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Biochemical and Antibacterial Properties of Bovine Mammary Secretion During Mammary Involution and at Parturition1
Biochemical and Antibacterial Properties of Bovine Mammary Secretion During Mammary Involution and at Parturition 1 B R I A N J. N O N N E C K E National Animal Disease Center ARS, U S D A P.O. Box 70 Ames, IA 50010 K. L A R R Y S M I T H
Department of Dairy Science Ohio Agricultural Research and Development Center The Ohio State University Wooster 44691 ABST R A C T
candy their inhibitory capacity within cow. However, reduction of growth inhibition was significant when data from the four cows were pooled.
Mammary secretions from four Holstein Friesian cows were collected during late lactation, early involution, and early in subsequent lactation. Changes of pH, concentration of serum albumin, immunoglobulin G, citrate, lactoferrin, and number of leukocytes in secretions were typical of milk from glands undergoing these physiological transitions. Whey prepared from a cow's secretions was evaluated for its capacity to inhibit growth of Escbericbia coli, 60-Lilly, and a coliform strain isolated from mammary secretions of that cow. Wheys from different glands of the same cow differed markedly in their capacity to inhibit growth of coliforms. Inhibition of both strains by the wheys increased significantly during the dry period and was maximal in wheys collected day 15 of the dry period and at parturition in the subsequent lactation. The coliform strain isolated from a specific cow was inhibited more than Escbericbia coli, 60-Lilly, by whey from the specific cow. Inhibition of the cow-specific coliform strain by the day 15 whey was reduced significantly after addition of ferric iron or sodium citrate. Addition of excess ferric iron or citrate to wheys collected at parturition did not alter signifi-
INTRODUCTION
Lactoferrin (Lf), a glycoprotein in mammary secretion, is a powerful chelator of iron with an affinity constant for iron of approximately 1036. Lactoferrin alone or in conjunction with specific antibody inhibits bacterial growth (1, 23) and may be a potent antimicrobial factor in the nonlactating or infected bovine mammary gland (1, 17, 18, 21). The antimicrobial activity of Lf has been attributed to its ability tenaciously to chelate iron, limiting the iron in the host's body fluids available for microbial metabolism (4). Coliform bacteria, a cause of bovine mastitis (8), employ at least two systems for sequestering iron from the iron-poor environment of the host (4). One system involves synthesis and secretion into the environment of the iron binding catechol, enterochelin, and a second, a citrate-mediated transport system, is induced by citrate in the growth medium. Citrate, a constituent of normal milk (16), and Lf undergo changes of concentration in milk during mammary involution, establishment of normal lactation, and mastitis. In vitro studies showed a negative correlation between molar ratio of citrate:lactoferrin (Cit:Lf) in a synthetic growth medium and degree of inhibition of coliform bacteria (1, 14). Based on these studies the molar ratio of Cit:Lf is elevated Received March 21, 1984. sufficiently in normal milk and colostrum to 1Salaries and research support provided by State limit the Lf-dependent inhibition of coliform and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio bacteria. However, the low molar ratio of citrate to Lf in secretion from the involuted State University. Journal Article No. 38-84.
1984 J Dairy Sci 67:2863--2872
2863
2864
NONNECKEAND SMITH
mammary gland suggests that Lf may inhibit effectively growth of coliform bacteria. Milk samples collected from individual glands of four cows during late lactation, early involution, and at parturition were evaluated for indicators of physiological changes of mammary function. Wheys derived from these samples were tested in vitro for their capacity to inhibit growth of E. coil, 60-Lilly, and a coliform strain isolated from the mammary secretion of each cow. MATERIALS AND METHODS Experimental Animals
Four purebred Holstein-Friesian cows, designated N1, N2, N3, and N4, were maintained under identical conditions, milked twice daily, and not dry cow-treated with antibiotics at the end of lactation. Milk was collected from individual glands of each animal 7 days prior to the dry period (D -- 7), at drying-off (D + 0), and 2 (D + 2 ) , 4 ( D + 4 ) , 6 (D + 6 ) , 8 ( D + 8 ) , and 15 (D + 15) days into the dry period, and in the subsequent lactation, at parturition (C + 0), and 7 days postpartum (C + 7). Each sample consisted of 3 to 5 ml of foremilk collected aseptically for bacteriological and cellular evaluation and 150 ml of milk for citrate and protein determinations and bacteriological growth studies. Bacteria in milk were identified according to recommendations of the National Mastitis Council (3). The total number of leukocytes in the secretions was determined by direct microscopic cell count (25) of a dried milk film (1 cm 2) stained with the Levowitz-Weber modification of the Newman-Lambert stain (24). Glassware
Glassware was prepared according to the procedures described in Experimental Tecbniques in Biocbemistry (2) to minimize iron contamination in the assays. Skim Milk and Whey Preparation
Whole milk was centrifuged at 30,000 x g for 30 min at room temperature and fat separated from skim milk by the fluid component decanted through cheesecloth. The pH of each skim milk was recorded. Whey was made by the skim milk equilibrated to 43°C and the pH Journal of Dairy Science Vol. 67, No. 12, 1984
adjusted to 4.5 by glacial acetic acid added. The precipitated casein was removed by centrifugation at 30,000 × g for 30 min, and pH of the whey was adjusted to the pH of original skim milk by addition of 1 M NaOH. Wheys were sterilized by filter (.45 g) and stored at 20°C until needed. Protein Quantitation
Quantitation of Lf, immunoglobulin G (IgG), and serum albumin (SA) in whey was by electroimmunodiffusion (EID) on cellulose acetate plates (Helena Labs) (19). Antisera consisted of 1.8 to 2.0% rabbit antibovine Lf, 4.0% rabbit anti-Fab fragment (antisera prepared by K. L. Smith), and 2.0% anti-SA (Miles Labs, Inc.). Standard curves were established from concentrations of .05 to 4.0 mg/ml for SA, .05 to .45 mg/ml for Lf, and .13 to .38 mg/ml for IgG. Citrate Determinations
Quantitation of citrate in milk was photometric (27). An individual assay consisted of 30 unknowns and five citrate standards of .0, .05, .075, .100, and .125 mg/ml of citrate, each prepared in duplicate. Optical density of standards plotted against their concentration resulted in a linear plot from which the concentration of citrate in the unknown could be interpolated. Bacterial Cu|tures
The coliform strains specific for cows were isolated from mammary secretions of individual cows during these experiments. Coliform strains were E. coli from cows N1 and N4, Enterobacter cloacae from cow N2, and Klebsiella pneumoniae from cow N3. Escbericbia coli, 60-Lilly, was obtained from E. J. Carroll (University of California, Davis, CA). Bacterial Growth Assay
A single colony of the test strain grown on blood agar (5% sheep blood in trypticase soy agar, Difco Labs) was inoculated into 5 ml of filter-sterilized bovine milk whey and incubated at 37°C for 24 h. One milliliter of this culture was inoculated into 5 ml of sterilized whey and incubated at 37°C for 12 h. After incubation the culture was diluted in sterile phosphate
INHIBITION OF COLIFORMS BY MILK WHEY buffered saline (PBS, .15 M, pH 6.8) to 102 cfu/lO #l of PBS. Sterile microtiter plates (120 mm X 80 ram) with 96 U-shaped wells (Cooke Lab. Products, Division of Syntach Labs) were used to support assays of bacterial growth inhibition. Of the 300-~tl capacity of each well, 250/~I was designated for filtered whey (without or with iron or citrate supplementation) and 10 /~1 for the bacterial inoculum (102 cfu of the test strain). Individual tests were prepared in triplicate. The prepared microtiter plate, covered with a sterilized lid, was incubated at 37°C for 12 h. After incubation, contents of individual wells were diluted serially in PBS, and the number of viable bacteria per milliliter was determined in triplicate by the drop method (12, 14) on blood agar.
2865
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RESULTS
The mean pH, SA, and IgG concentrations and mean number of leukocytes in the secretions of cows N1 to 4 are summarized in Table 1. The pH of the milk, initially 6.7 on D -- 7, increased significantly to a maximum of 7.6 on D + 15. At C + 0 pH had dropped significantly to 6.2, and by C + 7 was 6.6. Concentration of SA in the secretion increased significantly from D - 7 to D + 6 and reached a maximum of 3.10 mg/ml on D + 15. At C + 0 and C + 7 the concentration of SA in the milk had declined significantly to 1.74 a n d . 10 mg/ml. The IgG in milk increased significantly from .66 mg/ml on D -- 7 to 18.7 mg/ml on D + 15. However, the maximum concentration of IgG, 69.09 mg/ml, occurred at C + 0, and by C + 7 concentrations had returned to those typical of normal milk (.49 mg/ml). The increase of concentration of SA and IgG in the milk from D - 7 to D + 15 was progressive. Concentrations of SA, IgG, and numbers of leukocytes in secretions may have been influenced by infection status of mammary g!ands. However, no attempt was made to assess the influence of infection because of the small number of COWS.
Numbers of leukocytes in milk increased significantly from D -- 7 to D + 6 and remained elevated until C + 0 when they declined significantly. No further decrease of number of leukocytes was observed at C + 7. The concentration of Lf increased significantly from D -- 7 (.63 mg/ml) to D + 15 (13.5
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Journal of Dairy Science Vol. 67, No. 12, 1984
2866
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mg/ml) (Table 2); however, at C + 0 concentration had decreased significantly to 1.82 mg/ml and b y C + 7 was .12 mg/ml. The concentration of citrate decreased significantly from D -- 7 (1.54 mg/ml) to D + 15 (.50 mg/ml) (Table 2). A t C + 0 the amount of citrate increased significantly to 1.56 mg/ml and by C + 7 was 2.38 mg/ml, greater than at any time during flae study. The molar ratio o f Cit:Lf at D -- 7 was 1154; however, b y D + 15 of dry period molar ratio had declined significantly to 28 (Table 2). At C + 0 the molar ratio of Cit:Lf increased significatly to 380 and b y C + 7 was 9528. Whey in the bacterial growth assay was not supplemented nutritionally, because growth in the unsupplemented whey was considered a more valid indicator of inhibitory capacity o f the secretion. Growth o f test strains in unsupplemented whey was limited to a 102 to 103 times increase after 12 h of incubation compared to a 104 to 10 s increase in supplemented (supplemented with 17 mg/ml AOAC medium, Difco Lab) wheys (14). There were pronounced differences in growth of E. coli, 60-Lilly, in wheys from different cows sampled at the same time (Table 3). Overall, inhibition of E. coli, 60-Lilly, and the cow-specific strain increased significantly with the onset of the dry period, and inhibition was greatest at D + 15. Wheys collected at C + 0 were significantly more inhibitory than those wheys prepared from milk samples collected at D -- 7. However, the inhibitory capacity of wheys prepared from C + 7 milk samples was not significantly different from D -- 7 wheys. The cow-specific strains were more inhibited than E. coli, 60-Lilly, b y wheys prepared from a specific cow. Inhibition of cow-specific strains b y D + 15 and C + 0 wheys from cows N1 to 4, before and after supplementation of the wheys with ferric ammonium sulphate or sodium citrate, was determined (Tables 4 and 5). Iron or citrate supplementation of the D + 15 wheys from cows N1, N2, and N4 significantly reduced growth inhibition of the cow-specific strain. However, neither iron nor citrate supplementation of C + 0 wheys significantly modified their capacity to inhibit the cow-specific strain. When data from the four cows were pooled, the decrease of growth inhibition after supplementation of wheys was significant (P<.05).
TABLE 3. Bacterial growth inhibition i by whey collected during the experimental period. Time of milk collection 2 D +2
D+4
D+6
D+8
D+15
C+0
X SE
100 36
81 33
73 36
84 20
74 22
62 20
58 32
94 50
X SE
38 14
29 10
18 8
43 18
46 20
30 12
32 16
59 25
E. coil 60-Lilly
X SE
100 19
63 20
46 10
48 21
41 7
23 11
30 4
135 31
Cow-specific strain
.X SE
108 8
30 10
24 10
23 9
15 4
10 5
20 6
99 27
E. coli 60-Lilly
X SE
125 18
75 23
41 8
35 9
36 12
31 10
52 9
82 5
Cow-specific strain
.X SE
105 20
75 23
39 4
28 6
31 6
21 4
41 16
131 22
E. coli 60-Lilly
X SE
110 18
50 16
20 3
22 6
10 3
12 2
18 5
70 14
Cow-specific strain
X SE
120 6
47 19
26 4
13 3
10 5
5 2
3 1
77 29
E. coli 60-Lilly
X SE
108 a 6
67 b 7
45 b 11
47 b 13
40bc 13
32 c 11
40bc 9
95 a 14
Cow-specific strain
X SE
93 a 37
45 a 11
27 b 4
27 b 6
26 b 8
16 b 6
24 b 8
92 a 16
Bacterial strain
N1
E. coli 60-Lilly
Cow-specific strain
N2
N3
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TABLE 4. Effect of e x o g e n o u s ferric iron or s o d i u m citrate on the growth o f cow-specific coliform strain in w h e y s 15 days into dry period. O~ .a
Cow
00
Lactoferrin (mg/ml)
Molar ratio Cit:Lf
Unsupplemented
Growth (cfu/ml, Log10) !n w h e y Iron z supplemented
Citrate 2 supplemented
N1 (n = 4)
X SD
13.6 3.5
24 11
3.39 .31
4.44" .23
4.45* .12
N2
(n = 3)
X SD
12.4 3.4
55 62
2.81 .22
3.79* .27
3.80" .14
N3 (n = 4)
X' SD
8.9 1.2
23 14
3.02 .46
3.49 .36
3.38 .38
N4 (n = 4)
X SD
18.0 6.5
11 13
3.09 .40
4.63* .21
4.44" .22
All cows
.~ SD
13.2 3.7
28 19
3.08 .24
4.09* .54
4.02* .52
: Ferric a m m o n i u m sulphate added in excess of the a m o u n t to saturate e n d o g e n o u s lactoferrin. 2 Sufficient s o d i u m citrate added to increase t h e Cit:Lf (citrate:lactoferrin) molar ratio to 600. N u m b e r o f glands from each cow while provided secretion. *Denotes significant increase in growth after s u p p l e m e n t a t i o n (P<.05).
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TABLE 5. Effect of exogenous ferric iron or sodium citrate on the growth of cow-specific coliform strain in whey at parturition. Growth (cfu/ml, Loglo) in whey Cow
Lactoferrin (mg/ml)
Molar ratio Cit:Lf
Unsupplemented
Iron supplemented
Citrate 2 supplemented
N1 (n = 4) a
.X SD
1.9 .6
310 99
2.95 .89
3.38 .46
3.24 .51
N2 (n = 4)
.X SD
1.2 .2
563 115
3.14 .29
3.34 .10
3.49 .30
N3 (n = 4)
.X SD
2.3 .3
277 199
3.16 .14
3.24 .36
3.31 .20
All cows
,X SD
1.8 .5
357 138
3.07 .10
3.29* .08
3.33* .11
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2870
NONNECKE AND SMITH DISCUSSION
Significant changes occur in biochemical composition and antibacterial properties of bovine mammary secretion during early stages of involution and at parturition. The increase of SA, IgG, and Lf and decrease of citrate concentration were likely caused by reduction of secretory activity of the mammary gland and decline of milk volume, a phenomenon reported by others (22, 26). The increase of SA also reflects the increased permeability of the secretory epithelial barrier within the gland resulting in increased diffusion of serum proteins into the secretion (10). Elevated concentration of IgG in the D + 15 and C + 0 secretion was likely due to selective transport of IgG~ into secretion via receptors on the secretory epithelial cells (5, 7, 9). Concentration of Lf in normal bovine milk ranges from .1 to .3 mg/ml (19, 21). In our study, Lf increased significantly from .63 mg/ml at D -- 7 to 13.5 mg/ml on D + 15 of the dry period. Involution of the bovine mammary gland likely continues at least to day 30 of the dry period, at which time Lf reaches its maximum concentration (20 to 30 mg/ml) and is a major protein in secretion from the dry gland (21, 22). Schanbacher and Smith (20, 22) have postulated that Lf in the dry gland secretion originates from lysosomes of secretory calls and granulocytes of the involuting mammary gland. The decline of citrate during the dry period can be attributed to cessation of secretory activity in the gland. The increase of citrate at C + 0 and at C + 7 indicates reestablishment of normal lactation. Peaker and Linzell (16) have described the appearance of citrate in the secretion from the preparturient gland as a "harbinger" of lactogenesis. The significant increase of number of leukocytes in secretion from the involuting mammary gland (D + 4 through D + 15) has been reported by (8, 11). However, 72 h after cessation of milking, no further increase was observed. The primary function of leukocytes, mostly macrophages, during the early phase of involution may be to phagocytize cellular debris, fat, and casein generated during involution (8, 11). Compositional changes of the secretion during early involution and at parturition were
Journal of Dairy Science Vol. 67, No. 12, 1984
correlated with a significant increase of antimicrobial capacity of the corresponding whey. Although some wheys demonstrated pronounced inhibition for both the cow-specific strains and E. coli, 60-Lilly, none was bactericidal for either strain. However, the intact secretion (containing both humoral and cellular components) likely would be bactericidal (13, 15, 16, 26). The greater inhibition of the cow-specific coliform by wheys from the specific cow indicates immunological specificity of response, possibly due to the presence of specific antibody. A decline of the molar ratio of Cit:Lf has been associated with increased inhibition of mastitis-causing coliform bacteria growing in a synthetic medium (1). Our study demonstrated progressive increase of growth inhibitory capacity of wheys prepared from secretions sampled during the early phases of mammary involution, which was associated with a decline of the molar ratios of Cit:Lf and elevation of IgG concentration in secretions. Both Lf and IgG1 have been considered to contribute to antimicrobial action of bovine mammary secretion (13, 26). Addition of excess iron or citrate to the D + 15 wheys of cows N1, N2, and N4 caused marked reduction of their antimicrobial capacity, indicating that inhibition by unsupplemented wheys having comparatively low molar ratio of Cit:Lf was mediated partially by an iron-dependent system and that the cowspecific coliform may acquire iron either directly or by a citrate-mediated system (4). In contrast, the antimicrobial capacity of D + 15 wheys from cow N3 was moderately, but not significantly, reduced after supplementation with either exogenous iron or citrate. Growth inhibitory systems acting in the secretions o f cow N3 may consist of one or more of the noncellular defense active in bovine mammary secretions (18, 26). The degree of inhibition of the cow-specific coliform by wheys prepared from milk samples collected at C + 0 often exceeded that produced by the D + 15 wheys from the same animal. Addition of excess iron or citrate to the C + 0 wheys, as was done for the D + 15 samples, did not enhance significantly growth of the test strain in individual cow wheys. However, a significant enhancement of growth was evident when data for all four cows were
INHIBITION OF COLIFORMS BY MILK WHEY
pooled. The minimal effect of supplementation was not unexpected, because the Cit:Lf molar ratios of the C + 0 wheys were significantly greater than those of the D + 15 wheys and high enough to minimize the Lf-dependent growth inhibition (1, 12, 14, 22). The elevated concentration of IgG in these secretions may have inhibited the test strain, although high concentrations of specific antibody in colostrum are thought to inhibit complement binding, minimizing the antimicrobial effect of the complement-antibody system (18). The method of preparation of whey also may have depleted the nutritional value of the secretion, resulting in some growth inhibition by D + 15 and C + 0 wheys. In summary, secretion from the involuting and parturient mammary gland inhibits growth of mastitis-causing coliforms to a greater extent than secretion from the normal, lactating gland. During mammary involution concentration of noncellular antimicrobial factors (Lf and IgG) in the secretion increased significanly. Growth inhibitory properties of the D + 15 wheys were reduced significantly. Growth inhibitory properties of the D + 15 wheys were reduced significantly by addition of ferric iron or sodium citrate, suggesting the low molar ratio of Cit:Lf contributed to inhibitory properties of these wheys. The growth inhibitory properties o f t h e C + 0 wheys were affected moderately by addition of ferric iron or sodium citrate. This indicates an iron-independent antimicrobial system may be functioning in these secretions or that they may lack sufficient nutrients to support growth of the test strain. REFERENCES
1 Bishop, J. G., F. L. Schanbacher, L. C. Ferguson, and K. L. Smith. 1976. In vitro growth inhibition of mastitis-cansing coliform bacteria by bovine apoqactoferrin and reversal of inhibition by citrate and high concentrations of apoqactoferrin. Infect. Immunol. 14:911. 2 Brewer, J. M., A. J. Pesce, and R. B. Ashworth. 1974. Experimental techniques in biochemistry. Prentice-Hall, Inc., NJ. 3 Brown, R. W., D. A. Barnum, D. E. Jasper, J. S. McDonald, and W. O. Schultz. 1981. Microbiological procedures for use in the diagnosis of bovine mastitis. Natl. Mastitis Counc., Inc., Washington, DC, Carter Press, Inc., Ames. 4 Bullen, J. J., H. J. Rogers, and E. Griffiths. 1978. Role of iron in bacterial infection. Curr. Top. Microbiol. Immunol. 80:1.
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5 Dixon, F. J., W. O. Weigle, and J. J. Vacquez. 1961. Metabolism and mammary secretion of serum proteins in the cow. Lab. Invest. 10:216. 6 Ebethart, R. J., R, P. Natzke, F.H.S. Newbould, B. J. Nonnecke, and P. Thompson. 1979. Coliform m a s t i t i s - A review. J. Dairy Sci. 62:1. 7 Hammer, D. K., B. Kichhofen, and H. Malchow. 1969. Preferential absorption of a single bovine IgG type by isolated epithelial ceils of the mammary gland. Protides of the biological fluids. Pages 663-668 Proc. 16th Coll., Bruges. Pergamon Press, New York, NY. 8 Jensen, D. L., and R. J. Eberhart. 1981. Total and differential cell counts in secretions of the nonlactating mammary gland. Am. J. Vet. Res. 42:743. 9 Larson, B. L. 1958. Transfer of specific blood serum proteins to lacteal secretions near parturition. J. Dairy Sci. 41:1033. 10 Lascelles, A. K., and C. S. Lee. 1978. Involution of the mammary gland. In: Lactation, a comprehensive treatise. Vol. IV. B. L. Larson, ed. Academic Press, Inc., New York, NY. 11 McDonald, J. S., and A. J. Anderson. 1981. Total and differential somatic cell counts in secretions from noninfected bovine mammary glands: The early nonlactating period. Am. J. Vet. Res. 42: 1360. 12 Meynell, G. G., and E. Meynell. 1965. Theory and practice in experimental bacteriology. Cambridge Univ. Press, London, England. 13 Newby, T. J., C. R. Stokes, and F. J. Bourne. 1982. Immunological activities of milk. Vet. Immunol. Immunopathol. 3:67. 14 Nonnecke, B. J., and K. L. Smith. 1984. Inhibition of mastitis bacteria by bovine milk apo-lactoferrin evaluated by in vitro microassay of bacterial growth. J. Dairy Sci. 67:606. 15 Outteridge, P. J., and C. S. Lee. 1981. Cellular immunity in the mammary gland with particular reference to T, B lymphocytes and macrophages. The ruminant immune system. Adv. Exp. Med. Biol. 137:513. 16 Peaker, M., and J. L. Linzell. 1975. Citrate in milk: A harbinger of lactogenesis. Nature 253:464. 17 Reiter, B. 1978. Review of the progress of Dairy Science: Antimicrobial systems in milk. J. Dairy Res. 45:131. 18 Reiter, B., and A. J. Bramley. 1975. Defense mechanisms of the udder and their relevance to masitis control. Page 210 in Proceedings of a seminar on mastitis control. F. N, Dodd, T. K. Griffin, and J.R.G. Kingwell, ed. Int. Dairy Fed., Brussels. 19 Schanbacher, F. L., and K. L. Smith. 1974. Electroimmunodiffusion on cellulose acetate: A rapid method for analysis of bovine lactoferrin in chromatography effluents. Anal. Biochem, 59:235. 20 Schanbacher, F. L., and K. L. Smith. 1975. Formation and role of unusual whey proteins and enzymes: Relation to mammary function. J. Dairy Sci. 58:1048. 21 Smith, K. L., and S. P. Oliver. 1981. Lactoferrin: A component of nonspecific defense of the involuting bovine mammary gland. The ruminant immune
Journal of Dairy Science Vol. 67, No. 12, 1984
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NONNECKE AND SMITH
system. Adv. Exp. Med. Biol. 137:535. 22 Smith, K. L., and F. L. Schanbacher. 1977. Lactoferrin as a resistance factor to infection of the bovine mammary gland. J. Am. Vet. Med. Assoc. 170:1224. 23 Spik, G., A. Cheron, J. Montreuil, and J. M. Dolby. 1978. Baeteriostasis of a milk-sensitive strain of Escberiebia coli by immunoglobulins and ironbinding proteins in association. Immunology 35:663. 24 Standard Methods for the Examination of Dairy
Journal of Dairy Science Vol. 67, No. 12, 1984
Products 1972. 13th ed. Am. Publ, Health Assoc,, New York, NY. 25 Subcommittee of Screening Tests National Mastitis Council 1968. Direct microscopic somatic cell counts in milk. J. Milk Food Technol. 31: 344. 26 Watson, D. L. 1980. Immunological functions of the mammary gland and its secretion-comparative review. Aust. J. Biol. Sci. 33:403. 27 White, J. C., and D. T, Davies. 1963. The determination of citric acid in milk and milk sera. J. Dairy Res. 30:171.
Department of Dairy Science Ohio Agricultural Research and Development Center The Ohio State University Wooster 44691 ABST R A C T
candy their inhibitory capacity within cow. However, reduction of growth inhibition was significant when data from the four cows were pooled.
Mammary secretions from four Holstein Friesian cows were collected during late lactation, early involution, and early in subsequent lactation. Changes of pH, concentration of serum albumin, immunoglobulin G, citrate, lactoferrin, and number of leukocytes in secretions were typical of milk from glands undergoing these physiological transitions. Whey prepared from a cow's secretions was evaluated for its capacity to inhibit growth of Escbericbia coli, 60-Lilly, and a coliform strain isolated from mammary secretions of that cow. Wheys from different glands of the same cow differed markedly in their capacity to inhibit growth of coliforms. Inhibition of both strains by the wheys increased significantly during the dry period and was maximal in wheys collected day 15 of the dry period and at parturition in the subsequent lactation. The coliform strain isolated from a specific cow was inhibited more than Escbericbia coli, 60-Lilly, by whey from the specific cow. Inhibition of the cow-specific coliform strain by the day 15 whey was reduced significantly after addition of ferric iron or sodium citrate. Addition of excess ferric iron or citrate to wheys collected at parturition did not alter signifi-
INTRODUCTION
Lactoferrin (Lf), a glycoprotein in mammary secretion, is a powerful chelator of iron with an affinity constant for iron of approximately 1036. Lactoferrin alone or in conjunction with specific antibody inhibits bacterial growth (1, 23) and may be a potent antimicrobial factor in the nonlactating or infected bovine mammary gland (1, 17, 18, 21). The antimicrobial activity of Lf has been attributed to its ability tenaciously to chelate iron, limiting the iron in the host's body fluids available for microbial metabolism (4). Coliform bacteria, a cause of bovine mastitis (8), employ at least two systems for sequestering iron from the iron-poor environment of the host (4). One system involves synthesis and secretion into the environment of the iron binding catechol, enterochelin, and a second, a citrate-mediated transport system, is induced by citrate in the growth medium. Citrate, a constituent of normal milk (16), and Lf undergo changes of concentration in milk during mammary involution, establishment of normal lactation, and mastitis. In vitro studies showed a negative correlation between molar ratio of citrate:lactoferrin (Cit:Lf) in a synthetic growth medium and degree of inhibition of coliform bacteria (1, 14). Based on these studies the molar ratio of Cit:Lf is elevated Received March 21, 1984. sufficiently in normal milk and colostrum to 1Salaries and research support provided by State limit the Lf-dependent inhibition of coliform and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio bacteria. However, the low molar ratio of citrate to Lf in secretion from the involuted State University. Journal Article No. 38-84.
1984 J Dairy Sci 67:2863--2872
2863
2864
NONNECKEAND SMITH
mammary gland suggests that Lf may inhibit effectively growth of coliform bacteria. Milk samples collected from individual glands of four cows during late lactation, early involution, and at parturition were evaluated for indicators of physiological changes of mammary function. Wheys derived from these samples were tested in vitro for their capacity to inhibit growth of E. coil, 60-Lilly, and a coliform strain isolated from the mammary secretion of each cow. MATERIALS AND METHODS Experimental Animals
Four purebred Holstein-Friesian cows, designated N1, N2, N3, and N4, were maintained under identical conditions, milked twice daily, and not dry cow-treated with antibiotics at the end of lactation. Milk was collected from individual glands of each animal 7 days prior to the dry period (D -- 7), at drying-off (D + 0), and 2 (D + 2 ) , 4 ( D + 4 ) , 6 (D + 6 ) , 8 ( D + 8 ) , and 15 (D + 15) days into the dry period, and in the subsequent lactation, at parturition (C + 0), and 7 days postpartum (C + 7). Each sample consisted of 3 to 5 ml of foremilk collected aseptically for bacteriological and cellular evaluation and 150 ml of milk for citrate and protein determinations and bacteriological growth studies. Bacteria in milk were identified according to recommendations of the National Mastitis Council (3). The total number of leukocytes in the secretions was determined by direct microscopic cell count (25) of a dried milk film (1 cm 2) stained with the Levowitz-Weber modification of the Newman-Lambert stain (24). Glassware
Glassware was prepared according to the procedures described in Experimental Tecbniques in Biocbemistry (2) to minimize iron contamination in the assays. Skim Milk and Whey Preparation
Whole milk was centrifuged at 30,000 x g for 30 min at room temperature and fat separated from skim milk by the fluid component decanted through cheesecloth. The pH of each skim milk was recorded. Whey was made by the skim milk equilibrated to 43°C and the pH Journal of Dairy Science Vol. 67, No. 12, 1984
adjusted to 4.5 by glacial acetic acid added. The precipitated casein was removed by centrifugation at 30,000 × g for 30 min, and pH of the whey was adjusted to the pH of original skim milk by addition of 1 M NaOH. Wheys were sterilized by filter (.45 g) and stored at 20°C until needed. Protein Quantitation
Quantitation of Lf, immunoglobulin G (IgG), and serum albumin (SA) in whey was by electroimmunodiffusion (EID) on cellulose acetate plates (Helena Labs) (19). Antisera consisted of 1.8 to 2.0% rabbit antibovine Lf, 4.0% rabbit anti-Fab fragment (antisera prepared by K. L. Smith), and 2.0% anti-SA (Miles Labs, Inc.). Standard curves were established from concentrations of .05 to 4.0 mg/ml for SA, .05 to .45 mg/ml for Lf, and .13 to .38 mg/ml for IgG. Citrate Determinations
Quantitation of citrate in milk was photometric (27). An individual assay consisted of 30 unknowns and five citrate standards of .0, .05, .075, .100, and .125 mg/ml of citrate, each prepared in duplicate. Optical density of standards plotted against their concentration resulted in a linear plot from which the concentration of citrate in the unknown could be interpolated. Bacterial Cu|tures
The coliform strains specific for cows were isolated from mammary secretions of individual cows during these experiments. Coliform strains were E. coli from cows N1 and N4, Enterobacter cloacae from cow N2, and Klebsiella pneumoniae from cow N3. Escbericbia coli, 60-Lilly, was obtained from E. J. Carroll (University of California, Davis, CA). Bacterial Growth Assay
A single colony of the test strain grown on blood agar (5% sheep blood in trypticase soy agar, Difco Labs) was inoculated into 5 ml of filter-sterilized bovine milk whey and incubated at 37°C for 24 h. One milliliter of this culture was inoculated into 5 ml of sterilized whey and incubated at 37°C for 12 h. After incubation the culture was diluted in sterile phosphate
INHIBITION OF COLIFORMS BY MILK WHEY buffered saline (PBS, .15 M, pH 6.8) to 102 cfu/lO #l of PBS. Sterile microtiter plates (120 mm X 80 ram) with 96 U-shaped wells (Cooke Lab. Products, Division of Syntach Labs) were used to support assays of bacterial growth inhibition. Of the 300-~tl capacity of each well, 250/~I was designated for filtered whey (without or with iron or citrate supplementation) and 10 /~1 for the bacterial inoculum (102 cfu of the test strain). Individual tests were prepared in triplicate. The prepared microtiter plate, covered with a sterilized lid, was incubated at 37°C for 12 h. After incubation, contents of individual wells were diluted serially in PBS, and the number of viable bacteria per milliliter was determined in triplicate by the drop method (12, 14) on blood agar.
2865
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RESULTS
The mean pH, SA, and IgG concentrations and mean number of leukocytes in the secretions of cows N1 to 4 are summarized in Table 1. The pH of the milk, initially 6.7 on D -- 7, increased significantly to a maximum of 7.6 on D + 15. At C + 0 pH had dropped significantly to 6.2, and by C + 7 was 6.6. Concentration of SA in the secretion increased significantly from D - 7 to D + 6 and reached a maximum of 3.10 mg/ml on D + 15. At C + 0 and C + 7 the concentration of SA in the milk had declined significantly to 1.74 a n d . 10 mg/ml. The IgG in milk increased significantly from .66 mg/ml on D -- 7 to 18.7 mg/ml on D + 15. However, the maximum concentration of IgG, 69.09 mg/ml, occurred at C + 0, and by C + 7 concentrations had returned to those typical of normal milk (.49 mg/ml). The increase of concentration of SA and IgG in the milk from D - 7 to D + 15 was progressive. Concentrations of SA, IgG, and numbers of leukocytes in secretions may have been influenced by infection status of mammary g!ands. However, no attempt was made to assess the influence of infection because of the small number of COWS.
Numbers of leukocytes in milk increased significantly from D -- 7 to D + 6 and remained elevated until C + 0 when they declined significantly. No further decrease of number of leukocytes was observed at C + 7. The concentration of Lf increased significantly from D -- 7 (.63 mg/ml) to D + 15 (13.5
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Journal of Dairy Science Vol. 67, No. 12, 1984
2866
NONNECKE AND SMITH
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mg/ml) (Table 2); however, at C + 0 concentration had decreased significantly to 1.82 mg/ml and b y C + 7 was .12 mg/ml. The concentration of citrate decreased significantly from D -- 7 (1.54 mg/ml) to D + 15 (.50 mg/ml) (Table 2). A t C + 0 the amount of citrate increased significantly to 1.56 mg/ml and by C + 7 was 2.38 mg/ml, greater than at any time during flae study. The molar ratio o f Cit:Lf at D -- 7 was 1154; however, b y D + 15 of dry period molar ratio had declined significantly to 28 (Table 2). At C + 0 the molar ratio of Cit:Lf increased significatly to 380 and b y C + 7 was 9528. Whey in the bacterial growth assay was not supplemented nutritionally, because growth in the unsupplemented whey was considered a more valid indicator of inhibitory capacity o f the secretion. Growth o f test strains in unsupplemented whey was limited to a 102 to 103 times increase after 12 h of incubation compared to a 104 to 10 s increase in supplemented (supplemented with 17 mg/ml AOAC medium, Difco Lab) wheys (14). There were pronounced differences in growth of E. coli, 60-Lilly, in wheys from different cows sampled at the same time (Table 3). Overall, inhibition of E. coli, 60-Lilly, and the cow-specific strain increased significantly with the onset of the dry period, and inhibition was greatest at D + 15. Wheys collected at C + 0 were significantly more inhibitory than those wheys prepared from milk samples collected at D -- 7. However, the inhibitory capacity of wheys prepared from C + 7 milk samples was not significantly different from D -- 7 wheys. The cow-specific strains were more inhibited than E. coli, 60-Lilly, b y wheys prepared from a specific cow. Inhibition of cow-specific strains b y D + 15 and C + 0 wheys from cows N1 to 4, before and after supplementation of the wheys with ferric ammonium sulphate or sodium citrate, was determined (Tables 4 and 5). Iron or citrate supplementation of the D + 15 wheys from cows N1, N2, and N4 significantly reduced growth inhibition of the cow-specific strain. However, neither iron nor citrate supplementation of C + 0 wheys significantly modified their capacity to inhibit the cow-specific strain. When data from the four cows were pooled, the decrease of growth inhibition after supplementation of wheys was significant (P<.05).
TABLE 3. Bacterial growth inhibition i by whey collected during the experimental period. Time of milk collection 2 D +2
D+4
D+6
D+8
D+15
C+0
X SE
100 36
81 33
73 36
84 20
74 22
62 20
58 32
94 50
X SE
38 14
29 10
18 8
43 18
46 20
30 12
32 16
59 25
E. coil 60-Lilly
X SE
100 19
63 20
46 10
48 21
41 7
23 11
30 4
135 31
Cow-specific strain
.X SE
108 8
30 10
24 10
23 9
15 4
10 5
20 6
99 27
E. coli 60-Lilly
X SE
125 18
75 23
41 8
35 9
36 12
31 10
52 9
82 5
Cow-specific strain
.X SE
105 20
75 23
39 4
28 6
31 6
21 4
41 16
131 22
E. coli 60-Lilly
X SE
110 18
50 16
20 3
22 6
10 3
12 2
18 5
70 14
Cow-specific strain
X SE
120 6
47 19
26 4
13 3
10 5
5 2
3 1
77 29
E. coli 60-Lilly
X SE
108 a 6
67 b 7
45 b 11
47 b 13
40bc 13
32 c 11
40bc 9
95 a 14
Cow-specific strain
X SE
93 a 37
45 a 11
27 b 4
27 b 6
26 b 8
16 b 6
24 b 8
92 a 16
Bacterial strain
N1
E. coli 60-Lilly
Cow-specific strain
N2
N3
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TABLE 4. Effect of e x o g e n o u s ferric iron or s o d i u m citrate on the growth o f cow-specific coliform strain in w h e y s 15 days into dry period. O~ .a
Cow
00
Lactoferrin (mg/ml)
Molar ratio Cit:Lf
Unsupplemented
Growth (cfu/ml, Log10) !n w h e y Iron z supplemented
Citrate 2 supplemented
N1 (n = 4)
X SD
13.6 3.5
24 11
3.39 .31
4.44" .23
4.45* .12
N2
(n = 3)
X SD
12.4 3.4
55 62
2.81 .22
3.79* .27
3.80" .14
N3 (n = 4)
X' SD
8.9 1.2
23 14
3.02 .46
3.49 .36
3.38 .38
N4 (n = 4)
X SD
18.0 6.5
11 13
3.09 .40
4.63* .21
4.44" .22
All cows
.~ SD
13.2 3.7
28 19
3.08 .24
4.09* .54
4.02* .52
: Ferric a m m o n i u m sulphate added in excess of the a m o u n t to saturate e n d o g e n o u s lactoferrin. 2 Sufficient s o d i u m citrate added to increase t h e Cit:Lf (citrate:lactoferrin) molar ratio to 600. N u m b e r o f glands from each cow while provided secretion. *Denotes significant increase in growth after s u p p l e m e n t a t i o n (P<.05).
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TABLE 5. Effect of exogenous ferric iron or sodium citrate on the growth of cow-specific coliform strain in whey at parturition. Growth (cfu/ml, Loglo) in whey Cow
Lactoferrin (mg/ml)
Molar ratio Cit:Lf
Unsupplemented
Iron supplemented
Citrate 2 supplemented
N1 (n = 4) a
.X SD
1.9 .6
310 99
2.95 .89
3.38 .46
3.24 .51
N2 (n = 4)
.X SD
1.2 .2
563 115
3.14 .29
3.34 .10
3.49 .30
N3 (n = 4)
.X SD
2.3 .3
277 199
3.16 .14
3.24 .36
3.31 .20
All cows
,X SD
1.8 .5
357 138
3.07 .10
3.29* .08
3.33* .11
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2870
NONNECKE AND SMITH DISCUSSION
Significant changes occur in biochemical composition and antibacterial properties of bovine mammary secretion during early stages of involution and at parturition. The increase of SA, IgG, and Lf and decrease of citrate concentration were likely caused by reduction of secretory activity of the mammary gland and decline of milk volume, a phenomenon reported by others (22, 26). The increase of SA also reflects the increased permeability of the secretory epithelial barrier within the gland resulting in increased diffusion of serum proteins into the secretion (10). Elevated concentration of IgG in the D + 15 and C + 0 secretion was likely due to selective transport of IgG~ into secretion via receptors on the secretory epithelial cells (5, 7, 9). Concentration of Lf in normal bovine milk ranges from .1 to .3 mg/ml (19, 21). In our study, Lf increased significantly from .63 mg/ml at D -- 7 to 13.5 mg/ml on D + 15 of the dry period. Involution of the bovine mammary gland likely continues at least to day 30 of the dry period, at which time Lf reaches its maximum concentration (20 to 30 mg/ml) and is a major protein in secretion from the dry gland (21, 22). Schanbacher and Smith (20, 22) have postulated that Lf in the dry gland secretion originates from lysosomes of secretory calls and granulocytes of the involuting mammary gland. The decline of citrate during the dry period can be attributed to cessation of secretory activity in the gland. The increase of citrate at C + 0 and at C + 7 indicates reestablishment of normal lactation. Peaker and Linzell (16) have described the appearance of citrate in the secretion from the preparturient gland as a "harbinger" of lactogenesis. The significant increase of number of leukocytes in secretion from the involuting mammary gland (D + 4 through D + 15) has been reported by (8, 11). However, 72 h after cessation of milking, no further increase was observed. The primary function of leukocytes, mostly macrophages, during the early phase of involution may be to phagocytize cellular debris, fat, and casein generated during involution (8, 11). Compositional changes of the secretion during early involution and at parturition were
Journal of Dairy Science Vol. 67, No. 12, 1984
correlated with a significant increase of antimicrobial capacity of the corresponding whey. Although some wheys demonstrated pronounced inhibition for both the cow-specific strains and E. coli, 60-Lilly, none was bactericidal for either strain. However, the intact secretion (containing both humoral and cellular components) likely would be bactericidal (13, 15, 16, 26). The greater inhibition of the cow-specific coliform by wheys from the specific cow indicates immunological specificity of response, possibly due to the presence of specific antibody. A decline of the molar ratio of Cit:Lf has been associated with increased inhibition of mastitis-causing coliform bacteria growing in a synthetic medium (1). Our study demonstrated progressive increase of growth inhibitory capacity of wheys prepared from secretions sampled during the early phases of mammary involution, which was associated with a decline of the molar ratios of Cit:Lf and elevation of IgG concentration in secretions. Both Lf and IgG1 have been considered to contribute to antimicrobial action of bovine mammary secretion (13, 26). Addition of excess iron or citrate to the D + 15 wheys of cows N1, N2, and N4 caused marked reduction of their antimicrobial capacity, indicating that inhibition by unsupplemented wheys having comparatively low molar ratio of Cit:Lf was mediated partially by an iron-dependent system and that the cowspecific coliform may acquire iron either directly or by a citrate-mediated system (4). In contrast, the antimicrobial capacity of D + 15 wheys from cow N3 was moderately, but not significantly, reduced after supplementation with either exogenous iron or citrate. Growth inhibitory systems acting in the secretions o f cow N3 may consist of one or more of the noncellular defense active in bovine mammary secretions (18, 26). The degree of inhibition of the cow-specific coliform by wheys prepared from milk samples collected at C + 0 often exceeded that produced by the D + 15 wheys from the same animal. Addition of excess iron or citrate to the C + 0 wheys, as was done for the D + 15 samples, did not enhance significantly growth of the test strain in individual cow wheys. However, a significant enhancement of growth was evident when data for all four cows were
INHIBITION OF COLIFORMS BY MILK WHEY
pooled. The minimal effect of supplementation was not unexpected, because the Cit:Lf molar ratios of the C + 0 wheys were significantly greater than those of the D + 15 wheys and high enough to minimize the Lf-dependent growth inhibition (1, 12, 14, 22). The elevated concentration of IgG in these secretions may have inhibited the test strain, although high concentrations of specific antibody in colostrum are thought to inhibit complement binding, minimizing the antimicrobial effect of the complement-antibody system (18). The method of preparation of whey also may have depleted the nutritional value of the secretion, resulting in some growth inhibition by D + 15 and C + 0 wheys. In summary, secretion from the involuting and parturient mammary gland inhibits growth of mastitis-causing coliforms to a greater extent than secretion from the normal, lactating gland. During mammary involution concentration of noncellular antimicrobial factors (Lf and IgG) in the secretion increased significanly. Growth inhibitory properties of the D + 15 wheys were reduced significantly. Growth inhibitory properties of the D + 15 wheys were reduced significantly by addition of ferric iron or sodium citrate, suggesting the low molar ratio of Cit:Lf contributed to inhibitory properties of these wheys. The growth inhibitory properties o f t h e C + 0 wheys were affected moderately by addition of ferric iron or sodium citrate. This indicates an iron-independent antimicrobial system may be functioning in these secretions or that they may lack sufficient nutrients to support growth of the test strain. REFERENCES
1 Bishop, J. G., F. L. Schanbacher, L. C. Ferguson, and K. L. Smith. 1976. In vitro growth inhibition of mastitis-cansing coliform bacteria by bovine apoqactoferrin and reversal of inhibition by citrate and high concentrations of apoqactoferrin. Infect. Immunol. 14:911. 2 Brewer, J. M., A. J. Pesce, and R. B. Ashworth. 1974. Experimental techniques in biochemistry. Prentice-Hall, Inc., NJ. 3 Brown, R. W., D. A. Barnum, D. E. Jasper, J. S. McDonald, and W. O. Schultz. 1981. Microbiological procedures for use in the diagnosis of bovine mastitis. Natl. Mastitis Counc., Inc., Washington, DC, Carter Press, Inc., Ames. 4 Bullen, J. J., H. J. Rogers, and E. Griffiths. 1978. Role of iron in bacterial infection. Curr. Top. Microbiol. Immunol. 80:1.
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5 Dixon, F. J., W. O. Weigle, and J. J. Vacquez. 1961. Metabolism and mammary secretion of serum proteins in the cow. Lab. Invest. 10:216. 6 Ebethart, R. J., R, P. Natzke, F.H.S. Newbould, B. J. Nonnecke, and P. Thompson. 1979. Coliform m a s t i t i s - A review. J. Dairy Sci. 62:1. 7 Hammer, D. K., B. Kichhofen, and H. Malchow. 1969. Preferential absorption of a single bovine IgG type by isolated epithelial ceils of the mammary gland. Protides of the biological fluids. Pages 663-668 Proc. 16th Coll., Bruges. Pergamon Press, New York, NY. 8 Jensen, D. L., and R. J. Eberhart. 1981. Total and differential cell counts in secretions of the nonlactating mammary gland. Am. J. Vet. Res. 42:743. 9 Larson, B. L. 1958. Transfer of specific blood serum proteins to lacteal secretions near parturition. J. Dairy Sci. 41:1033. 10 Lascelles, A. K., and C. S. Lee. 1978. Involution of the mammary gland. In: Lactation, a comprehensive treatise. Vol. IV. B. L. Larson, ed. Academic Press, Inc., New York, NY. 11 McDonald, J. S., and A. J. Anderson. 1981. Total and differential somatic cell counts in secretions from noninfected bovine mammary glands: The early nonlactating period. Am. J. Vet. Res. 42: 1360. 12 Meynell, G. G., and E. Meynell. 1965. Theory and practice in experimental bacteriology. Cambridge Univ. Press, London, England. 13 Newby, T. J., C. R. Stokes, and F. J. Bourne. 1982. Immunological activities of milk. Vet. Immunol. Immunopathol. 3:67. 14 Nonnecke, B. J., and K. L. Smith. 1984. Inhibition of mastitis bacteria by bovine milk apo-lactoferrin evaluated by in vitro microassay of bacterial growth. J. Dairy Sci. 67:606. 15 Outteridge, P. J., and C. S. Lee. 1981. Cellular immunity in the mammary gland with particular reference to T, B lymphocytes and macrophages. The ruminant immune system. Adv. Exp. Med. Biol. 137:513. 16 Peaker, M., and J. L. Linzell. 1975. Citrate in milk: A harbinger of lactogenesis. Nature 253:464. 17 Reiter, B. 1978. Review of the progress of Dairy Science: Antimicrobial systems in milk. J. Dairy Res. 45:131. 18 Reiter, B., and A. J. Bramley. 1975. Defense mechanisms of the udder and their relevance to masitis control. Page 210 in Proceedings of a seminar on mastitis control. F. N, Dodd, T. K. Griffin, and J.R.G. Kingwell, ed. Int. Dairy Fed., Brussels. 19 Schanbacher, F. L., and K. L. Smith. 1974. Electroimmunodiffusion on cellulose acetate: A rapid method for analysis of bovine lactoferrin in chromatography effluents. Anal. Biochem, 59:235. 20 Schanbacher, F. L., and K. L. Smith. 1975. Formation and role of unusual whey proteins and enzymes: Relation to mammary function. J. Dairy Sci. 58:1048. 21 Smith, K. L., and S. P. Oliver. 1981. Lactoferrin: A component of nonspecific defense of the involuting bovine mammary gland. The ruminant immune
Journal of Dairy Science Vol. 67, No. 12, 1984
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NONNECKE AND SMITH
system. Adv. Exp. Med. Biol. 137:535. 22 Smith, K. L., and F. L. Schanbacher. 1977. Lactoferrin as a resistance factor to infection of the bovine mammary gland. J. Am. Vet. Med. Assoc. 170:1224. 23 Spik, G., A. Cheron, J. Montreuil, and J. M. Dolby. 1978. Baeteriostasis of a milk-sensitive strain of Escberiebia coli by immunoglobulins and ironbinding proteins in association. Immunology 35:663. 24 Standard Methods for the Examination of Dairy
Journal of Dairy Science Vol. 67, No. 12, 1984
Products 1972. 13th ed. Am. Publ, Health Assoc,, New York, NY. 25 Subcommittee of Screening Tests National Mastitis Council 1968. Direct microscopic somatic cell counts in milk. J. Milk Food Technol. 31: 344. 26 Watson, D. L. 1980. Immunological functions of the mammary gland and its secretion-comparative review. Aust. J. Biol. Sci. 33:403. 27 White, J. C., and D. T, Davies. 1963. The determination of citric acid in milk and milk sera. J. Dairy Res. 30:171.