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High Titer, Phage-Neutralizing Antibodies in Bovine Colostrum that Prevent Lytic Infection of Lactococcus lactis in Fermentations of Phage-contaminated Milk
High Titer, Phage-Neutralizing Antibodies in Bovine Colostrum that Prevent Lytic Infection of Lactococcus lactis in Fermentations of Phage-contaminated Milk B. L. GELLER,*,† J. KRAUS,* M. D. SCHELL,* M. J. HORNSBY,* J. J. NEAL,* and F. E. RUCH‡,1 *Department of Microbiology and The Center for Gene Research and Biotechnology, Oregon State University, Corvallis 97331-3804 †The Western Dairy Center, Utah State University, Logan 84322-8700 ‡Immucell Corporation, 56 Evergreen Drive, Portland, ME 04103
ABSTRACT Antibodies against six phages of Lactococcus lactis were produced in six bovine colostra. Each colostrum neutralized its homologous phage. In addition, each colostrum neutralized a different phage from the same species as its homologous phage, but either did not neutralize or weakly neutralized more distantly related lactococcal phages. The neutralization of heterologous phages correlated with the phage species but not with the strain on which the phage was grown. Blood serum from the same cows also neutralized homologous phages, but the titers were lower than that of the colostrum. Addition of colostrum to phage-contaminated milk prevented lysis of starter cultures of L. lactis. The titers of some of the colostra were sufficiently high that it may be economically practical to prepare antibodies from similar, high titer colostra for commercial use in factory bulk starter vats. ( Key words: Lactococcus lactis phage, lactococcal bacteriophage, colostrum, antibodies) INTRODUCTION Bacteriophage lysis of bulk starter cultures is the most common cause of failed milk fermentations in large, mechanized cheese factories. Strategies to prevent phage lysis have ranged from improved methods of sanitization to strain rotation (12). One proposed strategy of phage control is to immunize cows with lactococcal phages and then to add
the whey from the milk produced by these cows to bulk vats in cheese factories ( 7 ) . Antibodies in whey neutralize phages and prevent lysis of starter cultures in milk that has been contaminated by those phages (6, 7). Although this neutralization strategy has shown promise in laboratory tests, because of low antibody titers in whey, it has not been used commercially. A practical solution may be to produce colostrum with a high antibody titer against lactococcal phages. Colostrum is the first milk produced postpartum and is 200-fold more concentrated in immunoglobulins than bovine milk ( 5 ) . Although it is not legal in the US to add colostrum directly to milk (13), immunoglobulins prepared from colostrum might be added to milk to prevent bacteriophage infection. In this report, bovine colostra were prepared from cows that had been immunized with six lactococcal phages (i.e., two phages from each of three lactococcal phage species). Phages c2 and ml3 have heads with prolate morphology and are both of the c2 phage species. Phages p335 and p013 have small isometric heads and are both of the p335 phage species. Phages kh and 18-16 also have small isometric heads and are both of the 936 phage species. The phage-neutralizing titers of each colostrum were determined, and each colostrum was tested for its ability to prevent lysis of starter cultures in milk that had been contaminated with phages. MATERIALS AND METHODS Bacterial Strains, Phages, and Media
Received July 14, 1997. Accepted December 1, 1997. 1Present address: 159 Foreside Road, Falmouth, ME 04105. 1998 J Dairy Sci 81:895–900
Strains of Lactococcus lactis and bacteriophages were grown in M17 medium as described (11). Phages c2, ml3, and sk1 were propagated on L. lactis 895
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ssp. lactis C2, phages p335 and p013 on L. lactis ssp. lactis F7/2, phage kh on Lactococcus lactis ssp. cremoris KH, and phage 18-16 on citrate-utilizing Lactococcus lactis ssp. lactis 18-16. Phages were concentrated by precipitation with polyethylene glycol, purified on two CsCl gradients as described ( 2 ) , and dialyzed against 10 mM bis[2-hydroxyethyl]iminotris[hydroxymethyl]methane, pH 7.0, 50 mM NaCl, and 10 mM MgSO4. Glycerol was added to a final concentration of 20% (vol/vol), and the phages were frozen in liquid N2 and stored at –70°C. Protein concentration was determined by the Bradford method ( 3 ) . Immunization Six Holstein cows (500 to 700 kg) were each immunized during the dry cycle by two injections of phage at three immunization times. Each immunization included a subcutaneous injection in the neck and an intramuscular injection in the rump. The antigen was prepared for injection by mixing 0.4 to 0.7 mg of phage and 0.5 mg of Quil A (Sergent Pulp and Paper Co., Clifton, NJ) in 1.4 to 2.0 ml of phosphate-buffered saline with an equal volume of adjuvant A (Immucell Corp., Portland, ME). The time between injections was 2 to 3 wk. The third (final) immunization occurred at 1 to 10 d prepartum. Sera were collected on the day of the first (preimmune) and third (immune) injections. Phage-Neutralization Titer of Colostra and Sera Colostra and sera were diluted in M17 medium and mixed with an equal volume of phage at a concentration of 2 × 107 pfu/ml in M17 with 20% glycerol. The mixtures were incubated 1 h at 22°C. Immediately after incubation, the mixtures were diluted 2 × 10–4, and 100-ml aliquots were plated in duplicate on indicator strains as described (11). Control mixtures without colostrum or serum and others with colostrum or serum from cows that had not been immunized with phage were similarly prepared. Each experiment was repeated, and the titers are expressed as the mean values of the two experiments. The mean number of plaques from the duplicate plates that formed at each dilution was expressed as a percentage of the mean number of plaques from the duplicate plates without colostrum or serum. Plots of the data for percentage of neutralization versus log10 dilution were used to calculate the titer. The titer is expressed as the reciprocal of the dilution that neutralized 50% of the phage. Journal of Dairy Science Vol. 81, No. 4, 1998
Inhibition of Lysis in Milk Phages were diluted to final concentrations from 1 × 100 to 1 × 106 pfu/ml in sterile, reconstituted 11% (wt/vol) nonfat dry milk. Various dilutions of colostrum were added, and the mixtures were incubated 30 min at 30°C. A 1% volume of an overnight culture of the host strain was added, and the mixtures were incubated 18 h at 30°C. The pH of control mixtures without added phage or colostrum was 6.41 ± 0.05 ( n = 7 ) before incubation. After incubation, clotting was judged to occur if no liquid was released from the solid milk when the culture tube was tipped 90°. The pH of each mixture was measured with a glass electrode and pH meter (Beckman Instruments, Fullerton, CA). RESULTS Phage-Neutralizing Titers of Colostrum Colostrum was collected from six cows that had each been immunized with one of six different lactococcal phages. The phage-neutralizing titer of each colostrum was determined for each of the six phages. The colostrum from the cow that was immunized with phage c2 (hereafter referred to as c2 colostrum) was most effective against phage c2 (Figure 1A). The c2 colostrum was about four times less potent against phage ml3 and did not neutralize any of the small isometric-headed phages at the lowest dilution tested ( 1 × 10–2) . The titer of ml3 colostrum was highest (1.6 × 104) against phage ml3, followed by phage c2, which is of the same phage species (1.3 × 104; Figure 1B). Phage m13 had a 3-fold higher titer against phage c2 than did c2 colostrum against phage c2. The ml3 colostrum was essentially ineffective against all of the other phages tested although a titer slightly higher than the titer of colostrum from cows that were not immunized was measured against phage kh. The titers of p335 colostrum (Figure 1C) were the highest of any colostrum tested. The p335 colostrum was most effective against phages p335 and p013, which are of the same phage species (6.5 × 104 and 6.4 × 104, respectively). Neutralizing titers were about 100-fold and 20-fold higher against the small isometric-headed phages kh and 18-16, respectively, than those of colostrum from cows that were not immunized. The p335 colostrum was ineffective against prolate-headed phages c2 and ml3. The p013 colostrum had a profile similar to that of p335 anti-colostrum, except that its titers were lower
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Figure 1. Titers of anti-phage colostra. The phageneutralizing titers of each colostrum were measured against each of the phages indicated in the abscissa. Panels: A, c2 colostrum; B, m13 colostrum; C, p335 colostrum; D, p013 colostrum; E, kh colostrum; F, 18-16 colostrum; and G, colostrum from cows that had not been immunized with phage. The limit of detection was 1 × 102.
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by a factor of about 5- to 10-fold, depending on the phage (Figure 1D). The kh colostrum had a high titer (3.8 × 104) against phage kh, but a 30-fold weaker titer against phage 18-16, which is of the same phage species, and 30- to 50-fold lower titers against the small isometricheaded phages p335 and p013, which are not of the same phage species (Figure 1E). The kh colostrum did not neutralize prolate-headed phages c2 and ml3. The 18-16 colostrum was most effective against phage 18-16 (2.4 × 104) and less effective against the other small isometric-headed phages kh (2.2 × 103) , p013 (5.6 × 102) , and p335 (6.6 × 102; Figure 1F). The 18-16 colostrum did not neutralize the prolateheaded phages c2 and ml3. Colostrum from cows that had not been immunized did not reduce the number of plaques in any of the phage preparations at any dilution of colostrum from 1 × 10–2 to 1 × 10–6 (Figure 1G). Influence of Host Strain on Cross-Neutralization Phage sk1 is a 936 species phage (the same as phage 18-16) that propagates on the same strain as phage ml3. Phage sk1 was mixed separately with ml3 and 18-16 colostra, and the neutralization titers were determined. The neutralization titers of ml3 and 18-16 colostra against phage sk1 were 7 and 3.0 × 103, respectively. Thus, 18-16 colostrum was more effective in neutralizing phage sk1 than was ml3 colostrum, even though phages sk1 and ml3 were both propagated on the same strain and phage 18-16 was propagated on a different strain. Phage-Neutralizing Titers of Serum Titers of serum from each cow indicated that each serum neutralized its own phage (Table 1). However, the titer of each serum was less than the titer of the colostrum from the same cow. The titers of the sera
from cows vaccinated with the small isometric-headed phages p013, kh, and 18-16 were 3-to 6-fold less than those of the corresponding colostra. The serum titers against phage c2 and ml3 were about 300- and 30-fold lower, respectively, than the corresponding colostra. Prevention of Lysis in Milk Contaminated by Phage The abilities of ml3, p335, and kh colostra to prevent the lysis of starter cultures in milk that contained phage were determined by measuring the pH and clotting of the milk. In all experiments, the cultures of milk without phage formed a firm clot by 18 h, and the final pH was about 4.5. The cultures of milk without colostrum but with phage failed to clot, and the pH remained above 5.7. The ml3 colostrum at a dilution of 1 × 10–3 prevented lysis and enabled the milk cultures to acidify normally (pH = 4.65) and to clot firmly (Table 2). The ml3 anti-colostrum diluted to 1 × 10–4 or more did not prevent lysis, and the milk was not acidified or clotted. A reduction in the initial concentration of phage from 1 × 104 to 1 pfu/ml did not change the results. The p335 colostrum performed in a manner similar to that of ml3 colostrum, except that the lowest dilution required to prevent lysis was 2 × 10–4 (Table 2). A reduction in the initial phage concentration from 1 × 104 to 1 pfu/ml did not reduce the amount of colostrum necessary to restore clotting. The kh colostrum prevented lysis at a dilution of 5 × 10–4 but prevented lysis at 5 × 10–5 only when the initial phage concentration was reduced from 1 × 104 to 1 × 103 pfu/ml. When diluted to 5 × 10–6, the colostrum failed to prevent lysis. Lower initial concentrations of phage kh failed to prevent clotting in the absence of colostrum.
TABLE 1. Phage neutralization titers1 of blood sera. Serum against phage
X SD
c2
m13
p335
p013
kh
18-16
Preimmune
0.0122 0.004
0.56 0.09
NA3
2.0 0.6
13 9
3.8 0.8
<0.01
1Reciprocal
of dilution that neutralized 50% of phage in 1 h. titers expressed as the mean ( ±SD) × 103 ( n = 2). 3Sample not available. 2All
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DISCUSSION The phage-neutralizing titers of the colostra were qualitatively consistent with previous reports of phage-neutralizing antibodies (4, 9, 10, 14). There was significant cross-neutralization within each of the three phage species. There was less crossneutralization within phage species 936 than within the other two species, possibly because phages kh and 18-16 were grown on different strains, and each of the other two pairs was grown on the same strain. Phageneutralizing antibodies are directed against a variable receptor-binding protein in the tail of the phage, which recognizes a strain-specific receptor on the cell surface ( 8 ) . The phage-neutralizing titer of each colostrum was significantly lower against phages from heterologous phage species. Antibodies against the prolate-headed phages c2 and ml3 did not neutralize the other four small isometric-headed phages. However, colostra against the small-isometric headed phages p335, p013, kh, and 18-16 neutralized heterologous phages of the small isometric-headed morphology, albeit at a significantly lower titer. There was no crossneutralization between colostra from cows immunized with small isometric-headed phages and those immunized with prolate-headed phages. Although some of this reduction in titer may be attributable to the different strains used to grow the phage, the crossreactivity between the two species of small isometricheaded phages and their colostra indicates a similarity in receptor-binding proteins (despite their growth
on different strains). The neutralization of phage sk1 by kh colostrum, but not by ml3 colostrum, shows that neutralization is a function of the relatedness of the phages. The neutralization titers of colostra in this study were higher than those of the neutralization titers of whey or milk reported previously (6, 7). Duitschaever and Quinn ( 6 ) injected cows with a crude preparation of a lactococcal phage and found neutralizing titers of 0 to 2 × 101 in the whey. Erskine ( 7 ) vaccinated cows with phage that had been prepared by differential centrifugation and measured phage neutralization titers in the milk of 1 × 103. In the present report, cows were vaccinated with phages purified by CsCl gradients, and the phage neutralization titers of colostra were 3.8 × 103 to 6.5 × 104. Although differences existed in the methods used to measure and express the neutralization titers in each report, the titers can probably be compared because the effect of different incubation temperatures is approximately offset ( 1 ) by the difference in end points of neutralization. Thus, the phage-neutralizing activity of the colostra are 190- to 3250-fold higher than the activity in bovine whey reported by Duitschaever and Quinn ( 6 ) and 3.8- to 65-fold higher than the activity in bovine milk reported by Erskine ( 7 ) . Phage neutralization activities may also be compared by the ability to restore milk-clotting ability to a phage-infected starter culture. Duitschaever and Quinn ( 6 ) concluded that phage neutralization titers were too low to be of use in this manner. Erskine ( 7 )
TABLE 2. Acidification and clotting of milk that had been contaminated with phage as a function of added colostrum.
Colostrum
Colostrum dilution
m13 m13 m13 None None p335 p335 p335 None None kh kh kh kh kh None None
1 × 1 × 1 × ... ... 2 × 2 × 2 × ... ... 5 × 5 × 5 × 5 × 5 × ... ...
10–3 10–3 10–4 10–4 10–4 10–5 10–4 10–4 10–5 10–5 10–6
Phage
Initial phage concentration
Clot
pH
None m13 m13 m13 m13 None p335 p335 p335 p335 None kh kh kh kh kh kh
(pfu/ml) 0 1 × 104 1 × 100 1 × 104 1 × 100 0 1 × 104 1 × 100 1 × 104 1 × 100 0 1 × 104 1 × 103 1 × 104 1 × 103 1 × 104 1 × 103
+ + – – – + + – – – + + + – – – –
4.63 4.65 5.88 6.30 6.08 4.44 4.48 5.93 6.30 6.11 4.39 4.39 4.45 5.67 5.85 5.83 5.79
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found that a 1 × 10–2 dilution of neutralizing whey restored clotting by starter cultures only if the phage contamination was 100 pfu/ml or less. In the present report, colostrum restored the ability of starter cultures to clot phage-contaminated milk at a dilution of 1 × 10–3 to 5 × 10–5, even when the phage concentrations were 1 × 103 to 1 × 104 pfu/ml (Table 2). Similar experiments using heterologous colostrum revealed that the dilutions required for restoring the acidification and clotting of phage-contaminated milk were similar to the titers shown in Figure 1. These results show that phage infection can be inhibited in contaminated milk by high dilutions of colostrum containing high antibody titers. We estimate that the addition of immunoglobulins prepared from colostrum to the bulk vat in the cheese factory adds about $0.005 to $0.015/kg ($0.01 to $0.03/lb) to the cost of producing cheese. This estimate is based on the assumption that high titer colostrum can be consistently produced. Colostrum currently sells for about $0.75/kg ($3.00/gal), and we estimate the cost of immunization to be about $25 to $100 per cow, depending on the costs of producing phage for vaccination. The cost of preparing the immunoglobulin fraction from colostrum is estimated to be about $1.25/kg ($5/gal). Because each cow can produce about 20 kg ( 5 gal) of colostrum/yr, the additional costs of immunization and antibody preparation increase the cost of colostrum to about $3 to $7/ kg ($13 to $28/gal) of colostrum. In practice, the decision to add immunoglobulin preparations to the bulk vat will be one of economics. The cost of losses from starter lysis needs to be balanced against the cost of the immunoglobulins. We speculate that colostrum from cows immunized with lactococcal phage could be a practical source of immunoglobulins that could be added to the bulk starter vats to prevent phage-related problems. CONCLUSIONS High titer colostra from cows that were vaccinated with lactococcal phages neutralized their homologous phages. Neutralization of heterologous phages correlated with the species of phage and not the strain on which the phages were propagated. Colostrum
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from immunized cows prevented starter culture lysis when added to milk that had been contaminated with phage. ACKNOWLEDGMENTS We thank Joe Crabb (Immucell Corp.) for helping to prepare the manuscript and Janine Trempy (Oregon State University) for reading the manuscript. REFERENCES 1 Adams, M. H. 1959. Bacteriophages. Intersci. Publ., Inc., New York, NY. 2 Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1994. Preparing lambda DNA from phage lysates. Unit 1.13.1–1.13.3 Suppl. 10 in Current Protocols in Molecular Biology. Vol. 1. Green Publ. Assoc., Inc. and John Wiley & Sons, Inc., New York, NY. 3 Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254. 4 de Fabrizio, S. V., R. A. Ledford, Y.S.C. Shieh, J. Brown, and J. L. Parada. 1991. Comparison of lactococcal bacteriophage isolated in the United States and Argentina. Int. J. Food. Microbiol. 13:285–294. 5 Dixon, F. J., W. O. Weigle, and J. J. Vazquez. 1961. Metabolism and mammary secretion of serum proteins in the cow. Lab. Invest. 10:216–237. 6 Duitschaever, C. L., and P. J. Quinn. 1970. Antibody response of cows to Streptococcus lactis bacteriophage. J. Dairy Sci. 53: 1363–1366. 7 Erskine, J. M. 1964. A new laboratory method for preventing bacteriophage attack on cheese starter streptococci. J. Dairy Sci. 31:95–104. 8 Jarvis, A. W. 1978. Serological studies of a host range mutant of a lactic streptococcal bacteriophage. Appl. Environ. Microbiol. 36:785–789. 9 Jarvis, A. W. 1984. Differentiation of lactic streptococcal phages into phage species by DNA-DNA homology. Appl. Environ. Microbiol. 47:343–349. 10 Relano, P., M. Mata, M. Bonneau, and P. Ritzenthaler. 1987. Molecular characterization and comparison of 38 virulent and temperate bacteriophages of Streptococcus lactis. J. Gen. Microbiol. 133:3053–3063. 11 Terzaghi, B. E., and W. E. Sandine. 1975. Improved medium for lactic streptococci and their bacteriophages. Appl. Environ. Microbiol. 29:807–813. 12 Thunell, R. K., W. E. Sandine, and F. W. Bodyfelt. 1981. Phageinsensitive, multiple-strain starter approach to Cheddar cheese making. J. Dairy Sci. 64:2270–2277. 13 United States Department of Health and Human Services, Public Health Service/Food and Drug Administration. 1993. The Grade A Pasteurized Milk Ordinance. Publ. No. 229. US Govt. Printing Office, Washington, DC. 14 Wilkowske, H. H., F. E. Nelson, and C. E. Parmelee. 1954. Serological classification of bacteriophages active against lactic streptococci. Appl. Environ. Microbiol. 2:243–249.
ABSTRACT Antibodies against six phages of Lactococcus lactis were produced in six bovine colostra. Each colostrum neutralized its homologous phage. In addition, each colostrum neutralized a different phage from the same species as its homologous phage, but either did not neutralize or weakly neutralized more distantly related lactococcal phages. The neutralization of heterologous phages correlated with the phage species but not with the strain on which the phage was grown. Blood serum from the same cows also neutralized homologous phages, but the titers were lower than that of the colostrum. Addition of colostrum to phage-contaminated milk prevented lysis of starter cultures of L. lactis. The titers of some of the colostra were sufficiently high that it may be economically practical to prepare antibodies from similar, high titer colostra for commercial use in factory bulk starter vats. ( Key words: Lactococcus lactis phage, lactococcal bacteriophage, colostrum, antibodies) INTRODUCTION Bacteriophage lysis of bulk starter cultures is the most common cause of failed milk fermentations in large, mechanized cheese factories. Strategies to prevent phage lysis have ranged from improved methods of sanitization to strain rotation (12). One proposed strategy of phage control is to immunize cows with lactococcal phages and then to add
the whey from the milk produced by these cows to bulk vats in cheese factories ( 7 ) . Antibodies in whey neutralize phages and prevent lysis of starter cultures in milk that has been contaminated by those phages (6, 7). Although this neutralization strategy has shown promise in laboratory tests, because of low antibody titers in whey, it has not been used commercially. A practical solution may be to produce colostrum with a high antibody titer against lactococcal phages. Colostrum is the first milk produced postpartum and is 200-fold more concentrated in immunoglobulins than bovine milk ( 5 ) . Although it is not legal in the US to add colostrum directly to milk (13), immunoglobulins prepared from colostrum might be added to milk to prevent bacteriophage infection. In this report, bovine colostra were prepared from cows that had been immunized with six lactococcal phages (i.e., two phages from each of three lactococcal phage species). Phages c2 and ml3 have heads with prolate morphology and are both of the c2 phage species. Phages p335 and p013 have small isometric heads and are both of the p335 phage species. Phages kh and 18-16 also have small isometric heads and are both of the 936 phage species. The phage-neutralizing titers of each colostrum were determined, and each colostrum was tested for its ability to prevent lysis of starter cultures in milk that had been contaminated with phages. MATERIALS AND METHODS Bacterial Strains, Phages, and Media
Received July 14, 1997. Accepted December 1, 1997. 1Present address: 159 Foreside Road, Falmouth, ME 04105. 1998 J Dairy Sci 81:895–900
Strains of Lactococcus lactis and bacteriophages were grown in M17 medium as described (11). Phages c2, ml3, and sk1 were propagated on L. lactis 895
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ssp. lactis C2, phages p335 and p013 on L. lactis ssp. lactis F7/2, phage kh on Lactococcus lactis ssp. cremoris KH, and phage 18-16 on citrate-utilizing Lactococcus lactis ssp. lactis 18-16. Phages were concentrated by precipitation with polyethylene glycol, purified on two CsCl gradients as described ( 2 ) , and dialyzed against 10 mM bis[2-hydroxyethyl]iminotris[hydroxymethyl]methane, pH 7.0, 50 mM NaCl, and 10 mM MgSO4. Glycerol was added to a final concentration of 20% (vol/vol), and the phages were frozen in liquid N2 and stored at –70°C. Protein concentration was determined by the Bradford method ( 3 ) . Immunization Six Holstein cows (500 to 700 kg) were each immunized during the dry cycle by two injections of phage at three immunization times. Each immunization included a subcutaneous injection in the neck and an intramuscular injection in the rump. The antigen was prepared for injection by mixing 0.4 to 0.7 mg of phage and 0.5 mg of Quil A (Sergent Pulp and Paper Co., Clifton, NJ) in 1.4 to 2.0 ml of phosphate-buffered saline with an equal volume of adjuvant A (Immucell Corp., Portland, ME). The time between injections was 2 to 3 wk. The third (final) immunization occurred at 1 to 10 d prepartum. Sera were collected on the day of the first (preimmune) and third (immune) injections. Phage-Neutralization Titer of Colostra and Sera Colostra and sera were diluted in M17 medium and mixed with an equal volume of phage at a concentration of 2 × 107 pfu/ml in M17 with 20% glycerol. The mixtures were incubated 1 h at 22°C. Immediately after incubation, the mixtures were diluted 2 × 10–4, and 100-ml aliquots were plated in duplicate on indicator strains as described (11). Control mixtures without colostrum or serum and others with colostrum or serum from cows that had not been immunized with phage were similarly prepared. Each experiment was repeated, and the titers are expressed as the mean values of the two experiments. The mean number of plaques from the duplicate plates that formed at each dilution was expressed as a percentage of the mean number of plaques from the duplicate plates without colostrum or serum. Plots of the data for percentage of neutralization versus log10 dilution were used to calculate the titer. The titer is expressed as the reciprocal of the dilution that neutralized 50% of the phage. Journal of Dairy Science Vol. 81, No. 4, 1998
Inhibition of Lysis in Milk Phages were diluted to final concentrations from 1 × 100 to 1 × 106 pfu/ml in sterile, reconstituted 11% (wt/vol) nonfat dry milk. Various dilutions of colostrum were added, and the mixtures were incubated 30 min at 30°C. A 1% volume of an overnight culture of the host strain was added, and the mixtures were incubated 18 h at 30°C. The pH of control mixtures without added phage or colostrum was 6.41 ± 0.05 ( n = 7 ) before incubation. After incubation, clotting was judged to occur if no liquid was released from the solid milk when the culture tube was tipped 90°. The pH of each mixture was measured with a glass electrode and pH meter (Beckman Instruments, Fullerton, CA). RESULTS Phage-Neutralizing Titers of Colostrum Colostrum was collected from six cows that had each been immunized with one of six different lactococcal phages. The phage-neutralizing titer of each colostrum was determined for each of the six phages. The colostrum from the cow that was immunized with phage c2 (hereafter referred to as c2 colostrum) was most effective against phage c2 (Figure 1A). The c2 colostrum was about four times less potent against phage ml3 and did not neutralize any of the small isometric-headed phages at the lowest dilution tested ( 1 × 10–2) . The titer of ml3 colostrum was highest (1.6 × 104) against phage ml3, followed by phage c2, which is of the same phage species (1.3 × 104; Figure 1B). Phage m13 had a 3-fold higher titer against phage c2 than did c2 colostrum against phage c2. The ml3 colostrum was essentially ineffective against all of the other phages tested although a titer slightly higher than the titer of colostrum from cows that were not immunized was measured against phage kh. The titers of p335 colostrum (Figure 1C) were the highest of any colostrum tested. The p335 colostrum was most effective against phages p335 and p013, which are of the same phage species (6.5 × 104 and 6.4 × 104, respectively). Neutralizing titers were about 100-fold and 20-fold higher against the small isometric-headed phages kh and 18-16, respectively, than those of colostrum from cows that were not immunized. The p335 colostrum was ineffective against prolate-headed phages c2 and ml3. The p013 colostrum had a profile similar to that of p335 anti-colostrum, except that its titers were lower
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Figure 1. Titers of anti-phage colostra. The phageneutralizing titers of each colostrum were measured against each of the phages indicated in the abscissa. Panels: A, c2 colostrum; B, m13 colostrum; C, p335 colostrum; D, p013 colostrum; E, kh colostrum; F, 18-16 colostrum; and G, colostrum from cows that had not been immunized with phage. The limit of detection was 1 × 102.
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by a factor of about 5- to 10-fold, depending on the phage (Figure 1D). The kh colostrum had a high titer (3.8 × 104) against phage kh, but a 30-fold weaker titer against phage 18-16, which is of the same phage species, and 30- to 50-fold lower titers against the small isometricheaded phages p335 and p013, which are not of the same phage species (Figure 1E). The kh colostrum did not neutralize prolate-headed phages c2 and ml3. The 18-16 colostrum was most effective against phage 18-16 (2.4 × 104) and less effective against the other small isometric-headed phages kh (2.2 × 103) , p013 (5.6 × 102) , and p335 (6.6 × 102; Figure 1F). The 18-16 colostrum did not neutralize the prolateheaded phages c2 and ml3. Colostrum from cows that had not been immunized did not reduce the number of plaques in any of the phage preparations at any dilution of colostrum from 1 × 10–2 to 1 × 10–6 (Figure 1G). Influence of Host Strain on Cross-Neutralization Phage sk1 is a 936 species phage (the same as phage 18-16) that propagates on the same strain as phage ml3. Phage sk1 was mixed separately with ml3 and 18-16 colostra, and the neutralization titers were determined. The neutralization titers of ml3 and 18-16 colostra against phage sk1 were 7 and 3.0 × 103, respectively. Thus, 18-16 colostrum was more effective in neutralizing phage sk1 than was ml3 colostrum, even though phages sk1 and ml3 were both propagated on the same strain and phage 18-16 was propagated on a different strain. Phage-Neutralizing Titers of Serum Titers of serum from each cow indicated that each serum neutralized its own phage (Table 1). However, the titer of each serum was less than the titer of the colostrum from the same cow. The titers of the sera
from cows vaccinated with the small isometric-headed phages p013, kh, and 18-16 were 3-to 6-fold less than those of the corresponding colostra. The serum titers against phage c2 and ml3 were about 300- and 30-fold lower, respectively, than the corresponding colostra. Prevention of Lysis in Milk Contaminated by Phage The abilities of ml3, p335, and kh colostra to prevent the lysis of starter cultures in milk that contained phage were determined by measuring the pH and clotting of the milk. In all experiments, the cultures of milk without phage formed a firm clot by 18 h, and the final pH was about 4.5. The cultures of milk without colostrum but with phage failed to clot, and the pH remained above 5.7. The ml3 colostrum at a dilution of 1 × 10–3 prevented lysis and enabled the milk cultures to acidify normally (pH = 4.65) and to clot firmly (Table 2). The ml3 anti-colostrum diluted to 1 × 10–4 or more did not prevent lysis, and the milk was not acidified or clotted. A reduction in the initial concentration of phage from 1 × 104 to 1 pfu/ml did not change the results. The p335 colostrum performed in a manner similar to that of ml3 colostrum, except that the lowest dilution required to prevent lysis was 2 × 10–4 (Table 2). A reduction in the initial phage concentration from 1 × 104 to 1 pfu/ml did not reduce the amount of colostrum necessary to restore clotting. The kh colostrum prevented lysis at a dilution of 5 × 10–4 but prevented lysis at 5 × 10–5 only when the initial phage concentration was reduced from 1 × 104 to 1 × 103 pfu/ml. When diluted to 5 × 10–6, the colostrum failed to prevent lysis. Lower initial concentrations of phage kh failed to prevent clotting in the absence of colostrum.
TABLE 1. Phage neutralization titers1 of blood sera. Serum against phage
X SD
c2
m13
p335
p013
kh
18-16
Preimmune
0.0122 0.004
0.56 0.09
NA3
2.0 0.6
13 9
3.8 0.8
<0.01
1Reciprocal
of dilution that neutralized 50% of phage in 1 h. titers expressed as the mean ( ±SD) × 103 ( n = 2). 3Sample not available. 2All
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DISCUSSION The phage-neutralizing titers of the colostra were qualitatively consistent with previous reports of phage-neutralizing antibodies (4, 9, 10, 14). There was significant cross-neutralization within each of the three phage species. There was less crossneutralization within phage species 936 than within the other two species, possibly because phages kh and 18-16 were grown on different strains, and each of the other two pairs was grown on the same strain. Phageneutralizing antibodies are directed against a variable receptor-binding protein in the tail of the phage, which recognizes a strain-specific receptor on the cell surface ( 8 ) . The phage-neutralizing titer of each colostrum was significantly lower against phages from heterologous phage species. Antibodies against the prolate-headed phages c2 and ml3 did not neutralize the other four small isometric-headed phages. However, colostra against the small-isometric headed phages p335, p013, kh, and 18-16 neutralized heterologous phages of the small isometric-headed morphology, albeit at a significantly lower titer. There was no crossneutralization between colostra from cows immunized with small isometric-headed phages and those immunized with prolate-headed phages. Although some of this reduction in titer may be attributable to the different strains used to grow the phage, the crossreactivity between the two species of small isometricheaded phages and their colostra indicates a similarity in receptor-binding proteins (despite their growth
on different strains). The neutralization of phage sk1 by kh colostrum, but not by ml3 colostrum, shows that neutralization is a function of the relatedness of the phages. The neutralization titers of colostra in this study were higher than those of the neutralization titers of whey or milk reported previously (6, 7). Duitschaever and Quinn ( 6 ) injected cows with a crude preparation of a lactococcal phage and found neutralizing titers of 0 to 2 × 101 in the whey. Erskine ( 7 ) vaccinated cows with phage that had been prepared by differential centrifugation and measured phage neutralization titers in the milk of 1 × 103. In the present report, cows were vaccinated with phages purified by CsCl gradients, and the phage neutralization titers of colostra were 3.8 × 103 to 6.5 × 104. Although differences existed in the methods used to measure and express the neutralization titers in each report, the titers can probably be compared because the effect of different incubation temperatures is approximately offset ( 1 ) by the difference in end points of neutralization. Thus, the phage-neutralizing activity of the colostra are 190- to 3250-fold higher than the activity in bovine whey reported by Duitschaever and Quinn ( 6 ) and 3.8- to 65-fold higher than the activity in bovine milk reported by Erskine ( 7 ) . Phage neutralization activities may also be compared by the ability to restore milk-clotting ability to a phage-infected starter culture. Duitschaever and Quinn ( 6 ) concluded that phage neutralization titers were too low to be of use in this manner. Erskine ( 7 )
TABLE 2. Acidification and clotting of milk that had been contaminated with phage as a function of added colostrum.
Colostrum
Colostrum dilution
m13 m13 m13 None None p335 p335 p335 None None kh kh kh kh kh None None
1 × 1 × 1 × ... ... 2 × 2 × 2 × ... ... 5 × 5 × 5 × 5 × 5 × ... ...
10–3 10–3 10–4 10–4 10–4 10–5 10–4 10–4 10–5 10–5 10–6
Phage
Initial phage concentration
Clot
pH
None m13 m13 m13 m13 None p335 p335 p335 p335 None kh kh kh kh kh kh
(pfu/ml) 0 1 × 104 1 × 100 1 × 104 1 × 100 0 1 × 104 1 × 100 1 × 104 1 × 100 0 1 × 104 1 × 103 1 × 104 1 × 103 1 × 104 1 × 103
+ + – – – + + – – – + + + – – – –
4.63 4.65 5.88 6.30 6.08 4.44 4.48 5.93 6.30 6.11 4.39 4.39 4.45 5.67 5.85 5.83 5.79
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found that a 1 × 10–2 dilution of neutralizing whey restored clotting by starter cultures only if the phage contamination was 100 pfu/ml or less. In the present report, colostrum restored the ability of starter cultures to clot phage-contaminated milk at a dilution of 1 × 10–3 to 5 × 10–5, even when the phage concentrations were 1 × 103 to 1 × 104 pfu/ml (Table 2). Similar experiments using heterologous colostrum revealed that the dilutions required for restoring the acidification and clotting of phage-contaminated milk were similar to the titers shown in Figure 1. These results show that phage infection can be inhibited in contaminated milk by high dilutions of colostrum containing high antibody titers. We estimate that the addition of immunoglobulins prepared from colostrum to the bulk vat in the cheese factory adds about $0.005 to $0.015/kg ($0.01 to $0.03/lb) to the cost of producing cheese. This estimate is based on the assumption that high titer colostrum can be consistently produced. Colostrum currently sells for about $0.75/kg ($3.00/gal), and we estimate the cost of immunization to be about $25 to $100 per cow, depending on the costs of producing phage for vaccination. The cost of preparing the immunoglobulin fraction from colostrum is estimated to be about $1.25/kg ($5/gal). Because each cow can produce about 20 kg ( 5 gal) of colostrum/yr, the additional costs of immunization and antibody preparation increase the cost of colostrum to about $3 to $7/ kg ($13 to $28/gal) of colostrum. In practice, the decision to add immunoglobulin preparations to the bulk vat will be one of economics. The cost of losses from starter lysis needs to be balanced against the cost of the immunoglobulins. We speculate that colostrum from cows immunized with lactococcal phage could be a practical source of immunoglobulins that could be added to the bulk starter vats to prevent phage-related problems. CONCLUSIONS High titer colostra from cows that were vaccinated with lactococcal phages neutralized their homologous phages. Neutralization of heterologous phages correlated with the species of phage and not the strain on which the phages were propagated. Colostrum
Journal of Dairy Science Vol. 81, No. 4, 1998
from immunized cows prevented starter culture lysis when added to milk that had been contaminated with phage. ACKNOWLEDGMENTS We thank Joe Crabb (Immucell Corp.) for helping to prepare the manuscript and Janine Trempy (Oregon State University) for reading the manuscript. REFERENCES 1 Adams, M. H. 1959. Bacteriophages. Intersci. Publ., Inc., New York, NY. 2 Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1994. Preparing lambda DNA from phage lysates. Unit 1.13.1–1.13.3 Suppl. 10 in Current Protocols in Molecular Biology. Vol. 1. Green Publ. Assoc., Inc. and John Wiley & Sons, Inc., New York, NY. 3 Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254. 4 de Fabrizio, S. V., R. A. Ledford, Y.S.C. Shieh, J. Brown, and J. L. Parada. 1991. Comparison of lactococcal bacteriophage isolated in the United States and Argentina. Int. J. Food. Microbiol. 13:285–294. 5 Dixon, F. J., W. O. Weigle, and J. J. Vazquez. 1961. Metabolism and mammary secretion of serum proteins in the cow. Lab. Invest. 10:216–237. 6 Duitschaever, C. L., and P. J. Quinn. 1970. Antibody response of cows to Streptococcus lactis bacteriophage. J. Dairy Sci. 53: 1363–1366. 7 Erskine, J. M. 1964. A new laboratory method for preventing bacteriophage attack on cheese starter streptococci. J. Dairy Sci. 31:95–104. 8 Jarvis, A. W. 1978. Serological studies of a host range mutant of a lactic streptococcal bacteriophage. Appl. Environ. Microbiol. 36:785–789. 9 Jarvis, A. W. 1984. Differentiation of lactic streptococcal phages into phage species by DNA-DNA homology. Appl. Environ. Microbiol. 47:343–349. 10 Relano, P., M. Mata, M. Bonneau, and P. Ritzenthaler. 1987. Molecular characterization and comparison of 38 virulent and temperate bacteriophages of Streptococcus lactis. J. Gen. Microbiol. 133:3053–3063. 11 Terzaghi, B. E., and W. E. Sandine. 1975. Improved medium for lactic streptococci and their bacteriophages. Appl. Environ. Microbiol. 29:807–813. 12 Thunell, R. K., W. E. Sandine, and F. W. Bodyfelt. 1981. Phageinsensitive, multiple-strain starter approach to Cheddar cheese making. J. Dairy Sci. 64:2270–2277. 13 United States Department of Health and Human Services, Public Health Service/Food and Drug Administration. 1993. The Grade A Pasteurized Milk Ordinance. Publ. No. 229. US Govt. Printing Office, Washington, DC. 14 Wilkowske, H. H., F. E. Nelson, and C. E. Parmelee. 1954. Serological classification of bacteriophages active against lactic streptococci. Appl. Environ. Microbiol. 2:243–249.