Deana R. Jones

Comparison of dkgB‐linked intergenic sequence ribotyping to DNA microarray hybridization for assigning serotype to Salmonella enterica

Two DNA-based methods were compared for the ability to assign serotype to 139 isolates of Salmonella enterica ssp. I. Intergenic sequence ribotyping (ISR) evaluated single nucleotide polymorphisms occurring in a 5S ribosomal gene region and flanking sequences bordering the gene dkgB. A DNA microarray hybridization method that assessed the presence and the absence of sets of genes was the second method. Serotype was assigned for 128 (92.1%) of submissions by the two DNA methods. ISR detected mixtures of serotypes within single colonies and it cost substantially less than Kauffmann–White serotyping and DNA microarray hybridization. Decreasing the cost of serotyping S. enterica while maintaining reliability may encourage routine testing and research.

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.

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.

Modified pressure imaging for egg crack detection and resulting egg quality

Cracks in the shell surface compromise the primary barrier for external microbial contamination of the egg. Microcracks are very small cracks in the shell surface that are difficult to detect by human graders. New technology has been developed that uses modified pressure and imaging to detect microcracks in eggs. Research has shown the system to have an accuracy of 99.6% in detecting both cracked and intact eggs. A study was undertaken to determine if quality differences existed between modified pressure imaged and control eggs during extended cold storage. Three replicates were conducted with eggs stored at 4 degrees C for 5 wk with weekly quality testing. The physical quality factors monitored were Haugh units, albumen height, egg weight, shell strength, vitelline membrane strength and elasticity, and whole egg total solids. All measurements were conducted on individual eggs (12/treatments per replicate) each week with the exception of whole egg solids, which were determined from 3 pools (4 eggs each)/treatment per replicate each week. Percentage of whole egg total solids was the only significant difference (P < 0.05) between treatments (23.65% modified pressure imaged and 23.47% control). There was a significant difference (P < 0.05) for egg weight between replicates (60.82, 58.02, and 60.58 g for replicates 1, 2, and 3, respectively). Therefore, imaging eggs in the modified pressure system for microcrack detection did not alter egg quality during extended cold storage. Utilizing the modified pressure crack detection technology would result in fewer cracked eggs reaching the consumer, consequently enhancing food safety without affecting product quality.

Inactivation of Salmonella in Shell Eggs by Hot Water Immersion and Its Effect on Quality

Thermal inactivation kinetics of heat resistant strains of Salmonella Enteritidis in shell eggs processed by hot water immersion were determined and the effects of the processing on egg quality were evaluated. Shell eggs were inoculated with a composite of heat resistant Salmonella Enteritidis (SE) strains PT8 C405, 2 (FSIS #OB030832), and 6 (FSIS #OB040159). Eggs were immersed in a circulating hot water bath for various times and temperatures. Come-up time of the coldest location within the egg was 21 min. SE was reduced by 4.5 log at both hot water immersion treatments of 56.7 C for 60 min and 55.6 °C for 100 min. Decimal reduction times (D-values) at 54.4, 55.6, and 56.7 °C were 51.8, 14.6, and 9.33 min, respectively. The z-value was 3.07 °C. Following treatments that resulted in a 4.5 log reduction (56.7 °C/60 min and 55.6 °C/100 min), the surviving population of SE remained static during 4 wk of refrigerated storage. After processing under conditions resulting in 4.5 log reductions, the Haugh unit and albumen height significantly increased (P < 0.01) and yolk index significantly decreased (P < 0.05). The shell dynamic stiffness significantly increased (P < 0.05), while static compression shell strength showed no significant difference (P < 0.05). Vitelline membrane strength significantly increased (P < 0.05); although, no significant difference (P < 0.05) was observed in vitelline membrane elasticity. In summary, the hot water immersion process inactivated heat resistant SE in shell eggs by 4.5 log, but also significantly affected several egg quality characteristics.

Salmonella contamination in shell eggs exposed to modified-pressure imaging for microcrack detection

Microcracks in egg shells are a food safety risk and are difficult for professional human graders to detect. Modified-pressure imaging technology with 99.6% accuracy has been developed to detect microcracks. This study was conducted to determine whether the microcrack detection system would increase penetration of Salmonella into egg contents or lead to cross-contamination within the system. Thirty dozen grade A large white retail eggs were used for each of 3 replicates. Cracked eggs were removed and 72 eggs/replicate were dip inoculated in buffered peptone water containing 10(5) cfu/mL of nalidixic acid-resistant Salmonella Typhimurium (ST), whereas 144 eggs were dipped in sterile buffered peptone water. All eggs were incubated overnight at 25°C before imaging. Forty-five eggs of each treatment were imaged in the following order: control, inoculated, control. Imaged and nonimaged eggs from each treatment were used for cultural analysis of a shell rinse, shell emulsion, and contents sample for each egg. The ST levels were monitored on brilliant green sulfa agar with 200 mg/L of nalidixic acid. Egg contents were also enriched to determine the prevalence of ST in low levels. Salmonella Typhimurium was not detected on or in any of the control eggs, including the eggs imaged after the inoculated eggs. The highest level of ST was detected in inoculated shell emulsions (4.79 log cfu/mL). No differences in ST levels were found for any sample location between imaged and nonimaged inoculated eggs. Therefore, the modified-pressure imaging system for microcrack detection did not result in microbial cross-contamination or increase the level of microbial penetration in inoculated eggs. The imaging system can be used to assess eggs for cracks without negative food safety implications.

Recovery of Salmonella enterica serovar Enteritidis from hens initially infected with serovar Kentucky.

Salmonella enterica serovars Enteritidis and Kentucky differ greatly in epidemiology. We wanted to know if the non-pathogenic serotype Kentucky impacted the recovery of the pathogen Enteritidis from chickens. To explore this issue, 4 groups of hens were treated as follows: (i) hens were inoculated orally with Kentucky and injected intramuscularly 2 weeks later with Enteritidis, (ii) hens were contact infected with Kentucky and then with Enteritidis, (iii) hens were injected with Enteritidis only, and (iv) hens were contact infected with Enteritidis only. Hens exposed orally to serotype Kentucky received 10 exp9 CFU, and hens injected with serotype Enteritidis received 10 exp7 CFU intramuscularly. Contact infected hens were kept in rooms with deliberately infected hens. Droppings, cecal tonsils and 5 internal organs were sampled and cultured at 6, 13 and 20 days post-infection from the 4 groups. Egg production was monitored. Results suggest that non-pathogenic serotypes of Salmonella may mitigate recovery of Enteritidis from chickens exposed by contact. In summary, we show results from an initial experiment intended to investigate if multiple serotypes impact the ecology of pathogenic S. enterica on-farm.

Salmonella and Impact on Egg Production

There is a strong association between the incidence of human illness and the prevalence of Salmonella in commercial egg-producing poultry. Although most egg-associated disease is attributed to Salmonella Enteritidis, other serovars are sometimes implicated. Deposition of S. Enteritidis within the edible contents of eggs results from colonization of reproductive tissues in infected hens. Environmental conditions influence opportunities for Salmonella introduction, transmission, and persistence in laying flocks. Environmental influences are shaped by the various housing systems used in egg production. Substantial public and private resources have been invested in comprehensive S. Enteritidis testing and risk reduction programs for egg-laying chickens. A strategy involving multiple interventions throughout the egg production cycle is recommended as the most effective approach for controlling S. Enteritidis. Controlling temperature is critical for restricting Salmonella growth inside eggs.