Salmonella exposure to consumers from eggs has been a decades-long concern. In 2021, Salmonella Enteritidis was the most frequently isolated serovar in humans, with eggs the most common source.
The potential exposure occurs because S. Enteritidis can colonize in laying hens’ reproductive organs, from which it can move to the yolk or albumen before shell development and the egg is laid. There is also the unknown impact of S. Enteritidis in the egg-production sector’s ever-changing housing methods.
“A generation ago, nearly all laying hens were housed in conventional, multi-bird laying cages,” noted Richard Gast, poultry research microbiologist at USDA’s US National Poultry Research Center in Athens, Georgia.
As of 2022, 34.1% of laying hens were housed in some form of cage-free system, with the remaining 65.9% in conventional housing. “Poultry housing environments influence the likelihood of Salmonella infections in egg-laying hens,” he told participants of the 2025 American Association of Avian Pathologists annual meeting. “Conventional housing can impact Salmonella exposure through crowding and immunosuppressive stress, but free-range systems can increase the risk through exposure to environmental reservoirs and other birds.”
Gast developed two research projects to gain more perspective on Salmonella persistence, transmission and egg contamination among pullets and laying hens housed in an indoor cage-free system.
Experiment 1 methods
The objective for the first study was to assess S. Enteritidis colonization of the internal organs of layer pullets in cage-free housing. The birds were experimentally infected at either 9 weeks or 15 weeks of age and sampled at two intervals: 1 to 2 weeks post-inoculation or 21 to 22 weeks of age.
Layer pullets were reared on wood shavings in aviary-style pens and moved to experimental cage-free housing at either 9 weeks of age (trial 1) or 15 weeks (trial 2). The birds were evenly divided among four isolation rooms simulating commercial cage-free barns with community, kick-out nest boxes and perches. In each room, 72 pullets were placed on wood shavings at a density of 265 square inches of floor space.
The research team orally inoculated one-third of the pullets in each room with a 7.8 x 107 colony-forming unit (CFU) dose of a two-strain mixture of S. Enteritidis 1 day after arrival, identified by a colored leg band. “Uninoculated birds remained in each room to measure horizontal transmission,” Gast said.
At 6 and 12 days post-inoculation, 12 inoculated birds and 24 contact-exposed birds from each of the two rooms were euthanized. The researchers collected 5 to 10 grams each of the birds’ liver, spleen and intestinal tract to culture for S. Enteritidis. The remaining birds were similarly tested at 21 to 22 weeks of age; this time, however, the researchers collected ovary samples instead of livers.
Effect of inoculation timing
“For all three tissues, the frequency of recovering S. Enteritidis was significantly higher at 15 weeks than at 9 weeks,” Gast said. “This is likely due to less time between the 15-week inoculation and the 21- to 22-week sampling interval compared to the 9-week inoculation.”
As for horizontal contact, far more birds were infected following the 15-week inoculation than following the 9-week inoculation, he added.
In summarizing experiment 1, Gast noted that S. Enteritidis was isolated significantly more often from organ samples of pullets infected at 15 weeks of age than those infected at 9 weeks when tested at either of the two sampling periods.
Organ samples tested at 21 to 22 weeks of age yielded consistently lower S. Enteritidis frequencies than samples collected 1 to 2 weeks post-infection. “So, the inception of egg laying did not lead to any reappearance of infection,” he said.
The relatively high prevalence of S. Enteritidis in the ovaries of birds infected at 15 weeks of age and sampled at 21 to 22 weeks — the onset of egg production — has food-safety implications. “There is the potential for the egg to be contaminated due to reproductive tract infection,” Gast added.
Experiment 2 methods
In the second experiment, Gast wanted to assess environmental contamination and horizontal transmission of S. Enteritidis within groups of layer pullets in cage-free housing when infected just before sexual maturity.
The team set up three trials, involving 144 pullets raised on wood shavings in aviary-style pens. Those birds moved to the experimental cage-free facility at 15 weeks of age and were evenly distributed between two identical isolation rooms.
One week after placement, a portion of the pullets in each room was assigned to a trial, orally inoculated with 107 CFU of a two-strain mixture of S. Enteritidis and identified by a colored leg band:
- Trial 1 — 24 inoculated out of 72 hens (33%)
- Trial 2 — 12 inoculated out of 72 hens (17%)
- Trial 3 — 6 inoculated out of 72 hens (8%)
During the first 2 weeks post-inoculation, the researchers took samples at six intervals of the following:
- Wall-dust swab
- Nest-box swab
- Perch swab
- Flooring substrate drag swab
- Flooring substrate composite, consisting of wood shavings and litter material
At 2 weeks post-inoculation, the team selected and euthanized six to eight orally infected and 40 contact-exposed birds from each of the two rooms. They cultured 5 to 10 grams each of liver, spleen and the intestinal tract for S. Enteritidis.
Effect of percentage of inoculated birds
The team found very high horizontal transmission when 33% of the birds were inoculated, declining as the percentage of inoculated birds declined. “Also, when one-third of the birds were inoculated, nearly all of the environmental samples tested positive for S. Enteritidis,” Gast noted.
When 17% of the birds were inoculated, there was more variation in the environmental samples. Wall-dust swabs were the most effective sampling option, with flooring drag swabs the least effective.
At 8% inoculation, S. Enteritidis was found only in the floor substrate composite sample, with a lesser response from the nesting-box sample.
For experiment 2, Gast summarized that S. Enteritidis recovery from internal organs of horizontally exposed pullets and their cage-free environment declined significantly as the number of inoculated birds declined from 24 to 12 to 6, with the steepest decline between the last two groups.
Wall dust was the most effective environmental sample to detect S. Enteritidis for both the 33%- and 17%-inoculated groups. When they added the 8% group, the flooring substrate composite sample was the most efficient option. In all cases, the least efficient sample was the flooring substrate drag swab.
Overall observations
The two experiments showed that when egg-type pullets are infected with S. Enteritidis in the late-rearing period, the infection is more likely to persist into the early egg-production stage compared to an early infection.
When a higher portion — 33% to 17% — of pullets in cage-free housing were inoculated with S. Enteritidis, there was rapid environmental contamination and extensive horizontal transmission during the first 2 weeks after exposure.
“When a smaller portion (8%) of the pullets were inoculated, we observed less horizontal transmission and lower and slower development of environmental contamination,” Gast said.
The data from these two experiments can offer additional guidance as egg-laying producers plan Salmonella-control programs.
