J. R. Roberts

Effect of Egg Washing and Correlation between Eggshell Characteristics and Egg Penetration by Various Salmonella Typhimurium Strains

Salmonella is an important foodborne pathogen, causing an estimated 11,992 cases of infection in Australia per year. Egg or egg product related salmonellosis is a major concern for the egg industry. Worldwide, S. Typhimurium is one of the most common serovars identified in Salmonella food poisoning cases. The current study investigated the ability of five S. Typhimurium strains to penetrate washed and unwashed eggs using whole egg and agar egg penetration methods. All S. Typhimurium strains were able to penetrate eggshells and survive in egg albumen (at 20°C) according to whole egg penetration results. Polymerase Chain Reaction results demonstrated that S. Typhimurium strain 2 (103 and 105 CFU/mL), and strain 5 (103 and 105 CFU/mL) egg penetration was significantly higher (p<0.05) in washed eggs when compared to unwashed eggs. Statistical analysis of the agar penetration experiment indicated that S. Typhimurium was able to penetrate washed eggs at a significantly higher rate when compared to unwashed eggs (p<0.05). When compared to unwashed eggs, washed eggs also had significantly damaged cuticles. Statistical analysis also indicated that eggshell penetration by S. Typhimurium was related to various eggshell ultrastructural features such as cap quality, alignment, erosion, confluence, Type B bodies and cuticle cover.

Recovery of Salmonella and Escherichia coli from commercial egg shells and effect of translucency on bacterial penetration in eggs

This experiment was conducted to study the prevalence of Salmonella and Escherichia coli (E. coli). from the surface of egg shells, egg shell membranes or pores, and internal contents from unwashed eggs collected from commercial caged layer farms in Australia. Egg shell swabs, shell crush and egg internal contents (yolk and albumen) of an individual egg were processed for bacteriological examination. Salmonella spp. were not detected from any of the egg shell surfaces, egg shell crush or egg internal contents. Thirty five E. coli isolates were isolated from the egg shell surface. Ten E. coli strains were also isolated from shell crush. However, the internal contents of eggs appeared to be sterile. Polymerase chain reaction was performed on forty-five E. coli isolates using primers for heat stable enterotoxin genes A and B (STa and STb) and also for colicin V gene (cvaC). STa gene was detected in four E. coli isolates isolated from egg shell surfaces. All the E. coli isolates were negative for STb and cvaC genes. These data provide useful information regarding the prevalence of virulent E. coli and Salmonella spp. on and in unwashed eggs collected from layer farms. These data also suggest that unwashed eggs collected from caged layer farms are unlikely to be sources of Salmonella outbreaks. Egg shell translucency could be due to changes in the mammillary layer and mamillary cores during the early phases of egg shell formation and has the potential to increase the incidence of microcracks in egg shells, and hence, may be responsible for bacterial penetration. There was a significant correlation between egg shell translucency and egg shell penetration by Salmonella Infantis and E coli. Both strains of bacteria were able to penetrate the translucent egg shells even at very low doses. The penetration, however, was hindered in both translucent and non translucent eggs at 4 degrees C, as compared with room temperature which highlights the importance of storage of eggs at refrigerated temperatures.

The location of protoporphyrin in the eggshell of brown-shelled eggs

Protoporphyrin has been identified as the main eggshell pigment in brown-shelled eggs. However, there has been some uncertainty concerning the distribution of the pigment within the shell (and cuticle) in brown-shelled eggs. Most previous studies have suggested that the bulk of the shell pigment is deposited in the cuticle of the shell. The present study measured the levels of protoporphyrin in intact eggshells and in shells from which the cuticle had been removed, using eggs from flocks at 3 different ages. This enabled the calculation of the relative amount of protoporphyrin in the calcareous eggshell and the cuticle layer of the eggshell. The majority of the protoporphyrin pigment was located in the calcareous part of the eggshell (80-87%) with a minority contained within the cuticle (13-20%). These findings suggest that studies focused on maintenance of shell color in brown-shelled eggs need to consider the stage of egg formation at which the reduction in pigment deposition is occurring.

The effects of cortisol and testosterone on renal function in male Antechinus stuartii (Marsupialia)

Abstract Seasonal changes in the physiology of Antechinus stuartii result in complete male mortality after mating. The most important endocrine changes in males are large rises in plasma testosterone and cortisol concentrations. Glomerular filtration rate (GFR) in males declines coincident with high plasma testosterone and cortisol. In the present study GFRs were measured in males captured in May (when endogenous plasma testosterone and cortisol levels are low) and given depot injections of either saline, testosterone-only, cortisol-only or testosterone plus cortisol at doses designed to mimic plasma levels during the mating period. GFR decreased significantly with testosterone injection, independent of cortisol treatment. Urinary concentrations of sodium and chloride, and osmolality decreased significantly with cortisol treatment, although the addition of testosterone reversed the effect. Total urinary excretion of electrolytes was similar between groups. Plasma potassium levels significantly increased in testosterone plus cortisol treated males. Plasma sodium levels significantly increased and plasma chloride significantly decreased in all groups treated with cortisol. Water consumption significantly increased in all cortisol-treated males and food consumption significantly increased in all testosterone-treated males. The seasonal renal functional changes observed in A. stuartii were mimicked by testosterone administration.

Recovery of Salmonella from eggshell wash, eggshell crush, and egg internal contents of unwashed commercial shell eggs in Australia

The experiment was conducted to study the prevalence of Salmonella spp. on the eggshell surface, eggshell membranes or pores, and in egg internal contents from unwashed eggs collected from commercial caged layer farms in Australia. Eggshell rinsate, shell crush, and egg internal contents (yolk and albumen) of eggs were processed for Salmonella spp. Salmonella Infantis and Salmonella subspecies 1, serotype 4,12:d were isolated from the eggshell surface. Salmonella spp. were not isolated from any eggshell crush or egg internal contents. It would appear that the occurrence of Salmonella in the Australian egg industry is low.

LNA probe-based real-time RT-PCR for the detection of infectious bronchitis virus from the oviduct of unvaccinated and vaccinated laying hens.

In the present study, LNA-probe based real-time PCR was designed for the detection and absolute quantification of infectious bronchitis virus (IBV) from the oviduct of unvaccinated and vaccinated hens after IBV challenge. Using a recombinant plasmid standard, the detection limit of the reaction was found to be 10 copies and independent assay runs showed reproducible Ct values. Amongst the unvaccinated hens, the virus could be detected between 6 and 20 days post-infection (p.i.), with a peak of viral load between 10 and 14 days p.i. The virus was also detectable in the oviduct of vaccinated, challenged hens although the viral load was much lower compared to the viral load in the oviduct of unvaccinated, challenged hens. This indicates that rearing phase vaccination can offer significant protection of the fully functional oviduct against a pathogenic strain of IBV. The present test will be useful for the rapid identification of IBV directly from clinical samples. Most vaccination trials investigating the efficacy of vaccines for layer and breeder hens have been conducted based on the respiratory tract response. Evaluation of viral load from the oviduct of vaccinated and unvaccinated hens is an efficient method for assessing oviduct protection in commercial laying hens.

Egg quality and age of laying hens: implications for product safety

Eggs were collected from commercial caged layer flocks in early, mid, late and very late lay. Eggs were candled and scored for translucency. Cuticle cover was estimated using MST Cuticle Stain and a Konica Minolta hand-held spectrophotometer. Traditional measures of egg quality were determined using specialised equipment. Shell ultrastructural features were scored following plasma ashing of shell samples and viewing under a benchtop scanning electron microscope. Translucency score was significantly higher in late lay than for all other age groups. Shell quality declined with increasing flock age. However, the extent of cuticle cover on the egg shell was not significantly different among flock age groups. The incidence of shell ultrastructural features associated with good quality shells was lower for older flocks and incidence of ultrastructural features associated with poorer quality shells was higher for older flocks. Translucency score had a low correlation with the ultrastructural features of the mammillary layer.

Effects of egg shell quality and washing on Salmonella Infantis penetration

The vast majority of eggs in Australia are washed prior to packing to remove dirt and fecal material and to reduce the microbial contamination of the egg shell. The egg contents can be an ideal growth medium for microorganisms which can result in human illness if eggs are stored improperly and eaten raw or undercooked, and it is estimated that egg-related salmonellosis is costing Australia

Survey of Enterobacteriaceae contamination of table eggs collected from layer flocks in Australia

44 million per year. Egg shell characteristics such as shell thickness, amount of cuticle present, and thickness of individual egg shell layers can affect the ease with which bacteria can penetrate the egg shell and washing could partially or completely remove the cuticle layer. The current study was conducted to investigate the effects of egg washing on cuticle cover and effects of egg shell quality and cuticle cover on Salmonella Infantis penetration of the egg shell. A higher incidence of unfavorable ultrastructural variables of the mammillary layer such as late fusion, type B bodies, type A bodies, poor cap quality, alignment, depression, erosion and cubics were recorded in Salmonella penetrated areas of egg shells. The influence of egg washing on the ability of Salmonella Infantis on the egg shell surface to enter the egg internal contents was also investigated using culture-based agar egg penetration and real-time qPCR based experiments. The results from the current study indicate that washing affected cuticle cover. There were no significant differences in Salmonella Infantis penetration of washed or unwashed eggs. Egg shell translucency may have effects on Salmonella Infantis penetration of the egg shell. The qPCR assay was more sensitive for detection of Salmonella Infantis from egg shell wash and internal contents than traditional microbiological methods. The agar egg and whole egg inoculation experiments indicated that Salmonella Infantis penetrated the egg shells. Egg washing not only can be highly effective at removing Salmonella Infantis from the egg shell surface, but also allows subsequent trans-shell and trans-membrane penetration into the egg. Consequently, it is important to prevent recontamination of the egg after washing.

Prevalence of antibodies to Mycoplasma synoviae in laying hens and possible effects on egg shell quality

In the present study, eggs from commercial caged layer flocks at different stages of lay in Australia were collected. Enterobacteriaceae populations from eggshell surface and eggshell pore were enumerated and these populations characterized using API® Rapid 20E strips. The eggshell surface, eggshell pore and egg internal content samples were also processed for the isolation of Salmonella and these isolates were tested for the presence or absence of several virulence genes (prgH, sopB, spiC, orfL, invA, sifA, sitC, misL). Results indicated that there was no significant difference in total Enterobacteriaceae count on the eggs of the flock from early, mid or late lay flocks. Enterobacteriaceae isolates were of 11 different genera which included: Cedecea, Citrobacter, Enterobacter, Escherichia, Klebsiella, Kluyvera, Leclercia, Pantoea, Salmonella, Serratia and Yersinia. Out of all 153 identified Enterobacteriaceae isolates, the Escherichia genus was reported most frequently (60.78%). Results also indicated that overall there were 4.51% (14/310) Salmonella positive pooled samples. In this study, 14 Salmonella strains were isolated, serotyping confirmed that 12 out of them were Salmonella Infantis and the 2 others were Salmonella enterica subsp. enterica serovar 4,12:d: Polymerase chain reaction results indicated that all Salmonella Infantis isolates harboured invA, misL, orfL, prgH, sifA, sitC, sopB and spiC genes which suggests that Salmonella Infantis strains isolated from eggshell surface may have the capacity to invade and survive in macrophages.