The science behind the SPF egg industry.

With flu season approaching, it’s worth noting that the annual ounce of prevention we all should be rolling up our sleeves for, began with SPF chicken embryos for influenza vaccine seed production.

Specific Pathogen Free (SPF) eggs are from chicken flocks that have been confirmed to be free of specific diseases as required per the US Department of Agriculture (USDA) and the European Pharmacopoeia. SPAFAS, a small Connecticut company acquired by Charles River Laboratories in 1991 introduced the first commercial SPF flock in 1961.

Chicken embryos and fertile eggs have had a long history in the manufacturing of vaccines. In the early 1940’s, a yellow fever vaccine was produced using chicken embryos and was found to be contaminated with avian luekosis virus. By the 1960’s, progress had been made in recognizing and identifying avian diseases, the means by which infection happened, and if a pathogen was vertically transmitted from hen to eggs. With the understanding of avian pathogens, the idea of commercially producing SPF eggs to produce quality biologics was recognized. House design, husbandry, affordable diagnostics and flock managment evolved to ensure the flock remained negative for unwanted pathogens. While the SPF industry has been in existence for many decades, new biosecurity and testing measures assure that these valuable raw materials for vaccine manufacturing exceed the requirements to protect humans and our food supply.

SPF flocks are generally housed in filtered air positive pressure houses (FAPP), with some raised in isolators. An SPF flock is created after three successive generations, taking about a year and a half, without any biosecurity breaks to over 30 different pathogens—from Avian Influenza to Salmonella—as determined by weekly blood sampling. Unlike standard poultry flocks, SPF birds are not protected from diseases by vaccination. Becasue SPF flocks cannot have exposure to avian viruses or certain bacteria biosecurity is key.

Biosecurity is the most important factor in producing and supplying SPF eggs and products. Feed, water, bedding, and house materials are treated and/or disinfected to ensure pathogens do not enter the house. Employees follow strict gowning and house entry procedures, and visitors are restricted to certain areas on the farms. New advances in environmental testing of the houses and pest control have been implemented to further assure that the SPF flocks are disease-free.

Each SPF flock is tested weekly according to European Pharmacopoeia 5.2.2 and USDA Memo 800.65 and for decades the assays used to monitor the birds have remained unchanged. However, last year, CRL was the first to introduce Multiplexed Fluorometric Immunoassay (MFIA) technology for the earliest detection possible, allowing us to gauge how well our biosecurity efforts are working. This method tests for 14 pathogens in one assay and most importantly detects disease breaks earlier from onset of infection than most conventional methods. We also use two PCR tests to further assure the flocks are safe.

Technical Details

The MFIA is a serologic assay in which an array of uniquely-colored polystyrene microbeads can be coated with specific antigens and act as the solid phase of the immunoassay, much like the 96-well polystyrene plates used in the ELISA. Each unique color can be used to bind a unique antigen thus multiple antibody determinations can be achieved using a single sample in a single test well. When you add a sample addition to a well containing the coated microbeads, specific antibodies to any of the specific antigens, if present, will bind to the antigen on the microbeads.

After a washing step, a biotinylated anti-chicken immunoglobulin is added. If specific antibody is bound in the first step, the biotinylated anti-species immunoglobulin will be bound. A final addition of a fluorescent reporter molecule, streptavidin-phycoerythrin conjugate, is performed. The microbead contents of the wells are placed in a fluorometer. This device then analyzes each bead, identifying the unique color of each bead and simultaneously measuring any fluorescent signal.

These data are then compiled and analyzed by computer and a Mean Fluorescent Index (MFI) is calculated for each antigen on each serum sample. An MFI cutoff value is established and samples are classified as either negative, borderline, or positive based on the MFI. Thus, the basic steps and principles are very much like an indirect ELISA and IFA but with the advantage of multiplexing for multiple determinations in a single sample.

Why go to all this trouble? Human vaccines, including attenuated influenza virus vaccines, mumps and rubella, and yellow fever vacines, are prepared in SPF chicken eggs. Similarly, most all poultry vaccines are made using SPF eggs, cell cultures, and/or chickens. Vaccines play an important role in the health of humans and their feed source and SPF eggs are a key source for these vaccines.  Utilizing new technology to keep the SPF egg supply safe assures all of us the best chance for good health.