Control and monitoring of salmonella in egg-laying chickens

I. INTRODUCTION

Egg-associated Salmonella infection is a significant international public health problem. Internal contamination of eggs with S. Enteritidis has been the principal concern in North America and Europe, whereas external contamination with S. Typhimurium has been the predominant issue in Australia. Some strategies for controlling egg-borne Salmonella are designed to act with precision against epidemiologically important serovars, but others are applicable against all serovars. The incorporation of particular risk reduction practices into control programs is guided by their efficacy and cost-effectiveness under local conditions. This paper discusses the example of S. Enteritidis control in the United States to illustrate the utilization of both serovar-specific and serovar-independent strategies.

Over a period of nearly three decades, a high prevalence of S. Enteritidis infections in many regions of the world has been attributed to the consumption of contaminated eggs (Pires et al., 2014). Both retrospective epidemiological analysis and active disease surveillance have associated the incidence of human illness with the prevalence of S. Enteritidis in commercial egg-laying flocks (Arnold et al., 2014a). In a multi-national European survey (De Knegt et al., 2015), laying hens were found to be the most prominent reservoir for human salmonellosis, accounting for 42% of all cases (96% of which involved S. Enteritidis). Implementation of federal egg safety regulations in the USA was estimated to potentially reduce annual human health costs (due to 79,000 egg-related illnesses) by USD $1.4 billion (US Food and Drug Administration, 2009).

II. S. ENTERITIDIS INFECTION AND EGG CONTAMINATION

Young chicks are highly susceptible to colonization of the intestinal tract by S. Enteritidis, and infection introduced shortly after hatching can sometimes persist until maturity (Van Immerseel et al., 2004). Older hens are less susceptible and gut colonization usually declines steadily after exposure to the pathogen (Gast et al., 2011), although extended persistence is sometimes observed. Sustained intestinal carriage in even a small proportion of the hens in a flock could prolong opportunities for horizontal transmission and subsequent egg contamination. Fecal shedding of Salmonella by colonized birds is a leading source for continuing contamination of the poultry housing environment (Trampel et al., 2014). Mature hens infected with large oral doses of S. Enteritidis can sometimes shed this organism in their feces for several months (Gast et al., 2013b). In commercial flocks, the prevalence of Salmonella shedding in feces typically declines gradually after peaking just before egg laying begins (Gole et al., 2014), although the overall prevalence of fecal shedding in flocks may fluctuate considerably over time. Highly persistent fecal shedding of S. Enteritidis in a flock is not always indicative of the likelihood of systemic infection or egg contamination.

S. Enteritidis can invade past the intestinal tract to colonize livers and spleens of laying hens within just a few hours after they are orally infected (He et al., 2010). Colonization of internal organs declines steadily during the first few weeks after mature birds are exposed to S. Enteritidis, although infection may persist in some individuals (Gast et al., 2007b). Deposition of S. Enteritidis inside the edible interior contents of eggs results principally from colonization of reproductive organs (Gantois et al., 2009). However, a high frequency of reproductive organ invasion by Salmonella does not inevitably lead to egg contamination at a correspondingly high incidence (Gast et al., 2004). S. Enteritidis can invade both the ovary (the site of yolk maturation) and oviduct (the site of albumen secretion around descending yolks), and can thus be deposited in either of these major edible egg fractions (Gast et al., 2007b).

In experimentally infected hens, the likelihood of S. Enteritidis contamination inside eggs is directly related to the orally administered bacterial dose (Gast et al., 2013a). Exposure by horizontal contact has predictably led to the production of very few contaminated eggs. Nevertheless, the deposition of S. Enteritidis usually occurs at low frequencies and involves small initial populations of contaminants, even after inoculation with very large doses (Gast and Holt, 2000a). In commercial flocks, naturally occurring S. Enteritidis infections are often acquired via exposure to low pathogen doses from environmental sources or horizontal contact, and are accordingly associated with highly infrequent egg contamination (DeWinter et al., 2011). The location of Salmonella deposition within eggs is pivotal for determining whether the pathogen has an opportunity to multiply during storage. Egg yolk offers abundant nutrients that support rapid and prolific bacterial growth at warm temperatures (Gurtler and Conner, 2009), but albumen proteins can limit iron availability and disrupt bacterial membranes (Baron et al., 2016). S. Enteritidis is most often deposited in the albumen or on the exterior surface of the vitelline (yolk) membrane of contaminated eggs (Gast and Holt, 2001), and it can rapidly migrate across this membrane to access yolk nutrients at warm temperatures (Gast et al., 2010). However, prompt transfer of eggs to refrigeration temperatures can significantly diminish Salmonella penetration into and multiplication inside egg yolks (Gast et al. 2006).

III. EGG CONTAMINATION BY SALMONELLA STRAINS AND SEROVARS

Different Salmonella strains or serovars can vary significantly from each other in their characteristic consequences for infected laying hens. S. Enteritidis strains have sometimes differed very substantially in the frequencies at which they cause reproductive organ invasion and egg contamination after experimental infection of hens (Gast and Holt, 2000a). Strains which efficiently cause egg contamination have been differentiated from other environmental salmonellae by the expression of traits such as adherence to reproductive tract mucosa and survival in forming eggs. Among the responsible genes for these abilities are those found in the major pathogenicity islands, involved in cell wall or lipopolysaccharide structure, or related to stress responses (Raspoet et al., 2014). Deposition inside developing eggs may be the ultimate consequence of sequential expression of multiple phenotypic properties which are each necessary at specific stages of the overall infection process (Guard et al., 2010). Even between strains of a single S. Enteritidis phage type, the accumulation of small genetic changes may lead to divergence in abilities for invasion of reproductive tissues and eggs (Guard et al., 2011). However, individual strains have not been reported to have affinities for particular reproductive tract sites or locations inside eggs (Gast et al., 2007b). Although phage typing of S. Enteritidis isolates has been valuable for establishing epidemiological relationships, phage types have not been linked to any consistent pattern of abilities to cause egg contamination (Gantois et al., 2009).

The poultry housing environment may be a reservoir from which strains able to cause systemic infection and egg contamination may periodically emerge. Environmental S. Enteritidis isolates typically include a greater diversity of phage types than are recovered from contaminated eggs. The expression of diverse putative Salmonella virulence factors (including flagella, fimbria, outer membrane proteins, and iron uptake systems) can be influenced by environmental conditions such as pH and temperature. Impaired resistance to environmental stressors has been reported to reduce the pathogenicity of S. Enteritidis isolates for chickens (Shah, 2014). Additional pressure to select for expression of virulence properties is also exerted in the tissues of infected hens. Highly expressed genes in S. Enteritidis isolates from oviducts of infected hens were similarly highly expressed in isolates from eggs (Gantois et al., 2008). Repeated passage of S. Enteritidis strains through the internal organs of infected hens increased their association with egg contamination (Gast et al., 2003).

Although differences between individual Salmonella strains have been found regarding multiplication in egg yolks (and penetration through yolk membranes (Gast et al., 2007a), these properties do not seem to be associated with phage type. The frequency of S. Enteritidis migration into yolks can vary among eggs from genetically different commercial lines of laying hens (Gast et al., 2010). S. Enteritidis strains have been reported to survive better in egg albumen than isolates of other serovars (De Vylder et al., 2012). Reduced survival and growth in egg albumen were observed among S. Enteritidis strains which were sensitive to both acidic and oxidative stress (Shah et al., 2012).

Although a uniquely strong epidemiological association has been established between S. Enteritidis and egg-borne disease, some strains of other Salmonella serovars can invade reproductive organs of laying hens and cause egg contamination (Gast et al., 2011). In North America, egg contamination by S. Heidelberg (a common serovar in laying housing environments) has been implicated in occasional reports of human illness (Chittick et al., 2006). Likewise, S. Typhimurium has caused sporadic egg-transmitted disease in Australia (Moffatt et al., 2016). Other serovars which are prevalent in the housing environment of commercial laying flocks, such as S. Kentucky in the USA, are rarely linked to egg contamination. Experimental infection with serovars Heidelberg and Typhimurium has sometimes resulted in colonization of reproductive organs at relatively high frequencies (Gantois et al., 2008), but egg contamination has been observed far less often than for S. Enteritidis (Gast et al., 2011). This may be because S. Enteritidis adheres more strongly to reproductive tract mucosa, or because other serovars elicit more intense protective immune responses. Egg-associated illness in Australia appears to be primarily the consequence of external contamination of eggshells by S. Typhimurium (Moffatt et al., 2016).

IV. ENVIRONMENTAL INFLUENCES ON SALMONELLA IN LAYING FLOCKS

Environmental conditions in egg production facilities have a powerful influence on the opportunities for introduction and dissemination of pathogens in laying flocks (Trampel et al., 2014). Persistent environmental contamination is sometimes responsible for the transmission of infection into successive laying flocks over extended periods of time (Dewaele et al., 2012). Contaminated dust and feces can distribute Salmonella contamination widely throughout laying houses (Im et al., 2015). Severe rodent or insect infestations can amplify Salmonella levels sufficiently to threaten the efficacy of standard protocols for cleaning and disinfection of poultry facilities (Wallner-Pendleton et al., 2014).

Once introduced into laying flocks, rapid and extensive horizontal dissemination of salmonellae can be facilitated by direct contact between hens, ingestion of contaminated feed or feces, movement of personnel and equipment, and airborne circulation of contaminated dust and aerosols. Environmental stressors, such as excessive heat and deprivation of feed or water, can heighten the susceptibility of hens to horizontal transmission of infection (Okamura et al., 2010). Risk assessment for Salmonella in poultry is made difficult by the environmental complexity of commercial egg production facilities and management practices. Even within individual flocks, the prevalence of Salmonella infection often varies considerably over time (Wales et al., 2007). Among the risk factors most often associated with higher Salmonella prevalence are larger flock size, greater flock age, housing in older facilities, and multiple-age stocking (Denagamage et al., 2015).

V. HOUSING SYSTEMS AND SALMONELLA IN LAYING FLOCKS

The relative merits of different production housing systems for commercial laying hens have been actively debated in recent years in regard to their implications for issues as diverse as animal welfare, economic viability, and public health. The principal management system options include conventional laying cages (housing small groups of hens at relatively high stocking densities), enriched colony cages (providing lower stocking densities for larger hen groups plus environmental enhancements such as perches, nesting areas, and scratching pads), aviaries (allowing birds to move freely among multiple open levels of enriched cage and floor areas within houses), and free-range housing (offering greater opportunities for freedom of movement via varying degrees of access to outdoor forage or pasture areas). Each of these systems is associated with intrinsic facility design features and management practices which can affect the persistence and transmission of pathogens (Jones et al., 2015).

Although numerous studies have assessed the food safety consequences of different types of housing for egg-laying chickens, no overall consensus has emerged to suggest the superiority of any one system. Experiments comparing the prevalence of Salmonella environmental contamination, infection, or egg contamination attributable to housing flocks in various cage-based or cage-free systems have yielded a wide range of results. Some studies have reported higher frequencies of Salmonella infection from cage-based housing systems (Denagamage et al., 2015), other studies have associated cage-free housing systems with a higher incidence of egg and environmental Salmonella isolation (Jones and Anderson, 2013), and a third group of studies have not found differences in Salmonella prevalence between housing systems (Van Hoorebeke et al., 2011). A large field study conducted under commercial egg production conditions (Jones et al., 2015) identified no major differences between housing system in the presence of pathogens in the environment or on eggs, although each system had specific risk factors which posed unique management challenges (such as contaminated scratch pads in enriched cages and contaminated floor eggs in aviaries).

In experimentally infected hens, S. Enteritidis was isolated from internal organs at significantly higher overall frequencies from hens in conventional cages than in enriched colony cages (Gast et al., 2013b). This suggested that stress associated with some intrinsic characteristic of conventional cage housing (such as bird density or behavioral restriction) might compromise immunity, resulting in increased susceptibility to the systemic dissemination of S. Enteritidis infection. Chickens housed at high stocking densities have exhibited suppression of both humoral and cellular immunity and increased S. Enteritidis invasion of internal organs (Gomes et al., 2014). However, no difference was evident between conventional and enriched colony cage systems in the production of internally contaminated eggs by experimentally infected hens (Gast et al., 2014). Although systemic dissemination and reproductive organ invasion are necessary precursors to the deposition of S. Enteritidis inside eggs, the frequency at which they occur does not consistently not consistently predict the likelihood of subsequent egg contamination (Gast et al., 2011).

VI. SALMONELLA MONITORING IN LAYING FLOCKS AND EGGS

Monitoring for the presence of S. Enteritidis in hens, their environment, and eggs is a central component of most programs for controlling this pathogen. Testing serves both to identify flocks which potentially threaten public health and verifies the cost-effectiveness of investments made in risk reduction practices. Laying flock testing programs often focus exclusively on S. Enteritidis as the epidemiologically preeminent serovar associated with eggs. This approach may arguably represent a cost-effective use of limited resources for protecting public health, but monitoring for the emergence of previously infrequent or inconsequential serovars can also have important proactive value. The efficacy of testing for eradicating S. Enteritidis in poultry and eggs is limited by the continuing opportunities for reintroduction of the pathogen into flocks from diverse environmental sources of infection. Moreover, making decisions about the fate of flocks or eggs on the basis of testing results can be complicated by fluctuations over time in the prevalence of S. Enteritidis in the environment, hens, and eggs.

The presence of S. Enteritidis in the housing environment of laying flocks has been shown to correlate strongly with the likelihood that they will produce contaminated eggs. Because contaminated eggs are typically produced at very low frequencies by infected flocks, testing environmental samples for S. Enteritidis is often employed as a screening method to identify potentially infected flocks for further attention. Voided feces from infected hens are leading sources of laying house environmental contamination with Salmonella, although testing for fecal shedding does not always predict overall environmental sampling results (Wales et al., 2006). Dust samples have sometimes provided a higher frequency of S. Enteritidis isolation than fecal samples (Arnold et al., 2014b); testing both dust and feces may be superior to either sample individually. An assortment of environmental sampling methods have been used effectively in poultry facilities, including drag swabs, boot swabs, and the collection of litter material or dust from locations such as egg belts, fan blades, or nest boxes. Salmonella isolation and identification from environmental samples usually involves traditional selective enrichment culturing methods, followed by biochemical and serological confirmation, although rapid assays based on the recognition of specific genetic sequences or antigenic molecules are increasingly employed to identify particular strains with a high degree of precision.

The production of specific antibodies by infected chickens provides another option for detecting S. Enteritidis in laying flocks. Both serum and egg yolk antibodies can be detected, sometimes at high titers, for long periods of time after hens are exposed to S. Enteritidis (Gast et al., 2002). Testing for egg yolk antibodies to S. Enteritidis has been demonstrated to achieve a similar sensitivity for detecting infected flocks as did culturing environmental samples (Klinkenberg et al., 2011). Serological methods can be valuable as screening methods for flock infection because of their high detection sensitivity, but they have been infrequently applied in recent years because of concerns about antigenic cross-reactivity with other serovars and the persistence of a detectable antibody response long after active infection has been cleared.

The most definitive documentation that a laying flock poses a threat to public health risk is the isolation of Salmonella from the edible internal contents of eggs. Accordingly, egg culturing is a pivotal component in most S. Enteritidis monitoring protocols for laying flocks. However, even in flocks known to be infected with S. Enteritidis, egg contamination is infrequent, sporadic, and transient, thus limiting the diagnostic sensitivity of testing eggs for this pathogen (Gantois et al., 2009). Because salmonellae are generally found inside eggs at low frequencies and in very small cell concentrations, pools of the entire liquid contents (yolk plus albumen) of up to 20 eggs are often employed to keep sample numbers within logistically feasible limits. These egg contents pools may be pre-incubated or supplemented with an iron source to encourage expansion of initially small S. Enteritidis populations to more consistently detectable levels before continuing with traditional enrichment culturing methods (Gast and Holt, 2003). Eggshells are also sometimes cultured to detect external surface contaminants.

VII. SALMONELLA RISK REDUCTION IN EGG-LAYING FLOCKS

Controlling the prevalence of S. Enteritidis infection in commercial laying flocks is essential to reducing the risk of egg-transmitted illness for humans. The most promising overall strategy for achieving this objective involves the implementation of multiple interventions distributed throughout the egg production cycle (Trampel et al., 2014). Many risk reduction practices to control S. Enteritidis are similarly applicable against salmonellae of other serovars. Preventing Salmonella introduction into poultry facilities requires the enforcement of strict biosecurity measures, stocking with replacement pullets which are demonstrably uninfected, controlling populations of rodent and insect pests, and securing farms against access by wildlife. Some of these practices are also important for reducing persistence and horizontal dissemination of salmonellae within flocks. Indirect horizontal transmission of Salmonella to subsequent flocks via contaminated environmental sources can be minimized by thorough cleaning and disinfection of laying houses after depopulation.

Vaccination is a risk reduction practice that often has a serovar-specific target. Immunization of laying hens against S. Enteritidis seeks to reduce the susceptibility of individual birds to infection, vertical and horizontal transmission between birds, the overall pathogen load in poultry house environments, and the frequency of egg contamination. Both inactivated (killed) and attenuated (live) Salmonella vaccine products are commercially available, eliciting varying degrees of mucosal and systemic immunity. In experimental challenge studies, administration of either type of S. Enteritidis vaccine preparation to pullets or hens has typically reduced - but seldom altogether prevented - fecal shedding, organ invasion, and egg contamination (Trampel et al., 2014). Additionally, Salmonella vaccines sometimes fail to provide protection against infection when confronted with high challenge doses of the pathogen or when the immune responses of hens are reduced by stressors such as feed deprivation, water deprivation, or environmental heat (Barrow, 2007). Nevertheless, significantly lower frequencies of egg contamination and human S. Enteritidis infections have been attributed to the inclusion of vaccination as a component in risk reduction programs for commercial egg production (Cogan and Humphrey, 2003).

Egg refrigeration has often been identified as an important risk mitigation strategy to protect consumers against egg-borne transmission of S. Enteritidis infection to consumers. Although inadequate management of egg storage temperatures is uncommon in modern commercial egg production, one study concluded that nearly half of egg-associated illnesses were associated with such problems (DeWinter et al., 2011). Accordingly, many risk reduction plans include specifications for temperature control during egg storage. Because initial Salmonella populations inside eggs are usually very low, prompt refrigeration as soon as possible after egg are laid can prevent multiplication to higher levels more likely to cause human illness (Gast and Holt, 2000b). The effectiveness of egg refrigeration depends on the initial location and cell numbers of contaminants, the migration of bacteria or nutrients within eggs during storage, and the rate of achieving growth-limiting temperatures.

The currently applicable food safety regulations for shell production in the USA are provided by the rule for Prevention of Salmonella Enteritidis in Shell Eggs during Production, Storage, and Transportation (US Food and Drug Administration, 2009). This program mandates several specific risk reduction practices and a two-tiered monitoring program for egg-laying flocks. Commercial egg producers must develop a written S. Enteritidis prevention plan, purchase all replacement chicks from breeder flocks certified as uninfected, enforce comprehensive facility biosecurity, and stringently control rodents and insects. All eggs must be stored and transported under refrigeration at 7.2° C within 36 hours after laying (although equilibration to room temperature is allowed before processing to prevent egg sweating). Monitoring for S. Enteritidis in flocks is performed during both pullet rearing and egg laying. Samples are collected from the poultry housing environment and cultured to detect S. Enteritidis at 14-16 weeks of age from pullet flocks, at 40-45 weeks of age from laying flocks, and again at 4-6 weeks after induced molting. When S. Enteritidis is found during environmental monitoring of a flock, several lots of 1,000 eggs each are then also collected and cultured. Isolation of S. Enteritidis from any egg samples requires all eggs from that flock to be diverted for pasteurization until repeated negative egg testing results are achieved. Contaminated poultry houses must be thoroughly cleaned and disinfected between flocks. Regulations in the USA do not currently require the vaccination of egg-producing flocks against S. Enteritidis. This contrasts with the UK Lion Code of Practice for Shell Egg Producers, which mandates Salmonella immunization of egg-producing flocks in addition to risk reduction and monitoring (O’Brien et al., 2013).

VII. CONCLUSIONS

No single disease-control strategy appears to offer a completely effective solution to the complex problem of Salmonella in poultry flocks and eggs. In the United States, where S. Enteritidis has been the greatest concern, comprehensive efforts to assure the microbial safety of eggs have been built on a foundation of risk reduction practices: biosecurity, sanitation, pest control, and egg refrigeration. Vaccination has often used to augment these measures by inducing an additional protective barrier against infection. Flock and egg testing have been employed to identify situations requiring further attention and to evaluate the efficacy of risk reduction efforts. Problems with other serovars, such as S. Typhimurium in Australia, can present unique challenges to public health authorities and the egg industry. Nevertheless, many of the general underlying principles for Salmonella control in poultry flocks and eggs are internationally applicable.

Presented at the 29th Annual Australian Poultry Science Symposium 2018. For information on the latest and future editions, click here.