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.
The egg contents are an ideal growth medium for microorganisms that are hazardous to humans. It has been observed that the microflora of the eggshell is dominated by gram-positive bacteria, whereas gram-negative bacteria are best equipped to overcome the antimicrobial defenses of the egg content (De Reu et al., 2008). The egg industry in Australia is periodically implicated in cases of food poisoning, and eggs are potential sources of introduction of human pathogens into Australian kitchens (Fearnley et al., 2011). Cage laying systems are the major source of whole shell eggs for the supermarkets in Australia. Laying hens have a common opening for intestinal, urinary, and reproductive tracts, which could contribute to external eggshell contamination as the egg passes through this region. The contact between fecal material and the eggshell is often unavoidable and could potentiate the entry of microorganisms into the egg. Shell contamination most likely depends on either intestinal or oviduct infection; however, the process responsible for eggshell contamination by Salmonella-infected birds in not clear. Abnormalities in eggshells (thin shells, increased shell pore numbers, translucency) may potentiate the entry of food-borne pathogens into the eggs (De Reu et al., 2008; Chousalkar et al., 2010). The Australian poultry industry is considered free from Salmonellaenterica serovar Enteritidis, which is of major concern to the food industry all over the world. Cox et al. (2002) reported that Salmonella Infantis was the predominant Salmonella serovar in the Australian egg industry. Studies on microbial contamination of eggshells have been reported (Musgrove et al., 2004, 2005). It is difficult for bacteria to move across an intact good quality eggshell. However, small defects in the eggshell may provide means for the predominant bacterial species on the eggshell to penetrate and move into the egg contents (De Reu et al., 2006). It has also been found that bacterial contamination of air cells, shells, and egg contents is more common in eggs from older hens than from younger hens (Jones et al., 2004). In the present study, visually clean eggs collected from commercial egg farms from hens were tested for the presence of Salmonella spp.
MATERIALS AND METHODS
Visually clean eggs (n = 1,560, from 26 flocks), collected from the cage fronts of commercial layer farms, were processed for isolation of Salmonella spp. from the eggshell surface, eggshell crush, and internal contents. The farms included in this study had either HyLine or ISA Brown laying hens. Eggs were candled to ensure they were intact eggs without cracks.
Eggshell Surface Wash
Sixty eggs were collected from each farm. Six pooled eggs (60 eggs tested in groups of 6) were placed into 60 mL of sterile PBS in Whirl-Pak bags (NASCO, Silver Valley, ID) and rinsed by shaking for 2 min. Before rinsing, PBS was warmed to 42°C to facilitate bacterial recovery. After a rinse sample was obtained, each egg was removed and transferred to a different sterile bag. Rinsates and intact eggs were then stored at 4°C overnight if required.
Shell Crush Methodology
For the isolation of bacteria from eggshell pores, eggshells were processed as described by Musgrove et al. (2005). Briefly, after eggshell surface processing, each eggshell surface was dipped into 70% alcohol for 1 min to kill any bacteria on the outside of the shell and was allowed to air dry in a biosafety cabinet. The eggs were cracked open into a sterile container. The inside of the eggshell was then washed with sterile phosphate-buffered saline to remove the adhering egg albumen because of the antimicrobial activity of albumen. Shell and shell membranes of 6 pooled egg samples were transferred to a sterile bag and crushed gently. To each bag, 50 mL of either buffered peptone water or nutrient broth was added. The bag was incubated at 37°C overnight for further processing.
Egg Internal Contents
The egg internal contents collected in the sterile containers were thoroughly mixed. One milliliter of egg internal content was inoculated with 4 mL of either buffered peptone water or nutrient broth for further processing.
Characterization of Bacterial Isolates
The isolation of Salmonella was carried out as described earlier by Cox et al. (2002). To isolate Salmonella spp., the inoculated buffered peptone water (from shell surface, shell crush, and egg internal contents) was incubated at 37°C overnight, and 100 μL of this sample was inoculated into Rappaport Vassiliadis broth (Oxoid, Adelaide, Australia), which was then incubated at 42°C for 24 h. A loopful of the same sample was streaked onto xylose lysine deoxycholate agar (Oxoid) and bismuth sulphite agar (Oxoid) plates. Individual bacterial colonies were selected and subjected to Gram staining. Presumptive Salmonella colonies from bismuth sulphite or xylose lysine deoxycholate agar were selected and used to stab inoculate triple sugar iron agar slopes (Oxoid). After incubation at 37°C, the inoculated triple sugar iron agar slopes were examined at intervals of 24 h, up to 72 h, for typical Salmonella reactions. The presumptive Salmonella colonies were also tested by slide agglutination reaction using poly O and poly H antigens (Becton Dickinson and CO., North Ryde, Australia). Slopes of isolates were sent to the Institute of Medical and Veterinary Sciences (IMVS), Adelaide, Australia for Salmonella confirmation.
RESULTS AND DISCUSSION
Six Salmonella Infantis isolates and one Salmonella serotype 4,12:d isolate were obtained from the eggshell rinse of eggs collected from the flocks. Salmonella spp. were isolated from 5 out of 26 sampled flocks. Our finding regarding the predominance of Salmonella Infantis in the Australian layer industry is in agreement with Cox et al. (2002), who conducted longitudinal surveys on selected layer farms; however, the prevalence of Salmonella Infantis in the current study was low and based on egg sampling. Hence, further extensive survey work is essential.
Several studies conducted earlier found that Salmonella Infantis is a prevalent serovar within the poultry industry worldwide. A large Salmonella Infantis epidemic in broiler chickens, associated with a high incidence of human salmonellosis in Finland during 1975, implicated contaminated feed as a possible source of the infection (Raevuori et al., 1978). Salmonella Infantis was also reported as the second most common serovar isolated from layer flocks in Canada (Poppe et al., 1991) and Germany (Hinz et al., 1996). Salmonella Infantis outbreaks have not been recorded recently in Australia by OzFoodNet during the years 2004 to 2006 (OzFoodNet Working Group, 2005, 2006, and 2007). However, Salmonella Infantis was consistently a common serotype in human notifications in South Australia between 2005 and 2008 and a common serotype in 2 other Australian states in 2004 and 2006 (OzFoodNet Working Group, 2005, 2006, and 2007). Salmonella serotype 4,12:d is a rare serotype and has been isolated from rodent feces, which highlights the importance of rodent control.
More recently Salmonella Typhimurum (serovars 108, 9, and 44) outbreaks have been reported (OzFoodNet Working Group, 2009). The Australian egg industry has often been blamed for Salmonella outbreaks, although little research has been conducted to study the prevalence of Salmonella in table eggs. Daughtry et al. (2005) undertook a microbiological survey of commercial eggs in Australia to determine the prevalence of Salmonella contamination. During Daughtry’s study, eggshell wash or eggshell crush was not tested, and Salmonella spp. were not isolated from egg internal contents of 20,000 eggs sampled. Salmonella spp. were not isolated in our study conducted earlier (Chousalkar et al., 2010), which could be attributed to the small sample size (n = 500).
Genetic diversity of Salmonella spp. isolated during the present study needs further investigation. It is also essential to study the public health significance of these isolates. In the present study, shell rinse and crush methods were used to recover Salmonella spp. from commercial shell eggs as described earlier by Musgrove et al. (2004); however, Salmonella spp. were not isolated from eggshell crush or egg internal contents. Cox et al. (2002) observed that Salmonella Infantis strain inoculated onto the surface of the egg could penetrate the eggshell and had the potential to grow within the contents of the egg under experimental conditions. However, in their experiment, the dose of bacterial inoculation was very high and, moreover, eggs were washed in 70% ethanol before external inoculation, which may have damaged the cuticle, reducing its protective properties.
The current findings regarding the isolation of Salmonella spp. from eggshell contents and eggshell pores imply that an intact cuticle on an unwashed egg may prevent entry of bacteria across the eggshell. However controlled experiments are necessary to confirm this hypothesis. Previous research suggests that washing removes fecal material and reduces microbial load on the eggshell surface, which could ultimately reduce the likelihood of horizontal transmission occurring as well as reducing the potential for cross contamination during food handling and preparation. However, research has also shown that wet washing can damage the cuticle layer, thereby leaving pores exposed and potentiating bacterial penetration (Sparks and Burgess, 1993). Egg washing is widely used in many countries, including Australia (Hutchison et al., 2004). However, in most European countries, there is some debate about the benefits of washing eggs.
This research was conducted within the Poultry CRC, established and supported under the Australian Government’s Cooperative Research Centres Program. We acknowledge Rowly Horn (Rowly Horn Services, Sydney, Australia) for his help during sample collection.