Rupa Guraya

Colonization of Specific Regions of the Reproductive Tract and Deposition at Different Locations Inside Eggs Laid by Hens Infected with Salmonella Enteritidis or Salmonella Heidelberg

Abstract Internal contamination of eggs by Salmonella Enteritidis has been a significant source of human illness for several decades and is the focus of a recently proposed U.S. Food and Drug Administration regulatory plan. Salmonella Heidelberg has also been identified as an egg-transmitted human pathogen. The deposition of Salmonella strains inside eggs is apparently a consequence of reproductive tissue colonization in infected laying hens, but the relationship between colonization of specific regions of the reproductive tract and deposition in different locations within eggs is not well documented. In the present study, groups of laying hens were experimentally infected with large oral doses of Salmonella Heidelberg, Salmonella Enteritidis phage type 13a, or Salmonella Enteritidis phage type 14b. For all of these isolates, the overall frequency of ovarian colonization (34.0%) was significantly higher than the frequency of recovery from either the upper (22.9%) or lower (18.1%) regions of the oviduct. No significant differences were observed between the frequencies of Salmonella isolation from egg yolk and albumen (4.0% and 3.3%, respectively). Some significant differences between Salmonella isolates were observed in the frequency of recovery from eggs, but not in the frequency or patterns of recovery from reproductive organs. Accordingly, although the ability of these Salmonella isolates to colonize different regions of the reproductive tract in laying hens was reflected in deposition in both yolk and albumen, there was no indication that any specific affinity of individual isolates for particular regions of this tract produced distinctive patterns of deposition in eggs.

Salmonella Enteritidis Deposition in Eggs after Experimental Infection of Laying Hens with Different Oral Doses

The continuing attribution of human Salmonella Enteritidis infections to internally contaminated eggs has necessitated the commitment of substantial public and private resources to Salmonella Enteritidis testing and control programs in commercial laying flocks. Cost-effective risk-reduction requires a detailed and comprehensive understanding of how Salmonella Enteritidis infections in hens result in deposition of the pathogen inside eggs. The present study sought to resolve some incompletely defined aspects of the relationship between Salmonella Enteritidis oral-exposure dose levels in experimentally infected laying hens and the frequency and location of subsequent egg contamination. In two trials, groups of specific-pathogen-free hens were experimentally inoculated with oral doses of 10(4), 10(6), or 10(8) CFU of a phage type 4 Salmonella Enteritidis strain. Eggs were collected 5 to 23 days postinoculation, and the yolk and albumen of each egg were cultured separately to detect Salmonella Enteritidis contamination. Larger oral doses of Salmonella Enteritidis administered to hens were associated with significant increases in the frequencies of both yolk and albumen contamination. Moreover, Salmonella Enteritidis was found in the albumen of a far-higher proportion of contaminated eggs from hens given the largest dose than from the other two groups. Salmonella Enteritidis contamination was detected in 0.7% of yolk and 0.2% of albumen samples after inoculation of hens with 10(4) CFU, 4.0% of yolk and 1.7% of albumen samples after inoculation with 10(6) CFU, and 6.5% of yolk and 10.8% of albumen samples after inoculation with 10(8) CFU. These results demonstrate that oral-exposure doses of Salmonella Enteritidis for laying hens can significantly affect both the frequency and location of deposition of this pathogen inside eggs.

Effect of Refrigeration on In Vitro Penetration of Salmonella Enteritidis through the Egg Yolk Membrane

Internally contaminated eggs have been implicated as leading sources of transmission of Salmonella Enteritidis (SE) to humans. Although SE is not often deposited inside the nutrient-rich yolks of naturally contaminated eggs, penetration through the vitelline membrane to reach the yolk contents could result in rapid bacterial multiplication. In previous studies, such penetration has been observed occasionally at warm temperatures during experiments with in vitro egg contamination models. The present study was conducted to determine whether refrigeration affects the frequency of in vitro SE penetration of the egg yolk membrane. After inoculation of small numbers of SE onto the outside of the vitelline membranes of intact yolks, immediate refrigeration of contaminated samples prevented the penetration of SE into the egg yolk contents during 24 h of storage. However, SE penetrated inside the yolk contents in 4% of contaminated egg samples refrigerated after 2 h of storage at 30 degrees C, 15% of samples refrigerated after 6 h of storage at 30 degrees C, and 40% of samples stored at 30 degrees C for 24 h (48 samples per treatment group). These results highlight the value of prompt refrigeration for restricting the opportunities for SE to multiply to high numbers inside the yolks of contaminated eggs.

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.

Colonization of avian reproductive-tract tissues by variant subpopulations of Salmonella Enteritidis.

Abstract Leghorn hens were infected with Salmonella Enteritidis cultures of known genomic content and subpopulation characteristics to determine comparative abilities to colonize the avian reproductive tract. Group 1 received phage-type (PT)4 22079, which is a dimorphic subpopulation that can both contaminate eggs and form biofilm. Group 2 received a 90:10 mixture of monomorphic PT13a strains 21027 and 21046, which produce biofilm or contaminate eggs, respectively. Group 3 received a 10:90 mixture of the same two PT13a strains, respectively. Trials were repeated three times and a total of 30 hens per treatment group were infected. Dosage was by oral gavage and was calculated as 8.6 ± 2.01 × 107 colony-forming units per hen. Liver, spleen, and three different sections of oviduct (ovary, upper oviduct, and lower oviduct) were cultured per bird. Results were that all three groups had livers and spleens that were mostly positive (90.0% and 94.4% of 270 hens cultured, respectively). Reproductive-tract organs yielded 75 positives from 270 hens (27.8%), and treatment groups ranged from a low of 6.7% to a high of 76.7% positive cultures in any one trial. There was no significant difference between the numbers of positive reproductive-tract samples between treatment groups due to variance. These results suggest that the status of the reproductive tract at the time of infection may impact recovery of culture-positive tissue and contribute to variance. It is suggested that Salmonella Enteritidis cultures that vary in subpopulation composition have subtle differences in colonization of reproductive tissue that contribute to variance in egg contamination. Culture of non–reproductive-tract organs such as the liver and spleen was overall more reliable for detection of infected hens. The spleen was especially useful for detection because of its small size. Further research is needed to determine how sex hormones influence the infection pathway that results in egg contamination.

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.

Multiplication of Salmonella Enteritidis in Egg Yolks after Inoculation outside, on, and inside Vitelline Membranes and Storage at Different Temperatures

Prompt refrigeration to restrict bacterial growth is important for reducing eggborne transmission of Salmonella enterica serovar Enteritidis (SE). The nutrient-rich yolk interior is a relatively infrequent location for initial SE deposition in eggs, but migration across the vitelline membrane can result in rapid bacterial multiplication during storage at warm temperatures. The objective of the present study was to measure the multiplication of SE in yolks after introduction at three different locations and subsequent storage at a range of temperatures. Using an in vitro egg contamination model, approximately 100 CFU of SE was inoculated either inside yolks, onto the exterior surface of vitelline membranes, or into the adjacent albumen. After storage of samples from each inoculation group at 10, 15, 20, and 25°C for 24 h, SE was enumerated in yolks. For all three inoculation locations, the final SE levels in yolks increased significantly with increasing storage temperatures. At all storage temperatures, significant differences in SE multiplication were observed between inoculation sites (yolk inoculation>vitelline membrane inoculation>albumen inoculation). At 25°C, final log concentrations of 7.759 CFU of SE per ml (yolk inoculation), 2.014 CFU/ml (vitelline membrane inoculation), and 0.757 CFU/ml (albumen inoculation) were attained in yolks after storage. These results demonstrate that, even when the initial site of SE deposition is outside the egg yolk, substantial multiplication supported by yolk nutrients can occur during the first day of storage and the risk of bacterial growth increases at higher ambient storage temperatures.

In vitro penetration of Salmonella Enteritidis through yolk membranes of eggs from 6 genetically distinct commercial lines of laying hens

Although deposition of Salmonella Enteritidis inside yolks is less common than deposition in albumen or on the vitelline (yolk) membrane in naturally contaminated eggs laid by infected hens, bacterial migration into the yolk to reach its nutrient-rich contents could lead to extensive multiplication. The present study used an in vitro egg contamination model to assess the ability of small initial numbers of Salmonella Enteritidis to penetrate the vitelline membrane and multiply inside yolks of eggs laid by 6 genetically distinct commercial lines of hens during 24 h of storage at 30 degrees C. Eggs from each line were tested at 4 different hen ages by inoculation of approximately 100 cfu of Salmonella Enteritidis onto the outside of the vitelline membranes of intact yolks in plastic centrifuge tubes and then adding back the albumen into each tube before incubation. Overall, the frequency of penetration of Salmonella Enteritidis into the yolk contents of eggs from individual lines of hens ranged from 30 to 58% and the mean concentration of Salmonella Enteritidis in yolk contents after incubation ranged from 0.8 to 2.0 log(10) cfu/mL. For both of these parameters, values for one hen line were significantly higher than for 2 other lines, but no other differences were observed. Hen age did not have a significant effect on egg yolk penetration by Salmonella Enteritidis. These results indicate that opportunities for the migration and growth of small initial numbers of Salmonella Enteritidis to attain more dangerous levels inside contaminated eggs during storage at warm temperatures can sometimes vary between different lines of laying hens.

Multiplication of Salmonella Enteritidis on the Yolk Membrane and Penetration to the Yolk Contents at 30°C in an In Vitro Egg Contamination Model

Refrigeration to limit bacterial multiplication is a critical aspect of efforts to control the transmission of Salmonella enterica serovar Enteritidis (SE) to consumers of contaminated eggs. Although the nutrient-rich yolk interior is an uncommon location for SE contamination in freshly laid, naturally contaminated eggs, migration across the vitelline membrane could lead to rapid bacterial multiplication even when the initial site of deposition is outside the yolk. Multiplication on the yolk membrane (before, or in addition to, multiplication within the yolk contents) could be another source of increased risk to consumers. The present study used an in vitro egg contamination model to compare the abilities of four strains of SE to either multiply in association with the yolk membrane or migrate through that membrane to reach the yolk contents during 36 h of incubation at 30 degrees C. After inoculation onto the exterior surface of intact, whole yolks, all four SE strains penetrated the vitelline membrane to reach the yolk contents (at an overall frequency of 11.5%) after 12 h of incubation. The mean log concentration of SE was significantly higher in whole yolks (including yolk membranes) than in yolk contents at both 12 h (0.818 versus 0.167 CFU/ ml) and 36 h (2.767 versus 1.402 CFU/ml) of incubation. These results demonstrate that SE multiplication on the vitelline membrane may both precede and exceed multiplication resulting from penetration into the yolk contents during the first 36 h of unrefrigerated storage, reinforcing the importance of rapid refrigeration for protecting consumers from egg-transmitted illness.