Microbial populations from samples of six pooled eggs were enumerated for Enterobacteriaceae on violet red bile glucose agar (Oxoid, Australia) plates with overlay (purple-red colonies). Presumptive colonies were counted and reported as log cfu/mL of egg rinsate. Translucency tended to be higher in late lay flocks. The Enterobacteriaceae count on the egg shell surface was slightly higher in late lay flocks but was not significantly different from that of the flocks in early lay. 'Salmonella' Infantis was isolated from egg shell rinse. Samples were collected throughout the year 2010-2011. This study is on-going so, as the sample size increases, new insights will be obtained.

'Mycoplasma synoviae' ('M. synoviae') can cause respiratory disease, synovitis, peritonitis, egg apical abnormalities or a subclinical infection. The importance of 'M. synoviae' is well established in broilers but only a few studies have been conducted in layers. In the present study, the prevalence of 'M. synoviae' in commercial layer flocks was determined by ELISA using egg yolk antibodies. Subsequently, a possible correlation between the serological status of 'M. synoviae' and egg shell quality was also studied. In the flocks under study, seroprevalence of 'M. synoviae' was found to be 69 % (95 % confidence interval (CI) = 47 to 91). Statistical analysis showed that the vaccinated group (3.0 ± 0.1) had the highest translucency score as compared to infected (2.4 ± 0.1) and uninfected (2.5 ± 0.1) groups, whereas % shell reflectivity was highest in the infected group (31.41 ± 0.3) as compared to the other two groups. Shell breaking strength (39.5 ± 0.5 Newtons) and shell deformation (298.7 ± 3.8 μm) values were significantly lower in the infected group than in the uninfected and vaccinated groups. There was no significant difference among these three groups for egg quality parameters egg weight, egg shell weight, % egg shell, shell thickness.

In Australia, currently, all pullets reared for egg production are vaccinated by live attenuated strains of infectious bronchitis virus. Various vaccines and protocols to control this viral disease have been developed, although the severity of the disease varies from place to place and flock to flock. In the present trial, effects of vaccine strains on the oviduct of laying hens were assessed by determining the presence and persistence of viral load following experimental infection. There was no drop in egg production in any of the groups. Both A3 and Vic S vaccine strains were detected in the oviduct of vaccinated and unvaccinated hens, mainly on the 12th day p.i. Both the vaccines appeared to be safe for the oviduct.

Egg shell translucency, which can be due to changes in the mammillary cores and mamillary layer during the early phases of eggshell formation, has the potential to increase the incidence of microcracks in egg shells, and hence, may facilitate bacterial penetration. There was a significant correlation between egg shell translucency and egg shell penetration by 'Salmonella' Infantis. 'Salmonella' Infantis was able to penetrate translucent egg shells even at very low doses. The penetration, however, appeared to be hindered in both translucent and non-translucent eggs at 4°C, as compared with room temperature which highlights the importance of storage of eggs at refrigeration temperatures.

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°C, as compared with room temperature which highlights the importance of storage of eggs at refrigerated temperatures.

The effect of two vaccine strains of infectious bronchitis virus (IBV - VicS and A3 strains) on internal and external quality of eggs was studied in Isa Brown hens in full lay. Birds were either unvaccinated for IBV or had been vaccinated during rearing. The main effects of exposure to the vaccine viruses were that VicS resulted in paler coloured shells, mainly in the unvaccinated birds and the eggs from the hens challenged with VicS were more elongate than the other groups. These findings are consistent with our earlier findings with field strains of IBV and differ from those reported in the literature for different strains of IBV.

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 $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.

This study is an update of data presented previously (Roberts & Chousalkar, 2012) from a study which investigated egg quality at different stages of lay. Although eggs produced in Australia are considered medium to low risk for food borne illness, the egg industry in Australia is periodically implicated in cases of food poisoning. Egg shell defects may potentiate the movement of bacteria into the egg. Two egg shell characteristics were targeted: the extent of cuticle cover and the incidence of translucency. Eggs were collected from commercial caged layer flocks at different stages of lay: early (<25-40 wks), mid (40-55 wks), late (55-65 wks) and very late (>65 wks). Eggs were candled and scored for translucency. Cuticle cover was estimated using MST cuticle stain and a Konica Minolta hand-held spectrophotometer (L*a*b* colour scale). Traditional measures of egg quality were determined using specialised equipment (TSS, U.K.) Shell ultrastructural features were scored following plasma ashing of shell samples and viewing under a benchtop scanning electron microscope.

Two independent real time PCR assays were designed to detect and quantify T and N1/88 strains of infectious bronchitis virus (IBV) from faeces of experimentally-infected unvaccinated and unvaccinated laying hens. Vaccination VicS-A3-VicS during rearing can reduce the viral load in the faeces of vaccinated and T or N1/88 challenged hens. Both vaccinated and unvaccinated hens can shed T strain of IBV in faeces up to 9 weeks p.i. It is now possible to detect and differentiate N1/88 strain of IBV from vaccine strains. It is also possible to detect and quantify T and N1/88 strain of infectious bronchitis virus directly from faeces.

Egg or egg product related 'Salmonella' poisoning is a major concern for the Australian egg industry. Salmonellosis can be acquired by the ingestion of raw or undercooked eggs. 'Salmonella' Typhimurium (ST) is the most common serovar notified in Salmonella food poisoning cases in Australia. The objectives of the current study were to examine the effect of egg washing on the survival of 'Salmonella' on the eggshell surface, to investigate the penetration ability of four different 'Salmonella' Typhimurium phage types (ST PT), previously isolated from Australian layer farms, and to study the effect of egg washing on bacterial eggshell penetration. Our results indicated that there was no significant difference in survival of ST PTs on the egg shell surface of washed and unwashed eggs. Survival rate on inoculated eggshell surface was highest for ST PT 9 (83.33%) followed by ST PT 44 (53.33%), ST PT 193 (43.33%) and ST PT 170 (43.33%). All these phage types are able to penetrate the eggshell and they can survive in the egg internal contents at 20°C and 37°C in both washed and unwashed eggs. ST PT 44 penetration was significantly higher in washed eggs as compared to unwashed eggs. However, for other ST PT (PT 9, 170 and 193), we did not find any significant difference in the penetration of washed and unwashed eggs. The internal contents of whole eggs were most frequently contaminated by ST PT 44 (23.33%) followed by PT 170 (20%), PT 9 (10%) and PT 193 (10%). It was also found that there was no significant effect of incubation temperature (20°C and 37°C) on 'Salmonella' penetration.