Refrigeration of eggs influences the virulence of Salmonella Typhimurium.

Salmonella Typhimurium is a human pathogen associated with eggs and egg-derived products. In Australia, it is recommended that eggs should be refrigerated to prevent condensation that can enhance bacterial penetration across the eggshell. Except for the United States, the guidelines on egg refrigeration are not prescriptive. In the current study, in-vitro and in-vivo experiments were conducted to understand the role of egg storage temperatures (refrigerated vs ambient) on bacterial load and the virulence genes expression of Salmonella Typhimurium. The in-vitro egg study showed that the load of Salmonella Typhimurium significantly increased in yolk and albumen stored at 25 °C. The gene expression study showed that ompR, misL, pefA, spvA, shdA, bapA, and csgB were significantly up-regulated in the egg yolk stored at 5 °C and 25 °C for 96 h; however, an in-vivo study revealed that mice infected with egg yolk stored at 25 °C, developed salmonellosis from day 3 post-infection (p.i.). Mice fed with inoculated egg yolk, albumen, or eggshell wash stored at refrigerated temperature did not show signs of salmonellosis during the period of the experiment. Data obtained in this study highlighted the importance of egg refrigeration in terms of improving product safety.

Acidification and extended storage at room temperature of mayonnaise reduce Salmonella Typhimurium virulence and viability.

Despite food safety recommendations, raw egg-based foods, such as mayonnaise, are frequently identified as the source of Salmonella during outbreaks. Acidification and storage temperature have been linked with reduced bacterial culturability. Raw egg-based sauces stored at 25 °C have historically been linked with faster decline of Salmonella culturability than preparations stored at 5 °C. This study aimed to determine whether reduced culturability in acidified mayonnaise correlated with reduced in vitro bacterial motility, invasiveness and viability as well as disease-causing capacity in BALB/c mice. Acidification of mayonnaise and incubation at 25 °C for 4 h significantly reduced culturability of Salmonella Typhimurium DT9 but was dependent on initial bacterial load. Bacteria recovered from acidified mayonnaise exhibited reduced invasiveness into polarized cultured intestinal epithelial cells and 12 h post inoculation were no longer invasive suggesting a reduced capacity to cause disease. To confirm this, BALB/c mice were inoculated with Salmonella Typhimurium contaminated mayonnaise stored at 5 °C or 25 °C for 12, 24, 48, 72, and 96 h. Mice inoculated with mayonnaise incubated at 5 °C for 12 and 24 h exhibited mild to moderate disease symptoms; all other mayonnaise treatment groups did not exhibit disease symptoms. In acidified mayonnaise, Salmonella Typhimurium DT9 exhibited a global downregulation of metabolism, stress response, and virulence genes upon addition to mayonnaise. After 4 h of incubation at both 5 °C and 25 °C, however, the vast majority of genes were upregulated which was maintained over the 96-hour experiment suggesting that bacteria were severely stressed. Salmonella Typhimurium DT9 cells were isolated from mayonnaise samples and ATP production was quantified. At both 5 °C and 25 °C, ATP production decreased in acidified mayonnaise preparations. At 25 °C, ATP production decreased more rapidly than at 5 °C. After 24 h, ATP production of bacteria in mayonnaise stored at 25 °C was not significantly different from the dead control group. Thus, the current recommendation of only serving freshly prepared raw egg-sauces or refrigerating immediately after preparation, could be placing consumers at higher risk for contracting salmonellosis.

In vivo passage of Salmonella Typhimurium results in minor mutations in the bacterial genome and increases in vitro invasiveness.

Eggs and raw or undercooked egg-containing food items are frequently identified as the bacterial source during epidemiolocal investigation of Salmonella outbreaks. Multi-locus variable number of tandem repeats analysis (MLVA) is a widely used Salmonella typing method enabling the study of diversity within populations of the same serotype. In vivo passage, however, has been linked with changes in MLVA type and more broadly the Salmonella genome. We sought to investigate whether in vivo passage through layer hens had an effect on MLVA type as well as the bacterial genome and whether any mutations affected bacterial virulence. Layer hens were infected with either Salmonella Typhimurium DT9 (03-24-11-11-523) as part of a single infection or were co-infected with an equal amount of Salmonella Mbandaka. Salmonella shedding in both single and co-infected birds was variable over the course of the 16-week experiment. Salmonella Typhimurium and Salmonella Mbandaka were identified in feces of co-infected birds. Salmonella colonies isolated from fecal samples were subtyped using MLVA. A single change in SSTR-6 was observed in Salmonella Typhimurium strains isolated from co-infected birds. Isolates of Salmonella Typhimurium of both the parent (03-24-11-11-523) and modified (03-24-12-11-523) MLVA type were sequenced and compared with the genome of the parent strain. Sequence analysis revealed that in vivo passaging resulted in minor mutation events. Passaged isolates exhibited significantly higher invasiveness in cultured human intestinal epithelial cells than the parent strain. The microevolution observed in this study suggests that changes in MLVA may arise more commonly and may have clinical significance.

Correlating bacterial shedding with fecal corticosterone levels and serological responses from layer hens experimentally infected with Salmonella Typhimurium.

Salmonella Enteriditis and Salmonella Typhimurium are commonly isolated during egg-related outbreaks of salmonellosis and represent a significant international public health issue. In Australia, Salmonella Typhimurium is the most common serovar identified in egg product related foodborne outbreaks. While a number of studies have investigated Salmonella shedding and host responses to infection, they have been conducted over a short time period. The present study sought to characterise bacterial shedding and host responses to infection in hens infected with only Salmonella Typhimurium or co-infected with both Salmonella Typhimurium and Salmonella Mbandaka over a 16 week period. Salmonella shedding was quantified using the most probable number and qPCR methods and was highly variable over the course of the experiment. On day 1, fecal corticosterone metabolites in birds infected with Salmonella Typhimurium (674.2 ± 109.3 pg/mg) were significantly higher than control (238.0 ± 12.62 pg/mg) or co-infected (175.4 ± 8.58 pg/mg) birds. The onset of lay occurred between weeks 6-8 post-infection (pi) and Fecal corticosterone metabolite (FCM) concentrations increased in both control and co-infected birds. Antibody responses to infection were monitored in both serum and yolk samples. Salmonella Typhimurium specific antibody was lower in co-infected animals than monoinfected animals. Bacterial loads in internal organs were characterised to determine persistence. Spleen, liver and caecal tonsils were positive for bacteria in both groups, indicating that Salmonella was not cleared from the birds and internal organ colonization could serve as a reservoir for continued bacterial shedding.

Dynamics of Salmonella Shedding and Welfare of Hens in Free-Range Egg Production Systems.

The current study investigated the effect of environmental stressors (i.e., weather changes) on Salmonella shedding in free-range production systems and the correlations with behavioral and physiological measures (i.e., fecal glucocorticoid metabolites). This involved longitudinal and point-in-time surveys of Salmonella shedding and environmental contamination on four commercial free-range layer farms. The shedding of Salmonella was variable across free-range farms and in different seasons. There was no significant effect of season on the Salmonella prevalence during this investigation. In this study, the combined Salmonella most probable number (MPN) counts in environmental (including feces, egg belt, dust, nest box, and ramp) samples were highest in samples collected during the summer season (4th sampling, performed in February). The predominant serovars isolated during this study were Salmonella enterica serovar Mbandaka and Salmonella enterica serovar Typhimurium phage types 135 and 135a. These two phage types were involved in several egg product-related Salmonella outbreaks in humans. Multilocus variable-number tandem-repeat analysis (MLVA) results indicated that MLVA types detected from human food poisoning cases exhibited MLVA patterns similar to the strains isolated during this study. All Salmonella isolates (n = 209) were tested for 15 different genes involved in adhesion, invasion, and survival of Salmonella spp. We also observed variations for sopA, ironA, and misL There were no positive correlations between fecal corticosterone metabolite (FCM) and Salmonella prevalence and/or shedding in feces. Also, there were no positive correlations between Salmonella prevalence and Salmonella count (log MPN) and any of the other welfare parameters.IMPORTANCE In this study, the welfare of laying hens and Salmonella shedding were compared over a prolonged period of time in field conditions. This study investigated the long-term shedding of Salmonella serovars in a free-range egg production system. Given that there is increasing demand for free-range eggs, it is essential to understand the risks associated with such a production system.

Bacterial contamination of eggs and behaviour of poultry flocks in the free range environment.

The free range production system is becoming more common in Australia and is expected to increase. Free range hens are exposed to more stressors in comparison to hens from barn and cage systems and it is suggested that stress can increase bacterial shedding on eggs. The aims of this study were to examine the level of total bacteria and Enterobacteriaceae populations, as well as the presence of Salmonella and Campylobacter, in eggs collected from two free range flocks on two different farms and to conduct longitudinal observations of the behaviour and welfare of hens in the free range production system. Hen age (weeks) was shown to have a significant effect (increase) on the level of total bacteria on the egg shell surface and in shell pores, as well as having an effect on feather condition score. As the hens aged, the frequency of external visual egg characteristics increased, as did feather condition score (where feather condition was poorer). These observations indicate areas which should be investigated further to improve the food safety of eggs and optimise the welfare of free range hens.