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Poult Sci 2008. 87:912-917. doi:10.3382/ps.2007-00481
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IMMUNOLOGY, HEALTH AND DISEASE

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{dagger}, M. Yang{dagger} and M. E. Cook*,{dagger},1

* Department of Nutritional Science, and {dagger} Department of Animal Science, University of Wisconsin, Madison 53706

1 Corresponding author: mcook{at}wisc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The laying hen can produce antibodies to a wide array of antigens including viruses (Polson et al., 1980), bacteria (Ricke et al., 1988), fungi (Burdsall et al., 1990), proteins (Camenisch et al., 1999), and peptides (Cook, 2004). Furthermore, deposition of high quantities of antibodies in the egg has provided an opportunity for novel commercial uses of eggs (Cook, 2004; Kovacs-Nolan and Mine, 2004a,b; Berghman et al., 2005; Kovacs-Nolan et al., 2005). The extent by which eggs will be used as a source of antibodies will largely be determined by the quantity of antigen-specific antibodies deposited in the yolk (Tini et al., 2002).

Freund (1956) first described the value of incorporating Mycobacteria in paraffin-based oil with surfactants (Freund complete adjuvant; FCA) for the production of antibody when emulsified with soluble protein antigen (SPA) in aqueous solutions. In the last 50 yr, FCA has been the gold standard for generating high antibody titers (Warren et al., 1986; Schwarzkopk et al., 2001) and for inducing antibody-dependent autoimmune disease (Billiau and Matthy, 2001). Today FCA continues to serve as the benchmark for comparing other adjuvants for the production of antibody (Cooper, 1994). For example, in a study by Bollen et al. (1996), FCA was compared with Freund incomplete adjuvant (FCA without Mycobacteria) and Hunter’s TiterMax. In both rabbits and chickens, antibody titers were consistently highest in the FCA group as compared with the other adjuvant groups. Whereas many studies have investigated replacement strategies for FCA (Bollen et al., 1996), methods to improve upon FCA are lacking.

Pathogen-associated molecular patterns (PAMP) recognized by germline-encoded microbial recognition receptors play a significant role in orchestrating immune responses (Janeway and Medzhitov, 2002), and altering host recognition may provide a strategy of enhancing the adjuvant effect of FCA. Hence, the objective of this research was to examine the effect of whole cell bacteria species on the adjuvanticity of FCA as measured by egg antibody titer to SPA. Ferwerda et al. (2005) used knockout mice to demonstrate that host response to Mycobacteria is a nonredundant recognition mechanism involving Toll-like receptor (TLR) 2 and TLR-4 as well as the nucleotide-binding oligomerization domain 2. Both gram-negative and gram-positive killed whole cell bacteria preparations (bacterins) were used due to differences in TLR recognition of bacterin components demonstrated in mice (Takeuchi et al., 1999). Although the cellular constituents of bacteria involved in immune stimulation has yet to be fully elucidated, gram-positive bacterins were selected based upon the diamino acid in position 3 of the peptidoglycan stem peptide (Moreillon and Majcherczyk, 2003). Commercially available bacterins were used in this study because the preparations could have a practical application in the modification of FCA for egg yolk antibody synthesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Birds and Housing
Single-Comb White Leghorn (SCWL) laying hens (S& R Farms, Whitewater, WI), age 22 to 56 wk, used in egg antibody experiments were individually housed in cages. All procedures used in the following experiments were approved by Animal Care Committee at University of Wisconsin, Madison. In all experiments, 8 to 10 hens were randomly assigned to each vaccination treatment group. The sample size (n = 8) was based on a power test using data from past experiments and was found adequate to detect a 50% change in egg antibody content at P < 0.05. Hens had free access to feed and water. Diet consisted of a standard diet formulated to meet the nutritional requirements of laying hens.

Soluble Protein Antigen
The antigen selected for determining the effects of different adjuvants on antibody response was phospholipase A2 (PLA2) purified from porcine pancreas (Novozyme, Bagsvaerd, Denmark). The purity of the PLA2 was estimated to be greater than 90% based on SDS-PAGE conducted by our lab. Although any SPA could have been used for these studies, PLA2 was used for several reasons. First, PLA2 antigen is considered an immunogen because it has a molecular weight (~13 kDa) adequate to stimulate an immune response without requiring conjugation to a carrier protein. In addition, our laboratory has considerable experience studying PLA2, and PLA2 is used for commercial production of antibodies.

Experimental Design
In all experiments, hens were immunized according to methods modified from Schwarzkopf et al. (2001), and control hens were compared with treatment hens. Hens were injected i.m. into each breast and thigh with 0.25 mL of the appropriate primary injection (control or treatment). Seven days after primary immunization, all hens received identical booster injections in the same manner as primary injection (1 mL/bird with 0.25 mL/site). Therefore, treatment hens differed from control hens only at the primary injection.

Week 3 after primary immunization is representative of peak antibody titer in egg yolk (Schwarzkopk et al., 2001); therefore, eggs were collected once per week beginning on wk 3 and continuing until wk 10 for repeat testing by ELISA.

Adjuvant Preparations.
Primary and booster injections used in all experiments were water-in-oil emulsions (50:50) containing 3 mg/mL of PLA2 immunogen. Additionally, all primary injections (control and treatment) contained heat-killed Mycobacterium butyricum immunogens from FCA (DIFCO Laboratories, Detroit, MI). In addition to PLA2 and FCA, primary treatment injections contained various microbial bacterin immunogens. In brief, primary injections for control hens were made by emulsifying FCA with an equal volume of PBS containing a measured amount of lyophilized PLA2. Primary injections for treatment hens were made in the same manner except PLA2 was dissolved in various aqueous bacterins (Table 1Go) instead of PBS. 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 administered to all hens (control and treatment) were made in the same manner as the primary injections for control hens except Freund incomplete adjuvant (DIFCO Laboratories) replaced FCA.


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  		Table 1. Manufacturers of adjuvant additions to Freund complete adjuvant1
 
Two experiments were conducted to determine the adjuvant effect of combining E. coli bacterin (PBS Animal Health, Massillon, OH) with M. butyricum in FCA on antibody response of SCWL hens immunized against SPA. In the first experiment, hens of 2 strains (Bovan and Babcock) and 2 ages (26 and 48 to 52 wk of age) were used. The experiment was arranged as a 2 x 2 x 2 factorial (8 hens per treatment) where the 2 commercial strains of SCWL, approximately 5 or 10 mo of age, received their first immunization of PLA2 in FCA (control) or in FCA plus E. coli (treatment). To confirm the effects of combining E. coli adjuvant with FCA on hen antibody response to SPA, a second experiment was conducted with a single strain of 5-mo-old SCWL hens receiving control immunization or E. coli immunization as described above.

Two experiments were conducted in which SCWL hens received a primary immunization containing PLA2 antigen in PBS emulsified with FCA (n = 8) or antigen in S. aureus bacterin (PBS Animal Health) emulsified with FCA (n = 8). In a repeat experiment, an additional treatment was added to determine if the effect of S. aureus adjuvant addition was due to the lipoteichoic acid (LTA) component. The LTA treatment hens received PLA2 antigen plus 2 mg of S. aureus LTA (Sigma, St. Louis, MO) in PBS emulsified with FCA (n = 8).

A final experiment was conducted to determine adjuvant combination effect of other gram-positive bacterins with FCA. The SCWL hens received a primary control immunization containing PLA2 antigen in PBS emulsified with FCA (n = 10). Treatment immunizations contained antigen in Corynebacterium pseudotuberculosis (PBS Animal Health) emulsified with FCA (n = 10); or antigen in Streptococcus suis (PBS Animal Health) emulsified with FCA (n = 10).

ELISA.
Anti-PLA2 antibody content of egg yolk samples were measured by an ELISA developed in our lab. Briefly, a 96-well Nunc-Immuno Plate with MaxiSorp surface (Thermo Fischer Scientific, Waltham, MA) was coated overnight (100 µL per well) with PLA2 (ammonium sulfate suspension of PLA2 from porcine pancreas, ≥ 600 units/mg of protein; Sigma Aldrich, St. Louis, MO) diluted 1:300 in 50 mM sodium bicarbonate. After washing, the plate was blocked (175 µL per well) for at least 1 h with PBS containing 1% albumin from bovine serum (BSA, fraction 5 with ≥ 98% purity; Sigma Aldrich, St. Louis, MO). Water-extracted egg antibody samples were obtained by extracting liquid egg yolk (200 µL) with 1.8 mL acidified PBS (pH 5) overnight. The extraction mixture was centrifuged at 1,500 x g for 10 min, and the supernate was further diluted to 1:8,000 in PBS (pH ~7). In addition to the weekly egg yolk samples, an in-lab standard was applied to each ELISA plate. The standard applied to each plate consisted of a 2-fold serial dilution from 1:2,000 to 1:64,000 of water-extracted egg yolks from hens immunized against PLA2 in a previous trial. After coating, blocking, and washing the plate, duplicate samples and in-lab standard (100 µL/well) were incubated for 30 min on the plate followed by washing (6x). The detection antibody, goat anti-chicken IgG-Fc conjugated with horseradish peroxidase (Bethyl Laboratories, Montgomery, TX), was diluted 1:10,000 in PBS and added to the wells (100 µL/well) for 15 min followed by washing (8x). Substrate solution (50 mM sodium acetate) containing 0.1 mg/mL of tetramethyl benzidine and 3 mM H2O2 was added (120 µL per well) for color development (~5 min), and the enzymatic reaction was stopped by addition of 50µL per well 0.5 M H2SO4. Absorbance at 450 nm was measured (BioTek EL800 plate reader). Data expressed as Log2 titer were calculated by comparing samples with the in-lab standard. Titer was defined as the highest dilution of sample with an optical density equal to the standard diluted 1:64,000. The change in titer due to bacterin addition to FCA was calculated: [(treatment titer – control titer)/control titer)] x 100.

Statistical Analysis
Data collected from the experiments were analyzed by PROC GLM or PROC MIX procedure using SAS commercial statistical program (SAS Institute, Cary, NC; Littell et al., 1996). Data were analyzed according to the original design of each experiment, and main effects and interactions were reported. Because hens were obtained from commercial sources and comparisons between experiments could be confounded by uncontrolled environmental factors, data were analyzed within each experiment. Repeatable treatment effects were assessed by repeating the experiment. Probability of treatment difference was reported for each experiment as shown in each table and figure.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The E. coli bacterin added to FCA decreased egg yolk antibody titer to SPA by 23% (Log2 titer of 14.35 for FCA and 13.97 for FCA plus E. coli) in SCWL hens across different ages and strains (Table 2Go, P < 0.0001). There was no effect of SCWL strain on hen antibody response (P = 0.508); however, an effect of age approached significance when 52-wk-old hens had higher antibody response on d 28 after immunization than 26-wk-old hens (P = 0.056). No age effect was observed in later weeks (data not shown). In a second experiment where strain and age were not variables, suppressed antibody production associated with E. coli bacterin was observed over a period of 3 to 8 wk after the primary immunization of SCWL (Figure 1Go). The average weekly egg yolk antibody titer was decreased approximately 51% (P = 0.003) in egg yolks from hens injected with SPA in E. coli and FCA (Log2 titer = 13.29) as compared with SPA in PBS and FCA (Log2 titer = 14.32).


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  		Table 2. Effect of Escherichia coli addition to Freund complete adjuvant (FCA) on antibody response against soluble-protein antigen of Single Comb White Leghorn hens of different strains and age1
 

Figure 1
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  		Figure 1. Effect of Escherichia coli addition to Freund complete adjuvant (FCA) on hen egg yolk antibody titer. Log2 antibody titer of phospholipase A2 (PLA2) antibody in egg yolk samples collected weekly (3 to 8 wk) after primary immunization was determined by ELISA. Single-Comb White Leghorn hens received a primary immunization composed of soluble protein antigen (SPA) in PBS emulsified with FCA ({blacksquare}) or SPA in E. coli emulsified with FCA ({square}). Data expressed as mean ± SEM. Effect of E. coli addition at primary immunization was analyzed by SAS Mixed Procedure for repeated measures (P = 0.003).

 
When an adjuvant consisting of S. aureus bacterin plus FCA was used at the primary immunization, antibody to SPA (Log2 titer = 13.35) was increased 62% (P = 0.03) as compared with FCA alone (Log2 titer = 12.65) from 3 to 10 wk post primary immunization (Figure 2Go). In another experiment with limited data due to inconsistent egg production, the log2 antibody titer to SPA for the PBS (control), LTA, and S. aureus addition to FCA was 13.38, 12.93, and 13.80, respectively. No significant differences were found across wk 4 to 10 using repeated measures analysis (data not shown in figure format). On average, 3.9 eggs/wk were available for determining antibody titer of control and S. aureus-treated hens.


Figure 2
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  		Figure 2. Effect of Staphylococcus aureus addition to Freund complete adjuvant (FCA) on hen egg yolk antibody titer. Log2 antibody titer of phospholipase A2 (PLA2) antibody in egg yolk samples collected weekly (3 to 8 wk) after primary immunization was determined by ELISA. Single-Comb White Leghorn hens received a primary immunization composed of soluble protein antigen (SPA) in PBS emulsified with FCA ({blacksquare}) or SPA in S. aureus emulsified FCA ({square}). Data expressed as mean ± SEM. Effect of S. aureus addition at primary immunization was analyzed by SAS Mixed Procedure for repeated measures (P = 0.03).

 
Addition of C. pseudotuberculosis bacterin to FCA at the primary immunization of hens did not significantly increase (P = 0.13) antibody titers to SPA (Log2 titer = 13.88) as compared with the FCA control (Log2 titer = 13.4). However, a significant 51% increase (P = 0.04) in SPA antibody titer (Log2 titer = 13.99) was observed when S. suis bacterin was added to FCA across wk 3 to 10 post primary immunization (Figure 3Go).


Figure 3
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  		Figure 3. Effect of Corynebacterium pseudotuberculosis or Streptococcus suis addition to Freund complete adjuvant (FCA) on hen egg yolk anti-body titer. Log2 antibody titer of phospholipase A2 (PLA2) antibody in egg yolk samples collected weekly (3 to 10 wk) after primary immunization was determined by ELISA. Single-Comb White Leghorn hens received a primary immunization composed of soluble protein antigen (SPA) in PBS emulsified with FCA ({blacksquare}), S. suis emulsified with FCA ({square}), or C. pseudotuberculosis emulsified with FCA ({blacktriangleup}). Data expressed as mean ± SEM. Effect of S. suis or C. pseudotuberculosis addition at primary immunization was analyzed by SAS Mixed Procedure for repeated measures (P = 0.04, P = 0.13, respectively).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The effect of adjuvant combinations on hen antibody response was studied to improve the understanding of host immune response and to develop strategies for enhancing commercial egg antibody synthesis. The FCA is thought to enhance antibody response to SPA through stimulation of the innate immune system by Mycobacteria. Experiments were designed based on recent discoveries demonstrating that the innate immune system recognizes various bacterial components through TLR, which can modulate antibody response. Dendritic cells of the innate immune system recognize PAMP to distinguish pathogens from self (Janeway and Medzhitov, 2002). Additionally, the host is able to differentiate between gram-negative and gram-positive bacteria. The host antibody response to simultaneous PAMP exposure is not well defined; hence, the primary objective of our research was to determine if addition of killed whole cell bacteria to FCA could potentiate hen antibody response to SPA. Commercially available bacterins were used based on knowledge of the adjuvant effect of individual bacterin components or PAMP [lipopolysaccharide (LPS), peptidoglycan, and LTA]. Additionally, amendment of FCA with appropriate bacterin at the primary immunization could offer an inexpensive strategy for enhancing hen egg yolk antibody content to SPA.

In murine models, it has clearly been demonstrated that TLR-4 recognizes gram-negative bacterial LPS (Hoshino et al., 1999; Qureshi et al., 1999), whereas TLR-2 recognizes Mycobacteria (Means et al., 1999) and gram-positive bacterial LTA (Yoshimura et al., 1999). Because gram-negative bacteria stimulate through a different TLR than Mycobacteria, it was hypothesized that gram-negative E. coli would increase antibody response when combined with FCA. However, it was found that addition of E. coli bacterin to FCA decreased antibody response to SPA relative to that typically observed with FCA. These results are consistent with the finding that administration of E. coli LPS decreased hen antibody response to the SPA, bovine serum albumen (Parmentier et al., 1998). It would be interesting to determine if decreased antibody response to SPA in hens resulted from simultaneous stimulation of TLR-4 by E. coli LPS and of TLR-2 by FCA lipoproteins.

Considering recent discoveries demonstrating that gram-positive S. aureus and Mycobacteria both stimulate immunity through TLR-2, it was surprising to discover that S. aureus addition to FCA increased antibody response to SPA. In a repeat experiment, the possibility that the LTA component of S. aureus was responsible for the adjuvant combination effect was investigated. Induction of immune response by S. aureus LTA was mediated by TLR-2 in human embryonic kidney HEK293 cells (Schwandner et al., 1999). In a hen antibody experiment with apparent limited statistical power, it did not appear that LTA from S. aureus was the component responsible for increased antibody response when combined with FCA. In mice, LTA has been shown to induce cytokine release by monocytes similar to LPS (Morath et al., 2002) and therefore may not be responsible for enhanced antibody response.

In the final experiment, S. aureus or S. suis bacterins were found to potentiate antibody response to SPA when added to FCA at the primary immunization, but C. pseudotuberculosis addition to FCA had no effect. These findings were anticipated based on studies of gram-positive bacteria peptidoglycan. Moreillon and Majcherczyk (2003) reviewed the differences in the proinflammatory activity of peptidoglycan from gram-positive bacteria due to differences in the amino acid at position 3 of the peptidoglycan stem peptide. Mycobacteria tuberculosis and C. pseudotuberculosis contain diaminopimelic acid at this position while S. aureus and S. suis contain lysine.

Although our current studies did not investigate the mechanism of adjuvant combination effects, it is clear that simultaneous stimulation of the hen innate immune system with various PAMP recognized by TLR-2 enhanced antibody response to SPA. These results could be explained by the ability of TLR-2 to switch TLR partner to recognize diverse ligands and to initiate appropriate adaptive immune responses (Akira et al., 2001). Using cell culture models, Ozinsky et al. (2000) demonstrated that cooperation between TLR allows recognition of diverse PAMP. The homodimeric complex of TLR-4 recognizes LPS; however, the heterodimeric complex of TLR-2 with TLR-6 or with TLR-1 recognizes gram-positive peptidoglycan components (Ozinsky et al., 2000) or Mycobacteria lipoproteins (Takeuchi et al., 2002), respectively.

In summary, killed whole cell bacteria adjuvant additions to FCA containing various PAMP or TLR ligands modulated the adaptive immune response of the hen as measured by antibody response to vaccination. Escherichia coli adjuvant combination results suggest that commercial producers of egg antibody and veterinarians should be aware that combination vaccines can suppress antibody response. Improved vaccination strategies were designed by utilizing the mechanistic understanding of TLR determined in cell culture and murine models. In regards to commercial antibody production with laying hens, FCA has been known as the gold standard for maximum egg antibody production to SPA; however, combining gram-positive bacterins with FCA increased egg yolk antibody levels relative to antibody levels found when FCA was used alone. The potential exists for commercially available bacterins to serve as inexpensive adjuvant additions for the production of egg yolk antibody. When combined with FCA, gram-positive bacterins containing peptidoglycan structures or other PAMP dissimilar from FCA served as effective adjuvants for the production of egg antibody to SPA.


   ACKNOWLEDGMENTS
 
The authors express their appreciation to S&R Farms (Whitewater, WI) for providing laying hens, and to the staff at the University of Wisconsin Poultry Research Lab for support of research. Research was supported by National Center for Food Protection and Defense (NCFPD), Midwest Poultry Consortium, and aOvaTechnologies Inc. Mark E. Cook and Mingder Yang have an ownership interest in aOvaTechnologies, which has the rights to license the technology reported in this publication.

Received for publication November 26, 2007. Accepted for publication January 14, 2008.


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 DISCUSSION
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