Salmonella Enteritidis is responsible for a significant proportion of foodborne salmonellosis in the United States and continues to be attributable to table eggs despite increased federal oversight. Technologies, including feed additives, continue to be evaluated for preharvest application and their potential food safety benefits. Diamond V Original XPC, a Saccharomyces cerevisiae fermentation–based postbiotic (SCFP), was evaluated for its effectiveness in reducing Salmonella Enteritidis (SE) colonization in young layer pullets. A total of 40 day-old Hy-Line W-36 layer pullets were equally divided and randomly assigned to one of two dietary treatments, with SCFP or without SCFP (PCON), and orally gavaged on day 28 with SE at 106 CFU/mL. Another 20 day-old pullets were fed the same control feed without SCFP and blank inoculated on day 28 with 1 mL of sterile phosphate-buffered saline to serve as a negative control. Qualitative and quantitative analyses of cecal contents for Salmonella were performed for all birds on day 32. The prevalence of SE in the ceca of all directly challenged birds was 100%; however, the SE concentration in birds fed SCFP diet (3.35 log CFU/g) was significantly lower (P < 0.0001) than that of the PCON birds not fed SCFP (4.49 log CFU/g). The proportion of birds with enumerable SE concentrations was lower in SCFP-fed pullets (57.9%) than in the PCON pullets (95.0%). These data suggest that inclusion of SCFP in the diet may aid in the reduction of SE within the ceca of commercial laying hens and could serve as an additional preharvest food safety hurdle.
SCFP reduced Salmonella Enteritidis concentrations in the ceca of layer pullets.
SCFP reduced the proportion of ceca with enumerable Salmonella Enteritidis concentrations.
SCFP may be efficacious as a preharvest food safety hurdle in layer chickens.
Foodborne illnesses in the United States are caused by 31 known pathogens with an estimated 9.4 million cases annually (43). Nontyphoidal Salmonella causes an estimated 1.2 million illnesses, 23,000 hospitalizations, and 450 deaths (43). A proportion of these illnesses are associated with recognized foodborne illness outbreaks with known etiology and attribution to specific food groups. According to the Foodborne Disease Outbreak Surveillance System, 5,760 outbreaks occurred between reporting years 2009 and 2015, and Salmonella was the single confirmed pathogen in 896 outbreaks and 23,662 associated illnesses, of which 2,422 were directly attributed to eggs (9). The Interagency Food Safety Analytics Collaboration (26) reported that 811 outbreaks between 1998 and 2017 were caused by or suspected to be caused by Salmonella; >75% of illnesses were attributable to seven food categories, and chicken products (14.0%) and eggs (7.9%) constituted over 20% of outbreaks attributable to Salmonella.
The majority of clinical cases of salmonellosis transmitted by food are associated with specific Salmonella serovars. Luvsansharav et al. (33) reported on a novel method for determining the strength of serotype association with foodborne transmission called a foodborne relatedness measure based on U.S. public health data. These authors found that Salmonella serotypes Saintpaul, Heidelberg, and Berta had the highest foodborne relatedness measures followed by the next three most prevalent serotypes: Enteritidis, Typhimurium, and Newport. Salmonella enterica serovar Enteritidis has long been a recognized agent of foodborne illness primarily associated with consumption of table eggs, which led the U.S. Food and Drug Administration (46) to issue a regulatory directive, the Egg Safety Rule, to be implemented in 2009. The rule requires producers to implement preventive measures during egg production in poultry houses, specifies environmental monitoring and testing procedures, and requires cold chain controls for egg handling and transport (6, 23, 36, 46). To date, the egg industry has largely managed Salmonella risk in live production via farm management practices, vaccination strategies, and egg sanitization technologies (3, 16). Although these interventions have been effective in combination to some degree, the egg industry continues to seek additional food safety hurdles and technologies that can be implemented at the farm level, particularly as production practices evolve to meet consumer preferences (34, 49).
Feed additive technologies are often described as preharvest food safety interventions or are evaluated for such functionality. Broad diversity exists among these technologies and their modes of action for improving health, performance, immunity, or pathogen control in the gastrointestinal tracts of production animals (1, 25, 28, 35, 45). The majority of technologies fit uniquely into one of four generalized categories based largely on composition and functionality (although these definitions are still evolving): prebiotics, probiotics, synbiotics, or postbiotics (2, 21, 24). Postbiotics, a newer classification, are specifically the bioactive compounds resulting from fermentation by food-grade microorganisms (48).
Original XPC (SCFP; Diamond V, Cedar Rapids, IA) is a postbiotic product consisting of functional metabolites produced through a proprietary Saccharomyces cerevisiae fermentation process. In commercial poultry, this product has been reported to improve a variety of gut health, immunity, and production performance measures by lowering corticosterone, heterophil:lymphocyte ratios, and physical asymmetry during stress events; reducing intestinal lesions and improving immune function during Eimeria maxima and Eimeria tenella infections; improving feed conversion, growth, meat yield, and egg production; and possibly reducing colonization by foodborne pathogens such as Salmonella (11, 13, 14, 30, 32, 37, 39–41). Researchers using in vitro gut fermentation models with poultry donor microflora have also reported on the ability of SCFP and the microflora to synergistically influence Salmonella Typhimurium and Campylobacter jejuni levels (10, 38, 42).
The purpose of this study was to evaluate the effectiveness of SCFP in the feed of young layer pullets for reducing Salmonella Enteritidis colonization and concentration in pullet ceca.
MATERIALS AND METHODS
Animal husbandry and experimental design
Sixty-six day-old nonvaccinated Hy-Line W-36 female chicks (Hy-Line Hatchery, Spencer, IA) were randomly selected for the study. Twenty-two birds were allocated by live weight to each of three treatment rooms for group housing on fresh pine litter shavings (Building 29, College of Veterinary Medicine, Iowa State University, Ames), and animal care was provided throughout the study by the Laboratory Animal Resources staff in accordance with Institutional Animal Care and Use Committee approval (ISU IACUC 3-12-7327-G). Birds in each room were fed one of three dietary treatments until completion of the study on day 32 with ad libitum access to feed and water (Table 1). The negative control (NCON) and positive control (PCON) treatment groups were fed a standard commercial starter and grower layer pullet mash diet (Table 2) that contained no additional feed additives. The SCFP treatment group was fed the same commercial starter and grower mash diet, but it was supplemented with SCFP at 1.5 kg per metric ton (MT) in the starter diet from day 0 to day 21 and then reduced to 1.0 kg/MT in the grower diet from day 22 to day 32. All diets used in this study were supplied by a local integrator and screened for Salmonella before use. Photoperiods were 22 h light:2 h dark from day 1 to day 7, 21 h light:3 h dark from day 8 to day 14, 20 h light:4 h dark from day 15 to day 21, 19 h light:5 h dark from day 22 to day 28, and 18 h light:6 h dark from day 29 to day 32. On day 28, 20 birds per treatment were transferred to a biosafety level 2 environmentally controlled facility, placed into raised group-housed treatment pens (4 by 4 ft [1.2 by 1.2 m]; Livestock Infectious Disease Isolation Facility, College of Veterinary Medicine, Iowa State University), and orally challenged with Salmonella Enteritidis (SE), and ceca were harvested on day 32 for Salmonella analysis.
SE challenge preparation and administration
SE phage type 13a (Dr. Richard K. Gast et al., U.S. Department of Agriculture, Agricultural Research Service, Athens, GA) (20) was prepared according to Guard et al. (22). A frozen stock culture of SE was streak plated for isolation onto brilliant green agar and incubated for 16 h at 37 ± 1°C. A single colony was selected and transferred to 10 mL of brain heart infusion broth and incubated for 24 h at 37 ± 1°C, the mixture was centrifuged at ca. 5,590 × g for 10 min to pellet the cells, the cells were then resuspended in 10 mL of phosphate-buffered saline (PBS), and the cell population was estimated by measuring the optical density at 600 nm with a spectrophotometer (SmartSpec Plus, Bio-Rad, Hercules, CA). The primary culture was then diluted with sterile PBS to achieve challenge concentrations of ca. 1.0 × 106 CFU/mL, which were confirmed by spiral plating (Advanced Instruments, Norwood, MA) of dilutions on brilliant green and xylose lysine Tergitol 4 (XLT4) agar plates. On day 28, birds in the PCON and SCFP treatment groups were challenged by crop gavage with 1 mL of the 1.0 × 106 CFU/mL SE suspension, and birds in the NCON group were blank inoculated via crop gavage with 1 mL of sterile PBS.
On day 32, birds were euthanized with CO2 in accordance with IACUC approved procedures. Each bird was then placed onto its back, and the body cavity was aseptically opened with alcohol-flamed scissors. Both ceca were located and excised with a separate pair of sterile scissors, and cecal contents were combined into a single tube. All samples were maintained on ice and transported to the Veterinary Diagnostic Laboratory at the Iowa State College of Veterinary Medicine for microbiological analyses.
Starter and grower diets were screened for S. enterica before use in the experiment. Litter in the treatment pens was assessed by dabbing throughout the pen with a sterile gauze swab prehydrated with sterile skim milk on collection days 7, 14, 21, and 28. Samples were evaluated for SE with an immunoassay (RapidChek SELECT, Romer Labs, Newark, DE) according to the manufacturer's directions. Litter swabs were prepared in 100 mL of prewarmed 42°C proprietary primary medium, stomached, and incubated for 16 to 22 h at 42 ± 2°C, and 0.2 mL of the culture was transferred to tubes containing 2 mL of secondary medium and incubated for 16 to 22 h at 42 ± 2°C. After the second incubation step, test strips were inserted into the tubes and allowed to develop for 10 min and then interpreted.
Diets and cecal contents were qualitatively screened for SE according to the ISO 6579-1 qualitative detection method (27). Samples were prepared (1:10) in buffered peptone water (BPW; Difco, BD, Franklin Lakes, NJ) and incubated at 37 ± 1°C for 18 ± 2 h. These enriched samples were then selectively enriched by transferring 0.1 mL of BPW to 10.0 mL of modified semi-solid Rappaport-Vassiliadis agar, which was then incubated at 41.5 ± 1°C for 24 ± 3 h. Samples were then streak plated for isolation on XLT4 agar and incubated at 37 ± 1°C for 24 ± 3 h. At least one morphologically typical isolate per sample (i.e., black colonies with a clear edge) was confirmed as the challenge strain by poly-O and H antigen Salmonella latex agglutination (Difco, BD). One representative group D isolate from each treatment group was forwarded to the National Veterinary Services Laboratories (Ames, IA) for serotyping to further confirm SE.
In parallel with qualitative assessment, cecal contents were quantitatively assayed before enrichment by spiral plating 100 μL of sample serial dilutions to XLT4 agar utilizing the uniform deposition mode of the machine (Autoplate, Advanced Instruments), and plates were incubated at 37 ± 1°C for 24 ± 3 h. Colonies with typical morphology were enumerated (Qcount, Advanced Instruments), and estimates were log transformed for statistical analysis.
All data were analyzed with SAS version 9.4 (SAS Institute, Cary, NC). Cecal samples qualitatively positive for SE but nonenumerable by plate count were assigned an arbitrary value of approximately half of the approximated method limit of quantitation for statistical analyses (250 CFU/g). One cecal sample from the SCFP cohort was not reported by the laboratory and therefore was not included in the analysis. For cecal SE prevalence, a chi-square analysis was utilized with dietary treatment as the fixed effect. For cecal SE concentration, data were log transformed before analysis, and the MIXED function of SAS was utilized with dietary treatment as the main effect, and least-squares means adjusted for dietary treatment were calculated. A chi-square analysis with dietary treatment as the fixed effect was utilized to determine the proportion of samples with enumerable SE cecal concentrations. For all traits under investigation, results were considered significant at P < 0.05.
Qualitative testing of feed and litter indicated that birds in the treatment groups were negative for SE before they were challenged on day 28. SE was detected in 100% of the ceca collected from PCON and SCFP birds and 0% of NCON birds on day 32. Within-treatment least-squares means (95% confidence intervals [CIs]) for cecal SE concentrations were ca. 4.49 log CFU/g (3.92 to 5.06 log CFU/g) for PCON and 3.35 log CFU/g (2.77 to 3.94 log CFU/g) for SCFP. Of the PCON ceca examined, 95.0% (80.9 to 100.0%) of ceca had enumerable concentrations of SE above the limit of quantitation, whereas only 57.9% (43.4 to 72.4%) of SCFP samples were enumerable (Table 3). All isolates selected from within and between treatments were confirmed positive for the challenge SE strain.
SE is a foodborne pathogen of significant public health risk, and its association with the egg industry is of heightened importance. Therefore, feed additive technologies that additionally function as preharvest food safety hurdles while improving the health and performance of the bird may aid in the stepwise reduction of pathogen risk throughout the production continuum in a comprehensive food safety management plan.
The findings in this study suggest that, despite the high challenge dose, inclusion of SCFP in the diet of young layer pullets resulted in an overall mean reduction in SE colonization load of ca. 1.1 log CFU/g compared with the PCON (P < 0.0001). Inclusion of SCFP in the diet also resulted in a significant decrease (P < 0.0001) in the proportion of individual birds colonized with enumerable concentrations of SE, which could have important implication for overall flock load. These data suggest that SCFP can be an effective in-feed intervention against SE colonization within the ceca of young layer pullets. SCFP produced these observed effects within 4 days after high-dose direct SE challenge. Because the pullets were fed the respective treatment diets from day 1 to day 32, it is hypothesized that SCFP resulted in a more robust gut microflora and created an inhospitable environment for SE to colonize. This effect early in life may be important for managing the SE risk within a flock over the production cycle (19). Because SCFP in poultry has been reported to improve gut health and integrity, immune function, and microflora modulation compared with poultry on diets without SCFP, these effects are likely contributing factors to the findings of the present study.
Accurate assessment of the impact of in-feed preharvest interventions on the reduction of foodborne pathogens such as SE is complex and challenging due to a number of experimental and commercial considerations, particularly challenge dose and organism, administration or exposure routes (e.g., direct gavage, seeding models, and environmental seeding), production system type, sample type (e.g., feces, cloacal swab, ceca, and litter), animal age, temporal considerations, and laboratory analytical methodology (4, 7, 8, 12, 15, 18, 29, 31, 44). Challenge dose can influence colonization, concentrations, and transmission within various experimental models and subsequently the ability to detect and quantify Salmonella at various points in time from various sample types (7, 17). The direct high challenge dose (106 CFU per bird) utilized in the present study, although comparable to that used in other experimental models, is likely higher than typical exposure doses in real-world settings (5, 47). This difference may explain the fact that 100% of challenged birds remained qualitatively positive at harvest, which could be indicative of a type II error regarding the effect of SCFP on the qualitative reduction of SE. However, even with a high challenge dose, birds fed SCFP had lower concentrations of SE colonizing their ceca. This ability to quantitatively reduce SE under high-dose conditions suggests that birds exposed to low doses under commercial conditions may be able to completely shed SE or prevent SE from colonizing and may subsequently become SE free.
The data from this study suggest that flocks fed SCFP and their immediate environment may have an overall reduced environmental burden of SE associated with colonization and shedding by individual birds. Further research is warranted to understand the effects of various modes of exposure and dose on the magnitude and efficacy of the Salmonella reduction conferred by SCFP under experimental and commercial conditions.
The authors thank the late Dr. Darrell Trampel for his work on this study, without which it would not have been possible. The authors also acknowledge Melika Ibukic and all the other student assistants and staff that assisted with caring for animals, experimentation, and analytical processes. Authors affiliated with Diamond V provided the feed additive (SCFP) and financial support for the study to Iowa State University but were not associated with the execution of the research.