM anti-nutritional compounds like trypsin inhibitor and antigenic proteins (glycinin and β -conglycinin) can interfere in digestive functions and impair the nutrient digestibility (Jazi et al., 2018b). The fermentation of SBM with Bacillus subtilis, Aspergillus oryzae, Lactobacillus acidophilus, and Lactobacillus plantarum in the current study reduced trypsin inhibitor, β -conglycinin, and glycinin concentrations. The positive effect of fermentation microorganisms on hydrolysis of antigenic proteins of SBM may be attributed to Aspergillus oryzae and Bacillus subtilis ability in secreting proteases, including aminopeptidases, metalloproteinases serine, endopeptidases, and aspartic endopeptidases (Hong et al., 2004; Shi et al., 2017). As a novel nutritional strategy, we evaluated the efficacy of FSBM and compared it with XOS and LAC on performance and gut health under ST disease model.

Our results indicated that ST challenge reduced FI and BW and consequently impaired the FCR of the birds. These results corroborate with the studies of Shao et al. (2016) and Jazi et al. (2018a). These adverse effects have been attributed to the reduced appetite and nutrient absorption due to intestinal mucosal damage and redirecting nutrients for immune development following the activation of the immune system and inflammatory responses (Chalghoumi et al., 2009; Shao et al., 2016). The competition by Salmonella with the host for nutrients is another likely reason for the decrease in growth performance. In the present study, the tested feed additives of XOS and LAC alleviated growth performance loss and improved FCR in birds challenged by ST. Similarly, some studies have shown that probiotic supplementation can promote performance traits in chickens after inoculation of birds with pathogenic bacteria such as Salmonella (Park and Kim, 2014) and Escherichia coli (Wang et al., 2017). Improved growth performance in birds fed probiotic could be due to the inhibition of Salmonella colonization through production of probiotic metabolites with bactericidal and bacteriostatic properties that could protect the intestinal structure and barrier function against harmful bacteria as well as modulating the immune responses (Wang et al., 2017; Jazi et al., 2018a). Prebiotics like mannan oligosaccharide and XOS modulate the composition and functions of gut microflora by selectively stimulating the growth and/or activity of beneficial bacteria (Pourabedin et al., 2016; Ding et al., 2017). It was also reported that short-chain fatty acids (SCFA; end-product of fermentation of prebiotics), as an energy source for gut epithelial cells (Gadde et al., 2017), consequently improve gut health and growth performance. The results of this experiment indicated that the use of FSBM in broiler diets improved the growth performance following the challenge similar to the other

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Duodenum Villus height (VH) Crypt depth (CD) VH:CD ratio Jejunum Villus height (VH) Crypt depth (CD) VH:CD ratio Ileum Villus height (VH) Crypt depth (CD) VH:CD ratio

NC

FERMENTED SOYBEAN MEAL AND SALMONELLA

As for the effect of the LAB-based probiotics, it is well documented that LAB play an important role in the defense against infection by occupying binding sites in the intestinal mucous membrane and competing for nutrients with pathogens. Furthermore, LAB are able to the production of substances such as SCFA, bacteriocins, lactoferrin, and hydrogen peroxide which may exhibit inhibitory effects against the growth and colonization of the pathogens (Al-Khalaifah, 2018; Jazi et al., 2018a,b). For example, bacteriocins penetrate into the cell wall of pathogenic bacteria and disturb the electrolyte balance and thus destroy them. Other functions of the LAB include stimulating the mucin production and activating the immune responses to produce a defensive barrier against the pathogenic bacteria (Al-Khalaifah, 2018). Li et al. (2017) revealed that LAB-based probiotics supplementation by increasing the population of Lactobacillus and concentrations of SCFA such as lactate and butyrate can contribute to modulation of gut microbiota communities disrupted by Clostridium perfringens infection in birds. The resulted SCFA particularly butyrate modifies the invasive Salmonella by downregulating Salmonella pathogenicity gene expression responsible for adherence of salmonella to the epithelial cells (Gantois et al., 2006). Fermented feeds as a novel dietary feed can effectively modify the intestinal microbiota by supplying energy and nutrients to health-related bacterial species in the microbial community, as well as the supply of probiotics and their metabolites such as organic acids (Wang et al., 2018). Organic acids can reduce pathogenic bacteria either directly through the penetration into the bacterial cell-wall and produce H+ ions which in turn disrupt the enzyme activity of bacteria or indirectly through altering the gut pH (Suiryanrayna and Ramana, 2015; Shabani et al., 2019). Due to the existence of probiotic bacteria and high lactic acid in FSBM, feeding diets containing FSBM can increase the acidity of various sites of the gut and thus promote the acidophilic bacteria species such as LAB. Meanwhile, the proliferation of the LAB in the gut can suppress the colonization of harmful microbes, as previously discussed about probiotics. Some synergistic effects of the FF related to morphological and microbial parameters may be due to the various beneficial mechanisms exhibited by both probiotic and prebiotic properties of FF as suggested by Wang et al. (2018). It is suggested that microbial fermentation may change the organic substances into simpler and more effective compounds such as fatty acids, peptides, or amino acids and provide fermentable nutrients for useful gut microorganisms promoting their population and useful metabolites in a similar mode of action to prebiotics. The liver, spleen, and bursa of Fabricius are of the main systemic organs involved in inflammatory reactions such as acute phase protein production, antibody production, and lymphocyte activation, thereby, these organs can be affected by bacterial infections. In the

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two tested additives. Three reasons for the improvement of growth performance in birds fed with FSBM can be suggested. First, organic acids and LAB present in FF can reduce the pH and provide a favorable environment for the growth and colonization of beneficial bacteria and thereby improve the sanitary status and intestinal health (Ashayerizadeh et al., 2018; Jazi et al., 2018b). Second, given that antigenic compounds in soybean proteins may lead to some morphological changes in the intestine, a reduction in the amount of antigenic proteins during fermentation may prevent intestinal inflammation and improve the nutrient absorption area (Shi et al., 2017). Finally, the reduction of other anti-nutritional substances like trypsin inhibitor may enhance bioavailability of nutrients of FSBM (Seo and Cho, 2016). Noteworthy, FF due to the presence of organic acids, reduced gut pH, and conversion of pepsinogen to pepsin can ultimately facilitate protein breakdown (Suiryanrayna and Ramana, 2015). Therefore, it is not surprising that, in current study, the BW gain and feed efficiency of FSBM-fed birds compared to other treatments (except NC birds) were improved. Microbial culture samples of crop, ileum, and ceca, and internal organs of uninfected groups were detected negative for the presence of Salmonella, which indicates the absence of cross-contamination among the challenged and unchallenged groups and the successful sanitation and quarantine measures. Similar to the findings of other researchers (Chen et al., 2012; Shao et al., 2016) the present results indicated that ST challenge not only leads to the spread of Salmonella colonization throughout the gut, but also causes an invasion and dissemination to other internal organs (such as liver, spleen, and bursa of Fabricius), and reduces the counts of LAB. However, inclusion of XOS and LAC supplements and FSBM in broiler diets resulted in decrease of Salmonella colonization, gut pH, and internal organs invasion and an increase in the number of LAB in the gut digesta. In line with these results, previous studies have shown that dietary supplementation with prebiotic (Shao et al., 2013; Pourabedin et al., 2016), probiotic (Park and Kim, 2014; Jazi et al., 2018a), and FF (Heres et al., 2003; Ashayerizadeh et al., 2017) decreased Salmonella colonization in animals infected with Salmonella. Xylooligosaccharide similar to other functional oligosaccharides are not digestible in the upper parts of the gut. Instead, it is subjected to microbial fermentation in the hindgut and converted into SCFA, which play various roles in intestinal health and functions (De Maesschalck et al., 2015). The decreased number of Salmonella by XOS may be due to a reduction in pH created by SCFA and promoting the useful gut bacteria population that contribute to the competitive exclusion of pathogenic bacteria. Furthermore, XOS may serve as binding sites for pathogenic bacteria in the intestinal surface which can reduce the colonization of pathogenic bacteria (Ding et al., 2017).

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morphological changes such as villi atrophy and crypt hyperplasia (Shi et al., 2017). The second reason may be related to the decomposition of large proteins into small peptides after fermentation of SBM (Jazi et al., 2017). From this perspective, previous studies have shown that the supplementation of piglet diets with small peptides improves the morphological indices of the small intestine (Wang et al., 2003). In conclusion, this research demonstrated that the fermented SBM mitigated the growth performance suppression caused by Salmonella infection, with similar actions to probiotic and prebiotic supplements tested simultaneously. Feeding diets containing tested feed additives or FSBM had some beneficial effects on suppressing the Salmonella colonization and invasion, intestinal morphology, and immune response. Therefore, the present results suggest that FF could be applied as an effective and novel dietary strategy to ameliorate bacterial infections in broilers fed AB-free diets.

ACKNOWLEDGMENTS This project was financially supported by the Office of the Vice Chancellor for Research of the Mohaghegh Ardabili University, Ardabil, Iran (project number 252/97). The authors are grateful to Tak Genezist Company for supplying the probiotic product.

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present study, the higher relative weights of liver and bursa of Fabricius in challenged broilers could be related to Salmonella dissemination being spread to these organs. The H:L ratio is a reliable indicator of stress in birds (Zulkifli et al., 2000). The induced stress increases the heterophil count as well as H:L ratio while decreases the number of lymphocytes. Our results showed an increased count of heterophil and elevated H:L ratio in ST-infected chicks. Heterophils are the avian polymorphonuclear cells and of key components of the innate immune system. Increasing the heterophils count can indicate the activation of innate immune responses to counteract pathogenic bacteria through phagocytosis and antimicrobial activities, which involve the generation of reactive oxygen and nitrogen species, antimicrobial peptides and proteolytic enzymes (Pae et al., 2012). In the birds fed the diets containing XOS, LAC, and FSBM, count of heterophils, as well as H:L ratio significantly decreased as compared to the IC birds. The exact mechanisms by which LAB might exert their immunomodulatory activities remain unknown; however, it has been proposed that interactions between the LAB and the host immune system could lead to some immunomodulatory activities (Chen et al., 2012). Lactic acid bacteria may increase specific and nonspecific immune responses through enhancing the cytokine production by intraepithelial lymphocytes, activating macrophages, and increasing levels of antibodies (Al-Khalaifah, 2018). In this research, the ST challenge reduced the VH and VH:CD ratio in small intestinal sections, in line with the impaired growth observed in the same group of birds. These histological observations are consistent with a study by Shao et al. (2013). We also observed that feeding XOS, LAB, and FSBM diets increased VH and VH:CD ratio in the small intestine compared with the IC group. The positive impact of tested feed additives on intestinal morphology is likely attributed to their effects on the intestinal microbiota balance and microbial metabolites especially butyrate, which can influence the gut epithelial cell proliferation and increase the villus height (Ding et al., 2017; Jazi et al., 2018a). Furthermore, butyrate by stimulating goblet cells can promote production of mucins and improve intestinal development (De Maesschalck et al., 2015). Apart from improved microbial composition throughout the gut, two other probable reasons to explain the improvement in small intestinal morphology of birds fed FSBM are suggested. The first reason is again related to the reduction of anti-nutrient compounds such as trypsin inhibitor, β -conglycinin, and glycinin in FSBM. Some studies have shown that there is a negative correlation between trypsin inhibitor in SBM and VH (Zarkadas and Wiseman, 2005). Trypsin inhibitor also with the disturbance in activity of trypsin and chymotrypsin can adversely affect the digestive functions in the intestine. On the other hand, as previously mentioned, glycinin and β -conglycinin may lead to some

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