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Egg contamination with salmonella enteritidis

Controlling egg contamination with salmonella enteritidis by understanding its pathobiology

Published: October 20, 2011
By: Richard Gast and Jean Guard (United States Department of Agriculture, Agricultural Research Service, Egg Safety and Quality Research Unit, Russell Research Center)
Summary

For more than twenty years, public health authorities have reported the transmission of Salmonella Enteritidis to consumers of internally contaminated eggs produced by infected hens. Egg contamination is both a cause of food-borne human illness and a principal diagnostic criterion for identifying infected laying flocks. Improved characterization of bacterial attributes (phenotypic and genetic) responsible for flock infection and egg contamination would support S. Enteritidis control efforts. Salmonella deposition inside developing eggs results from colonization of reproductive tissues in systemically infected hens, especially the ovary and the upper oviduct. Both the yolk and albumen of developing eggs laid by infected hens can be contaminated by S. Enteritidis, with the initial site of deposition determined by which regions of the reproductive tract are colonized. The observed incidence of internal egg contamination with S. Enteritidis is typically low and involves small initial numbers of bacterial cells. Identifying the underlying genetic differences between egg-associated and non-egg-associated Salmonella strains has been a complex and difficult task. The complementary action of phenotypic properties relevant to different environmental contexts in the infected avian host, expressed by distinct bacterial subpopulations, may link the complicated series of events which leads to egg contamination. Flock testing and vaccination are among the most frequently discussed and widely applied strategies for controlling S. Enteritidis infection and egg contamination in breeding and laying flocks. Testing identifies infected flocks or contaminated eggs which require intervention responses and verifies the efficacy and cost-effectiveness of risk reduction practices. Vaccination reduces the susceptibility of poultry to Salmonella infection if risk reduction practices fail to prevent pathogen introduction into flocks. The potential effectiveness of both of these approaches depends on the extent to which they are guided by a thorough and realistic understanding of the course and consequences of S. Enteritidis infections in chickens.

Key Words: Salmonella Enteritidis, eggs, pathobiology, vaccination, flock testing

Introduction
For more than two decades, public health authorities throughout the world have reported the transmission of Salmonella enterica serovar Enteritidis (S. Enteritidis) to consumers of contaminated eggs produced by infected hens (Greig & Ravel, 2009). For example, epidemiological calculations in the United States have attributed more than 100,000 annual illnesses to contaminated eggs, although the estimated prevalence of S. Enteritidis in commercially produced table eggs is only 0.005%. A national regulatory plan for S. Enteritidis in egg-laying flocks in the United States was implemented in 2010, consistent with evolving international recognition that reductions in the frequency of illness due to S. Enteritidis have resulted from intensive commitments of resources by governments and egg producers to testing and risk reduction programs (Gast, 2008). Eggs contaminated by S. Heidelberg have also been implicated as food vehicles in human disease outbreaks in the United States, although far less often than S. Enteritidis
The environmental persistence of Salmonella in poultry houses creates continuous opportunities for laying hens to become infected with this pathogen, which can rapidly spread horizontally throughout flocks. Environmental contamination with S. Enteritidis can be intensified by severe rodent or insect infestations and can sometimes survive cleaning and disinfection regimens (Carrique-Mas et al., 2009). The deposition of S. Enteritidis in eggs is not only a leading cause of food-borne human illness, but also serves as the principal confirmatory diagnostic criterion for identifying infected commercial laying flocks. Advances in the characterization of bacterial attributes (both phenotypic and genetic) that lead to systemic infection and egg contamination would directly support efforts to control S. Enteritidis in egg-laying chickens (Gast, 2008).
S. Enteritidis Infection and Egg Contamination in Laying Hens
Invasion beyond the intestinal tract to internal organs such as the liver and spleen occurs within hours after oral exposure to S. Enteritidis, and creates the opportunity for subsequent reproductive tissue involvement and egg contamination (Gantois et al., 2009). Persistent intestinal colonization does not consistently correlate with either systemic infection or egg contamination by S. Enteritidis. The frequency of internal organ colonization by Salmonella typically declines steeply during the first several weeks after oral inoculation, so testing at longer post-inoculation intervals is generally not highly informative (Gast et al., 2007). Nevertheless, persistence of the pathogen in the tissues of even a small fraction of infected birds could still lead to egg contamination (Gast et al., 2009). Environmental stressors such as feed or water deprivation can also influence the opportunities for egg contamination to occur in infected flocks.
The deposition of S. Enteritidis in the contents of developing eggs results from colonization of reproductive tissues in systemically infected hens (Gantois et al., 2009), especially the ovary (site of yolk maturation and release) or the upper oviduct (site of albumen secretion around the descending yolk). However, high frequencies of reproductive tissue colonization have not always been associated with similarly high frequencies of egg contamination (Gast et al., 2007). Either the yolk or albumen (or both) of developing eggs laid by infected hens can be contaminated by S. Enteritidis, with the initial site of deposition determined according to which regions of the laying hen´´´´s reproductive tract are colonized. In most instances, the observed incidence of internal egg contamination following oral inoculation of hens with S. Enteritidis is relatively low and involves small initial numbers of bacterial cells, even when very large infecting doses are administered.  The initial site of S. Enteritidis deposition inside eggs is more often found in the albumen or on the outside of the vitelline (yolk) membrane than within the nutrient-rich interior of the yolk. However, studies using in vitro egg contamination models have shown that Salmonella can penetrate through yolk membranes and begin multiplication inside yolk contents during the first day of storage at warm temperatures (Gast et al, 2010).
The initial bacterial exposure dose is highly significant to the progression and outcome of systemic S. Enteritidis infections, as suggested by a strong association with the magnitude of both serum and egg yolk antibody responses. Both organ invasion and egg contamination are significantly reduced at lower infection doses (Gantois et al., 2009). Most experimental infection studies with S. Enteritidis have reported relatively modest incidences of egg contamination, even when very large oral doses were administered (Gast & Holt, 2000). Even lower frequencies of egg contamination are typically associated with naturally infected commercial poultry, likely due to both the infrequent prevalence of S. Enteritidis infection within flocks and the exposure of individual hens to relatively small bacterial doses (Ebel & Schlosser, 2000). Experimental horizontal contact transmission of S. Enteritidis, which may simulate naturally occurring infections more accurately, has produced intestinal colonization, organ invasion, and egg contamination at lower frequencies than is observed following large oral doses. Higher bacterial doses may cause an increased frequency and severity of pathological effects, but may also elicit stronger immune responses which promote the clearance of infection.
Egg Contamination by Different Salmonella Strains and Serotypes
Significant differences have been observed between S. Enteritidis strains in their potential to cause both reproductive organ invasion and egg contamination, but individual strains do not generally exhibit affinities for specific regions of the reproductive tract that are sufficient to yield distinctive patterns of deposition within eggs (Gast et al., 2007). Differences in the frequency at which they invade internal organs and contaminate eggs have been reported between Salmonella serotypes and even between strains of the same serotype, although some strains have been found to invade internal organs at high frequencies despite being associated with little or no deposition inside eggs (Gast et al., 2007). Experimental infection with S. Enteritidis strains has usually resulted in higher egg contamination frequencies in comparison to infection with strains of S. Heidelberg or S. Typhimurium, even when strains of all tested serotypes were isolated at similar frequencies from reproductive tissues (Gast et al., 2004).
Identifying the underlying genetic differences between egg-associated and non-egg-associated S. Enteritidis strains has been a complex and difficult task (Bottledoorn et al., 2010). An inherent ability to invade internal organs and contaminate eggs by some S. Enteritidis strains has been attributed to phenotypic properties including the production of high-molecular-mass lipopolysaccharide and growth to high cell density (Guard-Bouldin et al., 2004). Single-nucleotide genomic changes in a biofilm-negative S. Enteritidis phenotype led to an increased propensity for deposition inside of eggs laid by experimentally infected hens (Morales et al., 2007). In another study, specific genes were found to be highly expressed by S. Enteritidis isolates from both infected hens´´´´ oviducts and from eggs (Gantois et al., 2008). Selective pressures which are exerted in the tissues of infected hens may influence the expression of critical bacterial virulence factors, as shown by the increased ability of S. Enteritidis strains to cause egg contamination after repeated passages through infected hens (Gast et al., 2003). Also, environmental conditions such as pH and temperature can affect the expression of potential S. Enteritidis virulence factors such as flagella, fimbria, outer membrane proteins, and iron uptake systems. Stress has been hypothesized to play an important role in the induction of bacterial attributes that promote colonization of chicken oviducts and survival in egg albumen (Van Immerseel, 2010). The complementary action of phenotypic properties relevant to different environmental contexts in the infected avian host, expressed by distinct bacterial subpopulations, may link the complicated series of events occurring after initial intestinal colonization and which eventually leads to deposition inside eggs (Guard et al., 2010). Perhaps due to the complexity of the interconnected events which occur during the course of infection with this pathogen, efforts to genetically distinct lines of chickens with heightened resistance to Salmonella have been only partially successful (Beaumont et al., 2009).
S. Enteritidis Control in Egg-Laying Flocks
Vaccination and flock testing are among the most frequently discussed and widely applied strategies for controlling S. Enteritidis infections in laying flocks and the associated production of contaminated eggs. The potential effectiveness of both of these approaches is inherently determined by the extent to which their development and use is guided by a thorough and realistic understanding of the course and consequences of S. Enteritidis infections in chickens. Vaccine efficacy hinges on the elicitation of an immune response which is capable of interrupting the intricate sequence of events between initial pathogen exposure and deposition inside eggs. Likewise, refining testing protocols and applying testing results to make decision requires an understanding of how different critical parameters of infection are likely to be detected by particular sampling methods. Further characterization of the genetic and phenotypic attributes of Salmonella serotypes and strains which enable them to invade internal organs of laying hens, colonize reproductive tissues, and contaminate developing eggs is vital for identifying and differentiating individual isolates, defining epidemiological relationships between isolates, developing broadly and strongly protective vaccines, and developing effective testing strategies for detecting infected individuals and flocks.
Vaccination
The goals of vaccinating poultry flocks against Salmonella are to reduce the susceptibility of individual birds to infection, the horizontal transmission of infection within flocks, the vertical transmission of infection to the progeny of infected breeding flocks, the Salmonella load in poultry house environments (and corresponding opportunities for the transmission of infection to subsequent flocks), and the frequency of egg contamination. Available Salmonella vaccines include both inactivated (killed) and attenuated (live) products. Inactivated vaccines can be rapidly prepared from specific strains responsible for problems in a particular locale or in a particular commercial enterprise or industry, and multivalent killed vaccines containing mixtures of strains or serotypes can provide an expanded spectrum of protection. Attenuated vaccines can be easily and inexpensively administered (often in drinking water) and can induce a stronger and longer-lasting immune response because they more persistently present relevant antigens to the host immune system. Both killed and live vaccines have been documented to provide significant protection against S. Enteritidis infection and egg contamination in challenge studies, but neither type of vaccine has consistently been able to prevent infection altogether (especially against high challenge doses of the pathogen) or to effectively cross-protect against different serotypes (Barrow, 2007; Gast, 2008). Instances of poor performance by vaccines have been attributed to severe rodent control or sanitation problems in poultry houses, feed or water deprivation, or environmental stressors such as heat. Nevertheless, vaccination programs for egg-laying hens have been associated with significantly lower frequencies of egg contamination (Toyota-Hanatani et al., 2009) and human S. Enteritidis infections (Cogan & Humphrey, 2003). The most promising use for vaccination to support overall Salmonella control objectives may be as an individual contributing component within a comprehensive program of risk reduction practices, especially in application to highly susceptible flocks or flocks exposed to severe challenges from environmental sources, when epidemiologic evidence has led to heightened concerns about particular serotypes (such as S. Enteritidis).
Flock Testing
Testing has played an important but often controversial role in Salmonella control programs. Trace-back testing to identify and eradicate infected flocks which are responsible for human disease outbreaks has generally not been an especially effective control strategy because many Salmonella serotypes (including S. Enteritidis) can be continuously re-introduced into poultry houses and flocks from diverse environmental sources (Hogue et al., 1997). Making decisions (about the fate of either flocks or their eggs) based on testing results always involves a substantial degree of uncertainty because of both assay sensitivity issues and variations over time in the detectable parameters of infection (including fecal shedding and other measures of environmental contamination, antibody production, and egg contamination). One of the most prominent practical consequences of different bacterial exposure levels is the demonstrated direct relationship between experimental oral inoculation doses and the sensitivity of both serologic and bacteriologic detection of infection. Antibody detection can be very sensitive but is inherently historical and thus does not necessarily demonstrate active infection or ongoing egg contamination (Gast, 2008). The predictive usefulness of culturing eggs for Salmonella is severely restricted by the infrequent, transient, and sporadic nature of naturally occurring egg contamination (Gantois et al, 2009). Environmental samples are relatively easy to collect and process (Arnold et al., 2010), but only indirectly reflect the actual probability of egg contamination. In Salmonella control programs that use testing results to an appropriate response, the potential effectiveness of this response is heavily dependant on the type of testing questions that are asked. For example: what, when, and how many samples are collected and how are they tested? Testing within Salmonella control programs is essential not only to detect flocks which potentially pose a threat to public health, but also to verify that the investment of resources in risk reduction practices is ultimately cost-effective. Making decisions and selecting responses on the basis of serotype-specific testing results is useful (perhaps even essential) for responding to severe public health problems caused by specific individual serotypes, but a more serotype-independent testing approach can detect and respond to emerging problems (perhaps involving new reservoirs of infection or previously uncommon serotypes) before public health and economic impact becomes more severe.
Conclusions
No single response is likely to provide a complete solution to the complex public health and economic problems associated with Salmonella Enteritidis in poultry. Comprehensive control programs, based on a coordinated and sustained implementation of risk-reduction practices for both breeding and egg-producing flocks plus targeted testing to detect the pathogen in flocks and eggs, have yielded promising results in several nations. Testing identifies infected flocks or contaminated eggs which require intervention responses and provides essential verification of the efficacy and cost-effectiveness of risk reduction practices. Protective treatments such as vaccination can reduce the susceptibility of poultry to Salmonella infection if risk reduction practices fail to prevent the introduction of the pathogen into flocks. Whereas most of the prominent risk reduction practices are serotype-independent in their focus, vaccination can be especially valuable for enhancing the short-term responsiveness of control programs to address problems associated with specific serotypes of elevated significance such as S. Enteritidis.    
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Authors:
Richard Gast
USDA - United States Department of Agriculture
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Ganesh Kumar Dahal
Guybro Chemical
21 de mayo de 2012

Thank you very much for a good article on controlling egg contamination with Salmonella enteritidis which is common concern for public health.

Your advice on vaccination programme (Multivalent killed & attenuated vaccines) for breeders & commercial layers will be appreciated.

In our territories we have to rely on treatment of feed, water & litter of layers flock to prevent Salmonella enteritidis. Salmonella vaccines are still not in regular practice in commercial flocks.

Regards !

Dr.Thirumeignanam, D.,
26 de abril de 2012

Dear Dr.Richard,

Thank you your valuble article on Salmonella contamination in poultry by products specially egg.

Forum participants also rightly pointed out that prevention from feed sanitation/hygiene apart from vaccination.

Inclusion of feed additves such Salmonella growth inhibitors (e.g SalCURB) is neccessary for controlling the salmonella in feed and its raw materials (fish meal , bone and meat meal --animal byproducts etc).

Salmonella control is necessary in terms of both poultry and human health point of view.

Thanks and regards
Dr.Thirumeignanam
Animal Nutritionist

Nitin  Suryavanshi
26 de abril de 2012

Dear Dr Richard 
Pls suggest a planned vaccination program for salmonella control because the farms in developing countries lacks the basic facilities

Nitin  Suryavanshi
26 de abril de 2012

A very intresting and informative article. In addition to control steps of incoming raw material and vaccination and practicing sound pre harvest and post harvest operations as per the haccp standards, problem of salmonella is minimized, 

Food safety is prime duty of every units which should be followed...

Philippe Joly
26 de abril de 2012
Yes, it is true but the basis is first to respect a sanitation program. In some country like in Peru, I saw in one large farm with commercial birds and Parents introducing 10% of dry manure in their feed. In open houses, the contamination of the farm will arrive one day, we can be sure about that, and how many childs will be affected. Respect basic rules is the basis.
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