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Effect of Chronic Staphylococcal Mastitis on Mitogenic Responses of Bovine Lymphocytes1
Effect of Chronic Staphylococcal Mastitis on Mitogenic Responses of Bovine Lymphocytes 1 B R I A N J. N O N N E C K E and JAMES A. H A R P
US Department of Agriculture Agricultural Research Service National Animal Disease Center P.O. Box 70
Ames, IA 500;10
ABSTRACT
lesser extent the macrophage (7), in the pathogenesis of staphylococcal mastitis has been documented. Immunologically reactive lymphocytes have been demonstrated in milk (B. J. Nonnecke et al., 1985, unpublished data; 16) and dry gland secretion (6); however, the role of the lymphocyte in the pathogenesis of staphylococcal mastitis is not well described. Cell (lymphocyte)-mediated immune (CMI) responses have been demonstrated in staphylococcal infections of nonmammary tissue in a variety of animal species (1). In the cow (17) and guinea pig (14), CMI response to staphylococcal antigens is involved in milk leukocytosis and systemic response of a sensitized animal to antigenic challenge. Conceivably, the chronicity associated with staphylococcal mastitis may, in part, be due to an impairment of the immune system during infection. In the present study, we examined the effect of chronic staphylococcal mastiffs on the in vitro blastogenic responses of milk and peripheral blood lymphocytes to mitogenic stimulation.
We compared mitogenic responses of milk and peripheral blood lymphocytes from nonmastitic (control) cows and cows with experimentally induced staphylococcal mastitis in one gland. Milk lymphocytes from infected glands were essentially unresponsive to Concanavalin A, phytohemagglutinin-P, and pokeweed mitogen. Proliferative responses of milk lymphocytes from uninfected glands of infected cows were not as depressed as those from infected glands but were significantly less than those of milk lymphocytes from control cows. Proliferative responses of peripheral blood lymphocytes from control and infected cows to Concanavalin A and phytohemagglutinin-P were similar; however, peripheral blood lymphocytes from infected cows responded in reduced fashion to pokeweed mitogen compared with peripheral blood lymphocytes from control animals. These observations demonstrate that in vitro lymphocyte blastogenesis is markedly depressed during infection, suggesting that in vivo lymphocyte function is compromised, possibly contributing to the chronicity of staphylococcal mastitis.
MATERIALS AND METHODS Animals
INTRODUCTION
Staphylococcus aureus is considered a major cause of serious and often chronic infection in the bovine mammary gland (2, 11). The role of mammary leukocytes, especially the polymorphonuclear leukocyte (PMNL) (5, 15) and to a
Received April 29, 1985. No endorsements are herein implied. 1985 J Dairy Sci 68:3323-3328
Ten multiparous, lactating Holstein-Friesian cows from a closed herd at the National Animal Disease Center were used. Chronic staphylococcal mastiffs was established in a single gland of each of seven animals by infusing 2000 cfu of Staphylococcus aureus (American Type Culture Collection 27940) into the gland during the 1st mo of their second lactation. Viable staphylococci were present in milk from the infected glands for the duration of the lactation. Three cows not experimentally infected and free of naturally occurring mastitis served as controls.
3323
3324
NONNECKE AND HARP
L y m p h o c y t e blastogenesis studies were during the final 3 m o of the second lactation. Enrichment of Milk and Blood Lymphocytes
Milk and blood samples were collected at the morning milking from each of the 10 cows on 2 or more separate days during an 8-wk period. Individual quarter milk samples and a pooled milk sample were collected from each uninfected cow. Pooled milk from the three uninfected glands and a single sample from the infected gland were collected from each mastitic cow. A minimum of 2 L o f milk was diluted with three parts of physiologically warm (39°C) phosphate-buffered saline [(PBS) pH 6.8, .15 M]. Leukocytes were recovered from the diluted milk by continuous flow centrifugation (flow rate = 35 ml/min/cup, × 1,000 x g). Pelleted cells were resuspended in 100 ml PBS containing 100 units/ml penicillin G and 100 /~g/ml streptomycin and were washed once at 300 × g for 10 rain. Blood was collected by jugular venipuncture into tubes containing Alsever's solution and then diluted with two parts of PBS (pH 7.2,
.15M). Twelve milliliters of washed milk cells or diluted blood were overlayed on 3 ml of ficoll-diatrizoate [specific gravity 1.084 (13), Sigma Chemical Co.] in 15-ml conical tubes, and centrifuged 400 × g at 18°C for 40 min. Blood mononuclear bands were removed from each tube, washed (300 × g, 10 min) twice in Hank's balanced salt solution without Ca 2+ or Mg 2+, once in PBS, and resuspended in RPMI 1640 medium with 10 mM Hepes buffer, 2 mM L-glutamine, and antibiotics as described. Milk mononuclear cells were washed once in PBS and resuspended in RPMI 1640 medium.
phages (M~), or PMNL. Eosinophils and basophils (rarely observed) were included in the PMNL category. Number of leukocytes in enriched populations was determined using a hemocytometer, and viability was assessed by trypan blue exclusion (10). Lymphocyte Blastogenesis Assay
Multiple wells of a 96-well flat b o t t o m tissue culture plate (Falcon 3070, Becton-Dickinson) were seeded with 1.0 x 10 s viable milk or blood mononuclear leukocytes in 50 /al of RPMI 1640 medium with antibiotics. Additional components added to each well were 50 /~1 of fetal calf serum (heat-inactivated) in RPMI 1640 medium to give a final concentration in the assay of 10%, and 100 /ll of RPM! 1640 medium containing optimal dilutions (13) of phytohemagglutinin-P (PHA-P, 12.5 /~g/ml, Difco Laboratories), Concanavalin A (Con A), 7.8 /lg/ml (Sigma Chemical Co.), or pokeweed mitogen (PWM), 2.0 /.tg/ml, (Sigma Chemical Co.). Individual assays were prepared in triplicate. Culture plates were incubated for either 24 or 72 h at 39°C in a humidified atmosphere containing 5% carbon dioxide. After the initial 24 or 72 h incubation, 5 0 / l l (.5 /ICi) of [methyl-3H]thymidine were added to each well and plates were incubated an additional 18 h. Well contents were harvested onto glass fiber strips using a cell harvester (Model PHD, Cambridge Technology, Inc.) and the retained radioactivity counted in a liquid scintillation counter (Model LS-8000, Beckman Instruments). Differences between mean blastogenic responses were evaluated using Student's t test.
RESULTS Leukocyte Enumeration and Characterization
Number of leukocytes present in milk from individual glands and pooled samples was determined by the direct microscopic cell count method (12). These preparations were stained with Stat-Stain (Volu-Sol Medical Industries, Inc.). Blood and milk cell differential counts were determined from slides prepared by cytocentrifugation (Cytospin 2, Shandon Instruments) and stained with Stat-Stain. Cells were categorized as small mononuclear leukocytes (SML) (lymphocytes and monocytes), macroJournal of Dairy Science Vol. 68, No. 12, 1985
Cows experimentally infected in a single gland with S. a u r e u s developed chronic mastitis in that gland, which persisted throughout lactation. The infective strain repeatedly was isolated in pure culture from milk of infected glands. Milk production by infected glands of each cow was reduced relative to production in the previous lactation (J. S. McDonald, unpublished data). Uninfected glands of each mastitic cow, milked independently of the infected gland, remained free of infection throughout the study.
MASTITIS AND LYMPHOCYTE FUNCTION Milk and Blood Leukocyte Populations
Number of SML, M~, and PMNL/ml of milk from glands of control cows, uninfected glands of mastitic cows, and infected glands is in Table 1. Total number of leukocytes and number of SML, Mg~, and PMNL were significantly (P<.05) greater from infected glands than from uninfected glands. The greatest difference was associated with PMNL, which were at a concentration of 8.70 × 10S/ml from infected glands compared with .38 x 10S/ml and .75 × 105/ml in milk from control cows and uninfected glands of mastitic cows, respectively. No differences in number or types of leukocytes present in blood of infected and uninfected cows were detected. Enriched Mononuclear Leukocyte Populations
Mean -+ SE viability of enriched milk mononuclear leukocytes from infected and uninfected cows was 52 + 8.7%. Mean + standard error percentages of SML in enriched leukocyte populations originating from infected glands, uninfected glands of infected cows, and glands of uninfected cows were 56.4 + 8.4, 66.0 + 11.9, and 67.3 + 4.5%, respectively. Mean percentages of MO from these groups were 24.2 + 6.5, 25.9 + 12.3, and 25.9 -+ 4.7%. Similarly, mean percentages of PMNL from these groups were 19.4 + 7.9, 8.4 + 4.2, and 6.8 + .7%,
3325
respectively. The mononuclear leukocyte population recovered from the blood of all animals after ficoll-diatrizoate treatment was greater than 99.0% pure and 87.2% viable. Lymphocyte Blastogenesis
Blastogenic responses (X counts per minute -+ SE) of milk and blood lymphocytes cultured for 42 and 90 h in the absence or presence of three different mitogens are presented in Figures 1 and 2. Mitogen concentrations employed were those that provided optimal stimulation of blood lymphocytes from control animals. Responses of milk lymphocytes from control animals to the three mitogens were significantly (P<.05) greater at 42 and 90 h than responses of milk lymphocytes from uninfected or infected glands of infected cows. Milk lymphocytes from infected glands were essentially unresponsive to mitogenic stimulation at both 42 and 90 h, whereas lymphocytes from uninfected glands of the same animals were significantly (P<.05) more responsive to mitogens at both times, although not as responsive as milk lymphocytes from normal cows. The effect of chronic staphylococcal mastitis on milk lymphocyte mitogenesis was most pronounced for cultures stimulated by PHA-P. The PWM-stimulated blood lymphocytes from normal cows, compared with infected
TABLE 1. Geometric mean number of leukocytes present in milk from glands of uninfected cows, uninfected glands of mastitic cows, and glands experimentally infected with Staphylococcus aureus.
Source of milk
Leukocytes (× 10S/ml) MO2
SML1 ,X
SE
PMNL3
,X
SE
.X
SE
Unfected glands (n=24) (control cows)
1.06a
.15
.28 a
.12
.38a
.03
Uninfected glands (n=63) (infected cows)
.95a
.11
.39 a
.07
.75 b
.16
2.72b
1.84
1.98b
1.02
8.70 c
4.20
Infected glands (n=21)
a'b'CMeans with different superscripts within columns differ (P<.05). 1Small mononuclear leukocytes. 2Macrophage. 3Polymorphonuclear leukocytes. Journal of Dairy Science Vol. 68, No. 12, 1985
3326
NONNECKE AND HARP
"]° 30
I t
t°
28 26 24 22 2o 18
14
,°°...."° ~
42
90
42 90 INCUBATION TIME (h)
42
90
Figure 1. Incorporation of [3H]thymidine by milk lymphocytes from a) uninfected cows (n=6), b) uninfected glands of infected cows (n=21), and c) infected glands (n=21) after 42 and 90 h of incubation in vitro. Responses to the mitogens Concanavalin A (o-o), phytohemagglutinin-P (a-a), and pokeweed mitogen (e---e) are shown. Values in parentheses indicate blastogenesis of unstimulated lymphocyte cultures.
cows, incorporated significantly ( P < . 0 5 ) m o r e radiolabel at 90 h. Blood lymphocytes from uninfected animals incorporated more [3H]thymidine at 90 h than blood from infected animals, although the magnitude of responses of l y m p h o c y t e s to Con A and PHA-P was not significantly different (Figure 2). Blood lymphocytes from control or infected cows were more responsive to mitogenic stimulation than autologous milk lymphocytes, regardless of the mitogen employed. Differences were most pronounced for lymphocytes from blood of infected cows and lymphocytes from infected glands.
D I SCUSS I O N
Milk lymphocytes from mammary glands chronically infected with S. a u r e u s were hyporesponsive to mitogenic stimulation compared with milk lymphocytes from uninfected glands of the same animal or control cows. The Journal of Dairy Science Vol. 68, No. 12, 1985
magnitude of the proliferative response of a lymphocyte to mitogens is recognized as an indicator of the cellular immunocompetence (3, 9). Frequently viral, bacterial, and parasitic diseases are associated with reduced lymphocyte reactivity to mitogens (9). The almost complete abrogation of milk lymphocyte mitogenesis associated with mammary infection suggests a possible loss of immunocompetency within the infected gland. The chronic nature of a staphylococcal mastitis may be due to an impairment of the milk lymphocyte's capacity to respond to membrane receptor-mediated events such as antigen recognition. Protracted mastitis in a single gland was associated with a reduction in mitogenesis by lymphocytes from milk (uninfected gland) and blood of the infected cow indicating that effects of single-gland infection did not remain localized. It is important to determine whether infection-modified responses of milk and blood lymphocytes are associated with a reduction in immune capacity systemically as well as locally.
MASTITIS AND LYMPHOCYTE FUNCTION
140
3327
-b
120
'~ I00 E =. 80 60 40 2O 0
~--.o61
~.ts, .,)
I
42
I
1.~3-*.o9) I
90 42 INCUBATIONTIME (h)
1.92±
.66l
I
90
Figure 2. Incorporation of [3H]thymidine by peripheral blood lymphocytes from a) uninfected (n=6) and b) infected cows (n=21) after 42 and 90 h of incubation in vitro. Responses to the mitogens Concanavalin A (o-o), phytohemagglutinin-P (a-zx), and pokeweed mitogen (e---e) are shown. Values in parentheses indicate blastogenesis of unstimulated lymphocyte cultures.
Proliferative responses of tymphocytes to mitogens, unlike responses to antigen, are nonspecific (3, 8). Reduced mitogenesis of lymphocytes from mastitic cattle may be typical of mammary inflammation and not unique to mastitis caused by S. aureus. Antigen-specific reactivity of milk and blood lymphocytes from control and infected cows was tested using Protein A and a whole cell bacterin of the infective strain. Blastogenic responses of lymphocytes to these antigens were inconsistent (data not presented), possibly because the antigens employed in vitro did not have sufficient crossreactivity with those expressed b y the viable organism in vivo. Reduced mitogenic and inconsistent antigen-specific responses by milk and blood lymphocytes from infected cows might also indicate a general impairment of l y m p h o c y t e function due to mastitis. The PMNL, present in higher numbers in milk from infected than uninfected glands, may have contributed to reduced mitogenesis of lymphocytes from infected glands. Research (4) indicates that the myeloperoxidase H202 and halide system of the activated PMNL is cytotoxic for mononuclear leukocytes. L y m p h o c y t e mitogenesis is also suppressed by
reactive oxygen species (H202) produced by activated PMNL (18). Suppression appears to be due to the cytolethal effect of H20~ on the lymphocyte. In conclusion, an in vitro assay of lymphocyte function indicated that cows with chronic staphylococcal infection have impaired lymphocyte responses to mitogens. We are presently studying mechanisms responsible for impaired blastogenic responses of lymphocytes from infected cows. Further research is needed to determine if these effects are Specific to mastitis caused b y S. aureus. ACKNOWLEDGMENTS
The authors thank W. A. Hambly and B. Pesch for excellent technical assistance. Cows were infected with S. a u r e u s by J. S. McDonald. REFERENCES
1 Adlam, C., and C.S.F. Easom. 1983. Immunity and hypersensil~ivity to staphylococcal infection. Page 275 in Staphylococci and staphylococcal infections. Vol. 1. C.S.F. Easom and C. Adlam, ed. Academic Press, New York, NY. 2 Anderson, J. C. 1983. Veterinary aspects of staphylococci. Page 193 in Staphylococci and staphylococcal infections. Vol. 1. C.S.F. Easom and C. Journal of Dairy Science Vol. 68, No. 12, 1985
33 28
NONNECKE AND HARP
Adlam, ed. Academic Press, New York, NY. 3 Ashman, R. F. 1984. Lymphocyte activation. Page 267 in Fundamental immunology. W. E. Paul, ed. Raven Press, New York, NY. 4 Clark, R. A., and S. J. Klebanoff. 1977. Myeloperoxidase-H~O2-halide system: Cytotoxic effect on human blood leukocytes. Blood 50:65. 5 Colditz, I. G., and D. L. Watson. 1982. The role of humoral and cellular mediators in enhanced inflammatory reactions to staphylococcal infection in systemically immunized ewes. Microbiol. Immunol. 26:1171. 6 Concha, C., O. Hoimberg, and B. Morein. 1980. Characterization and response to mitogens of mammary lymphocytes from bovine dry gland secretion. J. Dairy Res. 47: 305. 7 Craven, N., and J. C. Anderson. 1984. Phagocytosis of Staphylococcus aureus by bovine mammary gland macrophages and intracellular protection from antibiotic action in vitro and in vivo. J. Dairy Res. 51:513. 8 Cunningham-Rundles, S., J. A. Hansen, and B. Dupont. 1976. Lymphocyte transformation in vitro in response to mitogens and antigens. Page 153 in Clinical immunobiology. F. H. Bach and R. A. Good, ed. Academic Press, New York, NY. 9 Kristensen, F., B. Kristensen, and S. Lazary. 1982. The lymphocyte test in veterinary immunology. Vet. Immunol. Immunopathol. 3:203. 10 Kruse, P. A., Jr., and M. K. Patterson, Jr. 1973. Tissue culture: methods and applications. Academic Press, New York, NY. 11 McDonald, J. S. 1977. Streptococcal and staphy-
Journal of Dairy Science Vol. 68, No. 12, 1985
12 13
14
15
16 17
18
lococcal mastiffs. J. Am. Vet. Med. Assoc. 170: 1157. National Mastitis Council, Subcommittee of Screening Test. 1968. Direct microscopic cell counts in milk. J. Milk Food Tecbnol. 31 : 344. Nonnecke, B. J., and M. E. Kehrli, Jr. 1985. Isolation of mononuclear cells from bovine milk by continuous-flow and density gradient centrifugation: Response of cells to mitogens. Am. J. Vet. Res. 46:1259. Nonnecke, B. J., and S. P. Targowski. 1984. The effect of local and parenteral vaccination on the response of the guinea pig mammary gland to staphylococcal challenge. J. Reprod. Immunol. 6: 365. Paape, M. H,, W. P. Wergin, A. J. Guidry, andW. D. Schultze. 1980. Phagocytic defense of the ruminant mammary gland. Page 555 in The ruminant immune system. J. E. Butler, ed. Plenum Press, New York, NY. Smith, J. W., and R. D. Schultz. 1977. Mitogen and antigen responsive milk lymphocytes. Cell. Immunol. 29:165. Targowski, S. P., and D. T. Berman. 1975. Leukocytic response of bovine mammary gland to injection of killed cells and cell walls of Staphylococcus aureus. Am. J. Vet. Res. 36:1561. Zoschke, D. C., and R. P. Messner. 1984. Suppression of human lymphocyte mitogenesis mediated by phagocyte-released inactive oxygen species: Comparative activities in normal and in chronic granulamatous disease. Clin. ImmunoL Immunopathol. 32:29.
US Department of Agriculture Agricultural Research Service National Animal Disease Center P.O. Box 70
Ames, IA 500;10
ABSTRACT
lesser extent the macrophage (7), in the pathogenesis of staphylococcal mastitis has been documented. Immunologically reactive lymphocytes have been demonstrated in milk (B. J. Nonnecke et al., 1985, unpublished data; 16) and dry gland secretion (6); however, the role of the lymphocyte in the pathogenesis of staphylococcal mastitis is not well described. Cell (lymphocyte)-mediated immune (CMI) responses have been demonstrated in staphylococcal infections of nonmammary tissue in a variety of animal species (1). In the cow (17) and guinea pig (14), CMI response to staphylococcal antigens is involved in milk leukocytosis and systemic response of a sensitized animal to antigenic challenge. Conceivably, the chronicity associated with staphylococcal mastitis may, in part, be due to an impairment of the immune system during infection. In the present study, we examined the effect of chronic staphylococcal mastiffs on the in vitro blastogenic responses of milk and peripheral blood lymphocytes to mitogenic stimulation.
We compared mitogenic responses of milk and peripheral blood lymphocytes from nonmastitic (control) cows and cows with experimentally induced staphylococcal mastitis in one gland. Milk lymphocytes from infected glands were essentially unresponsive to Concanavalin A, phytohemagglutinin-P, and pokeweed mitogen. Proliferative responses of milk lymphocytes from uninfected glands of infected cows were not as depressed as those from infected glands but were significantly less than those of milk lymphocytes from control cows. Proliferative responses of peripheral blood lymphocytes from control and infected cows to Concanavalin A and phytohemagglutinin-P were similar; however, peripheral blood lymphocytes from infected cows responded in reduced fashion to pokeweed mitogen compared with peripheral blood lymphocytes from control animals. These observations demonstrate that in vitro lymphocyte blastogenesis is markedly depressed during infection, suggesting that in vivo lymphocyte function is compromised, possibly contributing to the chronicity of staphylococcal mastitis.
MATERIALS AND METHODS Animals
INTRODUCTION
Staphylococcus aureus is considered a major cause of serious and often chronic infection in the bovine mammary gland (2, 11). The role of mammary leukocytes, especially the polymorphonuclear leukocyte (PMNL) (5, 15) and to a
Received April 29, 1985. No endorsements are herein implied. 1985 J Dairy Sci 68:3323-3328
Ten multiparous, lactating Holstein-Friesian cows from a closed herd at the National Animal Disease Center were used. Chronic staphylococcal mastiffs was established in a single gland of each of seven animals by infusing 2000 cfu of Staphylococcus aureus (American Type Culture Collection 27940) into the gland during the 1st mo of their second lactation. Viable staphylococci were present in milk from the infected glands for the duration of the lactation. Three cows not experimentally infected and free of naturally occurring mastitis served as controls.
3323
3324
NONNECKE AND HARP
L y m p h o c y t e blastogenesis studies were during the final 3 m o of the second lactation. Enrichment of Milk and Blood Lymphocytes
Milk and blood samples were collected at the morning milking from each of the 10 cows on 2 or more separate days during an 8-wk period. Individual quarter milk samples and a pooled milk sample were collected from each uninfected cow. Pooled milk from the three uninfected glands and a single sample from the infected gland were collected from each mastitic cow. A minimum of 2 L o f milk was diluted with three parts of physiologically warm (39°C) phosphate-buffered saline [(PBS) pH 6.8, .15 M]. Leukocytes were recovered from the diluted milk by continuous flow centrifugation (flow rate = 35 ml/min/cup, × 1,000 x g). Pelleted cells were resuspended in 100 ml PBS containing 100 units/ml penicillin G and 100 /~g/ml streptomycin and were washed once at 300 × g for 10 rain. Blood was collected by jugular venipuncture into tubes containing Alsever's solution and then diluted with two parts of PBS (pH 7.2,
.15M). Twelve milliliters of washed milk cells or diluted blood were overlayed on 3 ml of ficoll-diatrizoate [specific gravity 1.084 (13), Sigma Chemical Co.] in 15-ml conical tubes, and centrifuged 400 × g at 18°C for 40 min. Blood mononuclear bands were removed from each tube, washed (300 × g, 10 min) twice in Hank's balanced salt solution without Ca 2+ or Mg 2+, once in PBS, and resuspended in RPMI 1640 medium with 10 mM Hepes buffer, 2 mM L-glutamine, and antibiotics as described. Milk mononuclear cells were washed once in PBS and resuspended in RPMI 1640 medium.
phages (M~), or PMNL. Eosinophils and basophils (rarely observed) were included in the PMNL category. Number of leukocytes in enriched populations was determined using a hemocytometer, and viability was assessed by trypan blue exclusion (10). Lymphocyte Blastogenesis Assay
Multiple wells of a 96-well flat b o t t o m tissue culture plate (Falcon 3070, Becton-Dickinson) were seeded with 1.0 x 10 s viable milk or blood mononuclear leukocytes in 50 /al of RPMI 1640 medium with antibiotics. Additional components added to each well were 50 /~1 of fetal calf serum (heat-inactivated) in RPMI 1640 medium to give a final concentration in the assay of 10%, and 100 /ll of RPM! 1640 medium containing optimal dilutions (13) of phytohemagglutinin-P (PHA-P, 12.5 /~g/ml, Difco Laboratories), Concanavalin A (Con A), 7.8 /lg/ml (Sigma Chemical Co.), or pokeweed mitogen (PWM), 2.0 /.tg/ml, (Sigma Chemical Co.). Individual assays were prepared in triplicate. Culture plates were incubated for either 24 or 72 h at 39°C in a humidified atmosphere containing 5% carbon dioxide. After the initial 24 or 72 h incubation, 5 0 / l l (.5 /ICi) of [methyl-3H]thymidine were added to each well and plates were incubated an additional 18 h. Well contents were harvested onto glass fiber strips using a cell harvester (Model PHD, Cambridge Technology, Inc.) and the retained radioactivity counted in a liquid scintillation counter (Model LS-8000, Beckman Instruments). Differences between mean blastogenic responses were evaluated using Student's t test.
RESULTS Leukocyte Enumeration and Characterization
Number of leukocytes present in milk from individual glands and pooled samples was determined by the direct microscopic cell count method (12). These preparations were stained with Stat-Stain (Volu-Sol Medical Industries, Inc.). Blood and milk cell differential counts were determined from slides prepared by cytocentrifugation (Cytospin 2, Shandon Instruments) and stained with Stat-Stain. Cells were categorized as small mononuclear leukocytes (SML) (lymphocytes and monocytes), macroJournal of Dairy Science Vol. 68, No. 12, 1985
Cows experimentally infected in a single gland with S. a u r e u s developed chronic mastitis in that gland, which persisted throughout lactation. The infective strain repeatedly was isolated in pure culture from milk of infected glands. Milk production by infected glands of each cow was reduced relative to production in the previous lactation (J. S. McDonald, unpublished data). Uninfected glands of each mastitic cow, milked independently of the infected gland, remained free of infection throughout the study.
MASTITIS AND LYMPHOCYTE FUNCTION Milk and Blood Leukocyte Populations
Number of SML, M~, and PMNL/ml of milk from glands of control cows, uninfected glands of mastitic cows, and infected glands is in Table 1. Total number of leukocytes and number of SML, Mg~, and PMNL were significantly (P<.05) greater from infected glands than from uninfected glands. The greatest difference was associated with PMNL, which were at a concentration of 8.70 × 10S/ml from infected glands compared with .38 x 10S/ml and .75 × 105/ml in milk from control cows and uninfected glands of mastitic cows, respectively. No differences in number or types of leukocytes present in blood of infected and uninfected cows were detected. Enriched Mononuclear Leukocyte Populations
Mean -+ SE viability of enriched milk mononuclear leukocytes from infected and uninfected cows was 52 + 8.7%. Mean + standard error percentages of SML in enriched leukocyte populations originating from infected glands, uninfected glands of infected cows, and glands of uninfected cows were 56.4 + 8.4, 66.0 + 11.9, and 67.3 + 4.5%, respectively. Mean percentages of MO from these groups were 24.2 + 6.5, 25.9 + 12.3, and 25.9 -+ 4.7%. Similarly, mean percentages of PMNL from these groups were 19.4 + 7.9, 8.4 + 4.2, and 6.8 + .7%,
3325
respectively. The mononuclear leukocyte population recovered from the blood of all animals after ficoll-diatrizoate treatment was greater than 99.0% pure and 87.2% viable. Lymphocyte Blastogenesis
Blastogenic responses (X counts per minute -+ SE) of milk and blood lymphocytes cultured for 42 and 90 h in the absence or presence of three different mitogens are presented in Figures 1 and 2. Mitogen concentrations employed were those that provided optimal stimulation of blood lymphocytes from control animals. Responses of milk lymphocytes from control animals to the three mitogens were significantly (P<.05) greater at 42 and 90 h than responses of milk lymphocytes from uninfected or infected glands of infected cows. Milk lymphocytes from infected glands were essentially unresponsive to mitogenic stimulation at both 42 and 90 h, whereas lymphocytes from uninfected glands of the same animals were significantly (P<.05) more responsive to mitogens at both times, although not as responsive as milk lymphocytes from normal cows. The effect of chronic staphylococcal mastitis on milk lymphocyte mitogenesis was most pronounced for cultures stimulated by PHA-P. The PWM-stimulated blood lymphocytes from normal cows, compared with infected
TABLE 1. Geometric mean number of leukocytes present in milk from glands of uninfected cows, uninfected glands of mastitic cows, and glands experimentally infected with Staphylococcus aureus.
Source of milk
Leukocytes (× 10S/ml) MO2
SML1 ,X
SE
PMNL3
,X
SE
.X
SE
Unfected glands (n=24) (control cows)
1.06a
.15
.28 a
.12
.38a
.03
Uninfected glands (n=63) (infected cows)
.95a
.11
.39 a
.07
.75 b
.16
2.72b
1.84
1.98b
1.02
8.70 c
4.20
Infected glands (n=21)
a'b'CMeans with different superscripts within columns differ (P<.05). 1Small mononuclear leukocytes. 2Macrophage. 3Polymorphonuclear leukocytes. Journal of Dairy Science Vol. 68, No. 12, 1985
3326
NONNECKE AND HARP
"]° 30
I t
t°
28 26 24 22 2o 18
14
,°°...."° ~
42
90
42 90 INCUBATION TIME (h)
42
90
Figure 1. Incorporation of [3H]thymidine by milk lymphocytes from a) uninfected cows (n=6), b) uninfected glands of infected cows (n=21), and c) infected glands (n=21) after 42 and 90 h of incubation in vitro. Responses to the mitogens Concanavalin A (o-o), phytohemagglutinin-P (a-a), and pokeweed mitogen (e---e) are shown. Values in parentheses indicate blastogenesis of unstimulated lymphocyte cultures.
cows, incorporated significantly ( P < . 0 5 ) m o r e radiolabel at 90 h. Blood lymphocytes from uninfected animals incorporated more [3H]thymidine at 90 h than blood from infected animals, although the magnitude of responses of l y m p h o c y t e s to Con A and PHA-P was not significantly different (Figure 2). Blood lymphocytes from control or infected cows were more responsive to mitogenic stimulation than autologous milk lymphocytes, regardless of the mitogen employed. Differences were most pronounced for lymphocytes from blood of infected cows and lymphocytes from infected glands.
D I SCUSS I O N
Milk lymphocytes from mammary glands chronically infected with S. a u r e u s were hyporesponsive to mitogenic stimulation compared with milk lymphocytes from uninfected glands of the same animal or control cows. The Journal of Dairy Science Vol. 68, No. 12, 1985
magnitude of the proliferative response of a lymphocyte to mitogens is recognized as an indicator of the cellular immunocompetence (3, 9). Frequently viral, bacterial, and parasitic diseases are associated with reduced lymphocyte reactivity to mitogens (9). The almost complete abrogation of milk lymphocyte mitogenesis associated with mammary infection suggests a possible loss of immunocompetency within the infected gland. The chronic nature of a staphylococcal mastitis may be due to an impairment of the milk lymphocyte's capacity to respond to membrane receptor-mediated events such as antigen recognition. Protracted mastitis in a single gland was associated with a reduction in mitogenesis by lymphocytes from milk (uninfected gland) and blood of the infected cow indicating that effects of single-gland infection did not remain localized. It is important to determine whether infection-modified responses of milk and blood lymphocytes are associated with a reduction in immune capacity systemically as well as locally.
MASTITIS AND LYMPHOCYTE FUNCTION
140
3327
-b
120
'~ I00 E =. 80 60 40 2O 0
~--.o61
~.ts, .,)
I
42
I
1.~3-*.o9) I
90 42 INCUBATIONTIME (h)
1.92±
.66l
I
90
Figure 2. Incorporation of [3H]thymidine by peripheral blood lymphocytes from a) uninfected (n=6) and b) infected cows (n=21) after 42 and 90 h of incubation in vitro. Responses to the mitogens Concanavalin A (o-o), phytohemagglutinin-P (a-zx), and pokeweed mitogen (e---e) are shown. Values in parentheses indicate blastogenesis of unstimulated lymphocyte cultures.
Proliferative responses of tymphocytes to mitogens, unlike responses to antigen, are nonspecific (3, 8). Reduced mitogenesis of lymphocytes from mastitic cattle may be typical of mammary inflammation and not unique to mastitis caused by S. aureus. Antigen-specific reactivity of milk and blood lymphocytes from control and infected cows was tested using Protein A and a whole cell bacterin of the infective strain. Blastogenic responses of lymphocytes to these antigens were inconsistent (data not presented), possibly because the antigens employed in vitro did not have sufficient crossreactivity with those expressed b y the viable organism in vivo. Reduced mitogenic and inconsistent antigen-specific responses by milk and blood lymphocytes from infected cows might also indicate a general impairment of l y m p h o c y t e function due to mastitis. The PMNL, present in higher numbers in milk from infected than uninfected glands, may have contributed to reduced mitogenesis of lymphocytes from infected glands. Research (4) indicates that the myeloperoxidase H202 and halide system of the activated PMNL is cytotoxic for mononuclear leukocytes. L y m p h o c y t e mitogenesis is also suppressed by
reactive oxygen species (H202) produced by activated PMNL (18). Suppression appears to be due to the cytolethal effect of H20~ on the lymphocyte. In conclusion, an in vitro assay of lymphocyte function indicated that cows with chronic staphylococcal infection have impaired lymphocyte responses to mitogens. We are presently studying mechanisms responsible for impaired blastogenic responses of lymphocytes from infected cows. Further research is needed to determine if these effects are Specific to mastitis caused b y S. aureus. ACKNOWLEDGMENTS
The authors thank W. A. Hambly and B. Pesch for excellent technical assistance. Cows were infected with S. a u r e u s by J. S. McDonald. REFERENCES
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