Effect of Saccharomyces cerevisiae yeast products in reducing direct colonization and horizontal transmission of Salmonella Heidelberg in broilers

Effect of Saccharomyces cerevisiae yeast products in reducing direct colonization and horizontal... SUMMARY This study was conducted to evaluate the effectiveness of different levels of Saccharomyces cerevisiae yeast cell wall or a yeast culture in reducing the direct colonization and horizontal transmission of Salmonella Heidelberg in broiler chickens. At d of hatch, 2,000 male broiler chicks were randomly assigned into 5 treatment groups with 8 replicate pens per treatment. Birds were challenged with nalidixic acid-resistant S. Heidelberg either by direct inoculation of 107 cfu orally (seeders) or by horizontal transmission through inoculated penmates (contacts). Yeast cell wall (YCW) at 500 ppm decreased salmonella prevalence of the total infected birds (seeders and contacts) to 41.7% as compared to 54.2% in the untreated birds. The effect of YCW in reducing prevalence of positive birds was even greater when we considered the contact birds only; 32.5% in contact birds treated with 500 ppm as compared to 57.5% in the untreated group (P = 0.09). Furthermore, enumeration of S. Heidelberg colonization level in the cecum using the most probable number (MPN) method showed that YCW at 500 ppm reduced the bacterial load in the cecum of positive birds to 1.7 MPN/g as compared to 2.7 MPN/g in the control group. The reduction was statistically significant (P = 0.04) in the contact birds. The same effect was not seen in the yeast culture treatment. DESCRIPTION OF THE PROBLEM Salmonella are one of the most important zoonotic pathogens, resulting in millions of foodborne illnesses every yr in the United States and globally [1]. In the United States alone, non-thyphoid Salmonella affect around one million people every yr, resulting in an estimated death of 400 patients [2]. In a study ranking the importance of 14 major pathogens in 12 broad categories of food, resulting in 168 pathogen-food combinations, Salmonella ranked among the highest in terms of their combined impact on the total cost of illness and loss of quality-adjusted life yr (QALY) with an annual loss of $3.3 billion—17,000 QALY in the United States alone [3, 4]. Many cases of human salmonellosis are associated with consumption of contaminated poultry products [5]. Salmonella Enteritidis and S. Typhimurium are the 2 most common disease-causing serovars of Salmonella in humans [6]. In 2009, S. Heidelberg was the third-most common serovar isolated from chicken samples collected from retail shops and the fifth-most common serovar of Salmonella isolated from humans [7]. Since the incidental discovery of the ability of low dose antibiotic treatment to promote growth and performance of animals in the 1940s [8], antibiotic feed additives have been widely used in the poultry industry to promote gut health, increase feed efficiency, and improve performance [9]. However, with strong pressure from consumers and policy makers on the use of antibiotics in animal feed, the poultry industry is looking for alternatives to manage gut health and reduce pathogen pressure in young birds. Several strategies have been developed throughout the yr to reduce gastrointestinal tract infection in poultry at the production level, such as acidification of feed and water using organic acids, vaccination, and the use of alternative natural products in the form of pre- or probiotics [10]. In line with this, the use of yeast-based pre- and probiotics as alternatives to antibiotic feed additives has gained much attention recently. However, the effect of yeast cell wall (YCW)-based prebiotics on the health and performance of broiler chickens is still controversial. Some researchers reported positive effects on performance [11, 12] and gut health [13–15], while others conclude no beneficial effect [16–18]. Therefore, the objective of this study is to evaluate the effectiveness of different yeast products in reducing the direct colonization and horizontal transmission of S. Heidelberg in broiler chickens. MATERIALS AND METHODS Animals and Handling Procedure Two thousand (2,000) day-of-hatch Ross x Ross line 708 male broiler chicks [19] were obtained. Chicks were sexed, received routine vaccinations (HVT-SB1), and breeder flock number information recorded at the hatchery on the d of hatch. Birds were vaccinated with CocciVac-B52, a commercially available coccidiosis vaccine [20], at the recommended dose by spray cabinet on one d of age. No antibiotics were used during this study; birds were monitored daily for morbidity and mortality, and ill or dead birds were removed from the study. All animal procedures were conducted according to the guidelines provided by the Federation of Animal Science Societies for humane animal use [21]. Experimental Design and Treatment Groups Upon arrival at the research location, birds were randomly allocated to 40 floor pens (n = 50/pen) measuring 50 square feet in a modified conventional poultry house with solid sides and dirt floors that contained fresh pine shavings. Litter was not replaced during the study course. Pens, as experimental units, were assigned to one of the 5 treatment groups with 8 pens (replicates) of 50 birds each per treatment. Feed and water were provided ad libitum. The treatments include: Basal diet (T1) or basal diet containing a commercially available YCW, [22] at 125 ppm (T2), 250 ppm (T3), 500 ppm (T4), or a commercially available yeast culture (YC) [23] at 1,250 ppm (T5). The YCW used was derived from a single proprietary strain of Saccharomyces cerevisiae with a minimum guaranteed mannan content of 20% and β-glucan content of 20%. Commercial-type broiler diets (Table 1), formulated using commonly used local ingredients and calculated to meet or exceed NRC standards [24], were provided ad libitum in 3 different phases containing starter (0 to 21 d of age), grower (22 to 35 d of age), and finisher (36 to 43 d of age). Rations were fed as crumbles in the starter phase and as pellets during grower and finisher phases. Weights of feed filled into feeders were recorded. At the end of each feeding phase, all leftover feed was removed and individually weighed before it was replaced by a known amount of the next phase diet. Average daily feed intake during starter, grower, and finisher phases was calculated by subtracting the weight of feed remaining on d 21, 35, and 42 from total weight of feed added during that phase. Pen weights of broilers also were recorded on d 1, 35, and 42. Feed conversion ratios, adjusted for mortality [total feed consumption/(final live weight + total mortality weight of the birds)] for each treatment, were calculated for each phase. Table 1. Ingredients and nutrient composition of experimental diets1 (% as fed unless noted). Ingredients  Starter  Grower  Finisher  Soybean meal, dehulled  37.71  32.99  28.26  Fat, vegetable  2.18  1.98  1.81  Dicalcium phosphate  2.04  1.75  1.53  Calcium carbonate  1.02  0.98  0.74  Salt, plain (NaCl)  0.46  0.44  0.44  DL-methionine  0.30  0.22  0.19  L-lysine  0.17  0.16  0.13  Trace mineral2  0.08  0.08  0.08  Vitamin premix3  0.25  0.25  0.25  Ingredients  Starter  Grower  Finisher  Soybean meal, dehulled  37.71  32.99  28.26  Fat, vegetable  2.18  1.98  1.81  Dicalcium phosphate  2.04  1.75  1.53  Calcium carbonate  1.02  0.98  0.74  Salt, plain (NaCl)  0.46  0.44  0.44  DL-methionine  0.30  0.22  0.19  L-lysine  0.17  0.16  0.13  Trace mineral2  0.08  0.08  0.08  Vitamin premix3  0.25  0.25  0.25  1Control group was fed the basil diet. The other treatment diets were the same basal diet supplemented with 125 ppm YCW, 250 ppm, YCW, 500 ppm YCW, or 1,250 ppm YC, respectively. 2Trace mineral mix provided the following (per kg of diet): manganese (MnSO4•H2O), 60 mg; iron (FeSO4•7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4•5H2O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg. 3Vitamin mix provided the following (per kg of diet): Thiamin•mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; D-Ca pantothenate, 12 mg; vitamin B12 (cobalamin),12.0 μg; pyridoxine•HCL, 4.7 mg; D-biotin, 0.11 mg; folic acid, 5.5 mg; menadione sodium bisulfite complex, 3.34 mg; choline chloride, 220 mg; cholecalciferol, 27.5 ug; trans-retinyl acetate, 1892 ug; all-rac α tocopheryl acetate, 11 mg; ethoxyquin, 125 mg. View Large Table 1. Ingredients and nutrient composition of experimental diets1 (% as fed unless noted). Ingredients  Starter  Grower  Finisher  Soybean meal, dehulled  37.71  32.99  28.26  Fat, vegetable  2.18  1.98  1.81  Dicalcium phosphate  2.04  1.75  1.53  Calcium carbonate  1.02  0.98  0.74  Salt, plain (NaCl)  0.46  0.44  0.44  DL-methionine  0.30  0.22  0.19  L-lysine  0.17  0.16  0.13  Trace mineral2  0.08  0.08  0.08  Vitamin premix3  0.25  0.25  0.25  Ingredients  Starter  Grower  Finisher  Soybean meal, dehulled  37.71  32.99  28.26  Fat, vegetable  2.18  1.98  1.81  Dicalcium phosphate  2.04  1.75  1.53  Calcium carbonate  1.02  0.98  0.74  Salt, plain (NaCl)  0.46  0.44  0.44  DL-methionine  0.30  0.22  0.19  L-lysine  0.17  0.16  0.13  Trace mineral2  0.08  0.08  0.08  Vitamin premix3  0.25  0.25  0.25  1Control group was fed the basil diet. The other treatment diets were the same basal diet supplemented with 125 ppm YCW, 250 ppm, YCW, 500 ppm YCW, or 1,250 ppm YC, respectively. 2Trace mineral mix provided the following (per kg of diet): manganese (MnSO4•H2O), 60 mg; iron (FeSO4•7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4•5H2O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg. 3Vitamin mix provided the following (per kg of diet): Thiamin•mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; D-Ca pantothenate, 12 mg; vitamin B12 (cobalamin),12.0 μg; pyridoxine•HCL, 4.7 mg; D-biotin, 0.11 mg; folic acid, 5.5 mg; menadione sodium bisulfite complex, 3.34 mg; choline chloride, 220 mg; cholecalciferol, 27.5 ug; trans-retinyl acetate, 1892 ug; all-rac α tocopheryl acetate, 11 mg; ethoxyquin, 125 mg. View Large Salmonella Challenge A nalidixic acid resistant strain of S. Heidelberg was grown for 6 h in tryptic soy broth [25] and the number of cfu per milliliter determined by plating 10-fold serial dilutions of the bacterial suspension on xylose lysine tergitol-4 (XLT-4) medium containing 0.25ug/mL nalidixic acid [25]. The challenge dose was determined using optical density, and a dose of 5 × 107cfu/mL was used per chicken. On the first d, one-half of the birds (seeders) in a pen (n = 25) from each group were dyed and tagged and challenged by oral gavage with a 5 × 107 cfu S. Heidelberg, while the remaining half (n = 25) were left unchallenged (contacts). In this animal model [26–28], challenged chicks represented infected birds that may shed Salmonella in the environment (seeders), whereas unchallenged chicks represented birds that may become infected with Salmonella through horizontal contact with the contaminated environment (contacts). Sample Collection, Salmonella Isolation and Identification Environmental contamination by S. Heidelberg and potential horizontal transmission of the bacteria to non-inoculated penmates was determined at 14 and 42 d of age using boot sock swabs collected from all pens and cecal samples collected from representative birds, respectively, as described previously [28, 29]. A sterile pre-moistened boot sock swab [30] was placed onto one foot covered with a clean new plastic boot. Boot socks were removed and placed into a labeled sterile bag after walking around the interior perimeter of each of the pens. To determine horizontal transmission of S. Heidelberg among penmates, 10 birds from the non-challenged chickens (contact birds) from each pen were humanely euthanized by cervical dislocation and ceca aseptically removed for sampling. Ceca from each bird were put into a sterile plastic sample bag [31] individually, labeled, and stored on ice until transported to the onsite lab for Salmonella identification. Upon arrival at the laboratory, approximately 100 mL tetrathionate broth [25] was added to boot sock swab samples while ceca were weighed, and stomached in 50 mL of tetrathionate broth. A 1-mL aliquot was removed from each sample for most probable number (MPN) analysis into a test tube and mixed with tetrathionate broth. Samples were incubated overnight at 41.5°C, and a loop full of sample from each tube was struck onto XLT-4 agar plates [25] and incubated overnight at 37°C. Three black (H2S-positive) colonies were selected from each plate and tested for agglutination using Polyvalent-O Salmonella Specific Antiserum [32] to confirm Salmonella positive colonies. Salmonella Enumeration in Cecal Samples Using the Most Probable Number Method From each of the 10 cecal samples collected from horizontally exposed (contact) birds, a 1-mL sample of stomached tetrathionate broth was transferred into 3 adjacent wells in the first row of a 96-well, 2-mL deep plate [33]. A 10-fold serial dilution was prepared by adding a 0.1-mL aliquot of sample from the first row into 0.9-mL of tetrathionate broth [25] on the second row. Subsequent 10-fold dilutions were prepared in a similar way using a multichannel pipette, changing pipette tips between dilutions, until 10−5 dilution was reached for each sample. After incubation of the 96-well plates for 24 h at 42°C, 1-μL aliquot from each well was plated onto XLT-4 [25] agar plates containing nalidixic acid using a pin-tool replicator [34]. Plates were incubated at 37°C for 24 hours. Finally, the number of wells with black colonies (H2S-positive) was counted for each dilution, and the MPN of Salmonella calculations was performed as previously described [28]. Suspected colonies were picked and tested for agglutination using polyvalent-O Salmonella specific antiserum [32]. Data Management and Statistical Analysis Analyses were performed using commercially available statistical software (Stata v14.2) [35]. Boot sock Salmonella prevalence was compared among treatment groups using Fisher's exact test. Salmonella prevalence in ceca samples was compared among treatment groups and challenge status categories using generalized estimating equations (GEE) logistic models, and Salmonella MPN were compared using GEE linear models to account for the correlation between responses of birds from the same pen. GEE models were estimated using robust standard errors and an exchangeable working correlation structure. For the comparison of Salmonella MPN, samples with a negative culture result by the MPN method but a positive culture result by primary or secondary enrichment were arbitrarily assigned an MPN value equal to one half the minimum detection limit of the MPN assay. MPN values were log-transformed prior to statistical analysis. Post-hoc pairwise comparisons between treatments were performed using the Bonferroni procedure to limit the type I error rate to 5% over all comparisons. All statistical testing assumed a two-sided alternative hypothesis, and P < 0.05 was considered significant. RESULTS AND DISCUSSION Performance In this study, we compared 3 different concentrations of a commercially available Saccharomyces cerevisiae YCW and one concentration of a commercially available YC for their ability to lower both direct colonization of birds challenged at one d of age and reduce horizontal infection of unchallenged penmates with S. Heidelberg. In agreement with results from previous studies [16–18], we did not see any significant differences in either body weight or feed efficiency at either 35 or 42 d of age between the control and the supplemented group (Table 2). Similarly, there was no significant difference in performance between challenged and non-challenged birds. This is not surprising because the Salmonella serovar used for this challenge does not normally cause serious health issues in chickens. On the other hand, yeast cell products are widely reported to have a beneficial effect when administered to birds under some stressful conditions, including mycotoxin challenge [36], heat stress [37, 38], as well as pathogen and immune challenge [14, 39]. Table 2. Performance data at 35 and 42 d of age. Treatment  Feed intake  Adjusted feed conversion  Average weight gain  Percent mortality  35 d of age  Untreated  117.96  1.74  1.37  5.25  Yeast cell wall (125 ppm)  114.82  1.73  1.36  5.50  Yeast cell wall (250 ppm)  115.88  1.74  1.38  6.50  Yeast cell wall (500 ppm)  111.20  1.71  1.35  6.25  Yeast culture (1250 ppm)  114.23  1.73  1.37  6.00  42 d of age  Untreated  174.27  1.79  2.00  5.50  Yeast cell wall (125 ppm)  171.30  1.78  2.00  6.25  Yeast cell wall (250 ppm)  172.29  1.77  2.04  7.75  Yeast cell wall (500 ppm)  165.51  1.76  1.99  8.25  Yeast culture (1250 ppm)  167.96  1.77  2.00  8.00  Treatment  Feed intake  Adjusted feed conversion  Average weight gain  Percent mortality  35 d of age  Untreated  117.96  1.74  1.37  5.25  Yeast cell wall (125 ppm)  114.82  1.73  1.36  5.50  Yeast cell wall (250 ppm)  115.88  1.74  1.38  6.50  Yeast cell wall (500 ppm)  111.20  1.71  1.35  6.25  Yeast culture (1250 ppm)  114.23  1.73  1.37  6.00  42 d of age  Untreated  174.27  1.79  2.00  5.50  Yeast cell wall (125 ppm)  171.30  1.78  2.00  6.25  Yeast cell wall (250 ppm)  172.29  1.77  2.04  7.75  Yeast cell wall (500 ppm)  165.51  1.76  1.99  8.25  Yeast culture (1250 ppm)  167.96  1.77  2.00  8.00  View Large Table 2. Performance data at 35 and 42 d of age. Treatment  Feed intake  Adjusted feed conversion  Average weight gain  Percent mortality  35 d of age  Untreated  117.96  1.74  1.37  5.25  Yeast cell wall (125 ppm)  114.82  1.73  1.36  5.50  Yeast cell wall (250 ppm)  115.88  1.74  1.38  6.50  Yeast cell wall (500 ppm)  111.20  1.71  1.35  6.25  Yeast culture (1250 ppm)  114.23  1.73  1.37  6.00  42 d of age  Untreated  174.27  1.79  2.00  5.50  Yeast cell wall (125 ppm)  171.30  1.78  2.00  6.25  Yeast cell wall (250 ppm)  172.29  1.77  2.04  7.75  Yeast cell wall (500 ppm)  165.51  1.76  1.99  8.25  Yeast culture (1250 ppm)  167.96  1.77  2.00  8.00  Treatment  Feed intake  Adjusted feed conversion  Average weight gain  Percent mortality  35 d of age  Untreated  117.96  1.74  1.37  5.25  Yeast cell wall (125 ppm)  114.82  1.73  1.36  5.50  Yeast cell wall (250 ppm)  115.88  1.74  1.38  6.50  Yeast cell wall (500 ppm)  111.20  1.71  1.35  6.25  Yeast culture (1250 ppm)  114.23  1.73  1.37  6.00  42 d of age  Untreated  174.27  1.79  2.00  5.50  Yeast cell wall (125 ppm)  171.30  1.78  2.00  6.25  Yeast cell wall (250 ppm)  172.29  1.77  2.04  7.75  Yeast cell wall (500 ppm)  165.51  1.76  1.99  8.25  Yeast culture (1250 ppm)  167.96  1.77  2.00  8.00  View Large Bacterial Shedding, Environmental Contamination, and Prevalence of Infection Challenged birds were able to shed the bacteria and contaminate the environment, as all the pens tested for the growth of S. Heidelberg were positive (100%) on d 14 and 42 with no difference among treatment groups. There were no significant differences in the prevalence of S. Heidelberg-infected birds among treatment groups either in the direct S. Heidelberg challenged (seeder birds) or indirect challenged (contact birds) as assessed based on identification of the bacteria in cecal contents of birds (Table 3). However, the decrease in the prevalence of Salmonella-infected birds was numerically higher with YCW at 500 ppm as compared to all other groups, including the control group, having only 41.7% overall positive prevalence vs. control at 54.2%. The effect of YCW at 500 ppm in reducing the prevalence of Salmonella-infected birds was even greater in the contact birds, which was significant at the 90% level, but not at the 95% level (32.5 vs. 57.5%, P = 0.09); see also Figure 1. A previous in vitro study from our group has demonstrated the ability of YCW and live yeast to bind specific bacterial pathogens, including several species of Salmonella [40]. Therefore, binding could be the possible mechanism for the reduction of Salmonella in the intestine. On the other hand, a YC is a mixture of yeast and the media in which it was grown and as a result may not have a significant level of mannan-oligosaccharide to bind pathogens. In line with this, another study demonstrated reduced colonization of S. Enteritidis in broiler chickens with the addition of mannan-oligosaccharide, a component of YCW, to the diet [39]. A similar study demonstrated reduced fecal shedding of S. Enteritidis in broiler chickens with diets supplemented with mannan-oligoscaccharide [41]. Supplementation of water with D-mannose reduced the colonization of broiler chickens with S. Typhimurium [42]. All these data taken together, we can suggest that binding of Salmonella to the mannan-oligoscharides of the YCW is an important part of the mechanism by which YCW results in reduction of S. Heidelberg in the intestine of challenged birds. Figure 1. View largeDownload slide Dot plots of pen-level Salmonella prevalence by treatment and challenge status. Ten indirect challenged birds and 5 direct challenged birds were sampled in each of 8 pens per treatment group. Figure 1. View largeDownload slide Dot plots of pen-level Salmonella prevalence by treatment and challenge status. Ten indirect challenged birds and 5 direct challenged birds were sampled in each of 8 pens per treatment group. Table 3. Salmonella prevalence (%) in ceca samples collected on d 42. Treatment  Challenge status Indirect* (Contact birds)  Direct* (Seeder birds)  Total*  Untreated  46/80 (57.5)  19/40 (47.5)  65/120 (54.2)  Yeast cell wall (125 ppm)  51/80 (63.8)  24/40 (60.0)  75/120 (62.5)  Yeast cell wall (250 ppm)  49/80 (61.3)  21/40 (52.5)  70/120 (58.3)  Yeast cell wall (500 ppm)  26/80 (32.5)  24/40 (60.0)  50/120 (41.7)  Yeast culture (1250 ppm)  42/80 (52.5)  19/40 (47.5)  61/120 (50.8)  Treatment  Challenge status Indirect* (Contact birds)  Direct* (Seeder birds)  Total*  Untreated  46/80 (57.5)  19/40 (47.5)  65/120 (54.2)  Yeast cell wall (125 ppm)  51/80 (63.8)  24/40 (60.0)  75/120 (62.5)  Yeast cell wall (250 ppm)  49/80 (61.3)  21/40 (52.5)  70/120 (58.3)  Yeast cell wall (500 ppm)  26/80 (32.5)  24/40 (60.0)  50/120 (41.7)  Yeast culture (1250 ppm)  42/80 (52.5)  19/40 (47.5)  61/120 (50.8)  *Values given are Salmonella-positive birds over the total number of birds in each group or the respective percentage in parenthesis. Ten indirect challenged birds and 5 direct challenged birds were sampled in each of 8 pens per treatment group. Marginal percentages with a superscript in common do not differ with a level of significance of 5% over all comparisons. View Large Table 3. Salmonella prevalence (%) in ceca samples collected on d 42. Treatment  Challenge status Indirect* (Contact birds)  Direct* (Seeder birds)  Total*  Untreated  46/80 (57.5)  19/40 (47.5)  65/120 (54.2)  Yeast cell wall (125 ppm)  51/80 (63.8)  24/40 (60.0)  75/120 (62.5)  Yeast cell wall (250 ppm)  49/80 (61.3)  21/40 (52.5)  70/120 (58.3)  Yeast cell wall (500 ppm)  26/80 (32.5)  24/40 (60.0)  50/120 (41.7)  Yeast culture (1250 ppm)  42/80 (52.5)  19/40 (47.5)  61/120 (50.8)  Treatment  Challenge status Indirect* (Contact birds)  Direct* (Seeder birds)  Total*  Untreated  46/80 (57.5)  19/40 (47.5)  65/120 (54.2)  Yeast cell wall (125 ppm)  51/80 (63.8)  24/40 (60.0)  75/120 (62.5)  Yeast cell wall (250 ppm)  49/80 (61.3)  21/40 (52.5)  70/120 (58.3)  Yeast cell wall (500 ppm)  26/80 (32.5)  24/40 (60.0)  50/120 (41.7)  Yeast culture (1250 ppm)  42/80 (52.5)  19/40 (47.5)  61/120 (50.8)  *Values given are Salmonella-positive birds over the total number of birds in each group or the respective percentage in parenthesis. Ten indirect challenged birds and 5 direct challenged birds were sampled in each of 8 pens per treatment group. Marginal percentages with a superscript in common do not differ with a level of significance of 5% over all comparisons. View Large Bacterial Load Estimation in the Ceca of Salmonella Heidelberg-infected Birds There was no statistically significant difference in reducing the MPN of Salmonella in the ceca of positive birds. However, MPN were numerically lowest in the 500 ppm YCW-supplemented birds (log1.4 cfu/gram of cecal content) compared to all other treatment groups (Table 4). The effect of YCW in reducing MPN becomes statistically significant (P = 0.04) when the comparison among groups was made only within the contact birds, as shown in Figure 2. The box plots more concisely demonstrate the effect of YCW 500 ppm in decreasing the MPN of Salmonella in the indirect challenged (contact) birds. This is in agreement with a previous suggestion made by our group that Salmonella spp. were able to bind to mannose via the type-1 binding fimbriae, and hence Saccharomyces cerevisiae and cell wall from S.cerevisiae were shown to bind a variety of Gram-negative organisms, including Salmonella [40]. Finally, factorial analysis both for the prevalence of positive birds and MPN of Salmonella in cecal contents of positive birds did not show any significant interaction between the main effects (treatment and challenge method). Figure 2. View largeDownload slide Box plots of Salmonella MPN for culture-positive samples by treatment and challenge status. See Table 3 for sample sizes. Figure 2. View largeDownload slide Box plots of Salmonella MPN for culture-positive samples by treatment and challenge status. See Table 3 for sample sizes. Table 4. Geometric mean (95% CI) Salmonella MPN/g for culture-positive ceca samples collected on d 42.   Challenge status  Treatment  Indirect (Contact birds)*  Direct (Seeder birds)*  Untreated  2.3a (1.2, 4.4)  3.7 (1.9, 7.2)  Yeast cell wall (125 ppm)  2.9a (1.4, 6.0)  2.8 (1.9, 4.0)  Yeast cell wall (250 ppm)  3.4a (2.1, 5.4)  3.3 (1.3, 8.5)  Yeast cell wall (500 ppm)  1.4b (0.95, 2.1)  2.7 (1.5, 4.8)  Yeast culture (1250 ppm)  3.2a (1.3, 7.9)  2.4 (1.1, 5.4)    Challenge status  Treatment  Indirect (Contact birds)*  Direct (Seeder birds)*  Untreated  2.3a (1.2, 4.4)  3.7 (1.9, 7.2)  Yeast cell wall (125 ppm)  2.9a (1.4, 6.0)  2.8 (1.9, 4.0)  Yeast cell wall (250 ppm)  3.4a (2.1, 5.4)  3.3 (1.3, 8.5)  Yeast cell wall (500 ppm)  1.4b (0.95, 2.1)  2.7 (1.5, 4.8)  Yeast culture (1250 ppm)  3.2a (1.3, 7.9)  2.4 (1.1, 5.4)  *Values represent average values of MPN/g sample with lower and higher values of the 95% confidence interval. Ten indirect challenged birds and 5 direct challenged birds were sampled in each of 8 pens per treatment group. a,bMarginal means with a superscript in common do not differ with a level of significance of 5% over all comparisons. See Table 3 for sample sizes. View Large Table 4. Geometric mean (95% CI) Salmonella MPN/g for culture-positive ceca samples collected on d 42.   Challenge status  Treatment  Indirect (Contact birds)*  Direct (Seeder birds)*  Untreated  2.3a (1.2, 4.4)  3.7 (1.9, 7.2)  Yeast cell wall (125 ppm)  2.9a (1.4, 6.0)  2.8 (1.9, 4.0)  Yeast cell wall (250 ppm)  3.4a (2.1, 5.4)  3.3 (1.3, 8.5)  Yeast cell wall (500 ppm)  1.4b (0.95, 2.1)  2.7 (1.5, 4.8)  Yeast culture (1250 ppm)  3.2a (1.3, 7.9)  2.4 (1.1, 5.4)    Challenge status  Treatment  Indirect (Contact birds)*  Direct (Seeder birds)*  Untreated  2.3a (1.2, 4.4)  3.7 (1.9, 7.2)  Yeast cell wall (125 ppm)  2.9a (1.4, 6.0)  2.8 (1.9, 4.0)  Yeast cell wall (250 ppm)  3.4a (2.1, 5.4)  3.3 (1.3, 8.5)  Yeast cell wall (500 ppm)  1.4b (0.95, 2.1)  2.7 (1.5, 4.8)  Yeast culture (1250 ppm)  3.2a (1.3, 7.9)  2.4 (1.1, 5.4)  *Values represent average values of MPN/g sample with lower and higher values of the 95% confidence interval. Ten indirect challenged birds and 5 direct challenged birds were sampled in each of 8 pens per treatment group. a,bMarginal means with a superscript in common do not differ with a level of significance of 5% over all comparisons. See Table 3 for sample sizes. View Large CONCLUSION AND APPLICATIONS The challenge model, which utilizes direct challenged birds as seeders to infect the environment and contact birds, worked well, demonstrated by 100% prevalence of contaminated pens. YCW at 500 ppm decreased the prevalence of S. Heidelberg-positive birds, and this decrease was greater in contact birds as compared to the direct challenged birds (seeders). YCW was able to numerically decrease the bacterial load in the ceca of infected birds (both contact and seeder birds). However, the reduction was statistically significant (P < 0.04) only in the indirect challenged model (contact birds). In summary, the use of YCW as a prebiotic in broiler diets can decrease the rate of infection through horizontal transmission and cecal load of S. Heidelberg in contact birds, leading to lower contamination of chicken meat in the processing plants, effectively reducing the incidence of the zoonotic transmission of S. Heidelberg. Footnotes Primary Audience: Veterinarians, Nutritionists, Poultry Scientists, Producers, Researchers REFERENCES 1. Cosby D. E., Cox N. A., Harrison M. A., Wilson J. L., Buhr R. F., Fedorka-Cray P. J.. 2015. Salmonella and antimicrobial resistance in broilers: A review: Table 1. J. Appl. Poult. Res.  24: 408– 426. Google Scholar CrossRef Search ADS   2. Scallan E., Hoekstra R. M., Angulo F. J., Tauxe R. V., Widdowson M. A., Roy S. L., Jones J. L., Griffin P. M.. 2011. Foodborne illness acquired in the United States–Major pathogens. Emerg. Infect. Dis.  17: 7– 15. Google Scholar CrossRef Search ADS PubMed  3. Batz M. B., Hoffmann S., Morris J. G. Jr. 2012. 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Google Scholar CrossRef Search ADS PubMed  10. Vandeplas S., Dubois Dauphin R., Beckers Y., Thonart P., Thewis A.. 2010. Salmonella in chicken: Current and developing strategies to reduce contamination at farm level. J. of Food Prot.  73: 774– 785. Google Scholar CrossRef Search ADS   11. Hooge D. M. 2004. Meta-analysis of broiler chicken pen trials evaluating dietary mannan oligosaccharide, 1993-2003. Int. J. of Poult. Sci.  3: 163– 174. Google Scholar CrossRef Search ADS   12. Fowler J., Kakani R., Haq A., Byrd J. A., Bailey C. A.. 2015. Growth promoting effects of prebiotic yeast cell wall products in starter broilers under an immune stress and Clostridium perfringens challenge. J. Appl. Poult. Res.  24: 66– 72. Google Scholar CrossRef Search ADS   13. Xiao R., Power R. F., Mallonee D., Routt K., Spangler L., Pescatore A. J., Cantor A. H., Ao T., Pierce J. L., Dawson K. A.. 2012. Effects of yeast cell wall-derived mannan-oligosaccharides on jejunal gene expression in young broiler chickens. Poult. Sci.  91: 1660– 1669. Google Scholar CrossRef Search ADS PubMed  14. Zhang S., Liao B., Li X., Li L., Ma L., Yan X.. 2012. Effects of yeast cell walls on performance and immune responses of cyclosporine A-treated, immunosuppressed broiler chickens. Br. J. Nutr.  107: 858– 866. Google Scholar CrossRef Search ADS PubMed  15. Swiatkiewicz S., Arczewska-wlosek A., Jozefiak D.. 2014. Immunomodulatory efficacy of yeast cell products in poultry: A current review. Worlds Poult. Sci. J.  70: 57– 68. Google Scholar CrossRef Search ADS   16. Baurhoo B., Ferket P. R., Zhao X.. 2009. Effects of diets containing different concentrations of mannanoligosaccharide or antibiotics on growth performance, intestinal development, cecal and litter microbial populations, and carcass parameters of broilers. Poult. Sci.  88: 2262– 2272. Google Scholar CrossRef Search ADS PubMed  17. Martínez B. F., Contreras A. A., Gonzalez E. A.. 2010. Use of Saccharomyces cerevisiae cell walls in diets for two genetic strains of laying hens reared in floor and cages. Int. J. of Poult. Sci.  9: 105– 108. Google Scholar CrossRef Search ADS   18. 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  19. Blairsville, GA. 20. Merck, Madison, NJ. 21. FASS 2010. Guide for the Care and Use of Agricultural Animals in Research and Teaching Michigan State University and Texas Tech University. 22. Phileo Lesaffre Animal Care, Milwaukee, WI. 23. Diamond V, Cedar Rapids, IA. 24. NRC. 1994. Nutrient Requirements of Poultry . Ninth Revised Edition ed. The National Academies Press, Washington, DC. 25. Difco Laboratories, Detroit, MI. 26. Gast R. 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Effect of Saccharomyces cerevisiae yeast products in reducing direct colonization and horizontal transmission of Salmonella Heidelberg in broilers

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Applied Poultry Science, Inc.
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© 2018 Poultry Science Association Inc.
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1056-6171
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1537-0437
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10.3382/japr/pfy012
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Abstract

SUMMARY This study was conducted to evaluate the effectiveness of different levels of Saccharomyces cerevisiae yeast cell wall or a yeast culture in reducing the direct colonization and horizontal transmission of Salmonella Heidelberg in broiler chickens. At d of hatch, 2,000 male broiler chicks were randomly assigned into 5 treatment groups with 8 replicate pens per treatment. Birds were challenged with nalidixic acid-resistant S. Heidelberg either by direct inoculation of 107 cfu orally (seeders) or by horizontal transmission through inoculated penmates (contacts). Yeast cell wall (YCW) at 500 ppm decreased salmonella prevalence of the total infected birds (seeders and contacts) to 41.7% as compared to 54.2% in the untreated birds. The effect of YCW in reducing prevalence of positive birds was even greater when we considered the contact birds only; 32.5% in contact birds treated with 500 ppm as compared to 57.5% in the untreated group (P = 0.09). Furthermore, enumeration of S. Heidelberg colonization level in the cecum using the most probable number (MPN) method showed that YCW at 500 ppm reduced the bacterial load in the cecum of positive birds to 1.7 MPN/g as compared to 2.7 MPN/g in the control group. The reduction was statistically significant (P = 0.04) in the contact birds. The same effect was not seen in the yeast culture treatment. DESCRIPTION OF THE PROBLEM Salmonella are one of the most important zoonotic pathogens, resulting in millions of foodborne illnesses every yr in the United States and globally [1]. In the United States alone, non-thyphoid Salmonella affect around one million people every yr, resulting in an estimated death of 400 patients [2]. In a study ranking the importance of 14 major pathogens in 12 broad categories of food, resulting in 168 pathogen-food combinations, Salmonella ranked among the highest in terms of their combined impact on the total cost of illness and loss of quality-adjusted life yr (QALY) with an annual loss of $3.3 billion—17,000 QALY in the United States alone [3, 4]. Many cases of human salmonellosis are associated with consumption of contaminated poultry products [5]. Salmonella Enteritidis and S. Typhimurium are the 2 most common disease-causing serovars of Salmonella in humans [6]. In 2009, S. Heidelberg was the third-most common serovar isolated from chicken samples collected from retail shops and the fifth-most common serovar of Salmonella isolated from humans [7]. Since the incidental discovery of the ability of low dose antibiotic treatment to promote growth and performance of animals in the 1940s [8], antibiotic feed additives have been widely used in the poultry industry to promote gut health, increase feed efficiency, and improve performance [9]. However, with strong pressure from consumers and policy makers on the use of antibiotics in animal feed, the poultry industry is looking for alternatives to manage gut health and reduce pathogen pressure in young birds. Several strategies have been developed throughout the yr to reduce gastrointestinal tract infection in poultry at the production level, such as acidification of feed and water using organic acids, vaccination, and the use of alternative natural products in the form of pre- or probiotics [10]. In line with this, the use of yeast-based pre- and probiotics as alternatives to antibiotic feed additives has gained much attention recently. However, the effect of yeast cell wall (YCW)-based prebiotics on the health and performance of broiler chickens is still controversial. Some researchers reported positive effects on performance [11, 12] and gut health [13–15], while others conclude no beneficial effect [16–18]. Therefore, the objective of this study is to evaluate the effectiveness of different yeast products in reducing the direct colonization and horizontal transmission of S. Heidelberg in broiler chickens. MATERIALS AND METHODS Animals and Handling Procedure Two thousand (2,000) day-of-hatch Ross x Ross line 708 male broiler chicks [19] were obtained. Chicks were sexed, received routine vaccinations (HVT-SB1), and breeder flock number information recorded at the hatchery on the d of hatch. Birds were vaccinated with CocciVac-B52, a commercially available coccidiosis vaccine [20], at the recommended dose by spray cabinet on one d of age. No antibiotics were used during this study; birds were monitored daily for morbidity and mortality, and ill or dead birds were removed from the study. All animal procedures were conducted according to the guidelines provided by the Federation of Animal Science Societies for humane animal use [21]. Experimental Design and Treatment Groups Upon arrival at the research location, birds were randomly allocated to 40 floor pens (n = 50/pen) measuring 50 square feet in a modified conventional poultry house with solid sides and dirt floors that contained fresh pine shavings. Litter was not replaced during the study course. Pens, as experimental units, were assigned to one of the 5 treatment groups with 8 pens (replicates) of 50 birds each per treatment. Feed and water were provided ad libitum. The treatments include: Basal diet (T1) or basal diet containing a commercially available YCW, [22] at 125 ppm (T2), 250 ppm (T3), 500 ppm (T4), or a commercially available yeast culture (YC) [23] at 1,250 ppm (T5). The YCW used was derived from a single proprietary strain of Saccharomyces cerevisiae with a minimum guaranteed mannan content of 20% and β-glucan content of 20%. Commercial-type broiler diets (Table 1), formulated using commonly used local ingredients and calculated to meet or exceed NRC standards [24], were provided ad libitum in 3 different phases containing starter (0 to 21 d of age), grower (22 to 35 d of age), and finisher (36 to 43 d of age). Rations were fed as crumbles in the starter phase and as pellets during grower and finisher phases. Weights of feed filled into feeders were recorded. At the end of each feeding phase, all leftover feed was removed and individually weighed before it was replaced by a known amount of the next phase diet. Average daily feed intake during starter, grower, and finisher phases was calculated by subtracting the weight of feed remaining on d 21, 35, and 42 from total weight of feed added during that phase. Pen weights of broilers also were recorded on d 1, 35, and 42. Feed conversion ratios, adjusted for mortality [total feed consumption/(final live weight + total mortality weight of the birds)] for each treatment, were calculated for each phase. Table 1. Ingredients and nutrient composition of experimental diets1 (% as fed unless noted). Ingredients  Starter  Grower  Finisher  Soybean meal, dehulled  37.71  32.99  28.26  Fat, vegetable  2.18  1.98  1.81  Dicalcium phosphate  2.04  1.75  1.53  Calcium carbonate  1.02  0.98  0.74  Salt, plain (NaCl)  0.46  0.44  0.44  DL-methionine  0.30  0.22  0.19  L-lysine  0.17  0.16  0.13  Trace mineral2  0.08  0.08  0.08  Vitamin premix3  0.25  0.25  0.25  Ingredients  Starter  Grower  Finisher  Soybean meal, dehulled  37.71  32.99  28.26  Fat, vegetable  2.18  1.98  1.81  Dicalcium phosphate  2.04  1.75  1.53  Calcium carbonate  1.02  0.98  0.74  Salt, plain (NaCl)  0.46  0.44  0.44  DL-methionine  0.30  0.22  0.19  L-lysine  0.17  0.16  0.13  Trace mineral2  0.08  0.08  0.08  Vitamin premix3  0.25  0.25  0.25  1Control group was fed the basil diet. The other treatment diets were the same basal diet supplemented with 125 ppm YCW, 250 ppm, YCW, 500 ppm YCW, or 1,250 ppm YC, respectively. 2Trace mineral mix provided the following (per kg of diet): manganese (MnSO4•H2O), 60 mg; iron (FeSO4•7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4•5H2O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg. 3Vitamin mix provided the following (per kg of diet): Thiamin•mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; D-Ca pantothenate, 12 mg; vitamin B12 (cobalamin),12.0 μg; pyridoxine•HCL, 4.7 mg; D-biotin, 0.11 mg; folic acid, 5.5 mg; menadione sodium bisulfite complex, 3.34 mg; choline chloride, 220 mg; cholecalciferol, 27.5 ug; trans-retinyl acetate, 1892 ug; all-rac α tocopheryl acetate, 11 mg; ethoxyquin, 125 mg. View Large Table 1. Ingredients and nutrient composition of experimental diets1 (% as fed unless noted). Ingredients  Starter  Grower  Finisher  Soybean meal, dehulled  37.71  32.99  28.26  Fat, vegetable  2.18  1.98  1.81  Dicalcium phosphate  2.04  1.75  1.53  Calcium carbonate  1.02  0.98  0.74  Salt, plain (NaCl)  0.46  0.44  0.44  DL-methionine  0.30  0.22  0.19  L-lysine  0.17  0.16  0.13  Trace mineral2  0.08  0.08  0.08  Vitamin premix3  0.25  0.25  0.25  Ingredients  Starter  Grower  Finisher  Soybean meal, dehulled  37.71  32.99  28.26  Fat, vegetable  2.18  1.98  1.81  Dicalcium phosphate  2.04  1.75  1.53  Calcium carbonate  1.02  0.98  0.74  Salt, plain (NaCl)  0.46  0.44  0.44  DL-methionine  0.30  0.22  0.19  L-lysine  0.17  0.16  0.13  Trace mineral2  0.08  0.08  0.08  Vitamin premix3  0.25  0.25  0.25  1Control group was fed the basil diet. The other treatment diets were the same basal diet supplemented with 125 ppm YCW, 250 ppm, YCW, 500 ppm YCW, or 1,250 ppm YC, respectively. 2Trace mineral mix provided the following (per kg of diet): manganese (MnSO4•H2O), 60 mg; iron (FeSO4•7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4•5H2O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg. 3Vitamin mix provided the following (per kg of diet): Thiamin•mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; D-Ca pantothenate, 12 mg; vitamin B12 (cobalamin),12.0 μg; pyridoxine•HCL, 4.7 mg; D-biotin, 0.11 mg; folic acid, 5.5 mg; menadione sodium bisulfite complex, 3.34 mg; choline chloride, 220 mg; cholecalciferol, 27.5 ug; trans-retinyl acetate, 1892 ug; all-rac α tocopheryl acetate, 11 mg; ethoxyquin, 125 mg. View Large Salmonella Challenge A nalidixic acid resistant strain of S. Heidelberg was grown for 6 h in tryptic soy broth [25] and the number of cfu per milliliter determined by plating 10-fold serial dilutions of the bacterial suspension on xylose lysine tergitol-4 (XLT-4) medium containing 0.25ug/mL nalidixic acid [25]. The challenge dose was determined using optical density, and a dose of 5 × 107cfu/mL was used per chicken. On the first d, one-half of the birds (seeders) in a pen (n = 25) from each group were dyed and tagged and challenged by oral gavage with a 5 × 107 cfu S. Heidelberg, while the remaining half (n = 25) were left unchallenged (contacts). In this animal model [26–28], challenged chicks represented infected birds that may shed Salmonella in the environment (seeders), whereas unchallenged chicks represented birds that may become infected with Salmonella through horizontal contact with the contaminated environment (contacts). Sample Collection, Salmonella Isolation and Identification Environmental contamination by S. Heidelberg and potential horizontal transmission of the bacteria to non-inoculated penmates was determined at 14 and 42 d of age using boot sock swabs collected from all pens and cecal samples collected from representative birds, respectively, as described previously [28, 29]. A sterile pre-moistened boot sock swab [30] was placed onto one foot covered with a clean new plastic boot. Boot socks were removed and placed into a labeled sterile bag after walking around the interior perimeter of each of the pens. To determine horizontal transmission of S. Heidelberg among penmates, 10 birds from the non-challenged chickens (contact birds) from each pen were humanely euthanized by cervical dislocation and ceca aseptically removed for sampling. Ceca from each bird were put into a sterile plastic sample bag [31] individually, labeled, and stored on ice until transported to the onsite lab for Salmonella identification. Upon arrival at the laboratory, approximately 100 mL tetrathionate broth [25] was added to boot sock swab samples while ceca were weighed, and stomached in 50 mL of tetrathionate broth. A 1-mL aliquot was removed from each sample for most probable number (MPN) analysis into a test tube and mixed with tetrathionate broth. Samples were incubated overnight at 41.5°C, and a loop full of sample from each tube was struck onto XLT-4 agar plates [25] and incubated overnight at 37°C. Three black (H2S-positive) colonies were selected from each plate and tested for agglutination using Polyvalent-O Salmonella Specific Antiserum [32] to confirm Salmonella positive colonies. Salmonella Enumeration in Cecal Samples Using the Most Probable Number Method From each of the 10 cecal samples collected from horizontally exposed (contact) birds, a 1-mL sample of stomached tetrathionate broth was transferred into 3 adjacent wells in the first row of a 96-well, 2-mL deep plate [33]. A 10-fold serial dilution was prepared by adding a 0.1-mL aliquot of sample from the first row into 0.9-mL of tetrathionate broth [25] on the second row. Subsequent 10-fold dilutions were prepared in a similar way using a multichannel pipette, changing pipette tips between dilutions, until 10−5 dilution was reached for each sample. After incubation of the 96-well plates for 24 h at 42°C, 1-μL aliquot from each well was plated onto XLT-4 [25] agar plates containing nalidixic acid using a pin-tool replicator [34]. Plates were incubated at 37°C for 24 hours. Finally, the number of wells with black colonies (H2S-positive) was counted for each dilution, and the MPN of Salmonella calculations was performed as previously described [28]. Suspected colonies were picked and tested for agglutination using polyvalent-O Salmonella specific antiserum [32]. Data Management and Statistical Analysis Analyses were performed using commercially available statistical software (Stata v14.2) [35]. Boot sock Salmonella prevalence was compared among treatment groups using Fisher's exact test. Salmonella prevalence in ceca samples was compared among treatment groups and challenge status categories using generalized estimating equations (GEE) logistic models, and Salmonella MPN were compared using GEE linear models to account for the correlation between responses of birds from the same pen. GEE models were estimated using robust standard errors and an exchangeable working correlation structure. For the comparison of Salmonella MPN, samples with a negative culture result by the MPN method but a positive culture result by primary or secondary enrichment were arbitrarily assigned an MPN value equal to one half the minimum detection limit of the MPN assay. MPN values were log-transformed prior to statistical analysis. Post-hoc pairwise comparisons between treatments were performed using the Bonferroni procedure to limit the type I error rate to 5% over all comparisons. All statistical testing assumed a two-sided alternative hypothesis, and P < 0.05 was considered significant. RESULTS AND DISCUSSION Performance In this study, we compared 3 different concentrations of a commercially available Saccharomyces cerevisiae YCW and one concentration of a commercially available YC for their ability to lower both direct colonization of birds challenged at one d of age and reduce horizontal infection of unchallenged penmates with S. Heidelberg. In agreement with results from previous studies [16–18], we did not see any significant differences in either body weight or feed efficiency at either 35 or 42 d of age between the control and the supplemented group (Table 2). Similarly, there was no significant difference in performance between challenged and non-challenged birds. This is not surprising because the Salmonella serovar used for this challenge does not normally cause serious health issues in chickens. On the other hand, yeast cell products are widely reported to have a beneficial effect when administered to birds under some stressful conditions, including mycotoxin challenge [36], heat stress [37, 38], as well as pathogen and immune challenge [14, 39]. Table 2. Performance data at 35 and 42 d of age. Treatment  Feed intake  Adjusted feed conversion  Average weight gain  Percent mortality  35 d of age  Untreated  117.96  1.74  1.37  5.25  Yeast cell wall (125 ppm)  114.82  1.73  1.36  5.50  Yeast cell wall (250 ppm)  115.88  1.74  1.38  6.50  Yeast cell wall (500 ppm)  111.20  1.71  1.35  6.25  Yeast culture (1250 ppm)  114.23  1.73  1.37  6.00  42 d of age  Untreated  174.27  1.79  2.00  5.50  Yeast cell wall (125 ppm)  171.30  1.78  2.00  6.25  Yeast cell wall (250 ppm)  172.29  1.77  2.04  7.75  Yeast cell wall (500 ppm)  165.51  1.76  1.99  8.25  Yeast culture (1250 ppm)  167.96  1.77  2.00  8.00  Treatment  Feed intake  Adjusted feed conversion  Average weight gain  Percent mortality  35 d of age  Untreated  117.96  1.74  1.37  5.25  Yeast cell wall (125 ppm)  114.82  1.73  1.36  5.50  Yeast cell wall (250 ppm)  115.88  1.74  1.38  6.50  Yeast cell wall (500 ppm)  111.20  1.71  1.35  6.25  Yeast culture (1250 ppm)  114.23  1.73  1.37  6.00  42 d of age  Untreated  174.27  1.79  2.00  5.50  Yeast cell wall (125 ppm)  171.30  1.78  2.00  6.25  Yeast cell wall (250 ppm)  172.29  1.77  2.04  7.75  Yeast cell wall (500 ppm)  165.51  1.76  1.99  8.25  Yeast culture (1250 ppm)  167.96  1.77  2.00  8.00  View Large Table 2. Performance data at 35 and 42 d of age. Treatment  Feed intake  Adjusted feed conversion  Average weight gain  Percent mortality  35 d of age  Untreated  117.96  1.74  1.37  5.25  Yeast cell wall (125 ppm)  114.82  1.73  1.36  5.50  Yeast cell wall (250 ppm)  115.88  1.74  1.38  6.50  Yeast cell wall (500 ppm)  111.20  1.71  1.35  6.25  Yeast culture (1250 ppm)  114.23  1.73  1.37  6.00  42 d of age  Untreated  174.27  1.79  2.00  5.50  Yeast cell wall (125 ppm)  171.30  1.78  2.00  6.25  Yeast cell wall (250 ppm)  172.29  1.77  2.04  7.75  Yeast cell wall (500 ppm)  165.51  1.76  1.99  8.25  Yeast culture (1250 ppm)  167.96  1.77  2.00  8.00  Treatment  Feed intake  Adjusted feed conversion  Average weight gain  Percent mortality  35 d of age  Untreated  117.96  1.74  1.37  5.25  Yeast cell wall (125 ppm)  114.82  1.73  1.36  5.50  Yeast cell wall (250 ppm)  115.88  1.74  1.38  6.50  Yeast cell wall (500 ppm)  111.20  1.71  1.35  6.25  Yeast culture (1250 ppm)  114.23  1.73  1.37  6.00  42 d of age  Untreated  174.27  1.79  2.00  5.50  Yeast cell wall (125 ppm)  171.30  1.78  2.00  6.25  Yeast cell wall (250 ppm)  172.29  1.77  2.04  7.75  Yeast cell wall (500 ppm)  165.51  1.76  1.99  8.25  Yeast culture (1250 ppm)  167.96  1.77  2.00  8.00  View Large Bacterial Shedding, Environmental Contamination, and Prevalence of Infection Challenged birds were able to shed the bacteria and contaminate the environment, as all the pens tested for the growth of S. Heidelberg were positive (100%) on d 14 and 42 with no difference among treatment groups. There were no significant differences in the prevalence of S. Heidelberg-infected birds among treatment groups either in the direct S. Heidelberg challenged (seeder birds) or indirect challenged (contact birds) as assessed based on identification of the bacteria in cecal contents of birds (Table 3). However, the decrease in the prevalence of Salmonella-infected birds was numerically higher with YCW at 500 ppm as compared to all other groups, including the control group, having only 41.7% overall positive prevalence vs. control at 54.2%. The effect of YCW at 500 ppm in reducing the prevalence of Salmonella-infected birds was even greater in the contact birds, which was significant at the 90% level, but not at the 95% level (32.5 vs. 57.5%, P = 0.09); see also Figure 1. A previous in vitro study from our group has demonstrated the ability of YCW and live yeast to bind specific bacterial pathogens, including several species of Salmonella [40]. Therefore, binding could be the possible mechanism for the reduction of Salmonella in the intestine. On the other hand, a YC is a mixture of yeast and the media in which it was grown and as a result may not have a significant level of mannan-oligosaccharide to bind pathogens. In line with this, another study demonstrated reduced colonization of S. Enteritidis in broiler chickens with the addition of mannan-oligosaccharide, a component of YCW, to the diet [39]. A similar study demonstrated reduced fecal shedding of S. Enteritidis in broiler chickens with diets supplemented with mannan-oligoscaccharide [41]. Supplementation of water with D-mannose reduced the colonization of broiler chickens with S. Typhimurium [42]. All these data taken together, we can suggest that binding of Salmonella to the mannan-oligoscharides of the YCW is an important part of the mechanism by which YCW results in reduction of S. Heidelberg in the intestine of challenged birds. Figure 1. View largeDownload slide Dot plots of pen-level Salmonella prevalence by treatment and challenge status. Ten indirect challenged birds and 5 direct challenged birds were sampled in each of 8 pens per treatment group. Figure 1. View largeDownload slide Dot plots of pen-level Salmonella prevalence by treatment and challenge status. Ten indirect challenged birds and 5 direct challenged birds were sampled in each of 8 pens per treatment group. Table 3. Salmonella prevalence (%) in ceca samples collected on d 42. Treatment  Challenge status Indirect* (Contact birds)  Direct* (Seeder birds)  Total*  Untreated  46/80 (57.5)  19/40 (47.5)  65/120 (54.2)  Yeast cell wall (125 ppm)  51/80 (63.8)  24/40 (60.0)  75/120 (62.5)  Yeast cell wall (250 ppm)  49/80 (61.3)  21/40 (52.5)  70/120 (58.3)  Yeast cell wall (500 ppm)  26/80 (32.5)  24/40 (60.0)  50/120 (41.7)  Yeast culture (1250 ppm)  42/80 (52.5)  19/40 (47.5)  61/120 (50.8)  Treatment  Challenge status Indirect* (Contact birds)  Direct* (Seeder birds)  Total*  Untreated  46/80 (57.5)  19/40 (47.5)  65/120 (54.2)  Yeast cell wall (125 ppm)  51/80 (63.8)  24/40 (60.0)  75/120 (62.5)  Yeast cell wall (250 ppm)  49/80 (61.3)  21/40 (52.5)  70/120 (58.3)  Yeast cell wall (500 ppm)  26/80 (32.5)  24/40 (60.0)  50/120 (41.7)  Yeast culture (1250 ppm)  42/80 (52.5)  19/40 (47.5)  61/120 (50.8)  *Values given are Salmonella-positive birds over the total number of birds in each group or the respective percentage in parenthesis. Ten indirect challenged birds and 5 direct challenged birds were sampled in each of 8 pens per treatment group. Marginal percentages with a superscript in common do not differ with a level of significance of 5% over all comparisons. View Large Table 3. Salmonella prevalence (%) in ceca samples collected on d 42. Treatment  Challenge status Indirect* (Contact birds)  Direct* (Seeder birds)  Total*  Untreated  46/80 (57.5)  19/40 (47.5)  65/120 (54.2)  Yeast cell wall (125 ppm)  51/80 (63.8)  24/40 (60.0)  75/120 (62.5)  Yeast cell wall (250 ppm)  49/80 (61.3)  21/40 (52.5)  70/120 (58.3)  Yeast cell wall (500 ppm)  26/80 (32.5)  24/40 (60.0)  50/120 (41.7)  Yeast culture (1250 ppm)  42/80 (52.5)  19/40 (47.5)  61/120 (50.8)  Treatment  Challenge status Indirect* (Contact birds)  Direct* (Seeder birds)  Total*  Untreated  46/80 (57.5)  19/40 (47.5)  65/120 (54.2)  Yeast cell wall (125 ppm)  51/80 (63.8)  24/40 (60.0)  75/120 (62.5)  Yeast cell wall (250 ppm)  49/80 (61.3)  21/40 (52.5)  70/120 (58.3)  Yeast cell wall (500 ppm)  26/80 (32.5)  24/40 (60.0)  50/120 (41.7)  Yeast culture (1250 ppm)  42/80 (52.5)  19/40 (47.5)  61/120 (50.8)  *Values given are Salmonella-positive birds over the total number of birds in each group or the respective percentage in parenthesis. Ten indirect challenged birds and 5 direct challenged birds were sampled in each of 8 pens per treatment group. Marginal percentages with a superscript in common do not differ with a level of significance of 5% over all comparisons. View Large Bacterial Load Estimation in the Ceca of Salmonella Heidelberg-infected Birds There was no statistically significant difference in reducing the MPN of Salmonella in the ceca of positive birds. However, MPN were numerically lowest in the 500 ppm YCW-supplemented birds (log1.4 cfu/gram of cecal content) compared to all other treatment groups (Table 4). The effect of YCW in reducing MPN becomes statistically significant (P = 0.04) when the comparison among groups was made only within the contact birds, as shown in Figure 2. The box plots more concisely demonstrate the effect of YCW 500 ppm in decreasing the MPN of Salmonella in the indirect challenged (contact) birds. This is in agreement with a previous suggestion made by our group that Salmonella spp. were able to bind to mannose via the type-1 binding fimbriae, and hence Saccharomyces cerevisiae and cell wall from S.cerevisiae were shown to bind a variety of Gram-negative organisms, including Salmonella [40]. Finally, factorial analysis both for the prevalence of positive birds and MPN of Salmonella in cecal contents of positive birds did not show any significant interaction between the main effects (treatment and challenge method). Figure 2. View largeDownload slide Box plots of Salmonella MPN for culture-positive samples by treatment and challenge status. See Table 3 for sample sizes. Figure 2. View largeDownload slide Box plots of Salmonella MPN for culture-positive samples by treatment and challenge status. See Table 3 for sample sizes. Table 4. Geometric mean (95% CI) Salmonella MPN/g for culture-positive ceca samples collected on d 42.   Challenge status  Treatment  Indirect (Contact birds)*  Direct (Seeder birds)*  Untreated  2.3a (1.2, 4.4)  3.7 (1.9, 7.2)  Yeast cell wall (125 ppm)  2.9a (1.4, 6.0)  2.8 (1.9, 4.0)  Yeast cell wall (250 ppm)  3.4a (2.1, 5.4)  3.3 (1.3, 8.5)  Yeast cell wall (500 ppm)  1.4b (0.95, 2.1)  2.7 (1.5, 4.8)  Yeast culture (1250 ppm)  3.2a (1.3, 7.9)  2.4 (1.1, 5.4)    Challenge status  Treatment  Indirect (Contact birds)*  Direct (Seeder birds)*  Untreated  2.3a (1.2, 4.4)  3.7 (1.9, 7.2)  Yeast cell wall (125 ppm)  2.9a (1.4, 6.0)  2.8 (1.9, 4.0)  Yeast cell wall (250 ppm)  3.4a (2.1, 5.4)  3.3 (1.3, 8.5)  Yeast cell wall (500 ppm)  1.4b (0.95, 2.1)  2.7 (1.5, 4.8)  Yeast culture (1250 ppm)  3.2a (1.3, 7.9)  2.4 (1.1, 5.4)  *Values represent average values of MPN/g sample with lower and higher values of the 95% confidence interval. Ten indirect challenged birds and 5 direct challenged birds were sampled in each of 8 pens per treatment group. a,bMarginal means with a superscript in common do not differ with a level of significance of 5% over all comparisons. See Table 3 for sample sizes. View Large Table 4. Geometric mean (95% CI) Salmonella MPN/g for culture-positive ceca samples collected on d 42.   Challenge status  Treatment  Indirect (Contact birds)*  Direct (Seeder birds)*  Untreated  2.3a (1.2, 4.4)  3.7 (1.9, 7.2)  Yeast cell wall (125 ppm)  2.9a (1.4, 6.0)  2.8 (1.9, 4.0)  Yeast cell wall (250 ppm)  3.4a (2.1, 5.4)  3.3 (1.3, 8.5)  Yeast cell wall (500 ppm)  1.4b (0.95, 2.1)  2.7 (1.5, 4.8)  Yeast culture (1250 ppm)  3.2a (1.3, 7.9)  2.4 (1.1, 5.4)    Challenge status  Treatment  Indirect (Contact birds)*  Direct (Seeder birds)*  Untreated  2.3a (1.2, 4.4)  3.7 (1.9, 7.2)  Yeast cell wall (125 ppm)  2.9a (1.4, 6.0)  2.8 (1.9, 4.0)  Yeast cell wall (250 ppm)  3.4a (2.1, 5.4)  3.3 (1.3, 8.5)  Yeast cell wall (500 ppm)  1.4b (0.95, 2.1)  2.7 (1.5, 4.8)  Yeast culture (1250 ppm)  3.2a (1.3, 7.9)  2.4 (1.1, 5.4)  *Values represent average values of MPN/g sample with lower and higher values of the 95% confidence interval. Ten indirect challenged birds and 5 direct challenged birds were sampled in each of 8 pens per treatment group. a,bMarginal means with a superscript in common do not differ with a level of significance of 5% over all comparisons. See Table 3 for sample sizes. View Large CONCLUSION AND APPLICATIONS The challenge model, which utilizes direct challenged birds as seeders to infect the environment and contact birds, worked well, demonstrated by 100% prevalence of contaminated pens. YCW at 500 ppm decreased the prevalence of S. Heidelberg-positive birds, and this decrease was greater in contact birds as compared to the direct challenged birds (seeders). YCW was able to numerically decrease the bacterial load in the ceca of infected birds (both contact and seeder birds). However, the reduction was statistically significant (P < 0.04) only in the indirect challenged model (contact birds). 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Journal of Applied Poultry ResearchOxford University Press

Published: May 15, 2018

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