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Additions of Killed Whole Cell Bacteria Preparations to Freund Complete Adjuvant Alter Laying Hen Antibody Response to Soluble Protein Antigen

D. L. Trott^*, E. M. Hellestad, M. Yang and M. E. Cook^*,,1

^* Department of Nutritional Science, and Department of Animal Science, University of Wisconsin, Madison 53706

¹ Corresponding author: mcook{at}wisc.edu

Passive transfer of antibodies from hen to egg has value to both the producer of commercial polyclonal egg antibody and the producer of hatching eggs. Water-in-oil emulsions are commonly amended with immune stimulants such as Mycobacteria (e.g., Freund complete adjuvant; FCA) to increase antibody production to soluble protein antigens (SPA). Recent discoveries of the mechanisms by which microbial products act as adjuvants led us to hypothesize that additions of killed whole cell bacteria (bacterins) to FCA could improve antibody responses to SPA. All injections used in each experiment were water-in-oil emulsions (50:50) containing 3 mg/mL of phospholipase A₂ (PLA₂) immunogen. Additionally, all primary control and treatment injections contained heat-killed Mycobacterium butyricum immunogens from FCA. In addition to PLA₂ and FCA, primary treatment injections contained various microbial bacterin immunogens. Hence, the experimental treatment of all experiments was addition of a commercial source of microbial bacterin to FCA for the primary injection only. Booster injections were the same as the primary control injections except Freund incomplete adjuvant replaced FCA. Anti-body titers to PLA₂ in yolk were determined by ELISA. Bacterins tested as additives to FCA were Escherichia coli, Staphylococcus aureus, Streptococcus suis, and Corynebacterium pseudotuberculosis. Escherichia coli bacterin added to FCA decreased egg yolk antibody titer to SPA by 23% in hens of different ages and strains (P < 0.0001). In a second experiment, a 51% decrease in antibody production associated with E. coli bacterin was sustained for several weeks after the primary immunization (P = 0.003). Staphylococcus aureus or Streptococcus suis combined with FCA increased egg yolk antibody 62 and 51%, respectively (P < 0.05), and Corynebacterium pseudotuberculosis had no effect. In conclusion, the addition of bacterin to FCA can influence hen antibody response to SPA as measured in egg yolks. It is hypothesized that the difference in antibody production may be related to the composition of various pathogen associated molecular patterns in the primary injection.

Key Words: egg antibody • Freund adjuvant • pathogen-associated molecular patterns • vaccinology