Egg Industry Presentations

Controlling Contamination by Understanding Salmonella Enteritidis Infection in Laying Hens

(View the Online PowerPoint Presentation)

This summary of the paper delivered by Dr. Richard Gast was prepared to supplement the posted set of PowerPoint Slides
which are reproduced with his permission for the benefit of the U.S. egg production industry.

Dr. Richard K. Gast of the USDA-ARS Egg Safety and Quality Research Unit provided a comprehensive review of the epidemiology, diagnosis and prevention of Salmonella Enteritidis (SE) infection at the North Central Avian Diseases Conference.  This program was arranged concurrently with the 2012 Midwest Poultry Federation Convention in St. Paul, MN.

Dr. Richard K. Gast

Dr. Richard K. Gast


The FDA Final Rule on SE was motivated by the data which showed a steady increase in the incidence rate of egg-attributable SE infection from 1996 to 2010.  A risk assessment study conducted by USDA calculated that over 100,000 cases of egg-attributable SE occurred annually in the USA.  This value which was pivotal to the establishment of the FDA program contained assumptions which have since been questioned. Regardless of opposition, the Final Rule was compiled and introduced in July 2010.

Within weeks of inception of the Final Rule, an outbreak of SE involving 29 clusters was identified in 11 states involving almost 2,000 diagnosed cases. All the isolates derived from patients had a common pulse field gel electrophoresis pattern.  Fifteen of these clusters were traced back to an Iowa egg production complex owned and operated by either Jack DeCoster or his affiliate.  Subsequent on-site investigations on the affected farms yielded a high level of the implicated SE serovar. Isolates were recovered from manure drag swabs, aisles and equipment in houses, wash water from an in-line plant and from the common feed mill supplying farms in the complex.

In assessing control of egg borne Salmonella infection, it is necessary to consider both pre-harvest and post-harvest modalities.  Both approaches are complimentary and are necessary to ensure an egg supply free of infection.

Gast has identified the restraints to controlling SE in flocks including the fact that infection is clinically inapparent.  Reservoirs of infection comprise rodents and especially mice which perpetuate infection.  In-line complexes with multi-age flocks intensify the problem of lateral transmission due to close proximity of houses and movement of personnel.  Many houses constructed over 15 years ago have undergone deterioration allowing access by rodents and in some cases even wild birds.  Disinfection is difficult due to the preponderance of wood and damaged concrete which creates an ideal environment for persistence of Salmonella infection, especially on wet surfaces.  High-rise houses with up to nine months of manure provide an ideal habitat for rodents and SE infection can persist in damp manure.  Salmonella is invariably dust- borne and extraction fans can transfer infection among houses in a complex.

In reviewing experimental data Gast demonstrated that inoculation of chicks at day old will induce fecal shedding for at least 24 weeks.  Intestinal colonization can result in 40% to 80% of pullets excreting SE, especially following the stress of onset of production through peak output.  Controlled laboratory infection of hens by the oral route with 108 cfu SE will result in up to 70% of hens developing intestinal colonization and subsequent recovery from the liver and spleen. Hens exposed by contact demonstrate up to a 30% prevalence rate of organ infection.  Ovaries and oviducts can be infected in up to 25% of hens inoculated by the oral route and 15% of their contacts.  Despite organ infection, the extent and duration of vertical transmission through the egg is relatively low.  Two weeks after infection, 7% of shells, 25% of yolks and 30% of albumen may yield SE.  By four weeks after infection, only 1% of eggs from inoculated hens yield SE from the yolk.

In experiments to determine the difference among vertical transmission rate associated with SE isolates,  Gast demonstrated fairly consistent ovarian and oviduct infection in the 20% to 35% range for SE PT13a and PT14b.  Salmonella Heidelberg (which is not a Group-D isolate) showed a similar recovery rate from the ovary and oviduct as SE.  In contrast, recovery from eggs was much lower for Salmonella Heidelberg than for the SE strains. It was determined that PT13a had a higher level of recovery from eggs than other serovars attaining 7% for yolk and 5% from albumen.  It is noted that the hens which were used to determine this data were infected with extremely high doses of the infective organisms. Under commercial conditions the rate of infection of both organs and eggs would be expected to be lower than the values recorded.  An interesting observation related to the fact that when SE was passaged through hens under controlled conditions a two-fold increase in the rate of recovery from eggs was recorded. This suggested adaptation by the organism which may conceivably occur under commercial conditions. 

Refrigeration has long been recognized as a practical method of reducing the level of infection among consumers.  The effectiveness of this approach is influenced by the initial level of contamination and location of SE within the egg. The post-harvest determinants of infectivity comprise the duration of storage and the temperature gradient to which eggs are subjected after production extending through processing and packing and subsequent storage and transport.  It was demonstrated that after 24 hours of storage, eggs held at 77F yielded 108 cfu/ml yolk.  In contrast, storage at 50F resulted in recovery of 101 cfu/ml yolk.  The presence of SE in albumen was considerably lower than from yolk but the level increased exponentially with storage temperature over the 24 hour period.

Vaccination is regarded as a pivotal method of protecting flocks from SE infection.  Vaccines reduce the susceptibility of individual hens to infection, inhibit horizontal transmission within flocks and block vertical transmission to eggs.  Full immunity relies on both cellular (tissue) and systemic (antibody) responses.

Inactivated (killed) vaccines are innocuous in that there is no live organism and vaccines can be prepared rapidly from an isolate especially when autogenous bacterins are required.  Under controlled conditions, inactivated SE vaccine markedly reduced recovery of SE from liver and spleen tissue in inoculated hens.  Vaccination reduced the recovery of SE from the egg contents of hens challenged with a virulent SE culture compared to non-vaccinated controls.  Fecal excretion was also reduced by administration of an inactivated vaccine.  Under controlled conditions the USDA laboratory demonstrated equivalent protection using either a commercially available inactivated SE vaccine compared to an experimental product.  Both vaccines provided protection when compared to non-vaccinated controls.

Attenuated live mutant vaccines have been available for a number of years.  Mass administration is possible at relatively low cost compared to injection of individual hens with inactivated emulsions.  Live vaccines impart a high degree of cross-protection against antigentically different serotypes.  This is evidenced by the fact that the three licensed vaccines in the U.S. are based on mutant Salmonella Typhimurium which provides cross-protection against Salmonella Enteritidis.  Unfortunately live vaccines do not persist in the intestinal tract for prolonged periods. They most certainly protect highly susceptible chicks during the critical brooding period by competing with SE for receptor sites on the enterocytes lining the intestinal tract.  Live mutant ST vaccines provide a level of protection in molted hens against SE although protection is not absolute. Up to 30% of vaccinated-challenged hens excreted SE for up to 17 days as determined in an experiment.  In contrast non-vaccinated controls showed a 70% prevalence of shedding 17 days after infection.

Based on studies conducted in the U.S. and the EU it is evident that combining live attenuated Salmonella vaccines during early rearing into a program incorporating the subsequent administration of one or more inactivated SE emulsion vaccines will provide a high level of durable protection.

The FDA Final Rule implemented in July 2010 requires environmental testing at 14 to 16 weeks of age.  This is designed to prevent transfer of an infected flock to a multi-aged, in-line unit or even to a single-house farm. A second environmental test is required between 40 and 45 weeks of age since hens if exposed to SE would be subjected to stress associated with growth and attaining peak production.  A third assay of the environment of the flock is required 4 to 6 weeks after initiation of molt.

In reviewing alternative programs as required for participation in the Pennsylvania Egg Quality Assurance Program and the directives imposed by Eggland’s Best for their Franchisees it is considered necessary to confirm the negative-SE status of day-old chicks by sampling the excreta on chick box papers.  This precaution will prevent placing infected batches of chicks on pullet rearing farms, many of which operate as multi-age units.  There have been a number of cases of SE in pullets from infected parent flocks or from contaminated hatcheries. These cases incurred extensive disruption of operations and financial losses due the need to depopulate rearing farms and in some cases laying units.  It is advisable to conduct a terminal environmental assay approximately two weeks before a flock is depleted.  If positive, intensive decontamination can be cared out to prevent infection of the succeeding placement of presumably negative pullets.

The SE status of an infected flock can be determined by environmental swabs and examination of egg pools.  Due to the fact that all commercial egg producing flocks in the U.S. have been vaccinated, the presence of antibody using the ELISA technique cannot be used to absolutely differentiate between infected and non infected flocks. It is however possible to ascertain the antibody status of flocks by determining the ELISA titer of flocks. Data can be used to confirm successful immunization following administration of vaccine.

In his conclusion, Dr. Gast supported current industry practices mandated by egg quality assurance programs and the FDA Final Rule.  A coordinated approach involving biosecurity, exclusion and suppression of rodents, vaccination and environmental assays are all necessary pre-harvest procedures.  Sanitizing egg shells and refrigeration as soon as possible after collection (off-line) or packing (both in- and off-line) are necessary post-harvest procedures.  In the final analysis food service providers and consumers have a responsibility to ensure that eggs are thoroughly cooked and that cross contamination does not occur during preparation of meals.

(View the Online PowerPoint Presentation)