J. Dairy Sci. 86:146-151
© American Dairy Science Association, 2003.
Opsonic Activity of Serum and Whey from Cows Immunized with the Ferric Citrate Receptor1
A. J. Wise,
J. S. Hogan,
K. Takemura and
K. L. Smith
Department of Animal Sciences The Ohio State University Ohio Agricultural Research and Development Center, Wooster 44691
Corresponding author:
J. S. Hogan; e-mail:
hogan.4{at}osu.edu.
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ABSTRACT
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The effects of immunizing dairy cows with the ferric citrate receptor, FecA, on the opsonic activity of serum and whey were measured in a phagocytosis assay. Fifteen cows were assigned to five blocks of three cows based on date of expected parturition. Cows within a block were randomly assigned to one of three treatments: 1) FecA immunization, 2) immunization with a commercially available Escherichia coli J5 bacterin, and 3) unimmunized controls. Cows were challenged at approximately 21 DIM by intramammary infusion of E. coli 727 into one mammary quarter. Escherichia coli 727 were opsonized for the phagocytosis assay with either 10% heat-inactivated serum or 50% heat-inactivated whey collected from each cow at calving, immediately before challenge and 7 d after challenge. Cows immunized with FecA or the E. coli J5 bacterin had increased IgG titers against FecA and E. coli 727 compared with unimmunized control cows. However, sera and whey collected from cows immunized with FecA did not enhance opsonization of E. coli 727 compared with sera and whey from control cows. Immunization with the E. coli J5 bacterin increased opsonization of sera greater than immunization with FecA. Immunoglobulin M antibody titer against E. coli 727 in whey and phagocytic indexes were positively correlated. The phagocytic index of whey immediately before challenge and 7 d after challenge were negatively associated with peak bacterial counts in mammary quarters challenged with E. coli 727. Results of the current trial suggest that the immune response resulting from immunization with FecA did not enhance opsonization and in vitro phagocytosis of E. coli 727.
Key Words: Escherichia coli • FecA • phagocytosis
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INTRODUCTION
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Gram-negative bacteria isolated from bovine IMI expressed iron-regulated outer membrane proteins including the ferric citrate receptor, FecA (Todhunter et al., 1991b; Lin et al, 1999). Immunizing dairy cows with FecA from Escherichia coli enhanced antibody titers against the protein, but had minimal effect on clinical severity of mastitis following intramammary challenge (Takemura et al., 2002). Immunizing cows with Escherichia coli J5 bacterins enhanced antibody titers against heterologous bacterial strains and reduced the severity of clinical coliform mastitis (Hogan et al., 1999). The difference between efficacies of the FecA vaccine and the E. coli J5 bacterins may reside in the opsonic activity of serum and whey resulting from immunization. Effective phagocytosis by neutrophils depends on the opsonization of bacteria by the appropriate antibodies or complement fragments (Craven and Williams, 1985). The efficacy of E. coli J5 bacterins was related to the enhanced opsonic activity of serum and whey against heterologous Gram-negative strains (Hogan et al., 1992a). In addition to iron acquisition, the expression of iron-regulated outer membrane proteins and other cell surface changes may enhance the pathogenicity of E. coli by rendering the bacteria less susceptible to phagocytosis (Telang et al., 2001). The purposes of the current trial were to determine: 1) the opsonic activity of whey and serum from FecA immunized cows in a bacterial cell phagocytosis assay; and 2) investigate the possible relationships among in vitro phagocytic index and clinical parameters following intramammary challenge with E. coli.
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MATERIALS AND METHODS
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Experimental Cows
Fifteen cows in The Ohio Agricultural Research and Development Center dairy herd were used. Cows were challenged by intramammary infusion of Escherichia coli 727 in the trial detailed by Takemura et al. (2002). Experimental cows were from five complete blocks grouped by anticipated parturition dates. Cows within blocks were randomly assigned to one of three treatments: 1) FecA immunization; 2) immunization with a commercially available Escherichia coli J5 bacterin; and 3) unimmunized controls. Cows were immunized by subcutaneous injections 14 d before the end of lactation, intramammary infusion 7 d after the end of lactation, and subcutaneous injection 28 d after the end of lactation. FecA and E. coli J5 vaccines were as described by Takemura et al. (2002). Cows were challenged by intramammary infusion of E. coli 727 approximately 21 d after parturition. Sampling frequency and procedures following challenge were detailed in Takemura et al. (2002). Peak bacterial counts (colony-forming units per milliliter of milk) and peak rectal temperature following challenge were the clinical variables tested for associations with in vitro phagocytic variables.
Serum and Whey
The opsonic activity of serum and whey from each cow was measured in samples collected 14 d before dry off (D-14), at calving (C+0), immediately before intramammary challenge (E+0), and 7 d after intramammary challenge (E+7). Serum and whey samples were heat-inactivated at 56°C for 30 min and then stored frozen at –20°C. Bacteria were opsonized with 10% heat inactivated serum and 50% heat inactivated whey for 20 min at 20°C before use in neutrophil assays.
Antibody Titers
Antibody titer in sera and whey were determined by sandwich ELISA (Takemura et al., 2002). Briefly, the coating antigens were purified FecA and E. coli 727 cultured in tryptic soy broth (Beckton, Dickinson and Company, Sparks, MD). Isotypes were determined by goat anti-bovine IgG and goat anti-bovine IgM (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Titer data were expressed as the reciprocal of the dilution log10.
Bacteria
Escherichia coli 727 was the bacterial strain used in the bacterial phagocytosis assay. Escherichia coli 727 were cultured before the in vitro phagocytosis assay using conditions employed by Takemura et al. (2002) for culturing E. coli 727 in the intramammary challenge trial. Before use, E. coli 727 was stored in tryptic soy broth containing 20% glycerin at –70°C. An overnight culture of E. coli 727 was prepared by inoculating 0.1 ml of stock culture into 12 ml of tryptic soy broth and incubating overnight at 37°C on a gyratory shaker at 100 rpm. Twenty-four milliliters of tryptic soy broth were each inoculated with 0.2 ml of the overnight culture and incubated for 2.5 h at 37°C and 50 rpm on a gyratory shaker. The bacteria were centrifuged for 20 min at 5000 x g. The bacterial pellet was diluted in Hank’s balanced salts solution to approximately 40 x 106colony-forming units. Bacteria were plated on MacConkey agar to confirm the total number of E. coli.
Preparation of Blood Neutrophils
Blood was collected from the jugular veins of two cows (50 ml each) on a daily basis. Neutrophils were isolated according to Carlson and Kaneko (1973). Neutrophil viability was measured by trypan exclusion. The total number of cells was determined with the aid of a hemocytometer. A differential stain (Diff-Quick; Baxter Healthcare Corporation; Miami, FL) was also done to determine the total percentage of neutrophils. The number of viable neutrophils was determined as [(neutrophil viability) (the total number of cells) (the percentage of neutrophils)]. Viable neutrophil concentrations were adjusted to 40 x 106 viable neutrophils/ml of Hank’s balanced salts solution.
Phagocytic Assay
Assays were prepared as a 2:1 bacteria:neutrophil ratio. The assays were incubated for 1.5 h at 37°C and 100 rpm on a gyratory shaker. After incubation, the samples were diluted 2:1:1 as 50 µl of assay suspension: 25 µl of acridine orange (1.4 mg per 10 ml of PBS): 25 µl of crystal violet (5 mg per 10 ml of PBS). Wet mount slides were prepared and bacterial cells were counted in the first 25 neutrophils visible under the 1000X oil immersion lens as the microscope stage (Nikon Fluorescence Microscope; Garden City, NY) was moved from left to right on the cover slip (Hogan et al., 1992a). Samples from cows within a block and sampling period were assayed on the same day. The assays were in duplicate and replicated on separate days. The person reading and recording phagocytic results was masked relative to cow and treatments. Parameters measured were phagocytic index (number of intracellular bacteria/total number of neutrophils), the percentage of neutrophils phagocytizing (number of neutrophils with intracellular bacteria/total number of neutrophils), and the number of bacteria per positive neutrophil (number of intracellular bacteria/number of positive neutrophils).
Statistical Analysis
Differences among treatments in neutrophil assays were tested by least squares analysis of variance (SAS, 1999). The phagocytic indexes of sera and whey prior to treatment (D-14) were different and were included as a covariate to adjust for variability unrelated to vaccine treatment. Differences among treatments within sample periods C+0, E+0, and E+7 were tested by Tukey’s multiple comparison test. The correlations between phagocytic indices and antibody titers were quantified by Pearson’s correlation coefficients (SAS, 1999).
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RESULTS
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Opsonization
The opsonization of bacteria with serum collected immediately before challenge from E. coli J5-vaccinated cows resulted in a higher phagocytic index than serum from FecA-vaccinated cows (P = 0.09; Figure 1
). The percentage of neutrophils phagocytizing bacteria opsonized with serum from E. coli J5-vaccinated cows was greater than for bacteria opsonized with serum from FecA-vaccinated cows at E+0 (P < 0.05; Figure 1). No differences existed among treatments at E + 0 for the number of bacteria per phagocytizing neutrophil (Figure 1). Furthermore, no differences existed among treatments at C+0 and E+7 for phagocytic index, the percentage of positive neutrophils, or the number of bacteria per positive neutrophil.
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Figure 1. Phagocytic index (A), percentage of neutrophils phagocytizing bacteria (B), and number of bacteria per phagocytizing neutrophil (C) for Escherichia coli opsonized with 10% heat-inactivated serum from Escherichia coli J5-immunized cows (solid bars) (n=5), unimmunized controls (open bars) (n=5), and FecA immunized cows (diagonal hashed bars) (n=5). Sera were collected at calving (C+0), at the time of intramammary challenge (E+0), and 7 d after challenge (E+7). Dispersion bars represent standard error of means.
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The opsonization of bacteria with 50% heat-inactivated whey resulted in no differences among treatments at C+0, E+0, and E+7 for the phagocytic index, the percentage of positive neutrophils, or the number of bacteria per positive neutrophil (Figure 2).
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Figure 2. Phagocytic index (A), percentage of neutrophils phagocytizing bacteria (B), and number of bacteria per phagocytizing neutrophil (C) for Escherichia coli opsonized with 50% heat-inactivated whey from Escherichia coli J5-immunized cows (solid bars) (n=5), unimmunized controls (open bars) (n=5), and FecA-immunized cows (diagonal hashed bars) (n=5). Sera were collected at calving (C+0), at the time of intramammary challenge (E+0), and 7 d after challenge (E+7). Dispersion bars represent standard error of means.
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Phagocytic Index versus Antibody Titers
Whey IgM titers against FecA at E+0 and E+7 were not correlated with phagocytic index of bacteria (P > 0.05). The correlation coefficient between IgM antibody titer to E. coli 727 in whey (Figure 3) immediately before challenge, and the phagocytic index of E+0 whey opsonized bacteria was r = 0.51 (r2 = 0.26). At E+7, whey IgM titer against E. coli 727 and phagocytic index of whey were not correlated (P > 0.05). Whey IgG titers against FecA and E. coli 727 were not related to phagocytic indices at either E+0 or E+7. Correlations were not significant (P > 0.05) among the antibody titers of serum against either E. coli 727 or FecA (Figure 4) or the phagocytic index of serum.
Phagocytic Index versus Clinical Parameters
Mean (± SD) peak bacterial counts in milk for all cows following intramammary challenge was 3.5 ± 3.0 cfu/ml log10. Mean (± SD) peak rectal temperature for all cows following intramammary challenge was 40.9 ± 1.6° C. The phagocytic index of whey at E+0 and E+7 were negatively associated with peak bacterial counts after challenge. The correlation coefficient (n=15) between the phagocytic index of whey at E+0 and peak bacterial counts after challenge was r = –0.58 (r2 = 0.34). The correlation coefficient (n=15) between the phagocytic index of whey at E+7 and peak cfu after challenge was r = –0.53 (r2 = 0.28). Correlations were not significant (P > 0.05) among the phagocytic index of serum opsonized bacteria and peak rectal temperatures.
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DISCUSSION
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The opsonic properties of sera and whey were not enhanced in FecA-immunized cows. Opsonic properties were similar between sera collected from FecA-immunized cows and sera collected from unimmunized controls. Furthermore, opsonic properties did not differ among treatments for bacteria opsonized with heat-inactivated whey. However, opsonic properties were enhanced for sera collected from E. coli J5-immunized cows. The opsonization of bacteria with sera collected at challenge from E. coli J5-immunized cows resulted in a higher phagocytic index than sera collected at challenge from FecA-immunized cows. The percentage of PMNs phagocytizing bacteria opsonized with sera collected at challenge from E. coli J5-immunized cows was greater than for bacteria opsonized with sera collected at challenge from FecA-immunized cows. Similar results were observed in a phagocytosis assay by Hogan et al. (1992a) as sera from E. coli J5-immunized cows expressed enhanced opsonic activity over unimmunized controls.
The recognition of a single protein by anti-FecA sera may contribute to the decreased opsonic activity of sera collected from FecA-immunized cows versus sera collected from E. coli J5-immunized cows. Immunization with FecA stimulates an immune response in cows against a single protein found in the outer membrane of E. coli bacteria (Lin et al., 1999). Anti-FecA sera recognizes only the FecA protein and does not react with any other outer membrane protein of E. coli or Klebsiella pneumoniae (Lin et al., 1999). However, immunization with an E. coli J5 whole cell bacterin stimulates immunoglobulin production against a set of common core antigens of Gram-negative bacteria, thus providing protection against a variety of coliform bacteria (Hellman et al., 1997). The stimulation of immunoglobulins against a wide variety of antigens may enhance the opsonic activity of anti-E. coli J5 sera versus the opsonic activity of sera collected from FecA-immunized cows.
Increased antibody titers following immunization enhances opsonic activity of mammary secretions (Hogan et al., 1992a). Immunoglobulin M was the primary heat-stable opsonin for E. coli in mammary secretions collected from early lactation cows (Williams and Hill, 1982). Results of the present trial supported the hypothesis that IgM titers are closely related to the opsonic activity of whey. A positive correlation existed between the phagocytic index of whey and whey IgM titers against E. coli 727 in the present study. No correlation existed between the phagocytic index of sera versus sera IgG and IgM titers against FecA nor the phagocytic index of whey versus the whey IgG and IgM titers against FecA.
Previous trials have reported that in vitro phagocytosis and neutrophil activity of cows were related to in vivo host response to intramammary infections (Lohuis et al., 1990). In the current trial, E. coli 727 were cultured for the phagocytosis assay using the same conditions for culture before the intramammary challenge trial (Takemura et al., 2002). Identical culture conditions should have induced similar antigenic presentation for both the in vitro and in vivo experiments. In the present study, a relationship existed between the phagocytic index versus peak bacterial counts after challenge. The phagocytic index of whey at challenge versus peak bacterial count after challenge were negatively correlated. In addition, the phagocytic index of whey 7 d after challenge versus peak bacterial count after challenge also were negatively correlated. The severity of clinical signs following intramammary infusion of E. coli were related to peak bacterial counts in milk from challenged quarters (Hogan et al., 1999). Minimizing peak cfu counts in milk after challenge subsequently reduced the severity of clinical signs (Hogan et al., 1992b).
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SUMMARY
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The immunization of dairy cows with FecA elicited an immune response against FecA and E. coli 727 but failed to enhance the opsonic activity of sera and whey against E. coli 727. Results of the current trial support previous findings that IgM is a primary opsonin for E. coli in mammary secretions, and the in vitro opsonic activity of whey from a mammary quarter is inversely related to in vivo peak bacterial counts in mammary quarters challenged with E. coli.
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FOOTNOTES
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1 Salaries and research support were provided by state and federal funds appropriated to the Ohio Agricultural and Research Development Center, The Ohio State University. Manuscript number 30-02AS. 
Received for publication March 26, 2002.
Accepted for publication August 9, 2002.
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REFERENCES
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Carlson, G. P., and J. J. Kaneko. 1973. Isolation of leukocytes from bovine peripheral blood. Proc. Soc. Exp. Biol. Med. 142:853–856.[Medline]
Craven, N., and M. R. Williams. 1985. Defences of the bovine mammary gland against infection and prospects for their enhancement. Vet. Immunol. Immunopathol. 10:71–127.[Medline]
Hellman, J., E. M. Zanzot, P. M. Loiselle, S. F. Amota, K. M. Black, Y. Ge, J. T. Kurnick, and H. S. Warren. 1997. Antiserum against Escherichia coliJ5 contains antibodies reactive with outer membrane proteins of heterologous gram-negative bacteria. J. Infect. Dis. 176:1260–1268.[Medline]
Hogan, J. S., D. A. Todhunter, G. M. Tomita, K. L. Smith, and P. S. Schoenberger. 1992a. Opsonic activity of bovine serum and mammary secretion after Escherichia coliJ5 vaccination. J. Dairy Sci. 75:72–77.[Abstract]
Hogan, J. S., W. P. Weiss, D. A. Todhunter, K. L. Smith, and P. S. Schoenberger. 1992b. Efficacy of an Escherichia coliJ5 mastitis vaccine in an experimental challenge trial. J. Dairy Sci. 75:415–422.[Abstract]
Hogan, J. S., V. L. Bogacz, M. Aslam, and K. L. Smith. 1999. Efficacy of an Escherichia coliJ5 bacterin administered to primigravid heifers. J. Dairy Sci. 82:939–943.[Abstract]
Lin., J., J. S. Hogan, and K. L. Smith. 1999. Antigenic homology of the inducible ferric citrate receptor (FecA) of coliform bacteria isolated from herds with naturally occurring bovine intramammary infections. Clin. Diagnos. Lab. Immunol. 6:966–969.[Abstract/Free Full Text]
Lohuis, J.A.C.M., Y. H. Schukken, P.A.J. Henricks, R. Heyneman, C. Burvenich, J.H.M. Verheiden, and A. Brand. 1990. Preinfection functions of blood polymorphonuclear leukocytes and the outcome of experimental Escherichia coli mastitis in the cow. J. Dairy Sci. 73:342–350.[Abstract]
SAS User’s Guide: Statistics, Version 8 Edition. 1999. SAS Inst., Inc., Cary, NC.
Takemura, K., J. S. Hogan, and K. L. Smith. 2002. Efficacy of immunization with ferric citrate receptor (FecA) from Escherichia coli on induced coliform mastitis. J. Dairy Sci. (In Press).
Telang, S., E. Vimr, J. R. Mahoney, I. Law, H. Lundquist-Gustafsson, M. Qian, and J. W. Eaton. 2001. Strain-specific iron-dependent virulence in Escherichia coli. J. Infect. Dis. 184:159–165.[Medline]
Todhunter, D. A., K. L. Smith, and J. S. Hogan. 1991. Antibodies to iron-regulated outer membrane proteins of coliform bacteria isolated from bovine intramammary infections. Vet. Immunol. Immunopathol. 28:107–115.[Medline]
Williams, M. R., and A. W. Hill. 1982. A role of IgM in the in vitro opsonization of Staphylococcus aureus and Escherichia coli by bovine polymorphonuclear leucocytes. Res. Vet. Sci. 33:47–53.[Medline]
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TOP
ABSTRACT
INTRODUCTION
), and FecA-immunized cows (
). Samples were collected at calving (C+0), at the time of intramammary challenge (E+0), and 7 d after challenge (E+7).