Internal Organ Colonization by Salmonella Enteritidis in Layer Pullets Infected at Two Different Ages During Rearing in Cage-Free Housing.

The poultry-housing environment plays a significant role in the transmission and persistence of the egg-associated pathogen Salmonella Enteritidis in laying flocks. The commercial egg industry is in the midst of a transition toward cage-free housing, but the food safety ramifications of this shift are not yet certain. The present study assessed internal organ colonization by Salmonella Enteritidis in layer pullets reared in cage-free housing and infected at two different ages. Groups of 280 pullets were transferred from the rearing facility (at 9 wk of age in one trial and 15 wk in another) to a containment facility with four isolation rooms simulating commercial cage-free barns with perches and nest boxes (70 birds/room). Twenty-four pullets in each room were orally inoculated with Salmonella Enteritidis immediately after placement in the containment facility. At 1-2 wk postinoculation in each trial, samples of liver, spleen, and intestinal tract were collected from all birds in two rooms for bacteriologic culturing to detect Salmonella Enteritidis. At 21-22 wk of age, samples of spleen, ovary, and intestinal tract were similarly collected and tested from all birds in the remaining two rooms. Among samples collected at 1-2 wk postinoculation, Salmonella Enteritidis was isolated significantly more often from groups of pullets infected initially at 15 wk of age than from those infected at 9 wk (61% vs. 38% of livers, 59% vs. 31% of spleens, and 84% vs. 57% of intestines). Among samples collected at 21-22 wk of age, the frequency of recovery of Salmonella Enteritidis was again significantly greater in birds infected at 15 wk of age than in those infected at 9 wk (16% vs. 6% of spleens, 9% vs. 1% of ovaries, and 26% vs. 10% of intestines). These data suggest that Salmonella Enteritidis infections introduced into flocks during the later stages of pullet rearing have greater potential to persist into the early phase of egg production.

Nota de investigación- Colonización de órganos internos por Salmonella Enteritidis en pollitas de postura infectadas en dos edades diferentes durante la crianza en alojamiento sin jaulas. El ambiente en alojamientos avícolas juega un papel importante en la transmisión y persistencia del patógeno asociado a los huevos Salmonella Enteritidis en parvadas postura. La industria comercial del huevo se encuentra en medio de una transición hacia alojamientos sin jaulas, pero las ramificaciones de este cambio en la seguridad alimentaria aún no están determinadas. El presente estudio evaluó la colonización de órganos internos por Salmonella Enteritidis en pollitas de postura criadas en alojamientos sin jaulas e infectadas a dos edades diferentes. Se transfirieron grupos de 280 pollitas desde las instalaciones de cría (a las 9 semanas de edad en un ensayo y a las 15 semanas en un segundo ensayo) a una instalación de contención con cuatro salas de aislamiento que simulaban alojamientos comerciales sin jaulas con perchas y nidos (70 aves/sala). Veinticuatro pollitas en cada sala fueron inoculadas oralmente con Salmonella Enteritidis inmediatamente después de su colocación en la instalación de contención. En cada ensayo, de una a dos semanas después de la inoculación, se recolectaron muestras de hígado, bazo y tracto intestinal para cultivo bacteriológico de todas las aves en dos salas para detectar Salmonella Enteritidis. A las 21-22 semanas de edad, se recolectaron y analizaron de manera similar muestras de bazo, ovario y tracto intestinal de todas las aves en las dos salas restantes. Entre las muestras recolectadas entre una y dos semanas después de la inoculación, Salmonella Enteritidis se aisló significativamente con mayor frecuencia en grupos de pollitas infectadas inicialmente a las 15 semanas de edad que en aquellas infectadas a las 9 semanas (61% contra 38 % en los hígados, 59% contra 31% de bazos y 84 % contra 57% en intestinos). Entre las muestras recolectadas a las 21-22 semanas de edad, la frecuencia de recuperación de Salmonella Enteritidis fue nuevamente significativamente mayor en aves infectadas a las 15 semanas de edad que en aquellas infectadas a las 9 semanas (16% contra 6% de bazos, 9% contra 1% en ovarios y 26% contra 10% de los intestinos). Estos datos sugieren que las infecciones por Salmonella Enteritidis introducidas en las parvadas durante las últimas etapas de la cría de pollitas tienen un mayor potencial para persistir en la fase inicial de la producción de huevos.

Research Note: Internal organ colonization by Salmonella Enteritidis in experimentally infected layer pullets reared at different stocking densities in indoor cage-free housing.

Contamination of eggs by Salmonella has often been identified as a source of food-borne human illness. S. Enteritidis is deposited inside developing eggs when invasive infections of laying hens reach the reproductive organs. The susceptibility of hens in cage-based housing systems to S. Enteritidis has been associated with their stocking density, but the applicability of this information to extensive (cage-free) systems is uncertain. The present study assessed internal organ colonization by S. Enteritidis in egg-type pullets reared at 2 different stocking densities in cage-free housing. Pullets were reared at either 374 cm2 or 929 cm2 of floor space per bird. At 16 wk of age, 4 groups of 72 pullets were moved into isolation rooms simulating commercial cage-free barns; 1/3 of the pullets in 2 rooms were orally inoculated with S. Enteritidis immediately after transfer and pullets in 2 rooms were similarly infected at 19 wk. At 6 and 12 d postinoculation, the pullets were euthanized and samples of liver, spleen, and intestinal tract were removed for bacteriologic culturing. No significant differences (P > 0.05) in S. Enteritidis isolation frequencies from any tissue were observed between high and low density rearing groups following infection at either age. However, S. Enteritidis was found significantly (P < 0.05) more frequently among pullets infected orally at 19 wk than at 16 wk in spleens and intestines. Likewise, the frequency of S. Enteritidis isolation from all birds (inoculated plus contact-exposed) at 19 wk was significantly higher than at 16 wk in livers and spleens. This increased susceptibility to invasive S. Enteritidis infection at reproductive maturity emphasizes the importance of risk reduction at a critical stage in the egg production cycle.

Tissue Colonization and Egg and Environmental Contamination Associated with the Experimental Infection of Cage-Free Laying Hens with Salmonella Braenderup.

In 2018, a national recall of shell eggs in the United States occurred due to human illness caused by Salmonella Braenderup. Although previous studies have identified Salmonella Braenderup in laying hens and the production environment, little is known about the ability of this Salmonella serovar to infect laying hens and contaminate eggs. The objective of this study was to examine the invasiveness of Salmonella Braenderup in laying hens as well as its ability to persist in the production environment. Specific-pathogen-free laying hens (four trials; 72 hens/trial) were orally challenged with 107 colony-forming units of Salmonella Braenderup. On day 6 postinoculation, half of the challenged hens were euthanatized, and samples of ileocecal junction (sections above and below it, and portions of both ceca), liver, spleen, ovary, and oviduct tissues were collected and cultured for Salmonella Braenderup. Egg and environmental (nest box swaps and substrate (litter)) samples were collected days 7-20 postinoculation (Trials 1 and 2; excluding weekends) and days 7-27 postinoculation (Trials 3 and 4; excluding weekends) to detect Salmonella Braenderup. Recovery of Salmonella Braenderup was highest in ileocecal tissue samples (11.1%-33.3%; P < 0.05), with little to no recovery in other collected tissue samples. Salmonella Braenderup was detected in a small number of shell emulsions (0%-2.9%; P < 0.01) and recovered in Trial 1 at a high rate (92.5%; P < 0.0001) in the substrate composite samples; however, recovery of Salmonella Braenderup was low in the other egg and environmental samples. These trials indicate that Salmonella Braenderup is not an invasive Salmonella serovar for cage-free laying hens, especially when compared to serovars of concern to the egg industry. However, it may persist in the environment at low levels.

Colonización de tejidos y contaminación ambiental y de huevo asociados con la infección experimental de gallinas de postura libres de jaulas por Salmonella Braenderup. En 2018, se retiraron del mercado a nivel nacional en los Estados Unidos huevos con cascarón debido a una enfermedad en humanos causada por Salmonella Braenderup. Aunque estudios anteriores han identificado Salmonella Braenderup en gallinas de postura y en ambientes de producción, se conoce poco sobre la capacidad de esta serovariedad de Salmonella para infectar a las gallinas ponedoras y contaminar el huevo. El objetivo de este estudio fue examinar la capacidad de invasión de Salmonella Braenderup en gallinas ponedoras, así como su capacidad para persistir en el ambiente de producción. Se desafiaron oralmente a gallinas de postura libres de patógenos específicos (cuatro ensayos; 72 gallinas/ensayo) con 107 unidades formadoras de colonias de Salmonella Braenderup. El día seis después de la inoculación, la mitad de las gallinas desafiadas se sacrificaron y se recolectaron y cultivaron muestras de la unión ileocecal (secciones anteriores y posteriores de la misma y porciones de ambos ciegos), hígado, bazo, ovario y oviducto y se cultivaron para Salmonella Braenderup. Se recolectaron muestras de huevos y ambientales (hisopos de las cajas de nido y sustrato [cama] en los días 7 a 20 después de la inoculación (Pruebas 1 y 2; excluyendo los fines de semana) y en los días 7 a 27 después de la inoculación (Pruebas 3 y 4; excluyendo los fines de semana) para detectar Salmonella Braenderup. La recuperación de Salmonella Braenderup fue mayor en las muestras de tejido ileocecal (11.1%­33.3%; P < 0.05), con poca o ninguna recuperación en otras muestras de tejido recolectadas. Se detectó Salmonella Braenderup en un pequeño número de emulsiones de cascarones (0%­2.9%; P < 0.01) y se recuperó en el Ensayo 1 a una tasa alta (92.5%; P < 0.0001) en las muestras compuestas de sustrato; sin embargo, la recuperación de Salmonella Braenderup fue baja en las otras muestras de huevos y ambientales. Estos ensayos indican que Salmonella Braenderup no es un serovar de Salmonella invasivo para gallinas de postura sin jaulas, especialmente cuando se compara con los serovares de interés para la industria del huevo. Sin embargo, puede persistir en el medio ambiente en niveles bajos.

Serotype Screening of Salmonella enterica Subspecies I by Intergenic Sequence Ribotyping (ISR): Critical Updates.

(1) Background: Foodborne illness from Salmonella enterica subspecies I is most associated with approximately 32 out of 1600 serotypes. While whole genome sequencing and other nucleic acid-based methods are preferred for serotyping, they require expertise in bioinformatics and often submission to an external agency. Intergenic Sequence Ribotyping (ISR) assigns serotype to Salmonella in coordination with information freely available at the National Center for Biotechnology Information. ISR requires updating because it was developed from 26 genomes while there are now currently 1804 genomes and 1685 plasmids. (2) Methods: Serotypes available for sequencing were analyzed by ISR to confirm primer efficacy and to identify any issues in application. Differences between the 2012 and 2022 ISR database were tabulated, nomenclature edited, and instances of multiple serotypes aligning to a single ISR were examined. (3) Results: The 2022 ISR database has 268 sequences and 40 of these were assigned new NCBI accession numbers that were not previously available. Extending boundaries of sequences resolved hdfR cross-alignment and reduced multiplicity of alignment for 37 ISRs. Comparison of gene cyaA sequences and some cell surface epitopes provided evidence that homologous recombination was potentially impacting results for this subset. There were 99 sequences that still had no match with an NCBI submission. (4) The 2022 ISR database is available for use as a serotype screening method for Salmonella enterica subspecies I. Finding that 36.9% of the sequences in the ISR database still have no match within the NCBI Salmonella enterica database suggests that there is more genomic heterogeneity yet to characterize.

Equivalency of peroxyacetic acid to chlorine as a shell egg sanitizing rinse.

In the United States, all shell eggs processed under the USDA Agricultural Marketing Service voluntary grading standards must receive a shell sanitizing rinse of 100-200 ppm chlorine or its equivalent after leaving the washing process. A study was conducted to determine the concentration of peroxyacetic acid (PAA) which would be equivalent to 100-200 ppm chlorine (Cl) in reducing target organisms under the required washing conditions for shell eggs. Three isolates of Salmonella spp. (Enteritidis, Braenderup, and Typhimurium), as well as Enterobacter cloacae were used as inocula. Sanitizing treatments were negative control; deionized water; 100 and 200 ppm Cl; and 50-500 ppm PAA (7 concentrations). Considering all isolates tested, 100 and 200 ppm chlorine had 2.6 and 2.3 log cfu/mL cultural organisms remaining on shell surface; 50 and 100 ppm peracetic acid had 1.9 and 1.0 log cfu/mL cultural organisms remaining, respectively, compared with untreated control average of 3.8 log cfu/mL (P < 0.001). Salmonella Typhimurium was least resistant to shell sanitizer treatments. Peroxyacetic acid concentrations >250 ppm did not produce significant reductions in microbial populations as PAA concentration increased. Culturing for the prevalence of viable and injured organisms, 400-500 ppm PAA resulted in fewer eggs (P < 0.0001) being positive for Salmonella spp. E. cloacae was culturable via enrichment from 99.4% of inoculated eggs, regardless of sanitizer treatment. The results of this study indicate that 50-100 ppm PAA is equivalent to 100-200 ppm chlorine in reducing egg surface microorganisms. The use of 400-500 ppm PAA resulted in a lower incidence of viable, but not culturable, Salmonella spp. on the shell surface. E. cloacae resulted in almost 100% viable, but not culturable, organism recovery for all sanitizing treatments and should be considered as an indicator organism when studying processing facility sanitation procedures.

Tissue-, age- and dose-dependent changes in avian ß-defensin and LEAP2 mRNA abundance in the intestines of Salmonella Typhimurium challenged broilers.

Salmonella is a pathogen normally found in the gastrointestinal tract of poultry. The objective of this study was to determine changes in avian ß-defensin (AvBD) and liver-enriched antimicrobial peptide 2 (LEAP2) mRNA following Salmonella challenge. Day of hatch chicks were challenged with 106, 107 or 108 colony-forming units (cfu) of Salmonella typhimurium. There were dose-, tissue- and age-specific changes in AvBD and LEAP2 mRNA. At 1-day post-infection (dpi) there was a transient upregulation of AvBD1, 8, 10 and 12 mRNA in the 108 cfu group. At 5 dpi, all seven AvBD mRNA were downregulated in the ileum, while only AvBD1, 6, 10 and 11 mRNA were downregulated in the jejunum and AvBD6, 8, 10, 12 and 13 were downregulated in the cecum. At 7 dpi, there was downregulation of all seven AvBD mRNA in the duodenum and downregulation of selected AvBD in the jejunum, ileum and cecum. LEAP2 mRNA was downregulated at all doses of Salmonella in the cecum at 1 dpi and in the ileum at 5 dpi. In summary, Salmonella infection caused an initial upregulation followed by a downregulation of AvBD mRNA.