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. 2026 Sep 17;105(12):107794. doi: 10.1016/j.psj.2026.107794

Environmental contamination and internal organ colonization by Salmonella Enteritidis in experimentally infected cage-free layer pullets reared in-place or relocated after simulated transport

Richard K Gast a,⁎, Javier S Garcia a, Rupa Guraya a, Deana R Jones a, Anne L Hurley-Bacon a, Darrin M Karcher b
PMCID: PMC13625779  PMID: 42772136

Abstract

The production of contaminated eggs by laying hens infected with Salmonella Enteritidis continues to present an international public health challenge. This study assessed how 2 different cage-free layer pullet rearing plans influenced environmental contamination and internal organ colonization after experimental infection with S. Enteritidis at 2 different ages. In 2 trials, birds were orally inoculated either as chicks at 1 wk of age or as pullets at 10 wk of age. In each trial, half of the birds were maintained entirely in-place and half were subjected to a transport and relocation treatment at 14 wk of age, simulating the common commercial practice of moving pullets from rearing to laying houses at that time. Environmental samples collected from each room at intervals after infection and internal tissues harvested at 16-17 wk of age were cultured for S. Enteritidis. The most sensitive detection of S. Enteritidis in the housing environment was obtained using flooring substrate composite samples (46%), and significantly less efficient detection was achieved via dust sampling (14%). The overall frequency of S. Enteritidis recovery from environmental samples was significantly greater in rooms housing birds infected at 10 wk of age than in rooms of birds infected at 1 wk (55 vs. 21%) and a similar pattern was observed for S. Enteritidis recovery from internal organs. The simulated transport stress treatment applied in this study did not affect either subsequent environmental contamination or internal organ colonization with S. Enteritidis. Isolation of S. Enteritidis from environmental samples in rooms housing relocated birds was infrequent (6%). S. Enteritidis was recovered significantly more often from spleens and ceca of birds infected at 10 wk and kept in-place than from similarly infected birds that were relocated (15 vs. 6% and 18 vs. 7%, respectively). These results suggest that continuing exposure to contaminated housing environments by flocks maintained in-place has the potential to perpetuate S. Enteritidis infections during the final stages of rearing before attaining reproductive maturity and commencing egg production.

Keywords: Salmonella Enteritidis, Cage-free layer housing, Environmental contamination, Internal organ colonization, In-place rearing

Introduction

Food-borne transmission of Salmonella infection to consumers continues to pose a significant international public health challenge. The estimated costs associated with human salmonellosis in the USA exceeded $17 billion in 2023 (Hoffman et al., 2025). The contamination of eggs with Salmonella enterica serovar Enteritidis is among the leading contributors to this problem (Chanamé Pinedo et al., 2022; Sher et al., 2021). Trends in the occurrence of human S. Enteritidis cases have been shown to mirror the incidence of this pathogen in egg-laying flocks (van den Berg et al., 2025) and genetic analysis has linked isolates responsible for disease outbreaks to isolates found in laying hens (Denagamage et al., 2017). However, comprehensive programs for risk reduction in commercial egg production have demonstrated substantial effectiveness over time in mitigating the scope of the problem (Chousalkar et al., 2018). The edible contents of eggs generally become contaminated with S. Enteritidis following bacterial colonization of the intestinal tract, invasion to internal organs such as the liver and spleen (Zeng et al., 2018), and further dissemination to reproductive tissues (ovary or oviduct), resulting in deposition inside yolk or albumen during egg formation before oviposition (Gast et al., 2024a). Experimental infection studies have demonstrated that S. Enteritidis has a greater propensity to cause egg contents contamination than other serovars (Gast et al., 2007, 2021), but relatively small initial doses of this pathogen ingested by laying hens from environmental sources typically lead to low overall frequencies of contamination in commercially produced eggs (DeWinter et al., 2011; Esaki et al., 2013). Pullets near the point of reproductive maturity can be highly susceptible to S. Enteritidis so infections initiated at this age can often persist until the onset of egg production (Groves et al., 2021; Gast et al., 2024b). Experimental infection studies have demonstrated that Salmonella colonization of poultry is both age and dose dependent (Gast et al., 2013; Yue and Groves, 2025).

Most infected hens clear S. Enteritidis from their internal organs within a few weeks following initial exposure (Gast et al., 2007, 2011), but colonization can persist for months in some birds (Gast et al., 2015). Fecal shedding into the laying house environment by infected hens provides opportunities for further horizontal dissemination of the pathogen throughout the flock (Crabb et al., 2019; Gast et al., 2020), and persistent environmental contamination that survives cleaning and disinfection of depopulated facilities may lead to the transmission of infection into subsequent flocks (Dewaele et al., 2012; Trampel et al., 2014). The prevalence of S. Enteritidis in the housing environment of commercial laying flocks correlates positively with the probability that contaminated eggs will be produced (Arnold et al., 2014). Environmental conditions in poultry facilities, affected by both facility design features and management practices, exert a powerful influence on the potential for Salmonella introduction and distribution in laying flocks (Trampel et al., 2014). Rearing conditions for pullets have been reported to significantly affect organ invasion and horizontal transmission following experimental infection with S. Enteritidis (Gast et al., 2022). Risk assessments for laying hen flocks have associated a higher prevalence of S. Enteritidis with larger and older flocks housed in larger and older facilities (Denagamage et al., 2015; St. Amand et al., 2025). In recent years, the commercial egg industry has gradually shifted from nearly exclusive reliance on housing layers in conventional multiple-bird cages toward increased utilization of alternatives such as indoor multi-level aviaries, which are characterized by increased freedom of movement and enhancements such as perches, nesting areas, and open floor spaces with scratching pads. However, this transition has uncertain implications for egg microbial safety, as risk comparisons between different housing systems can be confounded by variations between farms in hen genetics, stocking density, facility design, pathogen contamination levels in environmental reservoirs, and biological vector populations (Holt et al., 2011). Different environmental microbiota profiles have been identified in cage and cage-free poultry management systems (Wilson et al., 2021). The distinctive inherent features of each system require correspondingly targeted risk reduction strategies to reduce Salmonella prevalence and persistence (Jones et al., 2015).

Standard commercial egg industry practice for many years has been to transfer pullets from rearing houses to laying houses at approximately 14-16 wk of age. For contemporary aviary-style hen facilities, this move can subject the pullets to stress during transportation (H&N International, 2026; Lohman Tierzucht, 2026) and also necessitates their adaptation to unfamiliar environmental complexity in the aviary (Pullin et al., 2024). An alternative to this traditional approach is to start and rear chicks entirely in-place in the laying house itself. However, the comparative microbiological consequences of conventional and in-place rearing (for both the birds and their housing environment) are not yet fully understood. Birds raised in-place are not subjected to potentially immunosuppressive transport stress which might increase their susceptibility to infection (or exacerbate existing infections). Conversely, these birds are not moved into clean, initially pathogen-free housing environments a few weeks before the critical age of reproductive maturity. The present study applied an experimental S. Enteritidis infection model to compare the microbiological consequences (for both housing environment contamination and bird infection) of rearing egg-type pullets either entirely in-place or with a simulated transport experience and relocation at 14 wk of age.

Materials and methods

Experimental housing of layer pullets

In each of 2 similar trials, 280 female H&N Nick Chicks (a strain used by the egg industry) were obtained at 1 day of age from a commercial hatchery (without vaccination against Salmonella infection) and transferred to the U.S. National Poultry Research Center (Athens, GA). The chicks were distributed evenly (140 per room) between 2 separate isolation rooms in an ABSL-2 containment facility with central heating and air conditioning. Each isolation room simulated space in a commercial cage-free barn with perches and wood shavings flooring substrate. Schedules for managing temperature, lighting, and feeding were as specified for this line of birds and typical for commercial egg production (H&N International, 2026). Feed was supplied as an antibiotic-free, ground-mash starter ration for chicks through 6 wk of age and a similar grower ration thereafter. Water was supplied ad libitum via automatic nipple-type drinkers. At 6 wk of age, the number of birds in each room was reduced to 127 to meet required standards for floor space availability per bird. The experimental protocol (USNPRC-2025-10) was approved by the Institutional Animal Care and Use Committee of the U.S. National Poultry Research Laboratory.

Pre-inoculation cloacal swab samples

Immediately before chicks or pullets in each containment room were inoculated, sterile cotton swabs were used to collect cloacal swab samples from 72 randomly selected birds per room to ensure the absence of Salmonella infection within the group. Each sample was transferred into 10 ml of buffered peptone water (BPW; Romer Labs, Newark, DE) and incubated for 24 h at 37̊°C. A 0.1 ml portion of each culture was then transferred into 10 ml of Rappaport-Vassiliadis (RV) broth (Acumedia, Neogen Corp., Lansing, MI) and incubated for 24 h at 42̊°C. A 10-μl portion from each of these broth cultures was then streaked onto brilliant green (BGN) agar (Acumedia) supplemented with 0.02 mg/ml of novobiocin (Sigma Chemical Co., St. Louis, MO). These plates were incubated for 24 h at 37̊°C and then examined for the presence of typical Salmonella colonies.

Experimental infection of layer chicks or pullets with S.Enteritidis

In each trial, all birds in each containment room were orally inoculated with a single measured dose of a 2-strain mixture (phage types 4 and 13a) of S. Enteritidis. Chicks in trial 1 were infected at 1 wk of age and pullets in trial 2 were infected at 10 wk of age. Both Salmonella strains were originally isolated from internal organs of naturally infected chickens in commercial settings. Each inoculum strain was resuscitated by transfer into tryptic soy broth (Acumedia) for 2 successive cycles of 24-h incubation at 37°C. After cell numbers in each incubated culture were estimated by determining its optical density at 600 nm, equal numbers of the 2 inoculum component strains were combined, and further serial dilutions in 0.85% saline were performed to achieve the desired final cell concentration. Plate counts on BGN agar indicated that the average final S. Enteritidis cell concentration in each oral inoculum dose (0.5 ml in trial 1 and 1.0 ml in trial 2) was approximately 1.0 × 107 cfu. All inoculations were performed at the same time of day, 24 h after the birds last received feed.

Chick and pullet housing environmental samples

Prior to chick placement in the containment facility in each trial, at 1 d prior to oral inoculation of chicks or pullets with S. Enteritidis, at 2, 4, 6, 8, 10, 12, 14, 15, and 16 wk of age in trial 1, and at 11, 12, 13, 14, 15, and 16 wk of age in trial 2, four types of environmental samples were collected from each room as described by Garcia et al. (2023) and cultured to detect Salmonella contamination associated with fecal shedding of the pathogen into the rearing house by infected birds. Each environmental test set consisted of 2 samples each of flooring substrate composite (comprised of gloved handfuls from the drinker/feeder and perch areas), drag swabs of flooring substrate, wall dust swabs, and perch swabs. Samples were collected in sterile plastic bags (Whirl-Park, Madison, WI) and transported to the laboratory where 10 ml of BPW was added to each swab sample and 250 ml of BPW was added to a 25g portion of each flooring substrate composite sample. All samples were then mixed by stomaching for 1 min. After incubation for 24 h at 37̊°C, 0.1 ml portions from each sample were transferred into 10 ml of RV broth and incubated for 24 h at 42̊°C. A 10-μl aliquot of each RV broth culture was then streaked onto BGN agar (Acumedia) and incubated for 24 h at 37̊°C. Typical Salmonella colonies on these plates were subjected to biochemical and serological confirmation (Waltman and Gast, 2016).

Simulated transport and relocation experience

At 14 wk of age in each trial, ½ of the pullets in each room were subjected to a transport and relocation treatment intended to simulate the potential stress associated with the common commercial egg industry practice of moving birds from pullet rearing facilities to laying houses at approximately this age. Pullets were caught 24 hr after last receiving feed and transferred into plastic coops (21-22 birds per coop). The coops were stacked on a hand cart which was manually pushed back and forth along a hallway for 5 minutes with vigorous jostling of the cart while loud traffic and air horn sounds were played next to the birds. The crates were then placed in a quiet room and the transport treatment was repeated 3 more times at hourly intervals. After the last transport experience, these pullets were relocated into clean rooms with fresh flooring substrate. The other ½ of pullets (not transported) remained in-place in the original rearing rooms. Following completion of the transport and relocation process, the final experimental design for trial 1 included 2 replicate rooms each of transported and non-transported pullets that had been infected with S. Enteritidis at 1 wk of age, and trial 2 similarly consisted of 2 replicate rooms each of transported and non-transported pullets infected at 10 wk.

Internal organ samples

At 16 wk of age in each trial, all birds in a pair of rooms (one housing birds maintained continuously in-place and the corresponding room housing their former pen-mates after transport/relocation at 14 wk) were euthanized and 58-60 birds from each room were randomly selected for bacteriologic culture of internal tissues. Portions (approximately 1-5 g) of the liver, spleen, and intestinal tract (including the ileocecal junction and adjacent regions of both ceca) from each pullet were aseptically removed, transferred to 20 ml of BPW, and mixed by stomaching for 30 sec. After incubation for 24 h at 37̊°C, a 1-ml portion of each culture was transferred to 9 ml of tetrathionate broth (Acumedia) and incubated for 24 h at 37̊°C. A 10-μl aliquot of each culture was then streaked onto BGN agar. Following incubation of these plates for 24 h at 37̊°C, typical S. Enteritidis colonies were subjected to biochemical and serological confirmation. Prior to the initial incubation of spleen samples, a 2 ml portion of each BPW culture was removed after homogenization by stomaching, serially diluted in 0.85% saline, and aliquots of the original and diluted culture were spread onto BGN agar for S. Enteritidis enumeration (with a minimum detection threshold of 20 cfu/g). This sampling procedure was repeated for pullets from the other pair of containment rooms (maintained in-place and transported/relocated) in each trial at 17 wk of age. All necropsies were performed at the same time of day, 24 h after the birds last received feed.

Statistical analysis

The statistical adequacy of the experimental design (treatment replicates and constituent bird numbers) was validated by a power analysis using StatMate 2.0 (GraphPad Software, San Diego, CA). Significant differences (P < 0.05) in the frequency of S. Enteritidis isolation between groups of birds orally inoculated with S. Enteritidis at different ages or kept under different housing regimens (maintained continuously in-place or transported/relocated), and between the 4 types of environmental samples were determined by Fisher’s exact test using Instat biostatistics software (GraphPad Software). Because the 2 replicate rooms within each trial never differed significantly in Salmonella recovery from any type of samples in this analysis, their results were combined for presentation in the text and tables. Significant interactions (P < 0.05) between inoculation age (1 wk or 10 wk) and rearing system (in-place or transported/relocated) in their effects on the frequency of S. Enteritidis recovery were assessed by linear logistic regression using jamovi statistics software (The jamovi group; https://www.jamovi.org).

Results

No pre-infection samples (from either the birds or their environment) were Salmonella-positive in either trial. During the first 3 wk after oral Salmonella inoculations were administered, S. Enteritidis was recovered from significantly (P = 0.037) more environmental samples from rooms containing pullets infected at 10 wk of age (70.8%; Table 1) than from rooms housing chicks infected at 1 wk of age (47.9%). Similarly, during the final 2 wk that all birds remained together in the original rearing rooms prior to the transport and relocation of ½ of them, the frequency of environmental isolation of S. Enteritidis in rooms of pullets infected at 10 wk (56.3%; Table 1) was significantly (P < 0.0001) greater than in rooms of pullets infected at 1 wk (0%). During the 2-wk period after the transport/relocation event (and immediately preceding necropsy to culture internal organs), the frequency of S. Enteritidis positivity in environmental samples from the original rearing rooms containing birds infected at 10 wk (28.1%; Table 1) remained significantly (P = 0.002) greater than the corresponding value for birds infected at 1 wk (0%). However, < 10% of environmental samples taken from rooms housing transported/relocated birds were positive for S. Enteritidis with no significant difference (P > 0.05) observed between groups infected at 1 wk or 10 wk. Flooring substrate composite samples provided the most effective detection of S. Enteritidis contamination among the 4 types of environmental samples (Table 2), with an overall recovery frequency (46.4%) which was significantly (P < 0.0001) greater than for either wall dust swabs (14.3%) or perch swabs (13.1%). Flooring substrate swabs yielded an intermediate (25.0%) frequency of S. Enteritidis isolation. The results for individual inoculation age groups followed these same trends.

Table 1.

Isolation of Salmonella Enteritidis from environmental samples collected at different time periods following oral infection of chicks or pullets reared in cage-free rooms with or without or simulated transport and relocation at 14 wk1.

-Salmonella-positive/total (%)
Infection age (rearing system) 1-3 wk post-infection 1-2 wk before transport/relocation 1-2 wk after transport/relocation
Infected at 1 wk (maintained in-place) 23/48 (47.9)a 0/32 (0)a 0/32 (0)a
Infected at 1 wk (transported/relocated) — — 1/32 (3.1)ab
Infected at 10 wk (maintained in-place) 34/48 (70.8)b 18/32 (56.3)b 9/32 (28.1)b
Infected at 10 wk (transported/relocated) — — 3/32 (9.4)ab
Open in a new tab
1

In 2 trials, groups of pullets housed in individual cage-free rooms of a disease containment facility were orally inoculated with approximately 1.0 × 107 cfu of a 2-strain mixture of S. Enteritidis at either 1 wk or 10 wk of age. At 14 wk of age, ½ of the pullets from each room were subjected to a simulated transport experience and moved to clean cage-free rooms. The other ½ of the pullets remained in-place in the original rearing rooms. 4 types of samples from the housing environment were collected at intervals after inoculation and cultured for S. Enteritidis.

a,b

Values in columns that share no common superscripts are significantly (P < 0.05) different.

Table 2.

Isolation of Salmonella Enteritidis from 4 types of environmental samples collected after oral infection of chicks or pullets during 16 wk of rearing in cage-free rooms1.

Salmonella-positive/total (%)
Environmental sample type Infected at 1 wk Infected at 10 wk All groups
Flooring substrate drag swab 9/48 (18.8)ab 12/36 (33.3)a
0/24 (0)b 		21/84 (25.0)a
Flooring substrate composite 14/48 (29.2)b 25/36 (69.4)b 39/84 (46.4)b
Perch swab 3/48 (6.3)a 8/36 (22.2)a 11/84 (13.1)a
Wall dust swab 3/48 (6.3)a 9/36 (25.0)a 12/84 (14.3)a
Open in a new tab
1

In 2 trials, groups of pullets housed in individual cage-free rooms of a disease containment facility were orally inoculated with approximately 1.0 × 107 cfu of a 2-strain mixture of S. Enteritidis at either 1 wk or 10 wk of age. At 14 wk of age, ½ of the pullets from each room were subjected to a simulated transport experience and moved to clean cage-free rooms. The other ½ of the pullets remained in-place in the original rearing rooms. 4 types of samples from the housing environment were collected (2 of each type per room) and cultured for S. Enteritidis at biweekly intervals after inoculation at 1 wk of age and weekly intervals after inoculation at 10 wk.

a,b

Values in columns that share no common superscripts are significantly (P < 0.05) different.

When internal organs were harvested from euthanized pullets at 16-17 wk of age (Table 3), S. Enteritidis was infrequently isolated from tissues of birds infected at 1 wk of age: of the 3 types of samples taken from these birds, spleens provided the highest overall frequency of recovery (at only 6.0%) and all positive spleen samples contained < 20 cfu/g of S. Enteritidis (the minimum enumeration threshold). No significant differences (P > 0.05) were identified in the frequencies of S. Enteritidis recovery from any of the 3 sampled internal tissues of birds infected at 1 wk between pullets maintained in the original rearing rooms and those subjected to transport/relocation. The combined frequencies of S. Enteritidis isolation from all internal organs of birds infected at 10 wk of age and maintained in the original rearing rooms (Table 3) were significantly (P < 0.0001) greater than from similarly housed birds infected at 1 wk. Among pullets infected at 10 wk, the frequencies of recovery of S. Enteritidis were significantly higher from birds maintained in-place in the original rearing rooms than from those subjected to transport/relocation for samples of both spleens (15.0 vs. 5.8%; P = 0.033) and ceca (17.5 vs. 6.7%; P = 0.016). Only 8 of 18 spleen samples from birds infected at 10 wk harbored more than the 20 cfu/g detection threshold for S. Enteritidis. The mean contamination level among these 8 positive spleen samples was 86.4 cfu/g (and the maximum count obtained was 120 cfu/g. Regression analysis indicated a significant interaction (P < 0.025) between the effects of infection age and rearing system on the isolation of S. Enteritidis; the higher post-infection frequencies of pathogen detection in both internal organ and environmental samples from groups infected at 10 wk were more pronounced in pullets that remained in-place than in transported/relocated groups.

Table 3.

Isolation of Salmonella Enteritidis from internal organs at 16-17 wk of age after oral infection of chicks or pullets reared in cage-free rooms with or without simulated transport and relocation at 14 wk1.

Maintained continuously in-place
 Transported/relocated at 14 wk
Liver Spleen Ileum/Ceca Liver Spleen Ileum/Ceca
Salmonella-positive/total (%)
Infected at 1 wk of age 4/116 (3.4)a 8/116 (6.9)a 6/116 (5.2)a 3/116 (2.6)a 6/116 (5.2)a 5/116 (4.3)a
Infected at 10 wk of age 12/120 (10.0)ab 18/120 (15.0)ac 21/120 (17.5)a 6/120 (5.0)b 7/120 (5.8)b 8/120 (6.7)bc
Open in a new tab
1

In 2 trials, groups of pullets housed in individual cage-free rooms of a disease containment facility were orally inoculated with approximately 1.0 × 107 cfu of a 2-strain mixture of S. Enteritidis at either 1 wk or 10 wk of age. At 14 wk of age, ½ of the pullets from each room were subjected to a simulated transport experience and moved to clean cage-free rooms. The other ½ of the pullets remained in-place in the original rearing rooms. All pullets were sampled for the presence of S. Enteritidis in their internal organs at 16-17 wk of age.

a,b,c

Values in rows that share no common superscripts are significantly (P < 0.05) different.

Discussion

Commercial laying flocks infected with S. Enteritidis usually produce contaminated eggs at very low frequencies, so testing for the presence of this pathogen in the poultry housing environment is often relied upon for the preliminary identification of flocks which merit additional scrutiny or remedial restrictions to protect public health (Gast et al., 2024a). Environmental sampling has been shown to have high predictive value for flock infection status, even when Salmonella prevalence is low (Apentag et al., 2020; Khan et a., 2024). Fecal shedding by infected hens is often an important initial source for loading S. Enteritidis into the laying house environment (Henzler et al., 1998; Sodagari et al., 2020) and salmonellae can persist in contaminated feces and dust in poultry facilities for extended periods of time (Im et al., 2015; McWhorter and Chousalkar, 2020). Bacterial survival in dust and flooring substrate materials is enhanced by high ambient moisture levels (McWhorter and Chousalkar, 2020; Pal et al., 2021a). At all principal post-infection environmental testing intervals in the present study (shortly after oral inoculation, just prior to the simulated transport of half of the birds, and after the transported birds were relocated), the overall frequency of isolation of S. Enteritidis from samples taken in rooms of birds infected at 10 wk of age was significantly higher than from birds infected at 1 wk of age. These results may have been due to the combined effects of a much greater initial amount of contaminated fecal material excreted into the housing environment by the larger pullets than by the much smaller chicks plus a shorter elapsed time between oral inoculation of the pullets and the pre- and post-transport sampling intervals. Statistical analysis confirmed that this effect of inoculation age on Salmonella isolation was influenced by rearing system as the higher frequencies of S. Enteritidis recovery following infection at 10 wk were especially evident among birds left in-place. Because the 2 infection-age groups were separately hatched and reared, unidentified variations between trials may also have contributed to the observed outcomes. S. Enteritidis was infrequently recovered from samples collected in rooms housing relocated birds (only 6% overall), even in groups infected at 10 wk of age.

A variety of sampling strategies have been successfully applied to detect Salmonella in flooring substrate or dust sources (including walls, egg belts, fan blades, feeding systems, and nest boxes). Dust has been reported as one of the most effective samples for Salmonella detection in both naturally and experimentally infected laying flocks (Chousalkar et al., 2016; McWhorter and Chousalkar, 2019; Gast et al., 2025). S. Enteritidis was found in circulating airborne dust among groups of experimentally infected hens (Gast et al., 2004). Detection of Salmonella in dust has been reported to increase over time in poultry flocks (Kim et al., 2024). A systematic review suggested that dust sampling was the most reliable Salmonella detection strategy for cage housing systems, whereas boot swabs of flooring substrate were preferable for non-cage housing systems (Pacholewicz et al., 2023). A recent study found that S. Enteritidis was most efficiently found in the environment of cage-free pullets during the first few week after experimental infection by composite samples of flooring substrate, although various dust samples and drag swabs of the flooring substrate also yielded positive results at high frequencies (Gast et al., 2025). In the present study, over the course of longer post-infection intervals for pullets in cage-free housing, flooring substrate composite samples once again provided the highest frequency of S. Enteritidis recovery, but samples of dust from walls and perches were significantly less useful for this purpose. A possible explanation for this observation is that the ongoing release of excreta from the birds might continually re-seed the flooring litter with salmonellae shed from infected individuals and contribute to moisture which promotes bacterial survival.

Persistent S. Enteritidis contamination of the poultry housing environment can perpetuate opportunities for extensive horizontal dissemination of infection throughout flocks and thus enhance the potential for egg contamination (Gast et al., 2011, 2014). In a previous experimental infection experiment, a higher frequency of environmental contamination was associated with a higher frequency of horizontal transmission of infection (Gast et al., 2025). Biological vectors such as rodents and insects can harbor and spread Salmonella and sometimes pose a threat to re-introduce the pathogen into egg laying facilities following depopulation and cleaning/disinfection (Wales et al., 2010; Sornplang et al., 2022). Circulating dust and aerosols can distribute bacteria widely around poultry houses, augmenting direct transmission of the pathogen between birds (Pal et al., 2021b; Khan et al., 2024). As a consequence of providing more access of hens to each other and to flooring substrate contaminated by fecal shedding, cage-free housing may support higher frequencies of horizontal transmission of Salmonella infection within flocks (Devylder et al., 2011). Less diverse intestinal microbiota have been reported in cage-free birds than in cage systems, potentially offering reduced protection against colonization by pathogens (Wiersema et al., 2021). A higher prevalence of S. Enteritidis in commercial laying houses was attributed to prior cage-free rearing of pullets (Garber et al., 2003). When internal tissues were sampled from pullets reared in cage-free conditions in the present study, very limited persistence of S. Enteritidis infection was observed in birds that had been orally infected at 1 wk of age but significantly more positive results were obtained from birds that had been more recently infected (at 10 wks of age). This trend was paralleled by the comparative frequencies of Salmonella detection in the housing environment of groups of birds infected either as chicks or pullets. Although prolonged S. Enteritidis infection has sometimes been reported following oral inoculation (especially for very young chicks), more rapid clearance from internal organs after exposure is more common (Gast and Holt, 1998; Gast et al., 2007).

Stress associated with transportation has been shown to increase shedding of Salmonella by poultry (Line et al., 1997; Marin and Lainez, 2009). Various stressors, including heat and crowding, can diminish humoral and cellular immune responses and increase susceptibility to Salmonella infection (Gomes et al., 2014; Quinteiro-Filho et al., 2017). In the current study, the experimental transport treatment did not increase the isolation of S. Enteritidis from the internal organs of sampled pullets, possibly as a consequence of an insufficient degree of immunosuppressive stress caused by the simulated transport experience. Indeed, S. Enteritidis was isolated from significantly fewer spleen and ceca samples from relocated birds infected at 10 wk of age than from the corresponding group maintained continuously in the original rearing rooms. Pullets moved into clean rooms after transport likely had much less opportunity for re-exposure to Salmonella from environmental sources in comparison to pullets maintained in rooms previously contaminated by housing infected birds, thereby also reducing bacterial carriage in internal organs of the relocated birds at the time of necropsy. These results, although not conclusively addressing the microbiological consequences of transporting pullets under commercial conditions (particularly considering a somewhat limited power of analysis based on only 2 replicates per treatment), demonstrate that rearing chicks and pullets continuously in-place may have associated risks for the perpetuation of S. Enteritidis infection (potentially until reproductive maturity and the commencement of egg production) as a consequence of continuing exposure to contaminated housing environments.

Author contributions

Richard Gast directed the conception and design of this work and the acquisition, analysis, and interpretation of data, and wrote the manuscript. Javier Garcia contributed to the conception and design of the study and to data acquisition and interpretation. Rupa Guraya contributed to experimental design and to data acquisition and interpretation. Deana Jones contributed to the conception and implementation of this project. Anne Hurley-Bacon contributed to study design, animal and facility resource management, and data acquisition. Darrin Karcher contributed to conception of the work, animal resource management, and data acquisition. All authors reviewed the manuscript critically for scientific content, reviewed and approved the final version of the manuscript, and agreed to be personally and publicly responsible for all aspects of the work.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We gratefully express appreciation for excellent technical assistance from Stephen Norris, Robin Woodroof, and Anna Thomas (U. S. National Poultry Research Center, Athens, GA). This study was supported by appropriated funds of the USDA Agricultural Research Service.

Footnotes

Primary Audience: Researchers, Veterinarians, Laying Flock Managers

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