Dust sprinkling as an effective method for infecting layer chickens with wild-type Salmonella Typhimurium and changes in host gut microbiota.

Role of dust in Salmonella transmission on chicken farms is not well characterised. Salmonella Typhimurium (ST) infection of commercial layer chickens was investigated using a novel sprinkling method of chicken dust spiked with ST and the uptake compared to a conventional oral infection. While both inoculation methods resulted in colonisation of the intestines, the Salmonella load in liver samples was significantly higher at 7 dpi after exposing chicks to sprinkled dust compared to the oral infection group. Infection of chickens using the sprinkling method at a range of doses showed a threshold for colonisation of the gut and organs as low as 1000 CFU/g of dust. Caecal content microbiota analysis post-challenge showed that the profiles of chickens infected by the sprinkling and oral routes were not significantly different; however, both challenges induced differences when compared to the uninfected negative controls. Overall, the study showed that dust sprinkling was an effective way to experimentally colonise chickens with Salmonella and alter the gut microbiota than oral gavage at levels as low as 1000 CFU/g dust. This infection model mimics the field scenario of Salmonella infection in poultry sheds. The model can be used for future challenge studies for effective Salmonella control.

A live attenuated Salmonella Typhimurium vaccine dose and diluent have minimal effects on the caecal microbiota of layer chickens.

Among the Salmonella reduction strategies in poultry production, one option is to use a Salmonella vaccine. The aim of vaccinating layer flocks is to reduce the shedding of wild-type Salmonella in the poultry environment, thereby reducing the contamination of poultry products (eggs and meat). Nutritive diluent and a higher dose of vaccine may enhance its colonization potential in the gut of chickens. In this study, a commercially available live attenuated vaccine (Vaxsafe® ST) was reconstituted in different media and delivered orally to day-old chicks at three different doses (107, 108, and 109 CFU/chick). Gut colonization of the vaccine strain and the effects of vaccination on gut microbiota were assessed in commercial-layer chickens. The vaccine diluent and dosage minimally affected microbiota alpha diversity. Microbiota beta diversity was significantly different (P < 0.05) based on the vaccine diluent and dose, which indicated that the vaccinated and unvaccinated chickens had different gut microbial communities. Differences were noted in the abundance of several genera, including Blautia, Colidextribacter, Dickeya, Enterococcus, Lactobacillus, Pediococcus, and Sellimonas. The abundance of Colidextribacter was significantly lower in chickens that received vaccine reconstituted in Marek's and water diluents, while Lactobacillus abundance was significantly lower in the water group. The highest vaccine dose (109 CFU/chick) did not significantly alter (P > 0.05) the abundance of microbial genera. Chicken age affected the microbiota composition more significantly than the vaccine dose and diluent. The abundance of Lactobacillus, Blautia, Caproiciproducens, Pediococcus, and Colidextribacter was significantly higher on day 14 compared with day 7 post-vaccination. The Salmonella Typhimurium vaccine load in the caeca was not significantly affected by diluent and vaccine dose; however, it was significantly lower (P < 0.0001) on day 14 compared with day 7 post-vaccination. Overall, the S. Typhimurium vaccine minimally affected the gut microbiota structure of layer chicks, whereas changes in microbiota were more significant with chicken age.

Salmonella in eggs and egg-laying chickens: pathways to effective control.

Eggs contaminated with Salmonella have been internationally significant sources of human illness for several decades. Most egg-associated illness has been attributed to Salmonella serovar Enteritidis, but a few other serovars (notably S. Heidelberg and S. Typhimurium) are also sometimes implicated. The edible interior contents of eggs typically become contaminated with S. Enteritidis because the pathogen's unique virulence attributes enable it to colonize reproductive tissues in systemically infected laying hens. Other serovars are more commonly associated with surface contamination of eggshells. Both research and field experience have demonstrated that the most effective overall Salmonella control strategy in commercial laying flocks is the application of multiple interventions throughout the egg production cycle. At the preharvest (egg production) level, intervention options of demonstrated efficacy include vaccination and gastrointestinal colonization control via treatments such as prebiotics, probiotics, and bacteriophages, Effective environmental management of housing systems used for commercial laying flocks is also essential for minimizing opportunities for the introduction, transmission, and persistence of Salmonella in laying flocks. At the postharvest (egg processing and handling) level, careful regulation of egg storage temperatures is critical for limiting Salmonella multiplication inside the interior contents.

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.

Research Note: Contamination of eggs by Salmonella Enteritidis and Salmonella Typhimurium in experimentally infected laying hens in indoor cage-free housing.

Contaminated eggs are a leading source of human Salmonella infections and this problem continues to challenge public health authorities and egg industries around the world. Salmonella invasion of the ovaries and oviducts of infected laying hens can result in bacterial deposition inside the edible portions of developing eggs. The introduction, persistence, and transmission of salmonellae in commercial egg-laying flocks are influenced by flock management practices, but the food safety ramifications of different types of laying hen housing remain unresolved. The present study assessed the frequency of internal contamination of eggs after experimental Salmonella Enteritidis and S. Typhimurium infection of laying hens in indoor cage-free housing. Groups of 72 hens were housed on wood shavings in isolation rooms simulating commercial cage-free barns with community kick-out nest boxes and perches and 1/3 of the hens in each room were orally inoculated with 8.0 × 107 cfu of 2-strain mixtures of either S. Enteritidis (2 rooms) or S. Typhimurium (2 rooms), and the entire internal contents of all eggs laid 5 to 30 d postinoculation in nest boxes or on the flooring substrate were cultured to detect Salmonella. Contaminated eggs were laid between 8 and 28 d postinoculation. The overall incidence of S. Enteritidis isolation from eggs (3.41%) was significantly (P = 0.0005) greater than S. Typhimurium (1.19%). The contamination frequencies associated with the 2 egg collection locations were not significantly different (P > 0.05). These results demonstrate that oral infection of a relatively small proportion of laying hens in indoor cage-free housing with invasive Salmonella serovars can result in the production of internally contaminated eggs at low frequencies over a period of nearly a month postinoculation.

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.

Research Note: Horizontal transmission and internal organ colonization by Salmonella Enteritidis and Salmonella Kentucky in experimentally infected laying hens in indoor cage-free housing.

The transmission of Salmonella to humans via contaminated eggs is an international public health concern. S. Enteritidis is deposited inside eggs after colonizing reproductive tissues of infected hens. Diverse housing facility characteristics and flock management practices influence Salmonella persistence and transmission in poultry, but the food safety consequences of different housing systems for laying hens remain unresolved. The present study compared the horizontal transmission of infection and invasion of internal organs during the first 2 wk after experimental S. Enteritidis and S. Kentucky infection of laying hens in indoor cage-free housing. Groups of 72 hens were housed in isolation rooms simulating commercial cage-free barns, and 1/3 of the hens in each room were orally inoculated with either S. Enteritidis (2 rooms) or S. Kentucky (2 rooms). At 6 d and 12 d postinoculation, 12 inoculated and 24 contact-exposed hens in each room were euthanized, and samples of liver, spleen, ovary, oviduct, and intestinal tract were removed for bacteriologic culturing. All orally inoculated hens were positive for intestinal colonization by S. Enteritidis at 6 d postinfection, and 70.8% of contact-exposed hens had become colonized by 12 d. S. Enteritidis was isolated from 100% of livers and 50.0% of ovaries from inoculated birds at 6 d and from 41.7% of livers and 10.4% of ovaries from contact-exposed birds at 12 d. The majority of both orally inoculated and contact-exposed hens were positive for intestinal colonization by S. Kentucky at 6 d, but S. Kentucky was found in other internal organs of both inoculated and contact-exposed hens significantly (P < 0.05) less often than S. Enteritidis at both sampling intervals. These results indicate that Salmonella infection can spread rapidly and extensively among hens in cage-free indoor housing, including a high frequency of internal organ involvement for invasive S. Enteritidis.

Pooling of Laying Hen Environmental Swabs and Efficacy of Salmonella Detection.

ABSTRACT: Environmental testing for Salmonella Enteritidis is required for U.S. shell egg producers with ≥3,000 hens on a farm. The egg producer assumes all costs for the mandatory testing. According to the U.S. Food and Drug Administration (FDA) Egg Rule, either manure scraper or drag swabs can be collected according to published guidelines and requirements. The present study was undertaken to determine the efficacy of Salmonella detection with one-, two-, and four-swab pools of either manure scraper or drag swabs. Resistant isolates of Salmonella serovars Enteritidis (1,000 ppm of streptomycin), Heidelberg (200 ppm of nalidixic acid [NA]), Typhimurium (200 ppm of NA), and Kentucky (200 ppm of NA) were utilized. Low (approximately 8.4 CFU) and high (approximately 84 CFU) levels of inocula were introduced onto a single swab within a pool. Single flocks from each conventional cage (manure scraper swabs) and cage-free barn (drag swabs) were monitored throughout the study at the ages required under the FDA Egg Rule. The highest and most consistent recovery of inoculum was found in single swab samples. For low dose inocula, recovery of isolates was low from single manure scraper swabs (57.9 to 29.2%) and decreased as more swabs were added to the pool. Recovery of isolates from manure scraper swabs was higher for high dose inocula, although Salmonella Heidelberg was outcompeted by the naturally occurring flora and had the lowest rate of recovery among the isolates tested. One- and two-swab pools of drag swabs had similar rates of recovery at both low and high doses for Salmonella Enteritidis, Salmonella Heidelberg, and Salmonella Typhimurium. When Salmonella Enteritidis and Salmonella Kentucky were combined in an inoculum, Salmonella Enteritidis was recovered at a much higher rate than was Salmonella Kentucky for all types of swabs and doses of inocula. Pooling of two drag swabs allowed for similar detection of low and high dose Salmonella, but the pooling of manure scraper swabs decreased detection of low dose Salmonella.

Contamination of eggs by Salmonella Enteritidis in experimentally infected laying hens of four commercial genetic lines in conventional cages and enriched colony housing.

Human illness caused by the consumption of eggs contaminated with Salmonella Enteritidis is a continuing international public health concern. This pathogen is deposited inside the edible contents of eggs as a consequence of its ability to colonize reproductive tissues in infected hens. Conditions in the housing environment can influence the persistence and transmission of avian Salmonella infections, but the food safety ramifications of different poultry management systems are not entirely clear. The present study assessed the deposition of S. Enteritidis inside eggs laid by groups of experimentally infected laying hens of 4 commercial genetic lines (designated as white egg lines W1 and W2 and brown egg lines B1 and B2). Groups of hens from each line were housed at 555 cm2 of floor space per bird in both conventional cages and colony units enriched with access to perches and nesting areas. All hens were orally inoculated with 5.75 × 107 cfu of a 2-strain S. Enteritidis mixture, and the internal contents of eggs laid 5 to 24 D post-inoculation were cultured to detect the pathogen. No significant differences in egg contamination frequencies were found between the 2 housing systems for any of the hen lines. Contaminated eggs were laid between 7 and 21 D post-inoculation at an overall frequency of 2.47%, ranging from 0.25 to 4.38% for the 4 hen lines. The frequency of S. Enteritidis recovery from egg samples was significantly (P < 0.05) lower for line B2 than for any of the other lines, and the egg contamination frequency for line W1 was significantly greater than for line W2. The overall incidence of contamination among white eggs (3.38%) was significantly higher than among brown eggs (1.56%). These results demonstrate that S. Enteritidis deposition inside eggs can vary between genetic lines of infected laying hens, but housing these hens in 2 different systems did not affect the production of contaminated eggs.

Colonization of internal organs by Salmonella Enteritidis in experimentally infected laying hens of four commercial genetic lines in conventional cages and enriched colony housing.

The prevalence of Salmonella Enteritidis in commercial egg-laying flocks is a prominent public health concern because contaminated eggs cause human illness. Deposition of this pathogen inside eggs results from bacterial colonization of reproductive tissues in infected hens. Environmental conditions can influence avian Salmonella infections, but the food safety consequences of different poultry housing systems remain uncertain. The present study assessed the invasion of internal organs by Salmonella Enteritidis in groups of experimentally infected laying hens of four commercial genetic lines (designated as white egg lines W1 and W2 and brown egg lines B1 and B2). Groups of hens from each line were housed at 555 cm2 of floor space per bird in both conventional cages and colony units enriched with access to perches and nesting areas. All hens were orally inoculated with 5.75 × 107 colony-forming units of a two-strain Salmonella Enteritidis mixture. At 6 to 7 d post-inoculation, hens were euthanized, and samples of liver, spleen, ovary, oviduct, and intestinal tract were removed for bacteriologic culturing. The frequency of Salmonella Enteritidis recovery from intestinal samples was significantly (P < 0.05) greater for the two white egg lines combined than for the two brown egg lines combined in both conventional cage (72.2% vs. 50.0%) and enriched colony housing systems (66.7% vs. 37.5%). The frequency of intestinal Salmonella Enteritidis isolation from line B1 was significantly higher from hens in conventional cages (47.2%) than in enriched colonies (22.2%), but no differences were observed for other hen lines. Line W1 yielded more positive intestinal samples than either brown egg line in conventional cages, and line B2 had fewer positive intestinal samples than all other lines in enriched colonies. There were no significant differences between hen lines or housing systems in Salmonella Enteritidis isolation from other internal organs. These results demonstrate that Salmonella Enteritidis colonization of the intestinal tract can vary between genetic lines of egg-laying hens and that some lines are subject to housing system influences on Salmonella susceptibility.

Prevalence, Persistence, and Antimicrobial Resistance of Campylobacter spp. from Eggs and Laying Hens Housed in Five Commercial Housing Systems.

Husbandry practices for laying hens in commercial egg production is a topic of interest from a social, economic, and regulatory standpoint. Animal welfare concerns regarding the use of conventional cages have arisen and consumer perceptions of hen welfare have led to a higher demand for cage-free eggs. The aim of this study was to assess the impact of housing systems on prevalence, persistence, and antimicrobial resistance (AMR) of Campylobacter from laying hens and shell eggs. A total of 425 samples were collected over a 10-month period from the North Carolina Layer Performance and Management Test and Campylobacter isolates were identified by serological, biochemical, and molecular tests. Genetic variability was evaluated using pulsed-field gel electrophoresis (PFGE) and AMR testing was performed. Prevalence of Campylobacter spp. ranged from 11.1% in the enrichable cages to 19.7% in the conventional systems. A greater prevalence of Campylobacter was found in the fecal swab samples from free-range birds compared with those of birds housed in the more intensive housing systems (p > 0.05). Overall, 72 isolates were confirmed as Campylobacter spp. by PCR. More than 90% of the isolates (n = 66) were identified as Campylobacter jejuni, followed by Campylobacter coli (n = 6). C. jejuni isolates displayed high levels of resistance to tetracycline (67%). Genetic variability of Campylobacter was high, with more than 20 PFGE patterns identified. Pattern "a" comprised 42% of isolates from all housing systems and was also the most persistent. This study suggests that housing systems of laying hens used for commercial shell egg production may impact the rate of Campylobacter shedding by layers. Isolation rates and tetracycline resistance levels of this pathogen are still of concern, emphasizing the need for well-implemented biosecurity measures on the farm.

Multiplication in Egg Yolk and Survival in Egg Albumen of Genetically and Phenotypically Characterized Salmonella Enteritidis Strains.

Prompt refrigeration of eggs to prevent the multiplication of Salmonella Enteritidis to high levels during storage is an important practice for reducing the risk of egg-transmitted human illness. The efficacy of egg refrigeration for achieving this goal depends on the interaction among the location of contamination, the ability of contaminant strains to survive or multiply, and the rate at which growth-restricting temperatures are attained. The present study assessed the significance of several characterized genetic and phenotypic properties for the capabilities of 10 Salmonella Enteritidis isolates to multiply rapidly in egg yolk and survive for several days in egg albumen during unrefrigerated (25°C) storage. The growth of small numbers of each Salmonella Enteritidis strain (approximately 101 CFU/mL) inoculated into egg yolk samples was determined after 6 and 24 h of incubation. The survival of larger numbers of Salmonella Enteritidis (approximately 105 CFU/mL) inoculated into albumen samples was determined at 24 and 96 h of incubation. In yolk, the inoculated Salmonella Enteritidis strains multiplied to mean levels of approximately 102.6 CFU/mL after 6 h of incubation and 108.3 CFU/mL after 24 h. In albumen, mean levels of approximately 104.6 CFU/mL Salmonella Enteritidis were maintained through 96 h. The concentrations of the various Salmonella strains after incubation in either yolk or albumen were distributed over relatively narrow ranges of values. Significant ( P < 0.01) differences observed among individual strains suggested that maintenance of the fimbrial gene sefD may have positive genetic selection value by improving fitness to grow inside egg yolk, whereas the antibiotic resistance gene blaTEM-1 tet(A) appeared to have negative genetic selection value by decreasing fitness to survive in egg albumen.

Serotyping of Salmonella Enterica Isolated from Mice Caught on US Poultry Farms 1995 through 1998.

In this study, a collection of Salmonella enterica subspecies obtained from live mice caught on 32 poultry farms in the Northeast US between 1995 to 1998 was evaluated to provide a historical reference for serotype distribution during a time when egg contamination by serotype Enteritidis was at its peak. Of 821 mice cultured, 157 were positive (19.1%). Seven mice harbored two serotypes of Salmonella. Nine serotypes were detected, eight of which are often associated with foodborne illness. The three most prevalent serotypes were Enteritidis, Heidelberg, and Typhimurium. Enteritidis and Typhimurium were obtained from both spleens and intestines without preference according to type of sample. In contrast, Heidelberg was isolated most often from intestines and Schwarzengrund was most often obtained from spleens. These results support that the house mouse Mus musculus was a risk factor for introduction of multiple pathogenic Salmonella serotypes in poultry raised in the Northeast US during the mid-1990s. Isolates were submitted to the Food and Drug Administration and draft genomes for 64 isolates of Salmonella enterica serovar Enteritidis data have been released through the National Center for Biotechnology Information via the GenomeTrakr network.

Frequency and Duration of Fecal Shedding of Salmonella Serovars Heidelberg and Typhimurium by Experimentally Infected Laying Hens Housed in Enriched Colony Cages at Different Stocking Densities.

Eggs contaminated with Salmonella Enteritidis are leading sources of human salmonellosis, but Salmonella Heidelberg and Salmonella Typhimurium are also egg-associated pathogens. The management practices and housing facilities characterizing different systems for housing commercial egg flocks can influence Salmonella persistence and transmission. Animal welfare aspects of poultry housing have been widely debated, but their food safety ramifications are not thoroughly understood. The present study assessed the effects of two different bird stocking densities on the frequency and duration of fecal shedding of strains of Salmonella Heidelberg and Salmonella Typhimurium in groups of experimentally infected laying hens housed in colony cages enriched with perching and nesting areas. In separate trials, laying hens were distributed into two groups housed in enriched colony cages at stocking densities of 648 and 973 cm2/bird, and a third group was housed in conventional cages at 648 cm2/bird. All hens were orally inoculated with doses of 108 colony-forming units (CFU) of either Salmonella Heidelberg or Salmonella Typhimurium. At eight weekly postinoculation intervals, samples of voided feces were collected from beneath each cage and cultured to detect Salmonella. Fecal shedding of Salmonella Heidelberg continued for 8 wk in all housing groups, but Salmonella Typhimurium shedding ceased after as little as 5 wk in enriched colony cages at low stocking density. After Salmonella Heidelberg infection, the overall frequency of positive fecal cultures for all sampling dates combined was significantly (P < 0.05) greater from either conventional cages (51.0%) or enriched colony cages (46.5%) at high stocking density than from enriched colony cages at low stocking density (33.3%). No significant differences in Salmonella Typhimurium fecal isolation were identified between housing groups. These results demonstrate that stocking density can affect intestinal colonization and fecal shedding in laying hens for some (but not necessarily all) Salmonella serovars or strains.

Frequency and Duration of Fecal Shedding of Salmonella Enteritidis by Experimentally Infected Laying Hens Housed in Enriched Colony Cages at Different Stocking Densities.

Human infections with Salmonella Enteritidis are often attributed to the consumption of contaminated eggs, so the prevalence of this pathogen in egg-laying poultry is an important public health risk factor. Numerous and complex environmental influences on Salmonella persistence and transmission are exerted by management practices and housing facilities used in commercial egg production. In recent years, the animal welfare implications of poultry housing systems have guided the development of alternatives to traditional cage-based housing, but their food safety consequences are not yet fully understood. The present study assessed the effects of different bird stocking densities on the frequency and duration of fecal shedding of S. Enteritidis in groups of experimentally infected laying hens housed in colony cages enriched with perching and nesting areas. In two trials, groups of laying hens were distributed at two stocking densities (648 and 973 cm2/bird) into enriched colony cages and (along with a group housed in conventional cages at 648 cm2/bird) orally inoculated with doses of 1.0 × 108 cfu of S. Enteritidis. At 10 weekly postinoculation intervals, samples of voided feces were collected from beneath each cage and cultured to detect S. Enteritidis. Fecal shedding of S. Enteritidis was detected for up to 10 weeks postinoculation by hens in all three housing treatment groups. The overall frequency of positive fecal cultures was significantly (P < 0.05) greater from conventional cages than from enriched colony cages (at the lower stocking density) for the total of all sampling dates (45.0 vs. 33.3%) and also for samples collected at 4-9 weeks postinfection. Likewise, the frequency of S. Enteritidis isolation from feces from conventional cages was significantly greater than from enriched colony cages (at the higher hen stocking density) for the sum of all samples (45.0 vs. 36.7%) and at 6 weeks postinoculation. Moreover, the frequency of S. Enteritidis fecal recovery from enriched colony cages at the higher hen stocking was significantly greater than from similar cages at the lower stocking density for all 10 sampling dates combined (39.4 vs. 33.3%). These results suggest that stocking density can affect S. Enteritidis intestinal colonization and fecal shedding in laying hens, but some other difference between conventional and enriched colony cage systems appears to exert an additional influence.

Colonization of internal organs by Salmonella serovars Heidelberg and Typhimurium in experimentally infected laying hens housed in enriched colony cages at different stocking densities.

Contaminated eggs produced by infected commercial laying flocks are often implicated as sources of human infections with Salmonella Enteritidis, but Salmonella serovars Heidelberg and Typhimurium have also been associated with egg-transmitted illness. Contamination of the edible contents of eggs is a consequence of the colonization of reproductive tissues in systemically infected hens. In recent years, the animal welfare implications of diverse poultry housing and management systems have been vigorously debated, but the food safety significance of laying hen housing remains uncertain. The present study evaluated the effects of 2 different bird stocking densities on the invasion of internal organs by Salmonella serovars Heidelberg and Typhimurium in groups of experimentally infected laying hens housed in colony cages enriched with perching and nesting areas. Laying hens were distributed at 2 different stocking densities (648 and 973 cm2/bird) into colony cages and (along with a group housed in conventional cages at 648 cm2/bird) orally inoculated with doses of 107 cfu of 2-strain cocktails of either Salmonella Heidelberg or Salmonella Typhimurium. At 5 to 6 d post-inoculation, hens were euthanized and samples of internal organs (cecum, liver, spleen, ovary, and oviduct) were removed for bacteriologic culturing. The overall frequency of Salmonella isolation from ceca after inoculation with strains of serovar Heidelberg (83.3%) was significantly (P < 0.001) greater than the corresponding value for strains of serovar Typhimurium (53.8%), whereas Salmonella was recovered significantly more often from both livers (85.2% vs. 53.7%; P < 0.0001) and spleens (78.7% vs. 56.5%; P = 0.0008) after inoculation with strains of serovar Typhimurium than strains of serovar Heidelberg. However, there were no significant differences (P > 0.05) between stocking densities or cage systems in the frequencies of isolation of either Salmonella serovar from any of the five sampled tissues. These results contrast with prior studies, which reported increased susceptibility to internal organ invasion by Salmonella Enteritidis among hens in conventional cages at higher stocking densities.

Metabolic parameters linked by phenotype microarray to acid resistance profiles of poultry-associated Salmonella enterica.

Phenotype microarrays were analyzed for 51 datasets derived from Salmonella enterica. The top 4 serotypes associated with poultry products and one associated with turkey, respectively Typhimurium, Enteritidis, Heidelberg, Infantis and Senftenberg, were represented. Datasets were partitioned initially into two clusters based on ranking by values at pH 4.5 (PM10 A03). Negative control wells were used to establish 90 respiratory units as the point differentiating acid resistance from sensitive strains. Thus, 24 isolates that appeared most acid-resistant were compared initially to 27 that appeared most acid-sensitive (24 × 27 format). Paired cluster analysis was also done and it included the 7 most acid-resistant and -sensitive datasets (7 × 7 format). Statistical analyses of ranked data were then calculated in order of standard deviation, probability value by the Student's t-test and a measure of the magnitude of difference called effect size. Data were reported as significant if, by order of filtering, the following parameters were calculated: i) a standard deviation of 24 respiratory units or greater from all datasets for each chemical, ii) a probability value of less than or equal to 0.03 between clusters and iii) an effect size of at least 0.50 or greater between clusters. Results suggest that between 7.89% and 23.16% of 950 chemicals differentiated acid-resistant isolates from sensitive ones, depending on the format applied. Differences were more evident at the extremes of phenotype using the subset of data in the paired 7 × 7 format. Results thus provide a strategy for selecting compounds for additional research, which may impede the emergence of acid-resistant Salmonella enterica in food.

Colonization of internal organs by Salmonella Enteritidis in experimentally infected laying hens housed in enriched colony cages at different stocking densities.

Epidemiologic analyses have linked the frequency of human infections with Salmonella enterica subspecies enterica serovar Enteritidis to the consumption of contaminated eggs and thus to the prevalence of this pathogen in commercial egg-laying flocks. Contamination of the edible contents of eggs by Salmonella Enteritidis is a consequence of the colonization of reproductive tissues in systemically infected hens. The animal welfare implications of laying hen housing systems have been widely debated, but no definitive consensus has yet emerged about the food safety significance of poultry housing options. The present study sought to determine the effects of two different bird stocking densities on the invasion of internal organs by Salmonella Enteritidis in groups of experimentally infected laying hens housed in colony cages enriched with perching and nesting areas. In two trials, groups of laying hens were distributed at two different stocking densities into colony cages and (along with a group housed in conventional cages) orally inoculated with doses of 1.0 × 10(7) cfu of Salmonella Enteritidis. At 5 to 6 d post-inoculation, hens were euthanized and samples of internal organs were removed for bacteriologic culturing. For both trials combined, Salmonella Enteritidis was recovered at a significantly (P < 0.05) greater frequency from hens in enriched colony cages at the higher stocking density than at the lower density from livers (75.0% vs. 51.4%) and ovaries (51.4% vs. 30.6%). However, spleens from hens in enriched colony cages at the higher stocking density were significantly less often positive for Salmonella Enteritidis than from hens in conventional cages at that same density (90.3% vs. 68.1%). These results suggest that stocking density can influence the susceptibility of hens to Salmonella Enteritidis, but other housing systems parameters may also contribute to the outcome of infections.