Effect of synbiotic supplementation on layer production and cecal Salmonella load during a Salmonella challenge

Effect of synbiotic supplementation on layer production and cecal Salmonella load during a Salmonella challenge A. Luoma,∗ A. Markazi,∗ R. Shanmugasundaram,∗ G. R. Murugesan,† M. Mohnl,‡ and R. Selvaraj∗,1 ∗

Department of Animal Sciences, Ohio Agricultural Research and Development Center, Wooster, OH 44691; † BIOMIN America Inc., San Antonio, TX; and ‡ BIOMIN Holding GmbH, Getzersdorf, Austria (P < 0.05) BW more than those in the un-supplemented groups. Birds fed synbiotics had 0.7, 17.8, 21.7, 3, and 4.2% higher (P < 0.05) hen d egg production (HDEP) at 19, 20, 21, 22, and 23 wk of age, compared to the birds without supplementation, respectively. After administering the SE challenge, supplemented birds had 3, 6.7, 4.3, 12.5, and 14.4% higher (P < 0.05) HDEP at 24, 25, 26, 27, and 28 wk of age, compared to the birds not supplemented, respectively. Irrespective of the vaccination status, birds fed synbiotics and challenged with SE had a lower (P < 0.05) SE cecal load compared to the un-supplemented groups. At 22 d post Salmonella challenge, birds supplemented, vaccinated, and challenged had the highest bile IgA content. It can be concluded that supplementation of the synbiotic product could be beneficial to layer diets as a growth promoter, performance enhancer, and for protection against SE infection.

ABSTRACT This study analyzed the inhibitory efR fects of a synbiotic product (PoultryStar me) on production parameters, intestinal microflora profile, and immune parameters in laying hens with and without a Salmonella challenge. The synbiotic product contained 4 probiotic bacterial strains (Lactobacillus reuteri, Enterococcus faecium, Bifidobacterium animalis, and Pediococcus acidilactici) and a prebiotic fructooligosaccharide. Layers were supplemented with the synbiotic from d of hatch to 28 wk of age. At 16 wk of age, birds were either vaccinated with Salmonella enterica Enteritidis (SE) vaccine or left unvaccinated. At 24 wk of age, a portion of the birds was challenged with 1 × 109 CFU of SE or left unchallenged, resulting in a 3 (vaccinated, challenged, or both vaccinated and challenged) X 2 (control and synbiotics) factorial arrangement of treatments. At 18 and 20 wk of age, birds fed synbiotics in both vaccinated and unvaccinated groups had increased

Key words: synbiotic, Salmonella, probiotics, cecal microflora 2017 Poultry Science 0:1–9 http://dx.doi.org/10.3382/ps/pex251

INTRODUCTION

Enteritidis-positive birds can cause human foodborne infections (Lillehoj et al., 2005). Salmonella colonizes within the intestines of poultry and can be spread through feces or infected intestinal contents spilling and contaminating the meat (Humphrey and Lanning, 1988). With the emergence of antibiotic-resistant Salmonella, treatment is becoming more difficult, and there has become a need for control of Salmonella in poultry flocks. Because most vaccines do not completely clear Salmonella colonization in chickens, the commercial poultry production industry attempts to control Salmonella infections by supplementing vaccination programs with probiotics or prebiotics (Davies and Breslin, 2003; Patterson and Burkholder, 2003). Furthermore, probiotics have gained interest in the poultry industry as alternatives to antibiotics due to concerns with antibiotic resistance (Gustafson and Bowen, 1997). Manipulation of the gut microbiota by supplementation of prebiotics and probiotics have shown promising results in controlling bacterial infections in poultry (Mead, 2000). Probiotics stimulate the immune responses (Koenen et al., 2004), competitively exclude pathogenic microbes (Nava et al., 2005), stimulate

Salmonella bacteria can lead to salmonellosis infection in humans, with symptoms including diarrhea, fever, and vomiting. In the United States, approximately 40,000 cases of salmonellosis are reported each year and acute salmonellosis in humans cause about 400 deaths a year (Fabrega and Vila, 2013). Salmonella enterica Enteritidis (SE) is one of the most isolated Salmonella serovars in chickens, although over 200 serovars of Salmonella are able to colonize the gastrointestinal tract of poultry (Gast, 2007). Salmonella typically are present in poultry as subclinical infections and hence the bird provides a source of contamination for human infection (Van Immerseel et al., 2005). Contaminated meat and eggs from S.  C 2017 Poultry Science Association Inc. Received February 22, 2017. Accepted August 16, 2017. 1 Corresponding author: [email protected] Author disclosures: This research was partially supported by Biomin, Hatch grant, and USDA-NIFA grant (# 2015–07001) awarded to RKS.

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LUOMA ET AL.

digestive enzymes (Saarela et al., 2000), and produce antibacterial substances. Prebiotics, which are often supplemented in conjunction with probiotics, are nondigestible carbohydrates that stimulate the growth of beneficial bacteria within the intestines of the host (Gibson and Roberfroid, 1995). Supplementation of prebiotics has been shown to modulate the gut microbiota by increasing specific beneficial bacteria as well as mimicking attachment sites of pathogens to reduce pathogenic bacterial adherence to the intestinal wall (Ija and Tivey, 1998). Although Salmonella can cause severe infections in humans, poultry with Salmonella infections remain asymptomatic over the entire lifespan, but the mechanism is not well understood (Van Immerseel et al., 2005). Immediately after a Salmonella infection, the innate immunity is activated, and proliferation of immune cells such as macrophages, heterophils, granulocytes, and dendritic cells is increased (Van Immerseel et al., 2002). The adaptive immunity is activated, as observed by distinct changes in the Tcell populations (Sasai et al., 2000), as well as increases in IgG and IgA antibodies 3 to 4 d post infection (Desmidt et al., 1998). T-regulatory cells, can suppress immune responses through producing antiinflammatory cytokines, IL-10, to down-regulate the host immune response against the pathogen and facilitate pathogen survival. The lipopolysaccharide of SE stimulates IL-10 production, and the single nucleotide polymorphism at the IL-10 locus is associated with S. Enteritidis burden (Ghebremicael et al., 2008). R The synbiotic product (PoultryStar me) analyzed in this study contained 4 probiotic bacterial strains (Lactobacillus reuteri, Enterococcus faecium, Bifidobacterium animalis, and Pediococcus acidilactici) and a prebiotic fructooligosaccharide. This study evaluated the effects of the synbiotics on production parameters, intestinal microflora profile, and immune parameters in layer hens with and without a Salmonella challenge.

MATERIALS AND METHODS Birds One-day-old (n = 384) White Leghorn chicks (HyLine North America, Johnstown, OH) were provided water and feed ad libitum, housed in battery cages (pullet and layer), and raised using standard animal husbandry practices. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at The Ohio State University. The birds were housed in pullet cages for the first 12 wk, after which they were transferred to layer cages. At wk 18, the light h were increased to 16 h to stimulate egg production.

Treatments The birds were fed a layer starter diet between 0 and 8 wk of age, a layer grower diet between 8 and 18 wk of age, and a layer finisher diet between 19 and 28 weeks. A total of 384 one-day-old layer chicks were randomly distributed to either a synbiotic supplemented group or a control group. Each treatment was replicated in 24 feeders of 8 chicks per replication (n = 24). The basal diet was a corn-soybean meal based diet and suppleR me, BIOMIN mented with the synbiotic (PoultryStar America Inc., San Antonio, TX). The synbiotic supplement was added to the feed at a rate of 1 g/kg for the first 3 wk, then reduced to 0.5 g/kg until the end of wk 23, and increased back to 1 g/kg inclusion until the end of the project at wk 28. At 14 wk of age, all the birds were weighed and birds in 16 out of 24 replicates per group for both groups (32 replicates in total) were vaccinated with a SE vaccine, resulting in a 2 (control and synbiotic supplementation) X 2 (vaccinated and non-vaccinated) factorial arrangement of treatments. At this point, the number of replications was 16 for each in the vaccinated group (n = 16) and 8 for each in the unvaccinated group (n = 8). Each replication had 8 pullets. Birds were vaccinated R subcutaneously with 0.3 cc of SE vaccine (Poulvac SE, Zoetis, Florham Park, NJ) at 14 wk of age, with a booster dose at 17 wk of age. Body weights were measured at 17, 18, and 20 wk of age. Eggs were collected and recorded daily from d of first egg until the end of the study. At 24 wk of age, half of the birds in the vaccinated groups (8 replicates per group) and all the birds that were not vaccinated (8 replicates per group) were challenged with 250 μl of 1 × 109 CFU SE. This resulted in a 3 (vaccinated, challenged, vaccinated+challenged) X 2 (control and synbiotic) factorial arrangement. The number of replications was 8 for all the treatment groups post Salmonella infection (n = 8). Each replication had 8 birds. A primary isolate of wild-type SE was used for the challenge. Salmonella Enteritidis was grown in tryptic soy broth for 12 hours. The cells were washed 3 times with 1X PBS by centrifugation (5,000 RPM), and the concentration of bacteria in the media was determined spectrophotometrically. The concentration of bacteria was further confirmed by serial dilution plating of the inoculum on Xylose Lysine Tergitol-4 (XLT) agar plates. On d -5, 3, 8, 10, 17, 22, 24, and 30 post challenge, one bird was randomly chosen from 6 out of the 8 replications for sample collection.

RNA Isolation, DNA Isolation, and Real-time PCR Single-cell suspensions of cecal tonsils were collected from 6 replication pens (n = 6) 5 d pre-challenge and 3, 8, 10, 17, 22, 24, and 30 d (n = 6) post challenge.

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SYNBIOTICS IMPACT ON LAYING HENS

One cecal tonsil from each bird was used, and a quarter of the total cells was suspended in 750 μl of Tri Reagent (Molecular Research Center, Cincinnati, OH). Chloroform (200 μl) was added to the solution, incubated for 5 min, and centrifuged at 11,000 X g for 15 minutes. After centrifugation, the top aqueous layer containing RNA was removed, added to isopropyl alcohol (500 μl), and incubated for 10 min to precipitate the RNA by further centrifugation at 13,000 X g. The supernatant was removed, and the pellet was washed in 75% ethanol. After centrifugation and drying, the pellets were dissolved in 20 to 200 μl of TE buffer, based on the size. The RNA concentration and quality was determined using the NanoDrop (Thermo Scientific, Waltham, MA). The mRNA was reverse transcribed into complementary DNA. Gene expressions of IL-10 and TNFα (LITAF) were analyzed by real-time PCR (iCycler, BioRad, Hercules, CA) using SyBr green (Qiagen, Valencia, CA). Gene expressions were normalized to the housekeeping gene β -actin. Fold change from the reference was calculated by the 2-ΔΔCT equation. The CT was determined by the iQ5 software (Biorad) when the fluorescence rose exponentially 2-fold times above background. The reference group was the group that had the lowest expression of the gene of interest. The total DNA was isolated from the cecal scrapings and cecal content as previously described (AmitRomach et al., 2004) with the modification of adding 600 μl lysis buffer (1 M Tris-HCl, 500 mM EDTA, 5 M NaCl, 20% SDS) and 32 μl Proteinase K, incubating for 5 min at 80◦ C, and extracting via isopropanol and 70% ethanol. Cecal microbiota (L. reutri, P. acidilacti, B. animalis, E. faecium, and S. enterica) were analyzed by real-time PCR as described earlier (Selvaraj et al., 2010). The threshold cycle (Ct) values were determined by iQ5 software (Bio-Rad, Hercules, CA) when the fluorescence rose exponentially 2-fold above background. The Ct values were normalized to the Ct values of the universal primer 16S. To evaluate the relative proportion of each examined bacterium, all Ct values were expressed relative to the Ct value of the universal primers, and proportions of each bacterial group are presented where the total of the examined bacteria was set at 100%, as described earlier (Amit-Romach et al., 2004).

Determination of Salmonella-specific IgA Concentrations An enzyme-linked immunosorbent assay (ELISA) was performed to determine Salmonella-specific IgA concentrations in the chicken plasma and bile. The concentrations of different regents were established using checkerboard titrations with dilutions of plasma, bile, antigens, and conjugates. Salmonella was grown as previously stated and lysed by glass beads sized 425 to 600 μm (Sigma, St. Louis, MO) in a TissueLyser LT (Qiagen Hilder, Germany) for 5 min at 50 1/s, 2 times to be used as an antigen to coat the wells of the

microtiter plates. Flat-bottomed 96-well microtitration plates were coated with 67.2 μl of the antigen (1.435 ug/ml) diluted in 0.1 M carbonate buffer, and incubated overnight at 4◦ C. The plates were washed 3 times with PBS-Tween 20 (50 mM Tris, pH 7.4, containing 150 mM sodium chloride and 0.05% Tween 20), and non-specific immune sites were blocked by PBS-Tween 20 in 2.5% nonfat dry milk incubated for 1 h at 37◦ C. The plasma was diluted to 1:10, and the bile was diluted to 1:200 in PBS-Tween 20–2.5% nonfat dry milk and added to the plates in duplicates (100 μl/well) and incubated for 1 h at room temperature. After washing, Horseradish peroxidase-labeled anti-chicken IgA (Novus Biologicals, Littleton, Co) diluted to 1:100,000 in PBS-Tween 20–2.5% nonfat dry milk was added to each well (100 μl/well) and incubated for 1 h at room temperature. After washing, peroxidase activity was revealed by adding 100 μl of Tetramethylbenzidine (TMB)-ELISA solution (3 M sodium acetate, TMB, hydrogen peroxide). The reaction was blocked by adding 100 μl of 2 M sulfuric acid, and the optical density (OD) value was measured at 490 nm in a microplate reader.

Statistical Analysis A 2-way ANOVA was used to examine the interaction effects of vaccination X synbiotic on dependent variables collected between 14 and 24 wk of age. A 2-way ANOVA was used to examine the interaction effects of vaccination/challenge X synbiotic on dependent variables collected between wk 24 and 28. When the interaction effects were not significant (P > 0.05), the main effects of the synbiotic were analyzed, if there were no main effects of vaccine/challenge. When interactions or main effects were significant (P < 0.05), differences between means were analyzed by Tukey’s least square means comparison.

RESULTS Effect of Synbiotic Supplementation on Body Weight Between 14 and 28 wk of Age There were significant interaction effects of vaccine and synbiotic supplementation on bird BW at 17 (P = 0.02), 18 (P = 0.03), and 20 (P = 0.01) wk of age (Figure 1). At 17 wk of age, birds supplemented with the synbiotics and not vaccinated were significantly heavier than those unvaccinated and un-supplemented. At 18 and 20 wk of age, birds fed the synbiotics in both the vaccinated and unvaccinated groups had increased BW more than those not supplemented. After 20 wk of age, BW did not differ significantly among the different treatment groups (data not shown).

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Body Weight (kg)

LUOMA ET AL.

Control

Control + Vaccine

1.6

Synbiotic

Synbiotic + Vaccine

1.4

A

1.2

C BC

A AB

BC C

B

A B

C C

1 0.8 0.6 0.4 0.2 0 Wk 17

Wk 18

Wk 20

Figure 1. Effect of synbiotic supplementation on bird BW. Birds were fed either basal diet (Control) or supplemented with the synbiotic product and vaccinated or not vaccinated with SE vaccine in a 2 (control and vaccinated) X 2 (control and synbiotic) factorial arrangement. The synbiotic product was fed from d of hatch, and birds were vaccinated with SE vaccine at 14 wk of age. At 17, 18, and 20 wk of age, average BW was measured. Bars (+SE) with no common superscript differ (P < 0.05). P-values: Week 17 Synbiotic X Vaccine P = 0.02, Synbiotic P < 0.01, Vaccine, P = 0.15. Week 18 Synbiotic X Vaccine P = 0.03, Synbiotic P < 0.01, Vaccine, P < 0.01. Week 20 Synbiotic X Vaccine P = 0.01, Synbiotic P < 0.01, Vaccine, P = 0.01.

80

Control

Synbiotic

70

A

B

Week HDEP (%)

60 B

50 40 A

30 B

20 10 0

A B

A

Wk 19

Wk 20

Wk 21

Wk 22

Wk 23

Figure 2. Effect of synbiotic supplementation on weekly hen d egg production (HDEP) pre-Salmonella challenge. Birds were fed either basal diet (Control) or supplemented with the synbiotic product from d of hatch through 23 wk of age and were vaccinated with SE vaccine at 14 wk of age in a 2 (control and vaccinated) X 2 (control and synbiotic) factorial design. Egg production was measured daily, and at 19, 20, 21, 22, and 23 wk of age, weekly HDEP was analyzed. Because there were no significant interaction or main effects of vaccine on egg production, the main effects of synbiotic supplementation were analyzed. Bars (+SE) with no common superscript differ (P < 0.05). P-values: Week 19 Synbiotic X Vaccine P = 0.68, Synbiotic P = 0.01, Vaccine, P = 0.68. Week 20 Synbiotic X Vaccine P = 0.42, Synbiotic P < 0.01, Vaccine, P = 0.09. Week 21 Synbiotic X Vaccine P = 0.29, Synbiotic P < 0.01, Vaccine, P = 0.05. Week 22 Synbiotic X Vaccine P = 0.69, Synbiotic P = 0.11, Vaccine, P = 0.03. Week 23 Synbiotic X Vaccine P = 0.59, Synbiotic P = 0.01, Vaccine, P = 0.2.

Effect of Synbiotic Supplementation on Weekly Hen d Egg Production Pre-Salmonella Challenge There were no significant interaction effects between vaccine and synbiotic supplementation on egg production at 19 (P = 0.68), 20 (P = 0.41), 21 (P = 0.29), 22 (P = 0.69), or 23 (P = 0.59) wk of age. There were no significant main effects of vaccination on egg production at 19 (P = 0.68), 20 (P = 0.09), 21 (P = 0.05), or 23 (P = 0.24) wk of age. At 22 wk of age, there was a significant main effect (P = 0.03) of vaccination on egg production. Because there were no significant interaction effects and the main effects of vaccination were not significant on egg production, the main effects of synbi-

otic supplementation was analyzed. Birds supplemented with the synbiotics produced more eggs, calculated as weekly hen d egg production (HDEP), compared to the un-supplemented birds between 19 and 23 wk of age. Birds fed the synbiotic had 0.7, 17.8, 21.7, 3, and 4.2% greater HDEP at 19, 20, 21, 22, 23 wk of age, compared to the birds not supplemented, respectively (Figure 2).

Effect of Synbiotic Supplementation on Weekly HDEP Post Salmonella Challenge There were no significant interaction effects between vaccination/challenge and synbiotic supplementation on egg production at 24 (P = 0.34), 25 (P = 0.34),

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Week HDEP (%)

SYNBIOTICS IMPACT ON LAYING HENS

100 90 80 70 60 50 40 30 20 10 0

Control

Synbiotic

B A

B

A

B A

Wk 24

Wk 25

Wk 26

WK 27

Wk 28

Figure 3. Effect of synbiotic supplementation on weekly hen d egg production (HDEP) post Salmonella challenge. Birds were fed either basal diet (Control) or supplemented with the synbiotic product from d of hatch through 28 wk, were either vaccinated or not vaccinated with SE vaccine at 14 wk of age, and challenged with either 0 or 1 × 109 CFU of SE at 24 wk of age in a 3 (vaccinated, challenged, vaccinated+challenged) X 2 (control and synbiotic) factorial design. Egg production was measured daily, and at 24, 25, 26, 27, and 28 wk of age, weekly HDEP was analyzed. Because there were no significant interaction or main effects of vaccine, the main effects of synbiotic supplementation were analyzed. Bars (+SE) with no common superscript differ (P < 0.05). P-values: Week 24 Synbiotic X Vaccine P = 0.34, Synbiotic P = 0.03, Vaccine, P = 0.48. Week 25 Synbiotic X Vaccine P = 0.34, Synbiotic P = 0.01, Vaccine, P = 0.95. Week 26 Synbiotic X Vaccine P = 0.13, Synbiotic P = 0.10, Vaccine, P = 35. Week 27 Synbiotic X Vaccine P = 0.63, Synbiotic P = 0.06, Vaccine, P = 0.46. Week 28 Synbiotic X Vaccine P = 0.86, Synbiotic P = 0.01, Vaccine, P = 0.86. Vaccine

Challenge

Vaccine+Challenge

Synbiotic+Vaccine

Synbiotic+Challenge

Synbiotic+Vaccine+Challenge

Relative Proportion (%)

0.12 A

0.1 0.08 0.06 0.04 0.02 0

B

B

B

B

A

B B

Day 8

B

B

B

B

Day 17

Figure 4. Effect of symbiotic supplementation on cecal P. acidilacti content load post Salmonella challenge. Birds were fed either basal diet (Control) or supplemented with the synbiotic product from d of hatch through 28 wk, were either vaccinated or not vaccinated with SE vaccine at 14 wk of age, and challenged with either 0 or 1 × 109 CFU of SE at 24 wk of age in a 3 (vaccinated, challenged, vaccinated+challenged) X 2 (control and synbiotic) factorial design. Cecal content samples were collected on d 8 and 17 post challenge, and the relative proportions of P acidilacti in the cecal content were measured by real-time PCR after normalizing to the total DNA content of the cecal content. The relative proportions of P acidilacti in the cecal content were measured by real-time PCR after normalizing to the total DNA content of the cecal content. The proportions of P. acidilicati are presented where the total of the examined bacteria was set at 100%. Bars (+SE) with no common superscript differ (P < 0.05). P-values: Day 8 Synbiotic X Treatment P = 0.01, Synbiotic P = 0.01, Treatment P = 0.01. Day 17 Synbiotic X Treatment P < 0.01 Synbiotic P = 0.01, Treatment P < 0.01.

26 (P = 0.13), 27 (P = 0.63), or 28 (P = 0.86) wk of age. There were no significant main effects of vaccination on egg production at 24 (P = 0.48), 25 (P = 0.95), 26 (P = 0.13), 27 (P = 0.46), or 28 (P = 0.86) wk of age. Because there were no significant interaction effects on egg production post Salmonella challenge and the main effects of vaccination were not significant, the main effects of synbiotic supplementation were analyzed. Birds supplemented with the synbiotics produced more eggs, calculated as weekly HDEP, compared to the group without the synbiotic supplementation between 24 and 28 wk of age. Birds fed the synbiotic had 3, 6.7, 4.3, 12.5, and 14.4% greater HDEP

at 24, 25, 26, 27, and 28 wk of age, respectively, compared to the birds not fed the synbiotic (Figure 3).

Effect of Synbiotic Supplementation on Supplemented Probiotic Strains Content in the Ceca Post Salmonella Challenge There were significant interaction effects between vaccination/challenge and synbiotic supplementation on P. acidilacti content at 8 (P = 0.01) and 17 (P < 0.01) d post Salmonella challenge (Figure 4). At 8 d post Salmonella challenge, birds fed the synbiotic

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LUOMA ET AL. Control

Relative Proportion (%)

0.004

Synbiotic

A

0.0035 0.003 0.0025 0.002 0.0015 0.001 0.0005

A

B

B

0 Day 10

Day 22

Figure 5. Effect of synbiotic supplementation on P. acidilacti cecal content load post challenge. Birds were fed either basal diet (Control) or supplemented with the synbiotic product from d of hatch through 28 wk, were either vaccinated or not vaccinated with SE vaccine at 14 wk of age, and challenged with either 0 or 1 × 109 CFU of SE at 24 wk of age in a 3 (vaccinated, challenged, vaccinated+challenged) X 2 (control and synbiotic) factorial design. Cecal content samples were collected on d 10 and 22 post challenge and the relative proportions of P acidilacti in the cecal content were measured by real-time PCR after normalizing to the total DNA content of the cecal content. The proportions of P. acidilicati are presented where the total of the examined bacteria was set at 100%. Bars (+SE) with no common superscript differ (P < 0.05). P-values: Day 10 Synbiotic X Treatment P = 0.42, Synbiotic P < 0.01, Treatment P = 0.42. Day 22 Synbiotic X Treatment P = 0.15, Synbiotic P < 0.01, Treatment P = 0.15.

and challenged with SE had a higher percentage of P. acidilacti in the ceca than all other treatment groups. At 17 d post Salmonella challenge, birds fed the synbiotic, vaccinated, and challenged with SE had a higher percentage of P. acidilacti in the ceca than all other treatment groups. There were no significant interaction effects between vaccination/challenge and synbiotic supplementation on cecal P. acidilacti content at 10 (P = 0.42) or 22 (P = 0.15) d post Salmonella challenge. There were no significant main effects of vaccination/challenge on cecal P. acidilacti content at 10 (P = 0.42) or 22 (P = 0.15) d post Salmonella challenge. Because there

(a)

Effect of Synbiotic Supplementation on SE Cecal Content Load Post Salmonella Challenge Salmonella Enteritidis was undetectable in the cecal content at d -5 post challenge. At 3 d post Salmonella challenge, SE was detected in the cecal content of all treatment groups, except the groups with no SE challenge (data not shown). At 10 d post Salmonella challenge, SE was undetectable in the cecal content of all treatment groups (data not shown). At 8 d post Salmonella challenge, there were significant interaction effects between vaccination/challenge and synbiotic supplementation on SE cecal content (P = 0.03) (Figure 7). Irrespective of the vaccination status, birds

(b)

Vaccine

Challenge

Vaccine+Challenge

Synbiotic+Vaccine

Synbiotic+Challenge

Synbiotic+Vaccine+Challenge

1.8

0.008

1.6

0.007

1.4

Relative Proportion (%)

Relative Proportion (%)

0.009

were no significant interaction effects and the main effects of vaccination/challenge were not significant, the main effects of synbiotic supplementation was analyzed. Birds supplemented with the synbiotic had increased cecal P. acidilacti content, compared to the group not supplemented. Birds fed the synbiotic had 0.003 and 0.0003% greater P. acidilacti content at 10 and 22 d post Salmonella challenge, respectively, compared to the birds not supplemented (Figure 5). There were significant interaction effects at 8 (P = 0.03) and 24 (P = 0.03) d post Salmonella challenge (Figure 6a and b) on L. reuteri in the cecal content. There were no significant main effects of vaccination/SE or symbiotic supplementation on L. reuteri load in the cecal content. At both 8 and 24 d post challenge, the group that was supplemented, vaccinated, and challenged had the highest L. reuteri amount in the cecal content than all other treatment groups. E. faecium was undetectable in the ceca in any of the treatment groups.

0.006 0.005 0.004 0.003

A

0.002 0.001

B

AB AB

B

Vaccine Vaccine+Challenge Synbiotic+Challenge

Challenge Synbiotic+Vaccine Synbiotic+Vaccine+Challenge A

1.2 1 0.8 0.6 0.4

B

B B

B

B

0.2

B

0

0 Day 8

Day 10

Day 24

Figure 6. Effect of synbiotic supplementation on L. reuteri cecal content load post challenge. Birds were fed either basal diet (Control) or supplemented with the synbiotic product from d of hatch through 28 wk of age, were either vaccinated or not vaccinated with SE vaccine at 14 wk of age, and challenged with either 0 or 1 × 109 CFU of SE at 24 wk of age in a 3 (vaccinated, challenged, vaccinated+challenged) X 2 (control and synbiotic) factorial design. Cecal content samples were collected on d 8 and 10 (a) and 24 (b) post challenge and the relative proportions of L. reuteri in the cecal content were measured by real-time PCR after normalizing to the total DNA content of the cecal content. The proportions of L. reuteri are presented where the total of the examined bacteria was set at 100%. Bars (+SE) with no common superscript differ (P < 0.05). P-values: Day 8: Synbiotic X Treatment P = 0.02, Synbiotic P = 0.41, Treatment P = 0.02. Day 10: Synbiotic X Treatment P = 0.67, Synbiotic P = 0.37, Treatment P = 0.28. Day 24: Synbiotic X Treatment P = 0.02, Synbiotic P = 0.02, Treatment P = 0.01.

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SYNBIOTICS IMPACT ON LAYING HENS Vaccine Vaccine+Challenge Synbiotic+Challenge

Challenge Synbiotic+Vaccine Synbiotic+Vaccine+Challenge

Relative Proportion (%)

0.25 A

0.2 0.15 0.1

AB

0.05

B B

B

B

0 Day 8 Figure 7. Effect of synbiotic supplementation on SE cecal content load post Salmonella challenge. Birds were fed either basal diet (Control) or supplemented with the synbiotic product from d of hatch through 28 wk, were either vaccinated or not vaccinated with SE vaccine at 14 wk of age, and challenged with either 0 or 1 × 109 CFU of SE at 24 wk of age in a 3 (vaccinated, challenged, vaccinated+challenged) X 2 (control and synbiotic) factorial design. Cecal content samples were collected on d 8 post challenge, and the relative proportions of SE in the cecal content were measured by real-time PCR after normalizing to the total DNA content of the cecal content. The proportion of SE is presented where the total of the examined bacteria was set at 100%. Bars (+SE) with no common superscript differ (P < 0.05). P-values: Day 8 Synbiotic X Treatment P = 0.03, Synbiotic P = 0.03, Treatment P = 0.04.

fed the synbiotic and challenged with SE had lower SE content compared to those not vaccinated and not supplemented. Among the SE-challenged birds, synbiotic supplementation and vaccination seemed to produce comparable SE loads.

Effect of Synbiotic Supplementation on Bile and Plasma Anti-Salmonella IgA Content Post Salmonella Challenge There were significant interaction effects between vaccinated/challenge and synbiotic supplementation on bile anti-Salmonella IgA content at 8 (P = 0.01) and 22 (P = 0.02) d post Salmonella challenge (Figure 8). At 8 d, birds challenged but not supplemented and unvaccinated had the highest IgA content. Among birds not challenged, synbiotic supplementation produced comparable bile IgA content as vaccination. At 22 d post Salmonella infection, birds synbiotic supplemented, vaccinated, and challenged had the highest bile IgA content. There were no significant interaction effects between vaccination/challenge and synbiotic supplementation on plasma anti-Salmonella IgA content at 8 (P = 0.11) or 22 (P = 0.38) d post Salmonella challenge (data not shown).

DISCUSSION In this study, synbiotic supplementation improved layer bird growth and egg production, as well as immune responses against an experimental SE infection, resulting in a lesser cecal Salmonella colonization. In agreement with previous studies (Bailey et al., 1991; Hinton et al., 1990; Tortuero, 1973), our study

showed an increase in BW of birds supplemented with probiotics and prebiotics. Synbiotic-supplemented birds had an increase in BW gain and an increase in egg production, and began laying eggs at an earlier age than the control birds. An increase in egg production has been found in similar studies involving layers supplemented with probiotics (Zhang et al., 2012). These effects are likely due to improved nutrient absorption due to the supplementation of the synbiotics. It has been shown (Dobrogosz et al., 1991; Chichlowski et al., 2007; Samanya and Yamauchi, 2002) that supplementation of prebiotics and probiotics can increase villi height and crypt depth in the intestine and therefore allow for greater surface area available for nutrient absorption. This study identified that supplementation with the synbiotic product increased relative percentage of P. acidilacti and L. reuteri in the ceca, showing that the supplemented probiotics strains survived the gut and colonized the ceca. Probiotics and prebiotics influence the intestinal microflora by increasing beneficial bacteria and decreasing pathogenic bacteria within the intestines due to competitive exclusion and production of antimicrobials (Lloyd et al., 1977; Barrow, 1991). These beneficial bacteria also could have contributed to the increase in total immunoglobulins and the observed increase in BW gain. Previous studies (Chateau et al., 1993; Oyarzabal and Conner, 1995; Mead, 2000) have concluded that the supplementation of prebiotics and probiotics in the diets of chickens can decrease pathogenic infections, including Salmonella. Our current study identified that although all the birds cleared the SE infection by 10 d post Salmonella challenge, synbioticsupplemented groups were able to clear the infection in fewer d than the un-supplemented groups. In addition,

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LUOMA ET AL.

Vaccine

Challenge

Vaccine+Challenge

Synbiotic+Vaccine

Synbiotic+Challenge

Synbiotic+Vaccine+Challenge

0.7

A

0.6

OD Value

0.5 AB

0.4

B

0.3 B

0.2 0.1

B

AB

AB AB AB

A

B B

0 Day 8

Day 22

Figure 8. Effect of synbiotic supplementation on bile anti-Salmonella IgA content post Salmonella challenge. Birds were fed either basal diet (Control) or supplemented with synbiotic product from d of hatch through 28 wk, were either vaccinated or not vaccinated with SE vaccine at 14 wk of age, and challenged with either 0 or 1 × 109 CFU of SE at 24 wk of age in a 3 (vaccinated, challenged, vaccinated+challenged) X 2 (control and synbiotic) factorial design. Bile samples were collected on d 8 and 22 post challenge and analyzed for Salmonella-specific IgA content through ELISA, and results are reported as average optical density (OD) values. Bars (+SE) with no common superscript differ (P < 0.05). P-values: Day 8 Synbiotic X Treatment P = 0.01, Synbiotic P = 0.84, Treatment P < 0.01. Day 22 Synbiotic X Treatment P = 0.02, Synbiotic P = 0.90, Treatment P = 0.07.

synbiotic-supplemented groups had lower overall SE colonization than the un-supplemented groups. This effect can be due to any of the previously proposed mechanisms of probiotics, including, but not limited to, competitive exclusion, stimulation of the immune response, and production of antimicrobial substances (Koenen et al., 2004; Saarela et al., 2000; Nava et al., 2005). It can be concluded that supplementation of the synbiotic product can be beneficial in decreasing SE infection of chickens. This study identified that unvaccinated and unsupplemented birds challenged with SE had the highest amount of IgA in the bile at 8 d post Salmonella infection. This is likely due to the continued presence of SE infection in these particular groups at 8 d post Salmonella infection, while the other groups had cleared the infection by that time point. Previous studies have found that IgA concentrations increase during Salmonella infection (Sheela et al., 2003). Interestingly, birds that were fed the synbiotic and challenged with SE, though having high amounts of IgA, had cleared the SE infection by 8 d post Salmonella infection. This suggests that the higher IgA in the synbiotic-supplemented group could have contributed to the SE clearance by limiting the replication and adhesion of the pathogen (Fukutome et al., 2001). It has been shown that the local humoral response plays an important role in the protection against Salmonella, since the pathogen typically invades mucosal epithelial cells following the initial intestinal infection (Tran et al., 2010). Other experimental Salmonella infections have shown increased IgA responses in intestinal secretions, peaking around 7 to 10 d post infection (Berthelot-Herault et al., 2003). The

relatively lower amounts of Salmonella-specific IgA in the plasma compared to the bile can be expected, because IgA is seen primarily in the bile and in mucosal secretions (Lebacq-Verheyden et al., 1974). We conclude from this study that supplementation of the synbiotic product used in this study to pullet and layer diets can improve growth and production performance, and protect against S. Enteritidis infection.

ACKNOWLEDGMENTS Animal husbandry help from K. Patterson (OSU), J. Sidle (OSU), J. Snell (OSU), and J. Welsh (OSU) are acknowledged.

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