How Does Salmonella Spread within the Australian Egg Layer Industry?

1. INTRODUCTION

Salmonella enterica subsp. enterica is the most common cause of foodborne human gastroenteritis, a disease characterised by gut inflammation and self-limiting diarrhoea in humans (Winter et al., 2010). It is estimated that gastroenteritis caused by Salmonella spp. accounts for 93.8 million human cases worldwide each year (Majowicz et al., 2010). Worldwide, contaminated food products of animal origin, particularly egg and egg products, are frequently implicated in outbreaks of human salmonellosis (Chousalkar and Gole, 2016). Salmonella Enteritidis (S. Enteritidis) and Salmonella Typhimurium (S. Typhimurium) phage types have dominated globally as the most common causes of human salmonellosis (Hendriksen et al., 2011). In Australia, human infections caused by S. Enteritidis occur largely as a consequence of overseas travel, with S. Typhimurium being a predominant cause of foodborne outbreaks linked to consumption of egg and egg products (Moffatt et al., 2016). Due to strict animal importing regulations and quarantine strategies of imported parent stock in Australia, S. Enteritidis has not been widely spread in commercial poultry flocks in Australia (Chousalkar et al., 2015). Recently, there have been some reports of S. Enteritidis isolation from egg and chicken meat farms in Queensland (Graham et al., 2018) and egg farms in New South Wales. Despite some reports of S. Enteritidis isolation from layer flocks and associated food borne outbreaks, egg products associated S. Typhimurium outbreaks remain the most frequently reported in Australia (OzFoodnet 2010, 2012). Numerous egg-related human Salmonella outbreaks have prompted significant interest amongst the public, public health authorities and industry. Thorough cooking of eggs can destroy most, if not all, bacteria present, including Salmonella, and cooked eggs pose low risk to human health. If egg products or food items are prepared from raw or lightly cooked egg contents, this will not destroy all Salmonella (if present). A rough estimate of the presence of Salmonella (all species, not just pathogenic) on eggs is greater than 1 in 20,000 (Arnold et al., 2014). Considering the estimated production of eggs in Australia and per capita consumption, the risk of foodborne illness in general is very low for humans consuming eggs. It has been demonstrated in a number of regions and studies that consumption of raw or lightly cooked eggs/egg preparations significantly increases the risk of human foodborne illness. This paper presents an overview of Salmonella epidemiology on laying farms and egg contamination.

II. EPIDEMIOLOGICAL INVESTIGATIONS AND TESTING OF FLOCKS FOR SALMONELLA

Layer hens have a common opening for the intestinal, urinary and reproductive tracts. Thus, external eggshell contamination with faecal material is often unavoidable. Although vertical transmission of Salmonella from bird to egg has been demonstrated (as has been shown for S. Enteritidis), it is generally accepted that horizontal transmission is also the most likely source of contamination of shell eggs (Gantois et al., 2009).

Currently, there is no nationwide prevalence data of Salmonella on egg farms. One of the major challenges in establishing prevalence is that shedding of S. Typhimurium from known positive hens is highly variable and can be influenced by stress experienced by hens on the farm (Gole et al., 2014a). Single time point sampling may not be sufficient to determine true prevalence. Therefore, longitudinal sampling of flocks is essential. A study conducted on commercial egg farms found that S. Typhimurium PT 9 was not detectable from egg contents, although the load of Salmonella spp on eggshell was up to log 6 CFU (Gole et al., 2014d). A Salmonella survey conducted on egg farms in Queensland reported that, S. Infantis was the most prevalent strain amongst egg layer flocks (Cox et al., 2002). Another microbiological survey conducted by New South Wales Food Authority on 49 egg farms in New South wales, showed that 20 % of the farms were positive for S. Typhimurium, whereas a survey conducted on 21 egg farms by Safe Food Queensland reported that 13.5 % of farms were positive for S. Typhimurium (Cuttell et al., 2014). Overall, the epidemiological investigations provide useful information on the distribution, ecology and changes in genome of Salmonella serovars. Conducting on-farm epidemiological investigations can be challenging due to the variable level of willingness from the farmers to participate in such studies. The epidemiological investigations concluded that the prevalence and/or shedding of Salmonella is influenced by flock size, flock management, shed controls, feeding practices, frequency of rodent population, production system, stage of lay, cleaning and disinfection practices adopted by a farm, level of biosecurity, exposure to wildlife vectors (birds and foxes), single aged vs multi-age flocks on farm, resting period between batches, frequency of sampling, type of samples, number of samples and timing of sampling (Denagamage et al., 2015). Epidemiological investigations (both cross sectional and epidemiological) are expensive due to high labour and Salmonella typing costs. In some countries, there is a requirement for more intense sampling as compared to others due to the risk of S. Enteritidis. In countries where S. Enteritidis is not endemic in commercial poultry flocks, such intensive sampling of the flock is not conducted on a regular basis. Salmonella positive status of faeces, egg belt and dust are significant predictors of eggshell contamination by Salmonella (Gole et al., 2014d). In a cage production system, the prevalence of Salmonella in faeces collected from the low tier cages was significantly higher compared with the samples from the high tier cages (Gole et al., 2014a) which could be attributed to the exposure of lower tiers of cages to the dust on the floor.

Birds raised in free-range production systems are potentially exposed to more environmental stressors than caged birds, including social stress and aggression, predation or thermal challenges. Free range flocks are more likely to be infected from their exposure to wildlife vectors (such as wild birds, foxes, etc) (Wales et al., 2007; Wales and Davies 2011). Such wild life vectors can not only spread infections from farm to farm or to the community but also play a significant role in introducing different genomic types of Salmonella to the flock (Chousalkar et al., 2016). Free-range flocks are exposed to several environmental stressors, which can ultimately result in increased Salmonella shedding in laying hens. Stress can have an immunosuppressive effect in laying hens that could influence Salmonella infection and shedding. Interestingly, there was no positive correlation between faecal corticosterone metabolites and the level of Salmonella shedding (Gole et al., 2017).

It is difficult to predict or determine whether eggs produced by free range and or free range organic production systems are “high risk” compared to caged production systems as the level and prevalence of Salmonella in the flock or on the farm is attributed to individual flock and/or farm management. More recently, a longitudinal study conducted on free-range farms from day old to end of the commercial life span indicated that birds are most likely to be exposed with Salmonella during production (McWhorter and Chousalkar, 2019).

S. Typhimurium and other serovars are able to survive/persist in the shed environment (such as in dust) so regular cleaning and or removal of dust from shed is important. Use of vaccination in multi-age flocks or single aged flock is “not an ultimate intervention” for reduction of Salmonella Typhimurium because of the complexities involved in achieving control, such as the efficacy of cleaning of sheds, the lack of resting periods between batches and the possible carryover of contamination from existing flocks. Hence implementation of more than one or several interventions strategies is essential (McWhorter and Chousalkar, 2018; Sharma et al., 2018). Throughout the life span of a laying hen, Salmonella can be introduced to a farm through various vehicles such as rodents, contaminated egg trays, people movement, and contaminated trucks, introduction of infected flock, contaminated feed or contaminated equipment (Chousalkar and Gole, 2016). In Australia, the number of backyard chickens is also on the rise. The biosecurity standards of backyard chickens is highly variable and Salmonella infected flocks could pose a significant risk to commercial flocks (Manning et al., 2015). Hence, regular cleaning, disinfection of equipment and a continuous review of on-farm biosecurity standards is critical.

III. SALMONELLA PENETRATION, SURVIVAL IN AND ON EGGS

Egg and eggshell quality could play a vital role in trans-shell penetration of Salmonella spp. A good quality eggshell significantly protects the internal contents from bacterial penetration. A cracked or damaged egg encourages bacteria to move across the eggshell, which may result in food poisoning. Eggs possess a cuticle, which acts as the first line of defence against bacteria (Gole et al., 2014c). This covers the external surface of the eggshell and functions to close the eggshell pores soon after lay to decrease bacterial penetration. In warm, freshly laid eggs, the cuticle does not mature for a period of time after lay, therefore leaving some pores open for Salmonella penetration (Miyamoto et al., 1998). Several surveys have been conducted on eggs to study the level of Salmonella contamination in and on retail or first grade eggs. The Salmonella contamination of table eggs was reviewed by (Martelli and Davies, 2012). The review concluded that S. Typhimurium is often not isolated from eggs compared with S. Enteritidis. This finding was later confirmed during field epidemiological investigation (Gole et al., 2014d) where eggs laid by S. Typhimurium flocks were not always contaminated. Egg-based Salmonella surveys provide some useful information on distribution of Salmonella serovars in different parts of the world, but the results of surveys are highly variable. Moreover, egg-based Salmonella surveys do not necessarily reflect the level or prevalence of Salmonella contamination at farm or bird level. Experimental investigations also reported a lack of correlation between egg contamination and duration of Salmonella shedding in faeces (could be interpreted as individual bird infection) (Gast et al., 2005; Pande et al., 2016a). The egg handling practices on or off farm could also influence the level of Salmonella contamination on eggshell.

Eggs have natural defence barriers for protection against the bacterial penetration. Egg albumen has many antibacterial properties but S. Enteritidis phage types have the ability to replicate in the oviduct, contaminate the developing egg, multiply in the albumen (Gantois et al. 2009) and several relevant genes associated with survival of S. Enteritidis in egg white have been identified (Clavijo et al., 2006). It has been hypothesised that S. Enteritidis uses the stress induced survival mechanism for survival in the egg white and colonise the oviduct (Van Immerseel, 2010). It has also been demonstrated that the virulence properties of S. Enteritidis are unaffected by the hostile environment within the egg albumen (Baron et al., 2004). In-vitro studies found that S. Typhimurium phage types have the ability to survive in the egg albumen, egg yolk and on the eggshell surface (Gantois et al., 2008; Gole et al., 2014b) and S. Typhimurium isolates possess genes associated with the survival of Salmonella in egg albumen (McWhorter et al., 2015). However, it is not clear whether the hostile environment within the egg albumen affects the virulence of S. Typhimurium. It is well established that penetration of Salmonella serovars is influenced by temperature, egg and eggshell quality, bacterial strain, pH and moisture (De Reu et al., 2006). In-vivo studies on the mechanism of egg contamination by S. Typhimurium provided variable results and this variation could be attributed to the dose, phage type, timing and route of infection (Wales and Davies, 2011). S. Typhimurium was localised in reproductive organs, egg internal contents tested negative (Okamura et al., 2010; Pande et al., 2016a), whereas another study found that egg internal contents were contaminated when hens were infected with S. Typhimurium by the aerosol route (Leach et al., 1999). S. Typhimurium has the ability to form a viable but non-culturable state (VBNC) and retain its invasive ability (Passerat et al., 2009). It could be hypothesised that some S. Typhimurium phage types enter this VBNC state up on exposure to fresh egg white and further studies are required to investigate this hypothesis. The presence of Salmonella on the eggshell underlines the importance of proper handling of eggs in the food industry as well as in the kitchen environment to avoid cross contamination of other food items.

IV. WHERE TO FROM HERE?

An increase in production and consumption of eggs and related Salmonella outbreaks in the Australia indicates that egg related Salmonella is likely to remain a concern for public health. Salmonella diagnostics, reporting and surveillance systems have improved over the years and will continue to improve in the years to come. Given the number of different emerging Salmonella serovars, a regular review of Salmonella control strategies from farm to fork is required. On farm epidemiological surveillance is essential to control and monitor the circulating Salmonella serovars and respective changes in genotype. Several other serovars other than S. Enteritidis and S. Typhimurium have been periodically implicated in egg related Salmonella outbreaks (Glass et al., 2016). Given the ability of Salmonella serovars to transfer or acquire genes via horizontal gene transfer with plasmids, transposons, and phages (Foley et al., 2013), the evolution of further virulent strains is not unexpected. The intervention practices such as vaccination and use of egg washing can reduce the load of Salmonella spp on the egg but not eliminate it. Salmonella serovars are not only able to survive in harsh environment in production and the supply chain but are able to quickly multiply if favourable environmental conditions are available. The implementation of regulations and policies for control of Salmonella on-farm has put onus on egg farmers and processors but end users also carry a significant responsibility of safe handling of eggs and egg products. Cultural diversity has contributed to a far wider selection of food, incorporating a greater range of raw foods of animal origin into our diet. The efficacy of messages such as 'cook eggs thoroughly' or 'wash your hands' will depend both on the ability to change consumer behaviour as well as where the risk can best be mitigated (Luber, 2009). It is also important to note that the food handling behaviour could be influenced by sex, income status and age of an individual (Patil et al., 2005). There is sufficient evidence to suggest that Salmonella spp are able to survive and persist on eggshells at varied levels. While egg handling procedures coupled with kitchen cleanliness limit the possibility of infection, only 10² CFU (colony forming units) of pathogenic strains of Salmonella are required to cause disease in humans (Fabrega and Vila, 2013). Salmonella serovars also have the ability to form biofilms on egg shells at ambient temperature (Pande et al., 2016b). Biofilms on the eggshell surface could act as a vehicle for contaminating food products, further spreading the pathogenic bacteria to other hosts risking public health. Continuing research is required to refine egg handling and hygiene practices.

To improve the reporting system, the general public also need to be more vigilant and aware about the onset of clinical symptoms, and the risk of spreading infection to very young, old or immunocompromised people. Such awareness could be implemented through general physicians/medical practitioners. Continuous dialogue between egg producers, regulators and researchers is critical to develop and implement the innovative Salmonella control strategies.

Presented at the 31th Annual Australian Poultry Science Symposium 2020. For information on the next edition, click here.