Characterization of highly pathogenic avian influenza virus in retail dairy products in the US.

In March 2024, clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (HPAIV) was detected in dairy cattle in the US, and it was discovered that the virus could be detected in raw milk. Although affected cow's milk is diverted from human consumption and current pasteurization requirements are expected to reduce or eliminate infectious HPAIV from the milk supply, a study was conducted to characterize whether the virus could be detected by quantitative real-time RT-PCR (qrRT-PCR) in pasteurized retail dairy products and, if detected, to determine whether the virus was viable. From 18 April to 22 April 2024, a total of 297 samples of Grade A pasteurized retail milk products (23 product types) were collected from 17 US states that represented products from 132 processors in 38 states. Viral RNA was detected in 60 samples (20.2%), with qrRT-PCR-based quantity estimates (non-infectious) of up to 5.4log1050% egg infectious doses per mL, with a mean and median of 3.0log10/mL and 2.9log10/mL, respectively. Samples that were positive for type A influenza by qrRT-PCR were confirmed to be clade 2.3.4.4 H5 HPAIV by qrRT-PCR. No infectious virus was detected in any of the qrRT-PCR-positive samples in embryonating chicken eggs. Further studies are needed to monitor the milk supply, but these results provide evidence that the infectious virus did not enter the US pasteurized milk supply before control measures for HPAIV were implemented in dairy cattle.IMPORTANCEHighly pathogenic avian influenza virus (HPAIV) infections in US dairy cattle were first confirmed in March 2024. Because the virus could be detected in raw milk, a study was conducted to determine whether it had entered the retail food supply. Pasteurized dairy products were collected from 17 states in April 2024. Viral RNA was detected in one in five samples, but infectious virus was not detected. This provides a snapshot of HPAIV in milk products early in the event and reinforces that with current safety measures, infectious viruses in milk are unlikely to enter the food supply.

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.

Persistence of fecal shedding of Salmonella Enteritidis by experimentally infected laying hens housed in conventional or enriched cages.

Salmonella Enteritidis can be deposited inside eggs laid by infected hens, so the prevalence of this pathogen in commercial egg-producing flocks is an important risk factor for human illness. Opportunities for the introduction, transmission, and persistence of salmonellae in poultry are potentially influenced by flock housing and management systems. Animal welfare concerns have spurred the development of alternatives to traditional cage-based housing. However, the consequences of poultry housing systems for food safety have not been fully resolved by prior research. The present study assessed the effects of two different housing systems (conventional cages and colony cages enriched with perching and nesting areas) on the persistence of fecal shedding of Salmonella Enteritidis by groups of experimentally infected laying hens. In each of two trials, 136 hens were distributed among cages of both housing systems and orally inoculated with doses of 10(8) cfu of Salmonella Enteritidis (phage type 13a in one trial and phage type 4 in the other). At weekly intervals, samples of voided feces were collected from beneath each cage and cultured to detect Salmonella Enteritidis. Fecal shedding of Salmonella Enteritidis was detected for up to 8 wk post-inoculation by hens housed in enriched colony cages and 10 wk by hens housed in conventional cages. For both trials combined, the frequency of positive fecal cultures was significantly (P < 0.05) greater for conventional cages than for enriched colony cages at 1 wk (84.7 vs. 71.5%), 2 wk (54.2 vs. 31.3%), 3 wk (21.5 vs. 7.6%), and 4 wk (9.7 vs. 2.8%) post-inoculation. These results demonstrate that the susceptibility of hens to intestinal colonization by Salmonella Enteritidis can differ between conventional and enriched cage-based production systems, although this effect does not necessarily translate into a corresponding difference in the longer-term persistence of fecal shedding.

Contamination of eggs by Salmonella Enteritidis in experimentally infected laying hens housed in conventional or enriched cages.

Both epidemiologic analyses and active disease surveillance confirm an ongoing strong association between human salmonellosis and the prevalence of Salmonella enterica subspecies enterica serovar Enteritidis in commercial egg flocks. The majority of human illnesses caused by this pathogen are attributed to the consumption of contaminated eggs. Animal welfare concerns have increasingly influenced commercial poultry production practices in recent years, but the food safety implications of different housing systems for egg-laying hens are not definitively understood. The present study assessed the effects of 2 different housing systems (conventional cages and colony cages enriched with perching and nesting areas) on the frequency of Salmonella Enteritidis contamination inside eggs laid by experimentally infected laying hens. In each of 2 trials, groups of laying hens housed in each cage system were orally inoculated with doses of 1.0 × 10(8) cfu of Salmonella Enteritidis. All eggs laid between 5 and 25 d postinoculation were collected and cultured to detect internal contamination with Salmonella Enteritidis. For both trials combined, Salmonella Enteritidis was recovered from 3.97% of eggs laid by hens in conventional cages and 3.58% of eggs laid by hens in enriched cages. No significant differences (P > 0.05) in the frequency of egg contamination were observed between the 2 housing systems.

Horizontal transmission of Salmonella Enteritidis in experimentally infected laying hens housed in conventional or enriched cages.

The majority of human illnesses caused by Salmonella Enteritidis are attributed to contaminated eggs, and the prevalence of this pathogen in commercial laying flocks has been identified as a leading epidemiologic risk factor. Flock housing and management systems can affect opportunities for the introduction, transmission, and persistence of foodborne pathogens in poultry. The animal welfare implications of different types of housing for laying hens have been widely discussed in recent years, but the food safety consequences of these production systems remain incompletely understood. The present study assessed the effects of 2 different housing systems (conventional cages and colony cages enriched with perching and nesting areas) on the horizontal transmission of experimentally introduced Salmonella Enteritidis infection within groups of laying hens. In each of 2 trials, 136 hens were distributed among cages of both housing systems and approximately one-third of the hens in each cage were orally inoculated with doses of 10(8) cfu of Salmonella Enteritidis (phage type 13a in one trial and phage type 4 in the other). At regular intervals through 23 d postinoculation, cloacal swabs were collected from all hens (inoculated and uninoculated) and cultured for Salmonella Enteritidis. Horizontal contact transmission of infection was observed for both Salmonella Enteritidis strains, reaching peak prevalence values of 27.1% of uninoculated hens in conventional cages and 22.7% in enriched cages. However, no significant differences (P > 0.05) in the overall frequencies of horizontal Salmonella Enteritidis transmission were evident between the 2 types of housing. These results suggest that opportunities for Salmonella Enteritidis infection to spread horizontally throughout laying flocks may be similar in conventional and enriched cage-based production systems.

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.

Inactivation of Highly Pathogenic Avian Influenza Virus with High-temperature Short Time Continuous Flow Pasteurization and Virus Detection in Bulk Milk Tanks.

Infections of dairy cattle with clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (HPAIV) were reported in March 2024 in the U.S. and viable virus was detected at high levels in raw milk from infected cows. This study aimed to determine the potential quantities of infectious HPAIV in raw milk in affected states where herds were confirmed positive by USDA for HPAIV (and therefore were not representative of the entire population), and to confirm that the commonly used continuous flow pasteurization using the FDA approved 72 °C (161°F) for 15 s conditions for high-temperature short time (HTST) processing, will inactivate the virus. Double-blinded raw milk samples from bulk storage tanks from farms (n = 275) were collected in four affected states. Samples were screened for influenza A using quantitative real-time RT-PCR (qrRT-PCR) of which 158 (57.5%) were positive and were subsequently quantified in embryonating chicken eggs. Thirty-nine qrRT-PCR positive samples (24.8%) were positive for infectious virus with a median titer of 3.5 log10 50% egg infectious doses (EID50) per mL. To closely simulate commercial milk pasteurization processing systems, a pilot-scale continuous flow pasteurizer was used to evaluate HPAIV inactivation in artificially contaminated raw milk using the most common legal conditions in the US: 72 °C (161°F) for 15 s. Among all replicates at two flow rates (n = 5 at 0.5 L/min; n = 4 at 1 L/min), no viable virus was detected. A mean reduction of ≥5.8 ± 0.2 log10 EID50/mL occurred during the heating phase where the milk is brought to 72.5 °C before the holding tube. Estimates from heat-transfer analysis support that standard U.S. continuous flow HTST pasteurization parameters will inactivate >12 log10 EID50/mL of HPAIV, which is ∼9 log10 EID50/mL greater than the median quantity of infectious virus detected in raw milk from bulk storage tank samples. These findings demonstrate that the US milk supply is safe when pasteurized.

Kinetics model comparison for the inactivation of Salmonella serotypes Enteritidis and Oranienburg in 10% salted liquid whole egg.

The goal of this study was to determine the inactivation kinetics of Salmonella in commercial 10% salted liquid whole egg (LWE) to assist the U.S. Department of Agriculture in writing new liquid egg pasteurization guidelines. Current data are not sufficient for predicting thermal inactivation kinetics of Salmonella spp. for use in updating pasteurization guidelines for many types of liquid egg products, including salted LWE (SLWE). This is, in part, due to variations in Salmonella strains and changes in the processing of liquid egg products that have arisen in the past 40 years. Pasteurization guidelines are currently being reevaluated in light of recent risk assessments. Heat-resistant Salmonella serovars Enteritidis and Oranienburg were composited and mixed into 10% SLWE, resulting in final populations of approximately 5.7-7.8 log colony-forming units (CFU)/mL. Inoculated egg was injected into glass capillary tubes, flame-sealed, and heated in a water bath at 60, 62.2, 63.3, 64.3, or 66°C. Contents were surface-plated and incubated at 37°C for 24 h. Survival curves were not log-linear (log levels versus time), but decreased rapidly, and after initial periods became linear. Asymptotic decimal reduction values at each temperature were calculated from survivor curves with a minimum inactivation of 5.0 log CFU/mL. The asymptotic thermal D-values for SLWE were 3.47, 2.23, 1.79, 1.46, and 1.04 min at 60, 62.2, 63.3, 64.3, or 66°C, respectively. The calculated thermal z-value was 11.5°C. A model that predicts lethality for given times and temperatures that was developed predicted that the current pasteurization requirements for 10% SLWE (i.e., 63.3°C for 3.5 min, or 62.2°C for 6.2 min) are not sufficient to inactivate 7 log CFU/mL of Salmonella and only achieve approximately 4 log CFU/mL inactivation. This model will assist egg-products manufacturers and regulatory agencies in designing pasteurization processes to ensure product safety.

Colonization of internal organs by Salmonella Enteritidis in experimentally infected laying hens housed in conventional or enriched cages.

More human illnesses caused by Salmonella enterica subspecies enterica serovar Enteritidis throughout the world have been linked to the consumption of contaminated eggs than to any other food vehicle. Deposition of this pathogen in the edible contents of eggs occurs when systemic infections of laying hens involve colonization of reproductive organs. In recent years, the consequences of different housing systems for laying flocks have become the focus of international attention from both animal welfare and public health perspectives. Nevertheless, many questions remain unresolved regarding the food safety implications of various laying hen production systems. The present study assessed the effects of 2 different housing types (conventional cages and colony cages enriched with perching, nesting, and scratching areas) on the invasion of internal organs by Salmonella Enteritidis in experimentally infected laying hens. In 2 trials, groups of laying hens housed in each cage system were orally inoculated with doses of 1.0 × 10(7) cfu of Salmonella Enteritidis. At 5 to 6 d postinoculation, hens were euthanized and samples of internal organs were removed for bacteriologic culturing. For both trials combined, Salmonella Enteritidis was recovered from 95.3% of cecal samples, with no significant differences observed between housing systems. However, Salmonella Enteritidis was detected at significantly (P < 0.05) higher frequencies from hens in conventional cages than from hens in enriched cages for samples of livers (96.9 vs. 75.0%), spleens (93.8 vs. 53.1%), ovaries (25.0 vs. 10.4%), and oviducts (19.8 vs. 2.1%). These results demonstrate that differences in housing systems for egg-laying flocks can affect the susceptibility of hens to colonization of internal organs by Salmonella Enteritidis.

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: 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.

A mathematical model of inactivation kinetics for a four-strain composite of Salmonella Enteritidis and Oranienburg in commercial liquid egg yolk.

The goal of this study was to develop a general model of inactivation of salmonellae in commercial liquid egg yolk for temperatures ranging from 58°C to 66°C by studying the inactivation kinetics of Salmonella in liquid egg yolk. Heat-resistant salmonellae (three serovars of Enteritidis [two of phage type 8 and one PT 13] and one Oranienburg) were grown to stationary phase in Tryptic Soy Broth and concentrated 10-fold by centrifugation. Each inoculum was added to liquid egg yolk and mixed thoroughly, resulting in a final population of ca. 7 log CFU/ml egg yolk. Inoculated yolk was injected into sterile glass capillary tubes, flame-sealed and heated in a water bath at 58, 60, 62, 64, and 66°C. Capillary tubes were ethanol sanitized, rinsed, and contents were extracted. Yolk was diluted, surface plated onto Tryptic Soy Agar+0.1% sodium pyruvate and 50 µg/ml nalidixic acid and incubated at 37°C for 24 h before colonies were enumerated. Decimal reduction values were calculated from survivor curves with a minimum inactivation of 6 log CFU/ml at each temperature. Survival curves (except for 66°C) featured initial lag periods before first order linear inactivation. Estimated asymptotic D-values were 1.83 min at 58°C, 0.69 min at 60°C, 0.26 min at 62°C, 0.096 min at 64°C and 0.036 min at 66°C. The estimate of the asymptotic z-value was ca. 4.7°C with standard error of 0.07°C. A linear relationship between the log(10) of the lag times and temperature was observed. A general kinetic model of inactivation was developed. The results of the study provide information that can be used by processors to aid in producing safe pasteurized egg yolk products and for satisfying pasteurization performance standards and developing industry guidance.

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.

Low Dose Infection of Hens in Lay with Salmonella enterica Serovar Enteritidis from Different Genomic Clades.

Salmonella enterica serovar Enteritidis is the leading cause of salmonellosis in people, and modeling of infections in chickens is used to identify intervention strategies. A review of 80 manuscripts encompassing 119 experiments indicated that the mean dose of infection was 108 CFU per bird. Experiments of less than 106 CFU were primarily conducted in immature birds. To address a lack of information on the impact of low dosages on the hen at lay, two experiments were conducted in triplicate. Experiment A addressed issues associated with vaccination; thus, hens were infected intramuscularly at 103, 105, and 107 CFU. For Experiment B, which was focused more on colonization and invasion, hens were infected orally with 5 × 103 CFU with 4 strains from different genomic clades. Samples from liver, spleen, ovarian pedicle, and paired ceca in both experiments were cultured 5, 6, 7, and 8 days postinfection. Eggshell microbiome taxa were assessed in Experiment B. Results indicated that dosages of 103 CFU in both experiments produced enough positive samples to be used within models. The intramuscular route resulted in approximately twice as many positive samples as the oral route. The kinetics of infection appeared to differ between low and high dosages suggestive of a J-curve response. These results could impact risk assessments if the hen at lay has a nonlinear response to infectious dose.

Infección por dosis baja en gallinas de postura con Salmonella enterica Serovar Enteritidis de diferentes clados genómicos. Salmonella enterica serovar Enteritidis es la principal causa de salmonelosis en las personas y el modelo de infecciones en pollos se utiliza para identificar estrategias de intervención. Una revisión de 80 manuscritos que abarca 119 experimentos indicó que la dosis media de infección fue de 108 unidades formadoras de colonias (UFC) por ave. Los experimentos de menos de 106 UFC se realizaron principalmente en aves inmaduras. Para abordar la falta de información sobre el impacto de las dosis bajas en gallinas de postura, se realizaron dos experimentos por triplicado. El experimento A abordó los problemas asociados con la vacunación; así, las gallinas se infectaron por vía intramuscular con 103, 105 y 107 UFC. Para el Experimento B, que se enfocó más en la colonización y la invasión, las gallinas se infectaron por vía oral con 5×103 UFC con 4 cepas de diferentes clados genómicos. Las muestras de hígado, bazo, pedículo ovárico y pares de sacos ciegos se cultivaron en ambos experimentos a los cinco, seis, siete y ocho días después de la infección. Los taxones del microbioma del cascarón de huevo se evaluaron en el Experimento B. Los resultados indicaron que las dosis de 103 UFC en ambos experimentos produjeron suficientes muestras positivas para ser utilizadas dentro de los modelos. La ruta intramuscular dio como resultado aproximadamente el doble de muestras positivas que la ruta oral. La cinética de la infección parece diferir entre las dosis bajas y altas que sugieren una respuesta tipo curva J. Estos resultados podrían afectar las evaluaciones de riesgo si las gallinas de postura tienen una respuesta no lineal a la dosis infecciosa.

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.

Can acceptable quality angel food cakes be made using pasteurized shell eggs? The effects of mixing factors on functional properties of angel food cakes.

Due to recent Salmonella outbreaks, the pasteurized shell egg market is rapidly growing. One objection to using pasteurized eggs is the belief that they will produce unacceptable angel food cakes. Eggs were pasteurized using a hot water immersion process (56.7°C for 60 min) similar to that used by industry. Angel food cakes were made from the pasteurized egg white (PEW) as well as from raw egg white (REW) for comparison. Meringues were made using three mixer speed settings (low, medium, and high) and three durations for each speed. Functional qualities such as egg foaming were evaluated. Angel food cakes were compared in terms of cake volume, texture profile, and color values. When the optimal processing factors used for REW were applied to PEW, an inferior meringue was formed. However, by increasing the mixing time for PEW by 200% at the highest speed, an acceptable meringue was formed. The best angel food cake prepared from PEW had a volume only 6.8% less than that of the best cake prepared from REW. Texture profile analyses showed that the best angel food cake made from PEW was 13% firmer, 7.4% less springy, and 62% chewier than that from REW. Color analyses showed that PEW made a slightly darker colored cake crust than REW, although there were no significant differences in the crumb color. Modifying the mixing conditions for PEW resulted in angel food cakes with quality similar to that of cakes made with REW, thus overcoming an objection to using safer pasteurized shell eggs.

Thermal Inactivation Kinetics of Three Heat-Resistant Salmonella Strains in Whole Liquid Egg.

The heat resistance of three heat-resistant strains of Salmonella was determined in whole liquid egg (WLE). Inoculated samples in glass capillary tubes were completely immersed in a circulating water bath and held at 56, 58, 60, 62, and 64°C for predetermined lengths of time. The recovery medium was tryptic soy agar with 0.1% sodium pyruvate and 50 ppm of nalidixic acid. Survival data were fitted using log-linear, log-linear with shoulder, and Weibull models using GInaFiT version 1.7. Based on the R2 and mean square error, the log-linear with shoulder and Weibull models consistently produced a better fit to Salmonella survival curves obtained at these temperatures. Contaminated WLE must be heated at 56, 60, and 64°C for at least 33.2, 2.7, and 0.31 min, respectively, to achieve a 4-log reduction of Salmonella; 39.0, 3.1, and 0.34 min, respectively, for a 5-log reduction; and 45.0, 3.5, and 0.39 min, respectively, for a 6-log reduction. The z-values calculated from the D-values were 3.67 and 4.18°C for the log-linear with shoulder and Weibull models, respectively. Thermal death times presented in this study will be beneficial for WLE distributors and regulatory agencies when designing pasteurization processes to effectively eliminate Salmonella in WLE, thereby ensuring the microbiological safety of the product.