Abstract Hatching eggs collected from resident broiler breeders at 48 wk of age were used to produce male and female chicks that were assigned sex separately to 96 new litter pens and fed either a 0 or 50 g/MT RFC (refined functional carbohydrate feed additive derived from yeast) diet. There were 24 replicate pens of 12 broilers each per diet per sex. Feed intake and BW were determined at 14, 28, and 42 d of age. Litter was sampled by pen using sterile socks at 35 d and tested for Salmonella spp. using an enzyme linked fluorescence assay method. Salmonella spp. was isolated from 7 of 48 control-fed broiler pens but no RFC-fed pens (P ≤ 0.05). Thereafter, 48 males and 48 females were selected based on litter Salmonella presence and RFC treatment. The cecas of these broilers were aseptically excised after feed withdrawal and lairage and tested for presence of Salmonella spp. There were 18 of the 48 control-fed broilers confirmed positive from litter-positive pens but none from litter-negative pens fed RFC. The serovar of litter and cecal Salmonella isolates was Salmonella enterica subsp. enterica serovar Senftenberg (S. Senftenberg). Female broilers that were fed RFC exhibited greater BW at 28 d (P ≤ 0.05) and 42 d (P ≤ 0.05) while RFC-fed males exhibited improved feed efficiency during the 15–28 d period (P = 0.06). These data demonstrated that dietary RFC reduced the prevalence of Salmonella in the litter and ceca of broilers when fed continuously while not being detrimental to broiler live performance. INTRODUCTION In the United States alone, non-typhoidal Salmonella spp. have resulted in as many as 1 million illnesses, 19,000 hospitalizations, and 380 deaths annually (Centers for Disease Control and Prevention, 2015). While poultry products have not been the only source of Salmonella contamination leading to these illnesses, data from the Interagency Food Safety Analytics Corporation (IFSAC) Project revealed that 10% of Salmonella outbreaks were attributed to chicken products and 12%, 9%, and 8% were attributed to seeded vegetables, beef, and pork, respectively (Golden and Boyer, 2015). Therefore, the loss of human life coupled with significant economic impacts due to illness, hospitalization costs, and response measures have made implementation of foodborne Salmonella control measures in the poultry industry of urgent importance. As worldwide demand for poultry products has continued to increase, the concurrent development of antimicrobial resistance has posed a significant threat to consumers and livestock and has caused reduced utilization of antibiotics in agriculture (Ganan et al., 2012; Gilbert, 2012; Spellberg et al., 2013). Further, the use of antibiotics as a Salmonella control measure has been discouraged due to limited effectiveness, potential for food product residue generation, and detrimental impacts on animal gut microflora that competitively exclude Salmonella (United States Department of Agriculture, 2009). Compounding the problems, overuse of antimicrobial disinfectants such as triclosans and/or phenols have also been shown to increase resistance among Salmonella serovars (Randall et al., 2004). Indeed, an increased number of Salmonella isolates that caused human illness have been reported to be resistant to multiple antimicrobial drugs (e.g., Salmonella Enterica serovar Typhimurium DT104) (Centers for Disease Control and Prevention, 1997; Glynn et al., 1998; Poppe et al., 1998). Poultry products have been shown to harbor a greater amount of antimicrobial-resistant Salmonella when compared to other meat products such as red meats, fish, and shellfish (D’Aoust et al., 1992), and emergence of resistant strains have made control of the pathogen using traditional means difficult. The use of yeast (Saccharomyces cerevisiae) in animal diets has become popular due to their nutritional benefits, immunological effects, and ready availability (Moyad, 2007, 2008). Further, mannan oligosaccharides (MOS) that have been derived from the cell wall of yeast have been reported to exhibit bacterial binding characteristics due to the mannose sugars in the MOS network (Oyofo et al., 1989a,b; Newman, 1994; Oyofo et al., 2015). Thus, MOS has been employed as a means to control microbial populations harbored by live poultry since they have been shown to agglutinate strains of both Escherichia coli (E. coli) and Salmonella spp. and reduce Salmonella spp. cecal colonization of broiler chicks (Oyofo et al., 1989a,b; Spring et al., 2000). To increase the effectiveness of this binding action, yeasts have been appropriately modified to selectively expose specific sugars responsible for MOS efficiency. Appropriate enzymatic hydrolysis of yeast has exposed D-mannose sugars held within the MOS complex to produce refined functional carbohydrates (RFC) that have activities against a range of gram-negative bacterial species. This action has been attributed to the presence of the Type 1 fimbriae on these species, which have been shown to be bound by D-mannose (Duguid, 1959; Duguid et al., 1966; Oyofo et al., 1989a,b). It was hypothesized that RFC formed irreversible agglutinates with bacteria such as Salmonella, which would then be eliminated from the intestinal tract in the feces and no longer be infectious (Walker et al., 2017). Previous research has indicated that RFC were able to reduce cecal Salmonella spp. colonization in turkeys during transport stress (Huff et al., 2013), reduce Salmonella prevalence of broiler breeder hen ceca throughout an egg production cycle (Walker et al., 2017), and reduce the incidence of Salmonella among broiler progeny of those breeder hens (Walker et al., 2017). It was of importance that the effects of RFC supplementation in broiler diets be investigated more thoroughly since broiler grow-out comprises a critical control point for Salmonella contamination. The objectives of the present study were to evaluate a minimal continuous dosage of dietary RFC as a means to control environmentally present Salmonella spp. in live broilers and to verify the absence of detrimental effects on live performance parameters and overall broiler health. MATERIALS AND METHODS The trial was conducted in accordance with the principles and specific guidelines of the Guide for the Care and Use of Agricultural Animals in Research and Teaching (FASS, 2010) and approved by the North Carolina State University Institutional Animal Care and Use Committee. Egg Collection and Incubation Broiler hatching eggs were collected from a 16-pen resident breeder flock at 48 wk of age and set in a Natureform Model NMC-2000 incubator (Natureform International, Jacksonville, FL) to embryonic day (E) 17 of incubation. At E 17, eggs were transferred into a Natureform Model NOM-45 incubator until E 21.5. Chick paper was placed in the bottom of each hatching basket to minimize vertical transfer of organic matter between egg trays that represented individual breeder flock pens. Eggs were initially incubated at 38.1°C dry bulb temperature that was gradually decreased to 36.7°C in the presence of 53% RH. Upon transfer, the wet bulb temperature was increased and the ventilation reduced to maintain a minimum RH of 67% to encourage growth of any bacteria present, which was thereafter adjusted as chicks began to hatch. As chicks were removed from the incubator at E 21.5, samples of chick paper lining the bottoms of all hatching baskets and 4 randomly selected egg shells from each hatcher tray of eggs were placed into sterile filter bags (VWR International, Radnor, PA) and assayed according to the enzyme-linked fluorescent assay (ELFA) protocol described below to determine presence of Salmonella. ELFA Analysis for Salmonella spp After samples had been collected in sterile filter bags and placed on ice for transport to a Biosafety Level 2 (BSL-2) facility, the weight was obtained and samples were enriched with a 1% buffered peptone water (BPW) solution to produce a 1 part sample: 9 parts BPW dilution. A Salmonella supplement (BioMérieux Product # 42,650) was then added directly to the enrichment solution so that 0.04 mL for every g of sample ratio was achieved (i.e., 1 mL of supplement for 225 mL of BPW). Samples were mechanically homogenized for 1 min before being incubated for 26–30 h at 37°C. After incubation, samples were removed based on their order collected and tested with the ELFA instrument (VIDAS® 30 Multiparametric Immunoassay Instrument, BioMérieux, Inc., Marcy-l'Étoile, France). This process entailed manually mixing the sample bag until the contents were homogenized, pipetting a 0.5 mL aliquot into the sample well of a reagent strip, and heat lysing the cells on a heat block (Vidas Heat and Go, BioMérieux Product # 93,554) for 5 min at 131°C. Samples were then allowed to cool for 10 min before being loaded into the instrument. The instrument functioned on the basis of a sandwich enzyme-linked immunosorbent assay (ELISA). A pipette tip designated for each sample had its inside coated with antibodies that recognized Salmonella antigens, namely somatic (O) lipopolysaccharide and flagellar (H) protein antigens to allow for detection of motile and non-motile strains of Salmonella. A series of washing phases then rid the sample of non-specific microorganisms and other debris in the sample matrix. The sample was then exposed to a solution containing a conjugate antibody. Antibodies were then cleaved in a specific enzymatic process, which resulted in release of fluorescent molecules that could be detected by spectrophotometry. Samples were determined positive for Salmonella spp. if the absorbance ratio surpassed a predetermined threshold that had been calculated based on a standard-control run, which was updated every 14 d. The positive control for the standard-control run consisted of purified and inactivated Salmonella receptors (the assay differential targets), preservative, and a protein stabilizer according to the manufacturer's instructions. All positive samples were confirmed with 2 types of selective media. These were typically Rapid Salmonella Agar (Bio-Rad Product #3,563,961; Hercules, CA 94,547) and XLT-4 agar (Oxoid Product #CM1061). Confirmation entailed obtaining a 10 μL disposable inoculation loop full of solution from the sample bag and streaking onto selective media until a pure culture was obtained. According to the manufacturer's instructions, the level of detection limit (LOD) for poultry fecal samples was 0.9 colony-forming units (CFU) per 25 g of sample with a confidence interval of 0.5–1.5 CFU. This was compared to conventional plating of the same sample type, which was found to be 1.0 CFU per 25 g of sample with a 0.6–1.5 CFU confidence interval. Broiler Feed Manufacture A starter broiler mash diet was manufactured by grinding corn through a hammermill (Model 1522, Roskamp Champion, Waterloo, IA) containing a pair of 2.4-mm screens followed by batching and blending in a twin shaft counterpoise ribbon mixer (Model TRDB126060, Hayes and Stolz, Fort Worth, TX) for 270 sec. A commercially available RFC additive (Aviator SCP; Arm and Hammer Animal Health, Princeton, NJ) that achieved a 50 g/MT final concentration was added to half of each diet at the expense of filler. Starter diets were steam-conditioned in a single pass conditioner for 30 sec at a temperature of 79.4°C before being pelleted using a 30 HP California Pellet Mill (Model PM1112–2, Crawfordsville, IN) that was equipped with a 4.4 × 35 mm die. Immediately thereafter, diets were cooled in a counter flow cooler (Model VK09 × 09KL, Geeland Counterflow USA, Inc, Orlando, FL) and crumbled with samples being collected periodically during bagging. A pelleted grower diet was manufactured by the same process without crumbling. All diets were manufactured at the North Carolina State University Feed Mill Education Unit and feed samples of both treatments were transferred to a BSL-2 laboratory after manufacture to be assayed for presence of Salmonella using the ELFA analysis. Broiler Experimental Facility After aseptic collection of chick paper samples, hatched broiler chicks were removed from hatching trays, sexed using the feather sexing method, counted, and maintained in chick boxes separate by breeder pen and sex until placement. Male and female chicks were transferred from the hatchery to a curtain-sided, heated, fan-ventilated housing facility on the same premises. The chicks were maintained separate in baskets until 12 male or female chicks (depending on assigned pen sex) were selected, group weighed, permanently identified with neck tags, and distributed among 96 floor pens containing new pine wood litter shavings. Each pen was 1.22 m wide by 1.83 m long, resulting in a total pen area of 2.23 m2 at a stocking density of 5.38 chicks/m2. Mesh screen was used to separate groups of 8 pens that had been blocked based on control or RFC diet feeding to minimize transfer of litter, dust, and other contaminants between pens. Pens contained one Plasson bell-type drinker and one tube feeder (Kuhl, Flemington, NJ). Three additional supplemental feeder flats were used until 5 d of age, with one being removed at that time and another being removed at 10 d to leave one remaining until 14 d. Feed was added and mounded on these trays as needed. At 14 d, remaining feed was screened from feeder lids back into the tube feeder. Tube feeders were shaken twice daily after 7 d to ensure consistent feed flow. An additional font drinker was used until 10 d of age. Litter temperatures were 36.7°C for the first 2 d. Ambient temperatures were thereafter reduced so that litter temperature was gradually reduced to 28.8°C by 7 d of age. House temperature was kept uniform throughout by use of upward directed fans placed in the center of the house. These fans operated continuously throughout the trial. Curtains were lowered (opened) only when ambient house temperatures approached 26.7°C after 14 d and then raised (closed) at night so that exhaust fan ventilation could be utilized. Lighting Program A photoperiod of 23 h of light was provided through 7 d by 18 W fluorescent lamps, followed by 22 h until 14 d, 21 h to 21 d, and 14 h beyond 22 d. Natural light alone was used during daylight hours after 7 d of age. Broiler Dietary Treatments Each broiler pen was assigned one of 2 dietary treatments resulting in 24 replicate pens per sex to complete the 2 factor design. Broiler diets were either a basal diet or the basal diet with 50 g/MT RFC addition. Broilers were provided, for ad libitum consumption, 900 g of starter feed initially, which was consumed in approximately 16 d, and grower feed thereafter. The starter feed was in crumble form and the grower feed was pelleted. Feed formulas are shown in Table 1. Diets were fed to 55 d of age. Table 1. Composition of broiler starter and grower diets. Ingredients Starter5 Grower6 (%) Corn 54.21 68.02 Soybean meal (48% CP) 34.76 23.33 Dicalcium phosphate (18.5% P) 1.52 1.46 Poultry by-product meal 5.04 4.03 Poultry fat 2.00 1.00 Limestone 0.63 0.62 Salt 0.50 0.50 Vermiculite filler 0.45 0.10 Choline chloride (60%) 0.20 0.20 Vitamin premix1 0.05 0.05 Mineral premix2 0.20 0.20 Selenium premix3 0.05 0.05 DL-Methionine 0.20 0.14 L-Lysine 0.06 0.14 L-Threonine 0.08 0.06 Coccidiostat4 0.05 0.05 Total 100.00 100.00 Calculated nutrient content Crude protein 24.00 19.00 Calcium 1.00 0.90 Available phosphorus 0.50 0.45 Lysine 1.32 1.05 Methionine 0.57 0.44 Threonine 0.88 0.70 Methionine + cysteine 0.95 0.77 Sodium 0.22 0.21 Metabolizable energy (kcal/g) 2.90 3.00 Ingredients Starter5 Grower6 (%) Corn 54.21 68.02 Soybean meal (48% CP) 34.76 23.33 Dicalcium phosphate (18.5% P) 1.52 1.46 Poultry by-product meal 5.04 4.03 Poultry fat 2.00 1.00 Limestone 0.63 0.62 Salt 0.50 0.50 Vermiculite filler 0.45 0.10 Choline chloride (60%) 0.20 0.20 Vitamin premix1 0.05 0.05 Mineral premix2 0.20 0.20 Selenium premix3 0.05 0.05 DL-Methionine 0.20 0.14 L-Lysine 0.06 0.14 L-Threonine 0.08 0.06 Coccidiostat4 0.05 0.05 Total 100.00 100.00 Calculated nutrient content Crude protein 24.00 19.00 Calcium 1.00 0.90 Available phosphorus 0.50 0.45 Lysine 1.32 1.05 Methionine 0.57 0.44 Threonine 0.88 0.70 Methionine + cysteine 0.95 0.77 Sodium 0.22 0.21 Metabolizable energy (kcal/g) 2.90 3.00 1Vitamin premix supplied the following per kg of diet: 6,614 IU vitamin A, 2,000 IU vitamin D3, 33 IU vitamin E, 0.02 mg vitamin B12, 0.13 mg biotin, 1.98 mg menadione (K3), 1.98 mg thiamine, 6.6 mg riboflavin, 11 mg D-pantothenic acid, 3.97 mg vitamin B6, 55 mg niacin, and 1.1 mg folic acid. 2Mineral premix supplied the following per kg of diet: manganese, 120 mg; zinc, 120 mg; iron, 80 mg; copper, 10 mg; iodine, 2.5 mg; and cobalt, 1 mg. 3Selenium premix provided 0.2 mg Se (as Na2SeO3) per kg of diet. 4Salinomycin (Sacox 60, Intervet/Merck; Millsboro, DE) supplied at 60 g of active ingredient per 907.19 kg of feed. 5There was 900 g of starter diet fed to approximately 16 d of age. 6Grower diet was fed from approximately 17 to 55 d of age. View Large Live Performance Data Broilers were group weighed by pen at placement, 14, 28, and 42 d of age. Feed consumption was determined by pen at 14, 28, and 42 d so that feed conversion ratio (FCR) could be calculated. Mortalities were removed and weighed twice daily so that their BW could be used in the FCR calculation. Litter Sampling At 35 d, a novel method was employed in which each of the 96 pens were sampled across their entire surface on an individual basis using a single pre-enriched sterile sock (Tubigrip Product # 1448) placed on a 7.62 cm-wide paint roller. This was a derivation of methods previously described by Buhr et al. (2007). Socks were cut into 15.24 cm sections, sterilized, placed in a sterile sampling bag (VWR International Product #10,048–882, Radnor, PA), and pre-enriched with 25 mL of a buffered peptone water solution prior to sampling. These socks were then aseptically fit over sections of sterilized PVC pipe that covered the roller, which then traversed the entire surface of each litter pen. One roller was used to sample broiler pens that had been fed control diets and another to sample those that had been fed the RFC diets. Further, PVC roller sheaths were sprayed with 80% ethanol between samples before each new sock was aseptically applied. A 1.22 m pole was attached to the rollers so that the entire surface of the pen could be rolled with the sock without having to enter the pen. Immediately upon collection, litter sample socks were returned to the sterile bag from which they came and placed in a cooler that had been filled with ice. The socks were transferred to a BSL-II microbiology lab where they were further enriched and homogenized according to an ELFA analysis protocol before being tested for the presence of Salmonella with the ELFA analysis. Broiler Cecal Sampling To confirm that litter Salmonella status was a reflection of the gastrointestinal tract Salmonella status of the broilers, bird ceca from Salmonella positive and negative pens were tested with ELFA methods at 44 d of age. A total of 48 broilers were selected based upon dietary RFC treatment and litter Salmonella presence at 35 d. There were 2 pens (12 male and 12 female birds) selected that had been on Salmonella-positive litter and fed a control diet as a positive control to assess the incidence of cecal Salmonella carriage due to litter contamination. Likewise, 2 additional pens were selected on the basis of being litter Salmonella-negative at 35 d and being fed the RFC diet to serve as negative controls. Birds were withdrawn from feed for approximately 12 h after which lairage was employed to mimic what would be typical with commercial broiler production. Birds were loaded by pen into transport crates and maintained outside of the housing facilities for an additional 3 h. Thereafter, birds were transferred to a poultry processing facility on the same premises where they were weighed, euthanized humanely via cervical dislocation, and had their cecas aseptically excised. The underside of each bird was sprayed with 80% ethanol before an incision was made with a sterile pair of scissors to access and excise the ceca. Gloves and surgical tools were changed between sampling broilers of the 2 treatments. The RFC-fed broilers were sampled first and control-fed broilers were sampled last. Cecas were placed in sterile filter bags (VWR International Product #10,048–882, Radnor, PA), pulverized with a rubber mallet to expel contents, and individually enriched so that Salmonella spp. presence could be determined following the same procedures used for litter samples. The entirety of these processes was repeated again at 55 d with 48 additional birds from litter positive and negative pens as described above. Salmonella Serotyping Representative litter and cecal Salmonella isolates were preserved at −80°C until the respective serovars were determined. Freezer cultures were incubated overnight on nutrient agar at 37°C to obtain a single colony, which was then suspended in a capped tube containing nutrient holding agar. Serovars were identified by the United States Department of Agriculture National Veterinary Services Laboratories (Ames, IA). Statistical Analysis The experiment was a 2 factor design within each sex. Feed consumption, BW, FCR, BW gain, and mortality data were analyzed as a randomized complete block design with the GLM procedure of JMP for ANOVA (SAS Institute, 2011). Means were partitioned with the LS Means procedure. Significance was determined by Tukey's HSD and Student's t-tests of significance and considered statistically significant at P ≤ 0.05 unless otherwise noted. The 35 d litter data were analyzed as the live performance data except that binomial data were analyzed independent of sex with the PROC FREQ procedure of SAS and significance was determined by the Chi-Square Likelihood Ratio. RESULTS Incubator and Feed Sampling Salmonella spp. was not isolated from the incubator chick paper samples that represented individual breeder pens. Likewise, Salmonella spp. was not isolated from egg shells or feed samples. Broiler Live Performance Results Female broilers fed RFC exhibited greater (P ≤ 0.05) BW at both 28 d and 42 d (Table 2). The FCR of male broilers fed RFC was improved (P = 0.06) during the 15- to 28-d period (Table 2). There were no significant effects on mortality (Table 2). Table 2. Male and female broiler live performance to 42 d of age as affected by feeding refined functional carbohydrates (RFC). Male Female RFC (g/MT)1 RFC (g/MT)1 0 50 SEM2 P-value 0 50 SEM2 P-value 14 d BW, g 544 545 3.89 0.86 518 519 3.92 0.96 28 d BW, g 1659 1680 11.53 0.21 1446b 1479a 9.56 0.02 42 d BW, g 3246 3279 21.71 0.28 2673b 2714a 14.51 0.05 1–14 d FCR, g:g 1.19 1.19 0.01 0.92 1.22 1.21 0.01 0.76 15–28 d FCR, g:g 1.53x 1.50y 0.01 0.06 1.57 1.55 0.01 0.32 29–42 d FCR, g:g 1.74 1.73 0.03 0.82 1.86 1.85 0.02 0.64 1–42 d FCR, g:g 1.59 1.58 0.02 0.80 1.66 1.66 0.02 0.99 1–42 d Mortality, % 3.52 2.50 1.10 0.51 2.57 2.33 1.25 0.89 Male Female RFC (g/MT)1 RFC (g/MT)1 0 50 SEM2 P-value 0 50 SEM2 P-value 14 d BW, g 544 545 3.89 0.86 518 519 3.92 0.96 28 d BW, g 1659 1680 11.53 0.21 1446b 1479a 9.56 0.02 42 d BW, g 3246 3279 21.71 0.28 2673b 2714a 14.51 0.05 1–14 d FCR, g:g 1.19 1.19 0.01 0.92 1.22 1.21 0.01 0.76 15–28 d FCR, g:g 1.53x 1.50y 0.01 0.06 1.57 1.55 0.01 0.32 29–42 d FCR, g:g 1.74 1.73 0.03 0.82 1.86 1.85 0.02 0.64 1–42 d FCR, g:g 1.59 1.58 0.02 0.80 1.66 1.66 0.02 0.99 1–42 d Mortality, % 3.52 2.50 1.10 0.51 2.57 2.33 1.25 0.89 x,yMeans within row with no common subscripts approached significance (P = 0.06). a,bMeans within row with no common subscripts differ significantly (P ≤ 0.05). 1RFC (Aviator SCP, Arm and Hammer Animal Nutrition, Princeton, NJ) was added to diets during mixing at the expense of vermiculite filler. 2Standard error of the mean (SEM) for n = 24 pens of 12 broilers each. View Large Litter Sampling At 35 d, the presence of Salmonella spp. was confirmed in 7 of the 48 broiler pens (14.58%) that had been fed a control diet and not isolated in pens fed RFC, resulting in a significant treatment effect (P ≤ 0.01; Table 3). Table 3. Incidence of Salmonella spp. presence in litter of sex-separate broiler pens at 35 d of age as affected by refined functional carbohydrates (RFC) inclusion in broiler diets. Broiler Treatment Litter Sampling Results1,2 RFC3 (g/MT) (%) 0 14.58 50 ND4 P-value 0.01 Broiler Treatment Litter Sampling Results1,2 RFC3 (g/MT) (%) 0 14.58 50 ND4 P-value 0.01 1Sampling of n = 48 pens per broiler treatment. 2Sampling of a 2.23 m2 broiler pen litter surface. 3RFC (Aviator SCP, Arm and Hammer Animal Nutrition, Princeton, NJ) was added to diets during mixing at the expense of vermiculite filler. 4ND = not detected. View Large Individual Ceca Sampling At 44 d, 11 broilers of the 24 sampled from broiler litter-positive pens that had been fed a control diet were confirmed positive for ceca Salmonella presence (45.8%). No broilers from litter-negative pens that were fed RFC were confirmed positive (Table 4). Upon repeating these sampling procedures at 55 d, 7 of the 24 broilers sampled from broiler litter-positive pens fed a control diet were confirmed positive for ceca Salmonella presence (29.2%), while there were no broilers from litter-negative pens that had been fed RFC confirmed positive (Table 4). Table 4. Prevalence of Salmonella spp. in male and female1 broiler ceca at 44 and 55 d of age as affected by refined functional carbohydrates (RFC) inclusion in broiler diets and litter Salmonella status. Broiler Treatment Ceca Salmonella Prevalence1,2 RFC3 44 d 55 d (g/MT) (%) (%) 0 45.83 29.17 50 ND ND4 Broiler Treatment Ceca Salmonella Prevalence1,2 RFC3 44 d 55 d (g/MT) (%) (%) 0 45.83 29.17 50 ND ND4 1Sampling of 12 males and females within each treatment. 2Sampling of n = 24 broilers per broiler treatment. 3RFC (Aviator SCP, Arm and Hammer Animal Nutrition, Princeton, NJ) were added to diets during mixing at the expense of vermiculite filler. 4ND = not detected. View Large Salmonella Serovar Replicate litter and ceca samples were determined to have Salmonella enterica serovar Senftenberg present. DISCUSSION It was important that the effects of RFC on broiler live performance when supplemented in the broiler diet be investigated within the scope of a typical integrated live production model. It was previously reported that RFC improved BW, feed intake, and feed efficiency when fed at 100 g/MT, and that feeding RFC in combination with a direct fed microbial or coccidiostat resulted in greater villus heights and increased nitrogen retention, respectively (Gomez and Angeles, 2011; Gomez et al., 2012). However, the RFC was only supplied to male broilers during the finishing period in each of these previous trials. The present trial evaluated a continuous dosage of 50 g/MT in the feed of male and female broilers. Female broiler BW was improved (P ≤ 0.05) at both 28 and 42 d of age (Table 4), as was male broiler BW FCR (P = 0.06) during the 15- to 28-d period (Table 4). Thus, the RFC did not negatively affect broiler live performance and in some cases improved it. Similarly, mortality was not significantly affected by broiler dietary RFC inclusion (Table 2). One challenge of supplementing yeast derivatives to animal feeds was the possibility of stimulating innate immunity and consequently diminishing growth and feed efficiency. This was evident in broilers that were not exposed to a microbial challenge (Huff et al., 2006). A comprehensive study investigating yeast cell walls, beta glucans, and mannoproteins in broiler diets found that live performance was not notably altered by including these in the feed but a consistent response of greater villus height was observed among all yeast derivative treatments, which could have consumed additional nutrients (Morales-López et al., 2009). The low dosage of RFC used in this experiment evidently was not sufficient to deteriorate BW gain or FCR. The immunomodulation provided by additives of fungal origin has been attributed to beta glucans, which have been characterized as conserved carbohydrate structures among fungi that elicited an immune response after recognition by metazoan carbohydrate receptors (Brown and Gordon, 2005). One result of this response was up-regulation of heterophil functionality, namely phagocytosis, bactericidal killing, and oxidative burst, in chicks fed a purified beta glucan diet (Lowry et al., 2005). The immature chickens of the aforementioned study were subsequently protected against Salmonella enterica serovar Enteritidis later in life. This may be envisioned as a similar scenario as that of this experiment given that the samples of hatching basket papers and egg shells proved negative for the presence of Salmonella spp. Thus, it may be presumed that the contamination found in these broilers arose from environmental contamination within the broiler house and that the RFC product may have helped a certain immunity develop. Furthermore, mannose sugars have been reported to bind directly to and sequester pathogens as discussed above. As evidenced in this study, a 50 g/MT RFC dosage did not diminish broiler live performance. Any nutrient-consuming immunostimulatory effects of the low dosage of this RFC additive were apparently offset by improvements in live performance due to a reduction in pathogen challenge. This phenomenon has been previously demonstrated to occur in turkeys, where RFC ameliorated diminished live performance when supplemented to animals that had been exposed to transport stress and an E. coli challenge (Huff et al., 2013). Due to the threat Salmonella poses to humans consuming poultry, the methodology employed for detection of Salmonella in poultry production environments must continue to be improved for the sake of both commercial and research entities alike. Salmonella sampling and enrichment methods were modified in this study to accommodate greater bacterial recovery rates, to reduce cross contamination, and to increase accuracy of the results in the present study. The novel litter sampling method used this trial was such an innovation in environmental detection methodology. Previously reported litter sampling methods required drag sponges or placement of a sterile sock on the boots of a person, who then walked on the litter to collect a pooled sample of organic material to be tested for Salmonella spp. prevalence (Skov et al., 1999; Buhr et al., 2007; Marin and Lainez, 2009; Mueller-Doblies et al., 2009). These methods were not entirely conducive to sampling multiple floor pen litter surfaces because they were laborious, increased likelihood of cross contamination, and increased the time between sample collection and further enrichment. Also, pre-enrichment of socks with buffered peptone water was not reported in past studies, but rather they were only moistened with a saline solution (Buhr et al., 2007). This step in the current methodology was designed to better facilitate bacterial recovery through immediate enrichment and increased affinity of the sock for organic matter on the litter surface. An ability to sample the entire litter surface without entering the pen also represented a litter sampling methodology that could be successfully employed in commercial and research facilities alike. Further, the ELFA technology utilized combined with confirmation by classic plate culture methods was found to be time and labor efficient when compared to plate culture methods alone. The feeding of RFC significantly reduced litter Salmonella incidence in RFC-fed broilers, as Salmonella were only isolated in the litter and cecas of broilers fed a control diet throughout the course of the experiment (Table 3). The litter Salmonella status was apparently reflective of the broiler gastrointestinal tract status, as demonstrated by the follow-up sampling of ceca from birds in positive and negative pens (Table 4). This was further confirmed by serotyping these isolates, as it was confirmed that multiple litter and ceca Salmonella isolates were S. Senftenberg. These findings confirmed the suspicion that a specific Salmonella serovar was responsible for the housing environment contamination and also reiterated the effectiveness of the litter sampling methods employed. Although not as common as other serovars, Salmonella enterica serovar Senftenberg has been known to pose a challenge to poultry production. This serovar has demonstrated the capability to be transmitted vertically (Liljebjelke et al., 2005) and resist desiccation in poultry production environments where it was able to persist despite cleaning and sanitation (Pedersen et al., 2008). It was also attributed to outbreaks in food commodities that affected humans in the United States between 1998 and 2008 (Jackson et al., 2013) and is able to be harbored by live poultry as confirmed by this study. Further, S. Senftenberg has been reported to be resistant to some traditional sanitation methods due to its biofilm formation ability (Youn et al., 2017), and a selected desiccation-resistant strain of S. Senftenberg was able to evade eradication by thermal treatment of litter (Chen et al., 2015). Given that the hatching basket papers and egg shells taken from the incubator were negative for Salmonella, it was likely that residual environmental contamination was responsible for the presence of Salmonella in both litter and ceca. In the present study, RFC supplemented at 50 g/MT was able to reduce prevalence of this Salmonella serovar in bird ceca and litter contamination despite its known resistance mechanisms. These results were in agreement with the results previously obtained in a broiler breeder study (Walker et al., 2017), which indicated that the mechanisms by which RFC (supplemented at the same 50 g/MT dosage) prevented intestinal and subsequent environmental Salmonella contamination were evident in both broiler breeder parent and their broiler progeny. Thus, the use of RFC at this dosage was an effective Salmonella control approach in broiler breeder and broiler flocks. Environmental persistence of Salmonella has been described as a significant challenge faced by poultry producers when attempting to eradicate the human pathogen from their flocks (Turnbull and Snoeyenbos, 1973; Davies and Wray, 1994, 1996; Rose et al., 2000). This study demonstrated that S. Senftenberg, which has previously demonstrated resistance to eradication in poultry housing, naturally proliferated in the gastrointestinal tracts and housing environment of broilers but could be controlled by continuous inclusion of 50 g/MT of RFC in the broiler diets. While the intention of RFC inclusion in the present study was to control Salmonella proliferation among broilers, it was also noted that RFC did not diminish broiler live performance thus making it practical and applicable for an industry that has relied on animal growth efficiency. Since control of Salmonella during production of feed can be costly and inefficient, it was important that it could be controlled in broilers so as to prevent transmission to subsequent production tiers and ultimately could lead to contamination at the processing plant and in processed poultry products. Global poultry production has shifted away from antibiotic usage in feeds as a means to control opportunistic pathogens so it has become imperative that alternative products such as RFC be investigated. The ability of such alternatives to eliminate Salmonella present in poultry housing facilities was also of critical importance. When relying on environmentally present Salmonella, it would be difficult to state that the RFC treatment completely eliminated Salmonella without exhaustive testing beyond the scope of this study. However, the present data clearly demonstrated the efficacy of the RFC in achieving substantial control of a resistant Salmonella serovar in broilers and their housing environment in near commercial conditions during a rearing cycle. REFERENCES Brown G. D., Gordon S.. 2005. Immune recognition of fungal β-glucans. Cell. Microbiol . 7: 471– 479. Google Scholar CrossRef Search ADS PubMed Buhr R. J., Richardson L. J., Cason J. A., Cox N. A., Fairchild B. D.. 2007. 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Poultry Science – Oxford University Press
Published: Apr 1, 2018
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