Although data derived from the FoodNet database indicated a relatively low incidence rate (see glossary) of 2.32 cases of Salmonella Enteritidis (SE) per 100,000 population in the United States during 2005, the plateau of approximately 30 outbreaks per year since 2002 is of concern to regulatory authorities.
The U.S. Centers for Disease Control and Prevention (CDC) has derived a mid-range estimate of 66 percent of incident (new) cases of SE as being attributable to consumption of contaminated eggs. Although subject to debate, the CDC has calculated that approximately 120,000 incident cases of SE are derived from eggs each year in the United States. This is attributed to persistence of infection in flocks in the Midwest and Western states and to a lower level of transmission to consumers in the Mid-Atlantic and Great Lakes regions.
A study conducted by the National Animal Health Monitoring System in 1999, prior to introduction of extensive vaccination, showed that 7 percent of layer houses were infected with SE. Deep-pit units, which represent the majority of in-line complexes erected during the past 20 years, showed a 13 percent prevalence of environmental contamination.
Based on the persistence of SE in the United States and a proven association with shell eggs, the U.S. Food and Drug Administration (FDA), which has jurisdiction over distribution of all food products in terms of the Federal Food, Drug and Cosmetics Act and the Public Health Service Act, has issued a proposed rule relating to "SE prevention measures". The proposed regulations are based on 21 CFR parts 16 and 118. Section 118.5 (a) of 21 CFR will require more frequent assay of the environment of flocks at critical intervals during the production cycle to identify potential infections. Positive assays would then lead to confirmatory tests on eggs from suspect flocks.
The current industry standard to detect environmental contamination requires drag swabs of accumulated droppings under cages or from egg belts. It is generally accepted that drag swabs are insensitive in their ability to detect environmental infection. This is in all probability due to the fact that surface contamination with SE may be non-detectable due to the low water activity of the crust covering acceptably dry "coned" manure.
In a recent peer reviewed article, data was published on the examination of 128 environmental drag swabs from two known SE positive flocks located in a Western and a Midwestern state, respectively.
Applying conventional culture procedures, only 23 percent of the swabs derived from manure or egg belts yielded SE.
A previous field diagnostic study conducted on a Midwest in-line complex demonstrating SE infection yielded five positive assays from 24 drag swabs from the four adjacent houses that were sampled. Given the relatively low sensitivity of drag swab assays to detect SE, it is evident that the UEP program based on sampling flocks only once at about two weeks before depletion has a high probability of generating a false positive result (not detecting environmental contamination).
Since the assay is conducted at the end of the production cycle, failure to detect exposure, which indicates intestinal colonization, may lead to possible vertical transmission during the peri-peak and peri-molt periods. This in turn creates the probability of food borne infection for consumers of undercooked eggs.
Increased frequency of environmental surveillance and more sensitive microbiological assay procedures will be mandated in the near future to identify flocks with intestinal SE colonization. This situation may result in transfer of the pathogen to eggs by trans-ovarial and trans-oviductal transmission if flocks are immunosuppressed or stressed.
Assay for SE
The approved FDA culture method requires a pre-enrichment phase in buffered peptone water followed by enrichment in either Tetrathionate broth or Rappaport-Vassiliadis medium. This is followed by plating on a selective medium such as Brilliant green agar, XLT-4 or Bismuth sulfite agar. Generally, XLT-4 is the preferred medium since it promotes the growth of SE colonies which are identified by their characteristic pink color with black centers.
Presumptive SE colonies are transferred to identifying media (Triple sugar iron agar and Lysine iron agar). Following 24 hours of incubation, the specific changes induced in the medium are indicative of SE. Provisional immunologic identification is carried out using the plate agglutination test procedure using serogroup-D antiserum.
The complete sequence of enrichment, isolation, and identification may take as long as seven to 10 days. Selecting presumptive positive SE colonies from isolation medium requires experience and is subject to inherent operator bias. This factor together with other sources of confounding associated with standard microbiological procedures may give rise to either false positive or false negative results.
Polymer Chain Reaction Assay
Recently, polymerase chain reaction technology (PCR) has been adapted to identify SE. The most current innovation is a real-time PCR system developed by microbiologists at the FDA. A fluorescent dye-labeled, gene-specific probe detects segments of the gene sefA which is specific to Salmonella group-D strains including SE. An advantage of the system is extreme sensitivity and the assay can detect one organism (live or dead) in the contents of 100 eggs.
The test incorporates high specificity in identifying SE and differentiating the organism from over 50 non-group-D Salmonella and non-Salmonella contaminants encountered in egg liquid including Shigella spp., Staphylococcus spp., and E. coli.
The procedure identified seven serotypes of SE including phage type 4 in addition to the more common pt13 and pt8. The real-time PCR technique is capable of providing a result within two days including enrichment prior to assay. The fact that the PCR procedure which has advantages over previous technology was developed by FDA suggests that this method may become a standard and approved procedure to confirm SE transmission.
The PCR technique is suitable for research, reference and regulatory establishments, but the cost, complexity and requirement for trained technicians will limit field diagnostic application, especially in company-operated and contract laboratories.
Immuno-Based Test Kits
A lateral flow test kit to detect SE has been developed by Neogen Corp., located in Lansing, Mich. (www.neogen.com). The test, marketed as the Reveal for SE, offers the obvious advantages of low cost, speed, sensitivity, specificity, and reproducibility. The test is highly correlated with results obtained using conventional microbiology incorporating double enrichment and highly selective isolation media. Sample enrichment is still required, especially when assaying eggs which may contain a small number of viable organisms. The test is performed by transferring a minute quantity of the enriched solution into the well of the indicator device. After application the solution diffuses along the solid medium. If SE antigen comprising live or dead organisms is present, binding will occur to specific antibody directed to the fimbrial protein, specific to group-D Salmonella spp. The antibody antigen complex which is conjugated to colloidal gold particles continues to move along the test strip and is detected in a second zone, resulting in the appearance of a visible line.
The kit incorporates a control to confirm correct function of the detector system which is indicated by a second line. If SE is present in the enrichment culture, two lines are visible, one representing the antigen-antibody complex and the second the control. A negative sample will have only the control line indicating correct function of the unit.
In extensive trials comparing the Reveal with conventional microbiological culture, the test kit demonstrated sensitivity of 83 percent, specificity of 94 percent, a positive predictive value of 76 percent, and an accuracy of 92 percent. False positives for SE can occur using the test system which cannot differentiate among other group-D Salmonella including S. Berta, S. Pullorum, and S. Gallinarum. With the exception of S. Berta, other group-D Salmonella are all epidemiologically significant.
Fortunately in the United States, neither S. Pullorum nor S. Gallinarum are encountered in commercial operations, and a positive result using the test kit is a substantial indication of the presence of SE. In any event, presumptive SE results should be verified by microbiology or PCR, and samples should be submitted to a designated reference laboratory such as the NVSL for confirmation. The Reveal test does, however, provide a rapid and reliable result to enable appropriate corrective action to be taken. It is reiterated that although sensitive and specific, the Reveal test can only detect SE which is present on the source swab derived from egg belts, fan blades or manure.
The entire detection system is subject to the limitations of sampling. A number of replicate samples, albeit pooled, may therefore be required to identify infected flocks. Naturally increasing the frequency and intensity of sampling will improve the sensitivity of detection. The cost of the Reveal kits including enrichment media is approximately $150 for 20 assays. This can be compared to a cost of $20-$30/microbiological assay as charged by certified commercial laboratories. Laboratory requirements for the Reveal for SE tests, including an incubator for enrichment, are minimal in comparison to a PCR installation which can cost in excess of $7,000 in addition to the purchase of reagents.
Neogen supplies the AccuScan reader which combines a digital camera and software. The reader provides an objective evaluation of the bands on the test kit, identifies flocks and stores data for download to a comprehensive database.
Take Home Message
Assay of flocks for the presence of SE in the environment or pools of liquid eggs will increase in intensity following adoption of the proposed FDA rule on SE. The Neogen Reveal for SE kit offers the opportunity to establish an inexpensive, practical in-company capability to identify SE-contaminated flocks and complexes. This will form the basis of appropriate action to eradicate infection or to serve as an internal surveillance program to detect SE in operations with no history of infection.