Veterinary Immunology and Immunopathology, 28 ( 1991 ) 107-115
107
Elsevier Science Publishers B.V., Amsterdam
Antibodies to iron-regulated outer membrane proteins of coliform bacteria isolated from bovine intramammary infections D.A. Todhunter, K.L. Smith and J.S. Hogan Ohio Agricultural Research and Development Center~OhioState University,Department of Dairy Science, Wooster,OH 44691, USA (Accepted 7 June 1990)
ABSTRACT Todhunter, D.A., Smith, K.L. and Hugan, J.S., 1991. Antibodies to iron-regulatedouter membrane proteins of coliform bacteria isolated from bovine intramammary infections. Vet. lmmunol, hn. munopathoL, 28: ! 07-115. Expression of iron-regulated outer membrane proteins (OMP) by Esdaerichia coli and Klebsiella pneumoniae initially isolated from bovine intramammary infections (IMI) was investigated. Additionally, the presenceofantibodies in bovine serum and mammary secretion directed against the ironregulated OMP was examined. Outer membrane proteins were separated by sodium-dodecylpolyacrylamide electrophoresis. Detection ofimmunoglobulin G directed against OMP was by immunoblotting. All Gram-negative bacteria expressed iron-regulatedOMP when grown in skim milk or tryptiease soy broth plus iron chelator, ot-c~'-dipyridyl.Immunoglobulin G directed against the ironregulatedOMP, as well as the major OMP and several other proteins, was detected in serum and milk of lactating cows with or without Gram-negative bacterial IMI. Antibody against the iron-regulated OMP was detected also in colostrum, secretion from the involuted gland, and in newborn calf serum 4 days after ingestingcolostrum.
INTRODUCTION
The dry period is a critical time in the dynamics of Gram-negative bacterial intramammary infections (IMI). Rate of Gram-negative bacterial IMI is 3 to 4 fold greater during the dry period than during lactating (Smith et al., 1985 ). Rate of new IMI is not constant across the dry period but elevated during the first 2 weeks of involution and the 2 weeks prepartum. Composition of mammary gland secretion changes markedly during involution. Concentrations of lactoferrin, serum albumin, and all immunoglobulin (Ig) classes increase (Schanbacher and Smith, 1975; Welty et al., 1976) while citrate and lactose concentrations decrease (Wheeloek et al., 1967; Lascelles and Lee, 1978). Data from mammary secretions obtained at various stages of involution dern0165-2427/91/$03.50
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onstrated that secretion became progressively more inhibitory to the in vitro growth ofEscherichia coli and Klebsiella spp. as involution progresses (Breau and Oliver, 1986; Oliver and Bushe, 1987). Secretion from the fully involuted gland inhibited the growth of several strains of Gram-negative bacteria (Todhunter et al., 1990). The iron-binding protein lactoferrin and Ig contributed in part to the inhibitory properties of secretion from the involuted gland. In an iron-restricted environment E. coli and Klebsiella spp. produce low molecular weight iron chelators (siderophores) and several outer membrane proteins (Hantke and Braun, 1975; Klebba et al., 1982; Chart and Griffiths, 1985 ). Some of these outer membrane proteins (OMP) are receptors for the ferric siderophore and are not synthesized under conditions of available iron, as the system is repressed in the presence of iron. E. coli, K. pneumoniae, Proteus spp., and Pseudomonas aeruginosa growing in vivo and in vitro, have been shown to produce iron-regulated OMP in the molecular weight range of 74 000 to 83 000 daltons (Pugsley and Reeves, 1976a,b; Klebba et al., 1982). Antibodies of the IgG class directed against the iron-regulated OMP have been detected in the serum of several animal species and humans demonstrating the antigenicity of the proteins (Griffiths et al., 1985; Shand et al., 1985; Bolin and Jensen, 1987). The objectives of this study were to ( 1 ) determine if E. coli and K. pneumoniae isolated initially from bovine IMI express iron-regulated OMP and (2) determine if antibody directed against the iron-regulated OMP is present in bovine serum and mammary secretions. MATERIALS AND METHODS
Bacterial strains and growth conditions Bacteria were E. coil and K. pneumoniae isolated from naturally occurring IMI in the OARDC dairy research herd, Wooster, OH. One E. coli strain (92) was obtained from Dr. J.S. McDonald, Ames, IA. All Gram-negative bacteria were stored on trypticase soy agar (BBL Microbiology Systems, Becton Dickinson and Co., Cockeysville, MD) at room temperature prior to use. Bacteria were grown in trypticase soy broth (TSB), TSB containing 300 aM o~-a'-dipyridyl ( Sigma Chemical Co., St. Louis, MO ), or skim milk. Milk was aseptically obtained from several cows, pooled, and centrifuged at 5000 ×g for 20 min at 4°C to remove somatic cells and fat. Gram-negative bacteria were incubated at 37°C for 18 h on a rotary shaker (200 rpm) in all growth media. Extraction and electrophoresis of outer membrane proteins Outer membrane proteins were extracted by the method of Shand et al. (1985) with slight modifications. Following incubation, bacteria were harvested by centrifugation at 2500 X g for 30 rain at 4°C and washed 3 × in 0.15
ANTIBODIESTO COLIFORMBACTERIAFROM BOVINEINFECTIONS
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M NaC1. Cells were suspended in deionized, distilled H20 and disrupted by sonication for 10 min. Sonicated bacteria were centrifuged at 5000 ×g for 10 min at 4°C. N-lauroylsarcosine sodium salt (Sigma Chemical Co. ) was added to the supernatant at a final concentration of 2% and incubated for 30 rain at room temperature. Outer membrane proteins were collected by centrifugation at 50 000Xg for 60 min at 4°C, washed 2 ×in deionized, distilled H:O, and stored at -70°C. Total protein was determined by the method of Lowry etal. (1951). Outer membrane proteins were separated by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) by the method of Lugtenberg et al. (1975) utilizing the discontinuous buffer system of Laemmli (1970). Stacking gels were 4% acrylamide (Bio-Rad Laboratories, Richmond, CA) in 0.125 M Tris, pH 6.8 and separating gels were 10% acrylamide in 0.375 M Tris, pH 8.8. Outer membrane proteins we,re prepared for electrophoresis by heating at 100°C for 5 min in 0.0625 M Tris, pH 6.8, 2% sodium dodecyl sulfate, 5% 2-p mercaptoethanol, and 10% glycerol. Molecular weight standards (Bio-Rad) were: phosphorylase B (92 500); bovine serum albumin (66 200); ovalbumin (45 000); carbonic anhydrase (31 000); soybean trypsin inhibitor (21 500) and lysozyme ( 14 400). Standards were prepared for electrophoresis as described above. Bromphenol blue tracking dye was added to achieve a final concentration of 0.00125%. A constant current of 30 mA/ gel was used. Gels were stained with 0.1% Coomassie blue R.250 in 40% (v/ v) methanol, 10% (v/v) glacial acetic and destained in 40% (v/v) methanol and 10% (v/v) glacial acetic acid.
Detection of antibodies by immunoblotting Outer membrane proteins were separated by SDS-PAGE and transferred to nitrocellulose sheets at 60 V overnight with cooling (Towbin et al., 1979). Buffer for electrotransfer was 0.025 M Tris, 0.192 M glycine, and 20% (v/v) methanol, pH 8.3. After transfer, nitrocellulose sheets were washed 4× for 15 min at 37°C in phosphate buffered saline (PBS) containing 0.3% polyoxyethylene sorbitan monolaurate (Batteiger et al., 1982). Washed nitrocellulose sheets were incubated overnight with gentle agitation in the presence of bovine antibody. Bovine antibody was diluted in PBS plus 0.3% polyoxyethylene sorbitan monolaurate (PBS-Tween). Nitrocellulose sheets were washed 3 X for 10 rain with PBS-Tween at room temperature and then incubated for I h with rabbit anti-bovine IgG conjugated to horse radish peroxidase (Sigma Chemical Co.) diluted in PBS-Tween. Nitrocellulose sheets were washed as described above and incubated for 5 to 10 rain with a substrate solution containing 30 rng of 4-chloro-l-naphthol dissolved in 10 ml methanol, 0.025 ml of H202 (30%), and PBS to a final volume of 60 ml.
1!0
D.A. TODHUNTERET AL.
Serum and mammary secretion immunoglobulin for immunoblots Blood and milk were obtained from five lactating cows with a Gram-negative bacterial IMI and five cows that were uninfected. Milk was obtained from the mammary quarter infected with Gram-negative bacteria and from one mammary quarter of uninfected cows. Milk was centrifuged at 16 000 ×g for 30 min at 4°C to remove fat and cells. Casein was precipitated from skim milk by rennet coagulation (4 g C a C O 3 + 1 mI saturated rennin/l secretion) and Ig was precipitated by addition of ammonium sulfate to 43% saturation. Blood was permitted to clot at room temperature for 4 h, centrifuged at 1800×g for 30 min, serum was decanted and stored at -20°C prior to use. Blood was also obtained from a newborn calf prior to colostrum ingestion, 4 days post-colostrum ingestion, and from a cow at 30 days of the dry period. Immunoglobulin was isolated also from a pooled source of dry cow secretion and from colostrum. Concentration of Ig from milk, dry cow secretion, and colostrum preparations was determined spectrophometrically at 280 nm with an extinction coefficient of 1.37. Immunoglobulin preparations were diluted with PBS to equal 30 mg/ml for milk Ig and 52 mg/ml for colostral and dry cow secretion Ig. Diluted mammary secretion Ig was stored at - 2 0 ° C prior to use. Blood serum and mammary secretion Ig were diluted in PBSTween for immunoblots immediately prior to use. RESULTS
The outer membrane profiles of two strains each ofE. coli and K. pneumoniae are shown in Fig. 1. High molecular weight proteins in the 75 000 to 85 000 molecular weight range were induced when bacteria were grown in iron re-
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Fig. 1. Outer membrane protein profiles of E. coil and Klebsiella pneumoniae separated by SDSPAGE and stained with Coornassie blue. Protein per lane was 20 Fg. Bacteria were grown in TBS or TBS plus 300/tM a-a'-dipyridyl (TSB-Fe). Lane contents were: ( 1) molecular weight ( × 103) standards; (2) E. coli 92, TSB; (3) E. coli 92, TSB-Fe; (4) E. coli 686, TSB; (5) E. coil 686, TSB-Fe; (6) K. pneumoniae 511, TSB; (7) K. pneumoniae 511, TSB,-Fe; (8) K. pneumoniae 575 TSB; (9) K. pneumoniae 575 TSB-Fe.
ANTIBODIES TO COLIFORM BACTERIA FROM BOVINE INFECTIONS
111
strictive medium (lanes 3, 5, 7, 9). The iron-regulated OMP were not detected in bacteria grown in TSB (lanes 2, 4, 6, 8). The profile of the ironregulated OMP ofE. coli 92 grown in skim milk (Fig. 2, lane 4) was similar to growth in trypticase soy broth plus a.a'-dipyridyl (lane 3 ). No iron-regu-
1 234 92.5 66.2 45
31 Fig. 2. Outer membrane proteins ofE. coli 92 separated by SDS-PAGE and stained with Coomassie blue. Protein per lane was 50 pg. E. coli 92 was grown in trypticase soy broth (lane 2), trypticase soy broth plus 300~gMa-a'.dipyridyl (lane 3), and skimmed milk (lane 4). Molecular weight ( × 10~) standards are in lane 1.
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Fig. 3. Immunoblots of separated outer membrane proteins of K. pneumoniae 511 reacted with bovine milk immunoglobulin (lanes 1-10). Lanes I-5 were milk immunoglobulin ( 1:50 dilution) obtained from five cows with a Gram-negative intramammary infection. Lanes 6-10 were milk immunoglobulin (1:10 dilution) obtained from uninfected cows. Approximate position of iron-regulated outer membrane proteins is indicated (--,).
112
D,A.TODHUNTERETAL.
6
2
3
4
7
8
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5
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Fig. 4. Immunoblots of separated outer membrane proteins ofEscherichia coli 92 (lanes 1-5) or K. pneumoniae 553 (lanes 6-10) reacted with the following antibody sources: lanes 1 and 6, immunoglobulin from skimmed dry cow secretion (I:100 dilution); lanes 2 and 7, dry cow serum ( !:I0 dilution);lanes 3 and 8, immunoglobulin from colostrum ( 1:I00 dilution); lanes 4 and 9, newborn calf serum ( 1:2 dilution); and lanes 7 and I0, calf serum obtained 4 days postcolostrum ingestion (1:2 dilution). Approximate position of iron-regulated outer membrane proteins is indicated (--,).
lated proteins were evident for E. coli 92 grown in trypticase soy broth (lane 2). Milk contained IgG antibody directed against the iron-regulated OMP and other OMP of K. pneumoniae (Fig. 3). Milk IgG from cows with a Gramnegative bacterial IMI (lanes 1-5) produced stronger reactions than from uninfected cows (lane 6-I0). None of the animals with a Gram-negative bacterial IMI were infected with the K. pneumoniae from which the OMP were isolated. Noticeable variation in reactivity to the OMP among cows was observed. Reactions of milk IgG against iron-regulated OMP from uninfected cows were difficult to detect in some animals. No IgG antibodies were detected against the iron-regulated OMP from bacteria that were grown in TSB. The presence of IgG antibody directed against the iron-regulated OMP of E. coli and K. pneumoniae was also detected in blood serum of all lactating cows tested. Serum immunoblots were similar to milk Ig. Dry, cow serum, dry cow secretion, and colostrum all contained IgG directed against the iron-regulated OMP and other OMP of E. coli 92 and K. pneumoniae 553 (Fig. 4). No IgG antibody against OMP ofE. coli and K. pneumoniae was detected in serum from a newborn calf prior to ingestion of colostrum (lanes 4 and 9). Immunoglobulin G antibodies against the iron.
ANTIBODIESTO COLIFORM BACTERIAFROM BOVINEINFECTIONS
i 13
regulated and major OMP were detected 4 days after ingesting colostrum (lanes 5 and 10). DISCUSSION
E. eoli and K. pneumoniae initially isolated from bovine IMI expressed ironregulated OMP under iron restrictive conditions. The same bacteria grown under iron sufficient conditions did not express iron-regulated OMP. Ironregulated OMP were also present in E. colt grown in skim milk. The number and molecular weights of OMP expressed by E. colt 92 grown in skim milk and TSB plus iron chelator were similar. Antibody of the IgG class that recognized the iron-regulated OMP of E. colt and K. pneumoniae was detected in serum, milk, dry cow secretion, and colostrum of dairy cattle. Antibody was present also that recognized the major OMP and several other outer membrane proteins. Transfer of IgG antibody to the OMP from dam to calf through colostrum was demonstrated. Antibodies to the iron-regulated OMP have been detected also in humans, turkeys, rabbits, mice, and guinea pigs (Griffiths et al., 1983; Shand et al., 1985; Bolin and Jensen, 1987). The antigenicity and molecular weight of the enterochelin receptor of E. colt, K. pneumoniae, and Salmonella typhimurium has been reported as highly conserved (Chart and Griffiths, 1985). The major OMP, including porins and outer membrane protein A, also have been demonstrated to show antigenic cross reactivity among genera of Enterobacteriaceae (Hofstra and Dankert, 1980). Antibodies were detected against the iron-regulated OMP orE. colt and K. pneumoniae in dairy cattle with no known history of infection by the Gram-negative bacterial strains used for isolation of OMP. Naturally occurring antibodies to the OMP of Enterobacteriaceae were proposed to have occurred by exposure through the gut (Griffiths et al., 1985). Cross reactivity of antibodies to the OMP of different Gram-negative bacterial genera was evident in dairy cattle. The role of antibody against the iron-regulated OMP and protection from infection with Gram-negative bacteria is unknown. Passive immunization with antibodies directed against the iron-regulated outer membrane protein receptors of E. colt and P. aeruginosa has indicated that such antibodies can lower mortality caused by those two Gram-negative organisms in turkeys and mice (Sokol and Woods, 1986; Bolin and Jensen, 1987). Possible functions of antibody directed against the iron-regulated OMP for protection against Gram-negative bacterial infection have included interference with iron uptake, opsonization form phagocytosis, and bactericida~ activity by complement activation. Immunoglobulin directed against the ferrichrome receptor has been shown to partially inhibit iron uptake in E. colt (Coulton, 1982). Imrnunoglobulin G directed against the ferripyochelin-binding protein of P. aeruginosa inhibited the binding and uptake of the iron chelator pyochelin in
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D.A. TODHUNTERET AL.
whole cells (Sokol and Woods, 1986). However, other studies have shown that the O-antigen and the capsule may prevent access of antibodies to OMP in E. coli and Klebsiella spp. (Vuopio.Varkila et aI., 1988; Williams et al., 1988). Presence of immunoglobulin in serum and mammary secretions directed against the iron-regulated outer membrane proteins indicates that these antibodies may have a possible role in the protection of the mammary gland against infections with Gram-negative bacteria. Immunoglobulin has been shown to enhance the bacteriostatic properties oflactoferrin for K. penumoniae, E. coli and K. oxytoca and these two components contribute in part to the bacteriostatie properties of dry cow secretion (Todhunter et al., 1990). Elucidation of a function of Ig directed against the iron-regulated outer membrane proteins possibly could lead to enhancement of the cow's resistance to Gram-negative bacterial IMI during the dry period. ACKNOWLEDGEMENTS
The authors thank Pamela Schoenberger, Sue Romig, Heidi Rennecker, and Lucinda Shock for technical assistance. Research was supported in part by USDA Special Grant No. 85-CRSR-2-2616. Salaries and research support provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript No. 81-90.
REFERENCES Batteiger, B., Newhall, W.J. and Jones, R.B., 1982. The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J. Immunol. Methods, 55: 297-307. Bolin, C.A. and Jensen, A.E., 1987. Passive immunization with antibodies against iron-regulated outer membrane proteins protects turkeys from Escherichia cclisepticemia. Infect. iraman., 55: 1239-1242. Brea~, W.C. and Oliver, S.P., 1986. Growth inhibition of environmental pathogens during physiological transitions ofthe bovine mammary, gland. Am. J. Vet. Res., 47:218-222. Chart, H. and Griffiths, E., 1985. Antigenic and molecular homology of the ferric enterobaetin receptor protein of Escherichia coll. J. Gen. Microbiol., 131:1503-1509. Coulton, J.W., | 982. The ferrichrome-iron receptor of Escherichia coil K- 12. Antigenicity of the fhuA protein. Biochim. Biophys. Acta, 717:154-162. Griffiths, E., Stevenson, P. and Joyce, P., 1983. Pathogenic Escherichia coil express new outer membrane proteins when growing in vivo. FEMS Microbiol. Lett., 16: 95-99. Griffiths, E., Stevenson, P., Thorpe, R. and Chart, H., 1985. Naturally occurring antibodies in human sera that react with the iron-regulated outer membrane proteins of Escherichia coll. Infect. Immun., 47:808-813. Hantke, K. and Braun, V., 1975. Membrane receptor dependent iron transport in Escherichia coll. FEBS Lett., 49: 301-305.
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Hofstra, H. and Dankert, J., 1980. Major outer membrane proteins: Common antigens in Enterobacteriaceae. J. Gen. Mierobioi., 119:123-131. Klebba, P.W., Mclntosh, M.A. and Neilands, J.B., 1982. Kinetics of biosynthesis of iron-regulated membrane proteins in Escherichia coli. J. Bacteriol., 149: 880-888. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. Lascelles, A.K. and Lee, C.S., 1978. Involution of the mammary gland. In: B.L. Larson (Editor), Lactation, A Comprehensive Treatise, IV. Academic Press, New York, NY, pp. I 15177. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Lugtenberg, B., Meijers, J., Peters, R., van der Hock, P. and van Alphen, L., 1975. Electrophoretie resolution of the 'major outer membrane protein' of Escherichia coil KI 2 into four bands. FEBS Lett., 58: 254-258. Oliver, S.P. and Bushe, T., 1987. Growth inhibition of Escherichia and Klebsiellapneumoniae during involution of the bovine mammary gland: Relation to secretion composition. Am, J. Vet. Res., 48: 1669-1973. Pugsley, A.P. and Reeves, P., 1976a. Characterization of Group B colicin-resistant mutants of Escherichia coli K-12: Coliein resistance and the role ofenterochelin. J. Bacteriol., 127: 218228. Pugsley, A.P. and Reeves, P., 1976b. Increased production of the outer membrane receptors for colicins B, D, and M by Escherichia coli under iron starvation. Biochem. Biophys. Res. Commun., 70: 846-853. Schanbacher, F.L. and Smith, K.L., 1975. Formation and role of unusual whey proteins and enzymes: Relation to mammary function. J. Dairy Sci., 58: 1048-1062. Shand, G.H., Anwar, H., Kadurugamuwa, J., Brown, M.R., Silverman, S.H. and Melling, J., 1985. In vivo evidence that bacteria in urinary tract infection grow under iron-restricted conditions. Infect. Immun., 48: 35-39. Smith, K.L., Todhunter, D.A. and Schoenberger, P.S., 1985. Environmental mastiffs: Cause prevalence, prevention. J. Dairy Sci., 68:1531-1553. Sokol, P.A. and Woods, D.E., 1986. Characterization of antibody to the ferripyochelin-binding protein ofpseudomonas aeruginosa. Infect. lmmun., 5 I: 896-900. Todhunter, D.A.: Smith, K.L. and Hogan, J.S., 1990. Growth of gram-negative bacteria in dry cow secretion. J. Dairy Sci., 73: 363-372. Towbin, H., Staehelin, L. and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354. Vuopio-Varkila, J., Karvonen, M. and Saxdn, H., 1988. Protective capacity of antibodies to outer-membrane components of Escherichia coli in a systemic mouse peritonitis model. J. Meal Microbiol., 25: 77-84. Welty, F.K., Smith, K.L. and Schanbacher, F.L., 1976. Lactoferrin concentration during involution of the bovine mammary gland. J. Dairy Sci., 59: 224-231. Wheelock, J.V., Smith, A., Dodd, F.H. and Lyster, F.L.J., 1967. Changes in the quantity and composition of mammary gland secretion in the dry period between lactation 1. The beginning of the dry period. J. Dairy Res., 34: 1-12. Williams, P., Lambert, P.A. and Brown, M.R.W., 1988. Penetration ofimmunoglobulins through the Klebsieila capsule and their effect on cell-surface hydrophobicity. J. Med. Microbiol., 26: 29-35.
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Elsevier Science Publishers B.V., Amsterdam
Antibodies to iron-regulated outer membrane proteins of coliform bacteria isolated from bovine intramammary infections D.A. Todhunter, K.L. Smith and J.S. Hogan Ohio Agricultural Research and Development Center~OhioState University,Department of Dairy Science, Wooster,OH 44691, USA (Accepted 7 June 1990)
ABSTRACT Todhunter, D.A., Smith, K.L. and Hugan, J.S., 1991. Antibodies to iron-regulatedouter membrane proteins of coliform bacteria isolated from bovine intramammary infections. Vet. lmmunol, hn. munopathoL, 28: ! 07-115. Expression of iron-regulated outer membrane proteins (OMP) by Esdaerichia coli and Klebsiella pneumoniae initially isolated from bovine intramammary infections (IMI) was investigated. Additionally, the presenceofantibodies in bovine serum and mammary secretion directed against the ironregulated OMP was examined. Outer membrane proteins were separated by sodium-dodecylpolyacrylamide electrophoresis. Detection ofimmunoglobulin G directed against OMP was by immunoblotting. All Gram-negative bacteria expressed iron-regulatedOMP when grown in skim milk or tryptiease soy broth plus iron chelator, ot-c~'-dipyridyl.Immunoglobulin G directed against the ironregulatedOMP, as well as the major OMP and several other proteins, was detected in serum and milk of lactating cows with or without Gram-negative bacterial IMI. Antibody against the iron-regulated OMP was detected also in colostrum, secretion from the involuted gland, and in newborn calf serum 4 days after ingestingcolostrum.
INTRODUCTION
The dry period is a critical time in the dynamics of Gram-negative bacterial intramammary infections (IMI). Rate of Gram-negative bacterial IMI is 3 to 4 fold greater during the dry period than during lactating (Smith et al., 1985 ). Rate of new IMI is not constant across the dry period but elevated during the first 2 weeks of involution and the 2 weeks prepartum. Composition of mammary gland secretion changes markedly during involution. Concentrations of lactoferrin, serum albumin, and all immunoglobulin (Ig) classes increase (Schanbacher and Smith, 1975; Welty et al., 1976) while citrate and lactose concentrations decrease (Wheeloek et al., 1967; Lascelles and Lee, 1978). Data from mammary secretions obtained at various stages of involution dern0165-2427/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
108
D.A,TODHUNTERETAL,
onstrated that secretion became progressively more inhibitory to the in vitro growth ofEscherichia coli and Klebsiella spp. as involution progresses (Breau and Oliver, 1986; Oliver and Bushe, 1987). Secretion from the fully involuted gland inhibited the growth of several strains of Gram-negative bacteria (Todhunter et al., 1990). The iron-binding protein lactoferrin and Ig contributed in part to the inhibitory properties of secretion from the involuted gland. In an iron-restricted environment E. coli and Klebsiella spp. produce low molecular weight iron chelators (siderophores) and several outer membrane proteins (Hantke and Braun, 1975; Klebba et al., 1982; Chart and Griffiths, 1985 ). Some of these outer membrane proteins (OMP) are receptors for the ferric siderophore and are not synthesized under conditions of available iron, as the system is repressed in the presence of iron. E. coli, K. pneumoniae, Proteus spp., and Pseudomonas aeruginosa growing in vivo and in vitro, have been shown to produce iron-regulated OMP in the molecular weight range of 74 000 to 83 000 daltons (Pugsley and Reeves, 1976a,b; Klebba et al., 1982). Antibodies of the IgG class directed against the iron-regulated OMP have been detected in the serum of several animal species and humans demonstrating the antigenicity of the proteins (Griffiths et al., 1985; Shand et al., 1985; Bolin and Jensen, 1987). The objectives of this study were to ( 1 ) determine if E. coli and K. pneumoniae isolated initially from bovine IMI express iron-regulated OMP and (2) determine if antibody directed against the iron-regulated OMP is present in bovine serum and mammary secretions. MATERIALS AND METHODS
Bacterial strains and growth conditions Bacteria were E. coil and K. pneumoniae isolated from naturally occurring IMI in the OARDC dairy research herd, Wooster, OH. One E. coli strain (92) was obtained from Dr. J.S. McDonald, Ames, IA. All Gram-negative bacteria were stored on trypticase soy agar (BBL Microbiology Systems, Becton Dickinson and Co., Cockeysville, MD) at room temperature prior to use. Bacteria were grown in trypticase soy broth (TSB), TSB containing 300 aM o~-a'-dipyridyl ( Sigma Chemical Co., St. Louis, MO ), or skim milk. Milk was aseptically obtained from several cows, pooled, and centrifuged at 5000 ×g for 20 min at 4°C to remove somatic cells and fat. Gram-negative bacteria were incubated at 37°C for 18 h on a rotary shaker (200 rpm) in all growth media. Extraction and electrophoresis of outer membrane proteins Outer membrane proteins were extracted by the method of Shand et al. (1985) with slight modifications. Following incubation, bacteria were harvested by centrifugation at 2500 X g for 30 rain at 4°C and washed 3 × in 0.15
ANTIBODIESTO COLIFORMBACTERIAFROM BOVINEINFECTIONS
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M NaC1. Cells were suspended in deionized, distilled H20 and disrupted by sonication for 10 min. Sonicated bacteria were centrifuged at 5000 ×g for 10 min at 4°C. N-lauroylsarcosine sodium salt (Sigma Chemical Co. ) was added to the supernatant at a final concentration of 2% and incubated for 30 rain at room temperature. Outer membrane proteins were collected by centrifugation at 50 000Xg for 60 min at 4°C, washed 2 ×in deionized, distilled H:O, and stored at -70°C. Total protein was determined by the method of Lowry etal. (1951). Outer membrane proteins were separated by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) by the method of Lugtenberg et al. (1975) utilizing the discontinuous buffer system of Laemmli (1970). Stacking gels were 4% acrylamide (Bio-Rad Laboratories, Richmond, CA) in 0.125 M Tris, pH 6.8 and separating gels were 10% acrylamide in 0.375 M Tris, pH 8.8. Outer membrane proteins we,re prepared for electrophoresis by heating at 100°C for 5 min in 0.0625 M Tris, pH 6.8, 2% sodium dodecyl sulfate, 5% 2-p mercaptoethanol, and 10% glycerol. Molecular weight standards (Bio-Rad) were: phosphorylase B (92 500); bovine serum albumin (66 200); ovalbumin (45 000); carbonic anhydrase (31 000); soybean trypsin inhibitor (21 500) and lysozyme ( 14 400). Standards were prepared for electrophoresis as described above. Bromphenol blue tracking dye was added to achieve a final concentration of 0.00125%. A constant current of 30 mA/ gel was used. Gels were stained with 0.1% Coomassie blue R.250 in 40% (v/ v) methanol, 10% (v/v) glacial acetic and destained in 40% (v/v) methanol and 10% (v/v) glacial acetic acid.
Detection of antibodies by immunoblotting Outer membrane proteins were separated by SDS-PAGE and transferred to nitrocellulose sheets at 60 V overnight with cooling (Towbin et al., 1979). Buffer for electrotransfer was 0.025 M Tris, 0.192 M glycine, and 20% (v/v) methanol, pH 8.3. After transfer, nitrocellulose sheets were washed 4× for 15 min at 37°C in phosphate buffered saline (PBS) containing 0.3% polyoxyethylene sorbitan monolaurate (Batteiger et al., 1982). Washed nitrocellulose sheets were incubated overnight with gentle agitation in the presence of bovine antibody. Bovine antibody was diluted in PBS plus 0.3% polyoxyethylene sorbitan monolaurate (PBS-Tween). Nitrocellulose sheets were washed 3 X for 10 rain with PBS-Tween at room temperature and then incubated for I h with rabbit anti-bovine IgG conjugated to horse radish peroxidase (Sigma Chemical Co.) diluted in PBS-Tween. Nitrocellulose sheets were washed as described above and incubated for 5 to 10 rain with a substrate solution containing 30 rng of 4-chloro-l-naphthol dissolved in 10 ml methanol, 0.025 ml of H202 (30%), and PBS to a final volume of 60 ml.
1!0
D.A. TODHUNTERET AL.
Serum and mammary secretion immunoglobulin for immunoblots Blood and milk were obtained from five lactating cows with a Gram-negative bacterial IMI and five cows that were uninfected. Milk was obtained from the mammary quarter infected with Gram-negative bacteria and from one mammary quarter of uninfected cows. Milk was centrifuged at 16 000 ×g for 30 min at 4°C to remove fat and cells. Casein was precipitated from skim milk by rennet coagulation (4 g C a C O 3 + 1 mI saturated rennin/l secretion) and Ig was precipitated by addition of ammonium sulfate to 43% saturation. Blood was permitted to clot at room temperature for 4 h, centrifuged at 1800×g for 30 min, serum was decanted and stored at -20°C prior to use. Blood was also obtained from a newborn calf prior to colostrum ingestion, 4 days post-colostrum ingestion, and from a cow at 30 days of the dry period. Immunoglobulin was isolated also from a pooled source of dry cow secretion and from colostrum. Concentration of Ig from milk, dry cow secretion, and colostrum preparations was determined spectrophometrically at 280 nm with an extinction coefficient of 1.37. Immunoglobulin preparations were diluted with PBS to equal 30 mg/ml for milk Ig and 52 mg/ml for colostral and dry cow secretion Ig. Diluted mammary secretion Ig was stored at - 2 0 ° C prior to use. Blood serum and mammary secretion Ig were diluted in PBSTween for immunoblots immediately prior to use. RESULTS
The outer membrane profiles of two strains each ofE. coli and K. pneumoniae are shown in Fig. 1. High molecular weight proteins in the 75 000 to 85 000 molecular weight range were induced when bacteria were grown in iron re-
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Fig. 1. Outer membrane protein profiles of E. coil and Klebsiella pneumoniae separated by SDSPAGE and stained with Coornassie blue. Protein per lane was 20 Fg. Bacteria were grown in TBS or TBS plus 300/tM a-a'-dipyridyl (TSB-Fe). Lane contents were: ( 1) molecular weight ( × 103) standards; (2) E. coli 92, TSB; (3) E. coli 92, TSB-Fe; (4) E. coli 686, TSB; (5) E. coil 686, TSB-Fe; (6) K. pneumoniae 511, TSB; (7) K. pneumoniae 511, TSB,-Fe; (8) K. pneumoniae 575 TSB; (9) K. pneumoniae 575 TSB-Fe.
ANTIBODIES TO COLIFORM BACTERIA FROM BOVINE INFECTIONS
111
strictive medium (lanes 3, 5, 7, 9). The iron-regulated OMP were not detected in bacteria grown in TSB (lanes 2, 4, 6, 8). The profile of the ironregulated OMP ofE. coli 92 grown in skim milk (Fig. 2, lane 4) was similar to growth in trypticase soy broth plus a.a'-dipyridyl (lane 3 ). No iron-regu-
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31 Fig. 2. Outer membrane proteins ofE. coli 92 separated by SDS-PAGE and stained with Coomassie blue. Protein per lane was 50 pg. E. coli 92 was grown in trypticase soy broth (lane 2), trypticase soy broth plus 300~gMa-a'.dipyridyl (lane 3), and skimmed milk (lane 4). Molecular weight ( × 10~) standards are in lane 1.
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Fig. 3. Immunoblots of separated outer membrane proteins of K. pneumoniae 511 reacted with bovine milk immunoglobulin (lanes 1-10). Lanes I-5 were milk immunoglobulin ( 1:50 dilution) obtained from five cows with a Gram-negative intramammary infection. Lanes 6-10 were milk immunoglobulin (1:10 dilution) obtained from uninfected cows. Approximate position of iron-regulated outer membrane proteins is indicated (--,).
112
D,A.TODHUNTERETAL.
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Fig. 4. Immunoblots of separated outer membrane proteins ofEscherichia coli 92 (lanes 1-5) or K. pneumoniae 553 (lanes 6-10) reacted with the following antibody sources: lanes 1 and 6, immunoglobulin from skimmed dry cow secretion (I:100 dilution); lanes 2 and 7, dry cow serum ( !:I0 dilution);lanes 3 and 8, immunoglobulin from colostrum ( 1:I00 dilution); lanes 4 and 9, newborn calf serum ( 1:2 dilution); and lanes 7 and I0, calf serum obtained 4 days postcolostrum ingestion (1:2 dilution). Approximate position of iron-regulated outer membrane proteins is indicated (--,).
lated proteins were evident for E. coli 92 grown in trypticase soy broth (lane 2). Milk contained IgG antibody directed against the iron-regulated OMP and other OMP of K. pneumoniae (Fig. 3). Milk IgG from cows with a Gramnegative bacterial IMI (lanes 1-5) produced stronger reactions than from uninfected cows (lane 6-I0). None of the animals with a Gram-negative bacterial IMI were infected with the K. pneumoniae from which the OMP were isolated. Noticeable variation in reactivity to the OMP among cows was observed. Reactions of milk IgG against iron-regulated OMP from uninfected cows were difficult to detect in some animals. No IgG antibodies were detected against the iron-regulated OMP from bacteria that were grown in TSB. The presence of IgG antibody directed against the iron-regulated OMP of E. coli and K. pneumoniae was also detected in blood serum of all lactating cows tested. Serum immunoblots were similar to milk Ig. Dry, cow serum, dry cow secretion, and colostrum all contained IgG directed against the iron-regulated OMP and other OMP of E. coli 92 and K. pneumoniae 553 (Fig. 4). No IgG antibody against OMP ofE. coli and K. pneumoniae was detected in serum from a newborn calf prior to ingestion of colostrum (lanes 4 and 9). Immunoglobulin G antibodies against the iron.
ANTIBODIESTO COLIFORM BACTERIAFROM BOVINEINFECTIONS
i 13
regulated and major OMP were detected 4 days after ingesting colostrum (lanes 5 and 10). DISCUSSION
E. eoli and K. pneumoniae initially isolated from bovine IMI expressed ironregulated OMP under iron restrictive conditions. The same bacteria grown under iron sufficient conditions did not express iron-regulated OMP. Ironregulated OMP were also present in E. colt grown in skim milk. The number and molecular weights of OMP expressed by E. colt 92 grown in skim milk and TSB plus iron chelator were similar. Antibody of the IgG class that recognized the iron-regulated OMP of E. colt and K. pneumoniae was detected in serum, milk, dry cow secretion, and colostrum of dairy cattle. Antibody was present also that recognized the major OMP and several other outer membrane proteins. Transfer of IgG antibody to the OMP from dam to calf through colostrum was demonstrated. Antibodies to the iron-regulated OMP have been detected also in humans, turkeys, rabbits, mice, and guinea pigs (Griffiths et al., 1983; Shand et al., 1985; Bolin and Jensen, 1987). The antigenicity and molecular weight of the enterochelin receptor of E. colt, K. pneumoniae, and Salmonella typhimurium has been reported as highly conserved (Chart and Griffiths, 1985). The major OMP, including porins and outer membrane protein A, also have been demonstrated to show antigenic cross reactivity among genera of Enterobacteriaceae (Hofstra and Dankert, 1980). Antibodies were detected against the iron-regulated OMP orE. colt and K. pneumoniae in dairy cattle with no known history of infection by the Gram-negative bacterial strains used for isolation of OMP. Naturally occurring antibodies to the OMP of Enterobacteriaceae were proposed to have occurred by exposure through the gut (Griffiths et al., 1985). Cross reactivity of antibodies to the OMP of different Gram-negative bacterial genera was evident in dairy cattle. The role of antibody against the iron-regulated OMP and protection from infection with Gram-negative bacteria is unknown. Passive immunization with antibodies directed against the iron-regulated outer membrane protein receptors of E. colt and P. aeruginosa has indicated that such antibodies can lower mortality caused by those two Gram-negative organisms in turkeys and mice (Sokol and Woods, 1986; Bolin and Jensen, 1987). Possible functions of antibody directed against the iron-regulated OMP for protection against Gram-negative bacterial infection have included interference with iron uptake, opsonization form phagocytosis, and bactericida~ activity by complement activation. Immunoglobulin directed against the ferrichrome receptor has been shown to partially inhibit iron uptake in E. colt (Coulton, 1982). Imrnunoglobulin G directed against the ferripyochelin-binding protein of P. aeruginosa inhibited the binding and uptake of the iron chelator pyochelin in
114
D.A. TODHUNTERET AL.
whole cells (Sokol and Woods, 1986). However, other studies have shown that the O-antigen and the capsule may prevent access of antibodies to OMP in E. coli and Klebsiella spp. (Vuopio.Varkila et aI., 1988; Williams et al., 1988). Presence of immunoglobulin in serum and mammary secretions directed against the iron-regulated outer membrane proteins indicates that these antibodies may have a possible role in the protection of the mammary gland against infections with Gram-negative bacteria. Immunoglobulin has been shown to enhance the bacteriostatic properties oflactoferrin for K. penumoniae, E. coli and K. oxytoca and these two components contribute in part to the bacteriostatie properties of dry cow secretion (Todhunter et al., 1990). Elucidation of a function of Ig directed against the iron-regulated outer membrane proteins possibly could lead to enhancement of the cow's resistance to Gram-negative bacterial IMI during the dry period. ACKNOWLEDGEMENTS
The authors thank Pamela Schoenberger, Sue Romig, Heidi Rennecker, and Lucinda Shock for technical assistance. Research was supported in part by USDA Special Grant No. 85-CRSR-2-2616. Salaries and research support provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript No. 81-90.
REFERENCES Batteiger, B., Newhall, W.J. and Jones, R.B., 1982. The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J. Immunol. Methods, 55: 297-307. Bolin, C.A. and Jensen, A.E., 1987. Passive immunization with antibodies against iron-regulated outer membrane proteins protects turkeys from Escherichia cclisepticemia. Infect. iraman., 55: 1239-1242. Brea~, W.C. and Oliver, S.P., 1986. Growth inhibition of environmental pathogens during physiological transitions ofthe bovine mammary, gland. Am. J. Vet. Res., 47:218-222. Chart, H. and Griffiths, E., 1985. Antigenic and molecular homology of the ferric enterobaetin receptor protein of Escherichia coll. J. Gen. Microbiol., 131:1503-1509. Coulton, J.W., | 982. The ferrichrome-iron receptor of Escherichia coil K- 12. Antigenicity of the fhuA protein. Biochim. Biophys. Acta, 717:154-162. Griffiths, E., Stevenson, P. and Joyce, P., 1983. Pathogenic Escherichia coil express new outer membrane proteins when growing in vivo. FEMS Microbiol. Lett., 16: 95-99. Griffiths, E., Stevenson, P., Thorpe, R. and Chart, H., 1985. Naturally occurring antibodies in human sera that react with the iron-regulated outer membrane proteins of Escherichia coll. Infect. Immun., 47:808-813. Hantke, K. and Braun, V., 1975. Membrane receptor dependent iron transport in Escherichia coll. FEBS Lett., 49: 301-305.
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Hofstra, H. and Dankert, J., 1980. Major outer membrane proteins: Common antigens in Enterobacteriaceae. J. Gen. Mierobioi., 119:123-131. Klebba, P.W., Mclntosh, M.A. and Neilands, J.B., 1982. Kinetics of biosynthesis of iron-regulated membrane proteins in Escherichia coli. J. Bacteriol., 149: 880-888. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. Lascelles, A.K. and Lee, C.S., 1978. Involution of the mammary gland. In: B.L. Larson (Editor), Lactation, A Comprehensive Treatise, IV. Academic Press, New York, NY, pp. I 15177. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Lugtenberg, B., Meijers, J., Peters, R., van der Hock, P. and van Alphen, L., 1975. Electrophoretie resolution of the 'major outer membrane protein' of Escherichia coil KI 2 into four bands. FEBS Lett., 58: 254-258. Oliver, S.P. and Bushe, T., 1987. Growth inhibition of Escherichia and Klebsiellapneumoniae during involution of the bovine mammary gland: Relation to secretion composition. Am, J. Vet. Res., 48: 1669-1973. Pugsley, A.P. and Reeves, P., 1976a. Characterization of Group B colicin-resistant mutants of Escherichia coli K-12: Coliein resistance and the role ofenterochelin. J. Bacteriol., 127: 218228. Pugsley, A.P. and Reeves, P., 1976b. Increased production of the outer membrane receptors for colicins B, D, and M by Escherichia coli under iron starvation. Biochem. Biophys. Res. Commun., 70: 846-853. Schanbacher, F.L. and Smith, K.L., 1975. Formation and role of unusual whey proteins and enzymes: Relation to mammary function. J. Dairy Sci., 58: 1048-1062. Shand, G.H., Anwar, H., Kadurugamuwa, J., Brown, M.R., Silverman, S.H. and Melling, J., 1985. In vivo evidence that bacteria in urinary tract infection grow under iron-restricted conditions. Infect. Immun., 48: 35-39. Smith, K.L., Todhunter, D.A. and Schoenberger, P.S., 1985. Environmental mastiffs: Cause prevalence, prevention. J. Dairy Sci., 68:1531-1553. Sokol, P.A. and Woods, D.E., 1986. Characterization of antibody to the ferripyochelin-binding protein ofpseudomonas aeruginosa. Infect. lmmun., 5 I: 896-900. Todhunter, D.A.: Smith, K.L. and Hogan, J.S., 1990. Growth of gram-negative bacteria in dry cow secretion. J. Dairy Sci., 73: 363-372. Towbin, H., Staehelin, L. and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76: 4350-4354. Vuopio-Varkila, J., Karvonen, M. and Saxdn, H., 1988. Protective capacity of antibodies to outer-membrane components of Escherichia coli in a systemic mouse peritonitis model. J. Meal Microbiol., 25: 77-84. Welty, F.K., Smith, K.L. and Schanbacher, F.L., 1976. Lactoferrin concentration during involution of the bovine mammary gland. J. Dairy Sci., 59: 224-231. Wheelock, J.V., Smith, A., Dodd, F.H. and Lyster, F.L.J., 1967. Changes in the quantity and composition of mammary gland secretion in the dry period between lactation 1. The beginning of the dry period. J. Dairy Res., 34: 1-12. Williams, P., Lambert, P.A. and Brown, M.R.W., 1988. Penetration ofimmunoglobulins through the Klebsieila capsule and their effect on cell-surface hydrophobicity. J. Med. Microbiol., 26: 29-35.
