Effect of Synbiotic Supplementation on Production Performances and Cecal Salmonella Load during a Salmonella Challenge

Abstract

This study analyzed the inhibitory effects of the synbiotic product “PoultryStar® me” on production parameters, intestinal microflora profile, and immune parameters in layer hens with and without a Salmonella challenge. The synbiotic product contained four probiotic bacterial strains and the prebiotic fructooligosaccharide. The probiotics were Lactobacillus reuteri, Enterococcus faecium, Bifidobacterium animalis, and Pediococcus acidilactici. PoultryStar® me was supplemented to layers from day of hatch to 28 weeks of age. Birds were then either vaccinated, challenged, or both vaccinated and challenged, resulting in a 3 X 2 (control of symbiotic) factorial design. At 24 weeks of age, birds that were to be challenged were gavaged with 1 x 109 CFU of Salmonella Enterica Enteritidis. At 18 and 20 wk of age, birds fed synbiotics in both vaccinated and unvaccinated group had higher (P < 0.05) body weight than that in the no synbiotic fed group. Birds fed synbiotics had 0.7%, 17.8%, 21.7%, 3%, and 4.2% higher (P < 0.05) hen day egg production (HDEP) at 19, 20, 21, and 23 wk of age, compared to the birds fed no synbiotics, respectively. At 24 wk of age, birds were challenged with 250 µl of 1 X 109 CFU S. enterica Enteritidis. Birds fed synbiotics 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 fed no synbiotics, respectively. Irrespective of the vaccination status, birds fed synbiotics and challenged with Salmonella had lower (P < 0.05) S. Enteritidis content compared to that in the no synbiotic supplemented and unvaccinated treatment group. At 22 d post-Salmonella challenge, birds in synbiotic supplemented, vaccinated and challenged with Salmonella had the highest bile IgA content. It can be concluded that supplementation of synbiotic product could be beneficial to layer diets as a growth promoter, performance enhancer, and for protection against S. enteritidis infection.

Introduction

Salmonella can lead to salmonellosis infection in humans, with symptoms including diarrhea, fever and vomiting. Approximately 40,000 cases of salmonellosis are reported each year in the United States, although the real number may be 30-fold greater. Acute salmonellosis in humans causes 400 deaths a year in the United States (Fabrega and Vila, 2013). Salmonella can be spread by feces or infected intestinal contents spilling and contaminating the meat (Humphrey et al., 1988). 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). Probiotics decrease pathogenic infection in both layers and broilers through competitive exclusion, production of antimicrobials, and modulation of the immune system. Prebiotics, which are often supplemented in conjunction to probiotics, are non-digestible carbohydrates that stimulate the growth of beneficial bacteria within the intestines of the host (Lee et al., 2016).

The synbiotic product (PoultryStar® me) analyzed in this study contained four probiotic bacterial strains (Lactobacillus reuteri, Enterococcus faecium, Bifidobacterium animalis, and Pediococcus acidilactici) and the prebiotic fructooligosaccharide. Our study analyzed the effects of the synbiotic product 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 (Hy-Line North America; Johnstown, OH) were provided ad libitum intake of water and feed, housed in battery cages (pullet and layer), and raised using standard animal husbandry practices. The feed formulations are provided in Figure 2. The experiment was 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 weeks after which they were transferred to layer cages. At week 18, the light hours were increased to 16 hours to bring them into production.

Treatments

The birds were fed a layer starter diet between 0-8 wk of age, a layer grower diet between 8-18 wk of age, and a layer finisher diet between 19 to end of the experiment. A total of 384 one-day-old layer chicks were randomly distributed to the synbiotic supplemented or control groups. Each treatment was replicated in 24 feeders of 8 chicks per replication (n=24). The basal diet was based on corn and soybean meal (Figure 2) and supplemented with the synbiotic (PoultryStar® me, Biomin, San Antonio, TX). The synbiotic supplement was added to the feed at a rate of 1 g/kg for the first 3 weeks, then reduced to 0.5 g/kg until the end of wk 23 of age, and increased back to 1 g/kg inclusion until the end of the project at wk 28 of age.

At 14 wk of age the birds were weighed. At 14 wk of age, 32 birds in both the synbiotic and control groups (64 birds in total) were vaccinated with a Salmonella vaccine resulting in a 2 (control and synbiotic supplementation) X 2 (vaccinated and non-vaccinated group) factorial arrangement of treatments. For pre-Salmonella infection, the number of replication was 16 for the vaccinated group (n = 16) and 8 for the unvaccinated groups (n = 8). Each replication had 8 pullets. Birds were vaccinated subcutaneously with 0.3 cc of Salmonella vaccine (Poulvac® SE, Zoetis, Florham Park, NJ) at 14 wk of age, with a booster dose at 17 wk of age. Body weight was measured at 17, 18, and 20 wk of age. Eggs were collected and recorded daily from day of first egg until the end of the project.

At 24 wk of age, half of the birds in the vaccinated groups and all the birds that were not vaccinated were challenged with 250 µl of 1 X 109 CFU Salmonella. This resulted in a 3 (vaccinated, challenged, vaccinated+ challenged) X 2 (control and synbiotic) factorial design. The number of replication was 8 for all the treatment groups post-Salmonella infection (n = 8). Each replication had eight birds.

A primary isolate of wild-type Salmonella enterica Enteritidis was used for the challenge. Salmonella was grown in tryptic soy broth for 12 h. The cells were washed three times with 1X PBS by centrifugation (5,000 RPM), and the concentration of the bacteria in the media was determined spectrophotometrically. The concentration of the bacteria was further confirmed by serial dilution plating of the inoculum on Xylose Lysine Tergitol-4 (XLT) agar plates.

Results

Effect of synbiotic supplementation on bird body weights between 14-28 wk of age

There were significant interaction effects of vaccine and synbiotic supplementation on bird body weight at 17 (P = 0.02), 18 (P = 0.03), and 20 (P = 0.01) wk of age (Figure 2). At 17 wk of age, birds supplemented with the synbiotic product and not vaccinated were significantly heavier than birds not supplemented and unvaccinated. At 18 and 20 wk of age, birds fed synbiotics in both vaccinated and unvaccinated group had higher body weights than that in the no synbiotic fed group. After 20 wk of age, body weight did not differ significantly among the different treatment groups (data not shown).

Effect of synbiotic supplementation on weekly hen day egg production (HDEP) 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), and 23 (P = 0.59) wk of age (Figure 2). There were no significant main effects of vaccine on egg production at wk 19 (P = 0.68), 20 (P = 0.09), 21 (P = 0.05), and 23 (P = 0.24) wk of age. At 22 wk of age, there was a significant main effect (P = 0.03) of vaccine on egg production. Because there were no significant interaction effects and the main effects of vaccine was not significant on egg production, the main effects of synbiotic supplementation was analyzed. Birds supplemented with synbiotic had higher number of eggs, as calculated as weekly HDEP, compared to the group with no synbiotic supplementation between 19-23 wk of age. Birds fed synbiotics 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 fed no synbiotics, respectively.

Effect of synbiotic supplementation on weekly hen day egg production (HDEP) post-Salmonella challenge

There were no significant interaction effects between vaccine/challenge and synbiotic supplementation on egg production at 24 (P = 0.34), 25 (P = 0.34), 26 (P = 0.13), 27 (P = 0.63), and 28 (P = 0.86) wk of age (Figure 3). There were no significant main effects of vaccine on egg production at 24 (P = 0.48), 25 (P = 0.95), 26 (P = 0.13), 27 (P = 0.46) and 28 (P = 0.86) wk of age. Because there were no significant interaction effects and the main effects of vaccine on egg production were not significant, the main effect of synbiotic supplementation was analyzed. Birds supplemented with synbiotic had higher number of eggs, as calculated as weekly HDEP, compared to the group with no synbiotic supplementation between 24-28 wk of age. Birds fed synbiotic had 3%, 6.7%, 4.3%, 12.5%, and 14.4% greater HDEP at 24, 25, 26, 27, and 28 wk of age, compared to the birds fed no synbiotic, respectively.

Effect of synbiotic supplementation on supplemented probiotic strains content in the ceca post-Salmonella challenge

There were significant interaction effects between vaccine/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 synbiotic and challenged with Salmonella had higher percentage of P. acidilacti in the ceca than all other treatment groups. At 17 d post-Salmonella challenge, birds fed synbiotic, vaccinated and challenged with Salmonella had higher percentage of P. acidilacti in the ceca than all other treatment groups.

There were no significant interaction effects between vaccine/challenge and synbiotic supplementation on cecal P. acidilacti content at 10 (P = 0.42) and 22 (P = 0.15) d post-Salmonella challenge (Figure 5). There were no significant main effects of vaccine/challenge on cecal P. acidilacti content at 10 (P = 0.42) and 22 (P = 0.15) d post-Salmonella challenge. Because there were no significant interaction effects and the main effect of vaccine/challenge was not significant, the main effect of synbiotic supplementation was analyzed. Birds supplemented with synbiotic had higher number of cecal P. acidilacti content, compared to the group with no synbiotic supplementation. Birds fed synbiotic had 0.003% and 0.0003% greater P. acidilacti content at 10 and 22 d post-Salmonella challenge, compared to the birds fed no synbiotics, respectively.

There were significant interaction effects at 8 (P = 0.03) and 24 (P = 0.03) d post-Salmonella challenge (Figure 6a, 6b) on L. reuteri in the cecal content. There were no significant main effects of vaccine/Salmonella or synbiotic 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 amount of cecal content L. reuteri than all other treatment groups.

Enterococcus faecium was undetectable in the ceca in any of the treatment groups.

Effect of synbiotic supplementation on S. Enteritidis cecal content load post-Salmonella challenge

Salmonella was undetectable in the cecal content at day -5 post challenge. At 3 d post-Salmonella challenge, Salmonella was detected in the cecal content of all treatment groups, except the groups with no Salmonella challenge (data not shown). At 10 d post-Salmonella challenge, Salmonella was undetectable in the cecal content of all treatment groups (data not shown). At 8d post-Salmonella challenge, there were significant interaction effects between vaccine/challenge and synbiotic supplementation on S. Enteritidis cecal content (P = 0.03) (Figure 7). Irrespective of the vaccination status, birds fed synbiotics and challenged with Salmonella had lower S. Enteritidis content compared to that in the no synbiotic supplemented and unvaccinated treatment group. Among the Salmonella challenged birds, birds in the synbiotic supplemented and unvaccinated treatment group had comparable S. Enteritidis content to the vaccinated group with no synbiotics.

Effect of synbiotic supplementation on bile and plasma anti-Salmonella IgA content post-Salmonella challenge

There were significant interaction effects between vaccine/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 in the unvaccinated and synbiotic un-supplemented group and challenged with Salmonella had the highest IgA content. Among birds with no Salmonella challenge, birds in synbiotic supplemented and unvaccinated treatment group had comparable bile IgA content to that in the vaccinated and synbiotic unsupplemented group. At 22 d post-Salmonella infection, birds in synbiotic supplemented, vaccinated and challenged with Salmonella had the highest bile IgA content.

There were no significant interaction effects between vaccine/challenge and synbiotic supplementation on plasma anti-Salmonella IgA content at 8 (P = 0.11) and 22 (P = 0.38) d post-Salmonella challenge (Figure 8).

Discussion

In agreement with previous studies (Bailey et al., 1991, Hinton et al., 1990, Tortuero 1973), our study showed an increase in performance of birds supplemented with probiotics and prebiotics. Synbiotic supplemented birds had an increase in body weight gain, increase in egg production, and began laying eggs at an earlier age than the control. An increase in egg production has been found in similar studies involving layers supplemented with probiotics (Zhang et al., 2012)

Our study found that supplementation with the synbiotic product increased relative percentage of P. acidilacti and L. reuteri in 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 such as lactic acid (Barrow 1991, Lloyd et al., 1977).

Previous studies (Chateau et al., 1993, Mead 2000, Oyarzabal and Conner, 1995) 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 Salmonella infection by 10 d post-Salmonella challenge, synbiotic supplemented groups were able to clear the infection in fewer days than the unsupplemented groups. In addition, synbiotic supplemented groups had lower overall Salmonella colonization than the unsupplemented groups. It can be concluded that supplementation of the synbiotic product can be beneficial in decreasing Salmonella infection of chickens.

This study identified that unvaccinated birds and synbiotic unsupplemented birds challenged with Salmonella had the highest amount of IgA in the bile at 8 d post-Salmonella infection. This is likely due to the continued presence of Salmonella infection in this particular group at 8 d post-Salmonella infection while the other groups had cleared the infection by that time point. Previous studies have found that IgA amounts increase during Salmonella infection (Sheela et al., 2003). Interestingly, birds that were fed synbiotics and challenged with Salmonella, though had high amounts of IgA, had cleared the Salmonella infection by 8 d post-Salmonella infection. This suggests that the higher IgA in synbiotic supplemented group could have contributed to the Salmonella clearance. The relatively lower amounts of Salmonella-specific IgA in the plasma compared to in 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 synbiotic product to layer diets can improve growth, improve production performance, and protect against S. Enteritidis infection.

Acknowledgements

Animal husbandry help from K. Patterson, J. Sidle, J. Snell, and J. Welsh, technical help from R. Shanmugasundaram, and product help from G. Raj Murugesan are acknowledged.

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