Leukocytes-Second
Line of Defense Against
Invading Mastitis Pathogens M. J. PAAPE, W. P. WERGIN, A. J. GUIDRY, and R. E. PEARSON U.S. Department of Agriculture Science and Education Administration Animal Physiology and Genetics Institute
Beltsville, MD 20705
ABSTRACT
In mammals, neutrophile polymorphonuclear leukocytes constitute one of the essential body defenses against disease. In a large mammal, such as the dairy cow, billions of neutrophils are mobilized to fight infection. For example, over 50 million neutrophils per milliliter milk are commonly in a mammary quarter inflicted with clinical mastitis. However, in spite of these numerous leukocytes, pathogenic organisms remain viable. Recent evidence indicates that bacteria are not eliminated from a diseased quarter because the phagocytic capacity of the neutrophils is reduced in the mammary gland. The morphology and physiology of the leukocyte is examined in this review in an attempt to explain why the phagocytic capacity of the neutrophil is reduced in the mammary gland of the bovine. INTRODUCTION
Phagocytosis is the process of recognition, ingestion, and digestion of foreign particles (bacteria, necrotic tissue, etc) by cells. The neutrophile polymorphonuclear (PMN)leukocytes and macrophages are the principal phagocytes in this role. In the lactating bovine udder, PMN leukocytes are the most numerous cells (17), whereas in the dry udder macrophages are the principal cells (19). Despite the importance of phagoeytosis as the major defense of the host against invading pathogens, the mechanisms and intricacies of this basic biologic process are not completely understood. The protective role of the PMN leukocytes in the lactating udder was shown by Jain et al.
Received December 19, 1977. 1979 J Dairy Sci 62:135-153
(16), who produced neutropenia in cows by injecting them with equine anti-bovine leukocyte serum. When udders were infected experimentally with Aerobacter aerogenes, the neutropenia led to unrestricted multiplication of the coliform bacteria within the infected quarters. In a similar study by Schalm et al. (50), udders of cows chronically infected with Stapbylococcus azLreus became gangrenous when neutropenia was produced with equine anti-bovine leukocyte serum. The PMN leukocytes make up most of the leukocytes in circulating blood of many animal species (Table 1). However, in the bovine, PMN leukocytes make up only 25% of the total leukocyte count (48). Nevertheless, when the size (g 682 kg) (23) and blood volume (8% body weight) (48) of mature lactating Holstein cows are considered, a potential pool of over 100 billion circulating mature PMN leukocytes are present to fight infection (Table 2). In addition to these circulating PMN leukocytes is a marginal pool of mature PMN leukocytes adhering to the walls of blood vessels (48). We can estimate the number of cells in this marginal pool by calculating the percentage of increase in PMN leukocytes after intravenous administration of adrenocorticotropin (ACTH) (Table 2). After injection of 250 IU of ACTH, Paape et al. (30) reported a 60% increase in mature PMN leukocytes (68 billion) in blood. Finally, in addition to the circulating and marginal pools of mature PMN leukocytes is a storage pool of mature and immature leukocytes in bone marrow (49). The number of PMN leukocyte storage pools that responds to an irritation in the mammary gland depends on the severity of the irritation and strength of the chemotactic agent. For example, an intramammary infusion of saline containing .1% oyster glycogen into two mammary quarters resulted in a milk leukocytosis of 17 x 109 PMN leukocytes/infused quarter (Table 3) 135
136
PAAPE ET AL.
TABLE 1. Comparative blood leukocyte counts for different species, a
Species
Total leukocyte count (cells/mm 3 )
Mature PMN leukocytes (%)
Total no. mature PMN leukocytes (cells/mm 3 )
Man Dog Cat Horse Pig Cow
7,780 11,500 12.500 9,000 16,000 8,000
47 61 60 52 29 25
3,600 7,000 7,500 4,700 5,500 2,000
aschalm et al. (47).
(37). Cytological e x a m i n a t i o n of these milk PMN l e u k o c y t e s indicated that 98% o f the cells were mature. The total and differential circulating l e u k o c y t e counts before and after infusion indicate that the ceils do n o t shift to the i m m a t u r e f o r m in circulating b l o o d (Tables 3 and 4) (40). These results suggest that the num-
TABLE 2. Storage pools of PMN leukocytes in the bovine.
Storage pool
Total no. mature PMN leukocytes X 109
Circulating Marginal Reserve (bone marrow) a Total available
101.0 68.2 ? over 169.2
a11.9% of leukocytes in bone marrow are mature PMN. Schalm and Lasmanis (48).
ber of mature PMN l e u k o c y t e s in the circulating and marginal pools is large enough to mobilize mature PMN l e u k o c y t e s into milk. Alternatively, infusion of .5 mg of Escbericbia coli e n d o t o x i n , a p o t e n t c h e m o t a c t i c agent, results in an excess of 53 X 106 PMN l e u k o c y t e s / ml of milk, depletes the three storage pools of m a t u r e PMN l e u k o c y t e s and causes release of i m m a t u r e PMN l e u k o e y t e s f r o m bone m a r r o w (Table 5) (39). Despite this capacity to mobilize tremendous numbers of mature and i m m a t u r e PMN l e u k o c y t e s f r o m three sources, the bovine m a m m a r y gland is highly susceptible to mastitis (27). N u m e r o u s PMN l e u k o c y t e s along with viable mastitis pathogens f r e q u e n t l y are shed f r o m infected quarters. Somatic cell counts (90% PMN leukocytes) in subclinically infected quarters averaged 700,000 cells/ml of milk; counts in clinically i n f e c t e d quarters average several million cellshnl of milk (90% PMN leuk o c y t e s ) (Table 6) (38). Despite these high leuk o c y t e counts, cows m a y remain subclinically
TABLE 3. Number of PMN leukocytes in primary and residual milk following infusion of saline containing oyster glycogen, a Milk fraction Parameter measured
Primary
Residual
Volume of milk (ml) Concentration of somatic cells (× 106/ml in milk) Total no. of somatic cells X 109 PMN leukocytes, % Immature PMN leukocytes, %
780 9.0 7.0 91.4 1.0
540 19.4 10.0 92.2 2.0
apaape et al. (37). Journal of Dairy Science Vol. 62, No. 1, 1979
SYMPOSIUM: BOVINE MAST1TIS
13 7
TABLE 4. Total and differential circulating leukocyte count before and after intramammary infusion of saline containing .1% oyster glycogen,a Parameter measured (per mm 3 in blood)
Before infusion
After infusion
Total circulating leukocytes
7,106
6,754
3,503 295
3,911 335
2,533 0 40 638
2,079 40 0 379
Differential circulating leukocytes Lymphocytes Monocytes Neutrophils Segmented Band Juvenile Eosinophils aEach value represents the mean from 3 animals.
infected in the same quarter for several lactations, and antibiotics still are required to cure clinically infected animals. This paper explores reasons why the PMN leukocytes, which greatly outnumber the infectious organisms in milk, are unable to eliminate effectively mastitis pathogens from the mammary gland.
C Y T O L O G Y OF THE B O V I N E PMN L E U K O C Y T E
The fine structure of a bovine PMN leukocyte isolated from blood is illustrated in Fig. 1. The ultrastructure of this cell is similar to that for other species (5). The cell is bounded by a plasma membrane which has receptors that bind immunogtobulin molecules or certain components of complement (53). Binding of immunoglobulins (primarily IgG series) and corn-
plement (C3b) allows the PMN leukocytes to recognize bacteria that have become opsonized or coated with immunoglobulins or complement. A prominent multilobed nucleus is the most conspicuous organelle in the cytoplasm. It has been suggested (14) that the multilobed shape of this organelle allows the PMN leukocyte to move through small openings so that cells easily can enter tissue to perform their specialized role of phagocytosis. In addition to the nucleus, two distinct types of cytoplasmic granules are azurophilic or primary granules and specific or secondary granules. Both types of granules contain bactericidal substances that enable PMN leukocytes to kill and digest bacteria. Finally islets of glycogen appear in the cytoplasm. During phagocytosis, glycogen is a source of glucose that flows through the hexose monophosphate shunt and produces NADPH. Hydrogen peroxide, an end product of NADPH
TABLE 5. Leukocyte response following intramammary infusion of .5 mg of Escbericbia coli endotoxin,a Hours relative to infusion
Somatic cells
Juvenile
0 8 18
(× 106/ mlin milk) .057 37.261 53.776
0 406 1,287
Blood PMN leukocytes Band
Mature
(per mm 3 in blood) 61 379 804
2,389 662 1,544
apaape et al. (39). Journal of Dairy Science Vol. 62, No. 1, 1979
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TABLE 6. Milk somatic cell counts and infectious status of the mammary gland, a
GLUCOSE-6 -PHOSPHATE
F
V0s.
I-.2°qr,,
Infectious status Uninfected
6-PHOSPHOGLUCONAT£
[ t
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02
RZBULOSE 5-PHOSPHATE RIB(]SE 5 -PHOSPHATE
139
Somatic cells/ml milk
Corynebacterium boris infected
40,000 150,000
Pathogen infected Subclinical Clinical
700,000 millions
apaape, M. J., and W. D. Schultze (38).
F o r a review of this system, the reader is referred to Sbarra et al. (47). PHAGOCYTOSIS BY B O V I N E PMN L E U K O C Y T E
Phagocytosis can be divided into several stages. The first stage consists of c o n t a c t between the PMN l e u k o c y t e and foreign particle and adherence of the particle to the surface of the l e u k o c y t e (Fig. 2). This stage is f o l l o w e d by the m o v e m e n t of pseudopods around the particle (Fig. 3 and 4). A f t e r the particle is surr o u n d e d by pseudopods, it is pulled into the interior of the cell as a p h a g o s o m e that is limited by a m e m b r a n e (Fig. 5). Inside the cell, cytoplasmic granules migrate toward and fuse their m e m b r a n e s with that of the p h a g o s o m e to f o r m the phagolysosome (Fig. 6). The particle subsequently is digested in the p h a g o l y s o s o m e where catabolic products are prevented f r o m leaking out into the cytoplasm. As a result, the cell is p r o t e c t e d f r o m self destruction. Phagocytosis of bacteria by l e u k o c y t e s in milk has been observed in vitro by several inves-
tigators. However, only a few compare the phagocytic ability of PMN l e u k o c y t e s of milk with that of PMN l e u k o c y t e s of blood. Wisniowski et al. (60) r e p o r t e d that PMN leukocytes isolated f r o m milk were p o o r e r phagocytes than PMN l e u k o c y t e s isolated f r o m blood. This observation was later c o n f i r m e d by K e n t and N e w b o u l d (18), whose microscopic e x a m i n a t i o n of internalized or adhered staphylococci indicated that 44% of the PMN leukocytes of blood c o n t a i n e d 1.7 S. aureus whereas only 30% of the PMN l e u k o c y t e s of milk contained 1.07 S. aureus. More recently, Russell and Reiter (46) r e p o r t e d that the n u m b e r of staphylococci killed by PMN l e u k o c y t e s isolated f r o m milk was significantly less than the n u m b e r killed by PMN l e u k o c y t e s isolated f r o m blood. T h e y attributed this reduced bactericidal activity to a deficiency by PMN leukocytes of milk to p h a g o c y t o s e and to kill intracellularly. Several suggestions have been proposed to
FIG. 1. Transmission electron micrograph of a polymorphonuclear (PMN) leukocyte isolated from bovine blood. The cell, which is limited by the plasma membrane, contains a lobulated nucleus (N 1 to N 4), specific (S) and azurophitic (A) granules, and aggregations of glycogen (G). The azurophilic granules are stained more intensely than the surrounding cytoplasm because the leukocyte was incubated with diaminobenzidine (DAB) (8) before postfixation with osmium tetroxide. As a result of this procedure, an electron dense deposit, indicative of peroxidase activity, formed in the granules. × 22,000. FIG. 2. Scanning electron micrograph of a PMN leukocyte that had been incubated with yeast cells for 10 min. Pseudopods (arrows) of the leukocyte (L) have made contact with a single yeast (Y) cell. X9,000. FIG. 3. Scanning electron micrograph of a leukocyte and several yeast cells. The plasma membrane (arrows) of the leukocyte (L) has begun to surround one of the yeast cells (Y). X 8,000. FIG. 4. Scanning electron micrograph of a yeast cell (Y) that is partly engulfed by advancing pseudopod membranes of a leukocyte. X 11,000. FIG. 5. Scanning electron micrograph of a PMN leukocyte that was incubated with yeast cells for 60 rain. The leukocyte (L), which was cryofractured after incubation, appears to contain three yeast (Y) cells that are internalized in phagocytic vacuoles. X 9,000. Journal of Dairy Science Vol. 62, No. 1, 1979
140
Journal of Dairy Science Vol. 62, No. 1, 1979
PAAPE ET AL.
SYMPOSIUM: BOVINE MASTITIS
141
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142
PAAPE ET AL.
TABLE 7. Percentage of milk PMN leukocytes isolated from milk containing fat globules, a PMN containing fat globules (%) Fat globules/PMN (no.)
TABLE 8. Effect of fat globules on phagocytosis, a staphylococcus a~re~s
68 2.1
aMean of 10 cows.
explain the reduced phagocytic and bacterial properties of PMN leukocytes of milk. Naidu and N e w b o u l d (24) f o u n d that PMN l e u k o c y t e s of milk c o n t a i n e d 38% less glycogen than PMN leukocytes of blood. Adding glucose to media containing PMN leukocytes of milk and staphylococci apparently increases phagocytosis (26). Wisniowski et al. (60) suggested that deficiency of opsonins in milk could be a factor contributing to the reduced phagocytosis by PMN leukocytes. To test this hypothesis, N e w b o u l d (26) added i m m u n e serum to skimmed milk and observed an increase in the percentage of PMN l e u k o c y t e s that ingested staphylococci. However, Russell and Reiter (46) failed to stimulate phagocytosis of staphylococci by PMN leukocytes of milk after adding normal bovine serum to skimmed milk. As a result, they concluded that the reduced phagocytosis of PMN leukocytes of milk was n o t due to a deficiency of opsonins in milk. To clarify this a p p a r e n t discrepancy, Guidry et al. (12) prepared w h e y f r o m cows that were i m m u n i z e d with heat kiIled S. a u r e u s . When PMN l e u k o c y t e s of milk and S. a u r e u s were incubated in vitro in this whey, phagocytosis increased by 62%. Absorption of the i m m u n e w h e y with h o m o l o g o u s S. a u r e u s r e m o v e d the opsonizing effects of immunization.
Studied material
phagocytosed (%)
Serum Whole milk Skimmed milk Serum plus cream Skimmed milk plus cream
80 44 74 56 63
aEach value represents the mean of 11 cows. Paape et al. (33).
The reduced phagocytic ability of PMN l e u k o c y t e s of milk also has been attributed to interference from c o m p o n e n t s in the milk (33, 45). Light microscopic observations of PMN l e u k o c y t e s of milk, which were isolated after infusion of a sterile irritant into the m a m m a r y gland, indicated that 68% of the PMN leukocytes contained on the average 2.1 phagocytosed fat globules (Table 7) (59). The presence of fat globules in PMN l e u k o c y t e s of milk has been c o n f i r m e d subsequently by a positive staining reaction with Oil Red 0 (Fig. 7 and 8). Fat globules also have been identified in PMN l e u k o c y t e s of milk by transmission electron m i c r o s c o p e (TEM) (2, 15, 41, 44) (Fig. 9). Studies of in vitro phagocytosis indicate that the fat globules in milk exert an inhibitory effect on phagocytosis (Table 8) (33). Phagocytosis decreases to 44% w h e n S, a u r e u s and PMN leukocytes o f blood are incubated in
FIG. 6. Part of a PMN leukocyte isolated from milk and incubated in vitro with S t a p b y l o c o c c u s aureus for 60 rain. The leukocyte contains two bacterial cells (SA). The cell that lies to the right has been engulfed recently by a pseudopod assembly. On the left, a bacterium, which appears to be digested partially, is internalized in a phagolysosome that is surrounded by specific granules (S). X 28,000. FIG. 7 and 8. Light micrographs of PMN leukocytes that were isolated from milk. In Fig. 7, the cells, which were stained with hematoxylin, show clear circular areas (arrows). When similar cells were stained with Oil Red 0 and counterstained with hematoxylin, as in Fig. 8, no clear areas were present. These results indicate that the clear areas in Fig. 7 represent phagocytosed fat globules. × 3,500. FIG. 9. PMN leukocyte containing four fat globules (FG), which was isolated from milk. The leukocyte had been incubated in diaminobenzidine to localize peroxidase activity as described for Fig. 1. As a result of this procedure, positive reaction products were observed in the azurophilic granules (A) and around the periphery of the fat globules (arrows). × 16,000. FIG. 10. Portion of a PMN leukocyte isolated from milk. The cell contains numerous phagosomes (P) that appear to be filled with casein micelles. × 20,000. Journal of Dairy Science Vol. 62, No. 1, 1979
SYMPOSIUM: BOVINE MASTITIS
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Journal of Dairy Science Vol. 62, No. 1, 1979
144
PAAPE ET AL.
TABLE 9. Phagocytosis and intracellular killing of whole milk and skimmed milk. a
S t a p b y l o c o c c u s aureus
Total surviving S.
by PMN leukocytes incubated in
aureus c
Intracellular Studied material b
Arithmetic mean (CFU X 104)
Whole milk Skimmed milk
.50
.14
Extracellular Log mean d
Arithmetic mean
Log mean d
(%)
(CFU)
(CFU X 106)
(%)
(CFU)
5.2
5.51 e
1.4
5.10 f
2.03 .35
21.7 3.1
5.97 e 5.43 f
aRepresents the mean from 8 separate determinations run in duplicate. Paape et al. (32). bStudied material (.8 ml) was in a total volume of 2 ml with PBS and incubated for 60 rain at 37 C. CTotal number of S. aureus and PMN leukocytes (0 h) was 10.7 × 106 CFU and 100 × 106 PMN leukocytes. dMeans in a column with no superscript letter in common differ from each other (P
Line of Defense Against
Invading Mastitis Pathogens M. J. PAAPE, W. P. WERGIN, A. J. GUIDRY, and R. E. PEARSON U.S. Department of Agriculture Science and Education Administration Animal Physiology and Genetics Institute
Beltsville, MD 20705
ABSTRACT
In mammals, neutrophile polymorphonuclear leukocytes constitute one of the essential body defenses against disease. In a large mammal, such as the dairy cow, billions of neutrophils are mobilized to fight infection. For example, over 50 million neutrophils per milliliter milk are commonly in a mammary quarter inflicted with clinical mastitis. However, in spite of these numerous leukocytes, pathogenic organisms remain viable. Recent evidence indicates that bacteria are not eliminated from a diseased quarter because the phagocytic capacity of the neutrophils is reduced in the mammary gland. The morphology and physiology of the leukocyte is examined in this review in an attempt to explain why the phagocytic capacity of the neutrophil is reduced in the mammary gland of the bovine. INTRODUCTION
Phagocytosis is the process of recognition, ingestion, and digestion of foreign particles (bacteria, necrotic tissue, etc) by cells. The neutrophile polymorphonuclear (PMN)leukocytes and macrophages are the principal phagocytes in this role. In the lactating bovine udder, PMN leukocytes are the most numerous cells (17), whereas in the dry udder macrophages are the principal cells (19). Despite the importance of phagoeytosis as the major defense of the host against invading pathogens, the mechanisms and intricacies of this basic biologic process are not completely understood. The protective role of the PMN leukocytes in the lactating udder was shown by Jain et al.
Received December 19, 1977. 1979 J Dairy Sci 62:135-153
(16), who produced neutropenia in cows by injecting them with equine anti-bovine leukocyte serum. When udders were infected experimentally with Aerobacter aerogenes, the neutropenia led to unrestricted multiplication of the coliform bacteria within the infected quarters. In a similar study by Schalm et al. (50), udders of cows chronically infected with Stapbylococcus azLreus became gangrenous when neutropenia was produced with equine anti-bovine leukocyte serum. The PMN leukocytes make up most of the leukocytes in circulating blood of many animal species (Table 1). However, in the bovine, PMN leukocytes make up only 25% of the total leukocyte count (48). Nevertheless, when the size (g 682 kg) (23) and blood volume (8% body weight) (48) of mature lactating Holstein cows are considered, a potential pool of over 100 billion circulating mature PMN leukocytes are present to fight infection (Table 2). In addition to these circulating PMN leukocytes is a marginal pool of mature PMN leukocytes adhering to the walls of blood vessels (48). We can estimate the number of cells in this marginal pool by calculating the percentage of increase in PMN leukocytes after intravenous administration of adrenocorticotropin (ACTH) (Table 2). After injection of 250 IU of ACTH, Paape et al. (30) reported a 60% increase in mature PMN leukocytes (68 billion) in blood. Finally, in addition to the circulating and marginal pools of mature PMN leukocytes is a storage pool of mature and immature leukocytes in bone marrow (49). The number of PMN leukocyte storage pools that responds to an irritation in the mammary gland depends on the severity of the irritation and strength of the chemotactic agent. For example, an intramammary infusion of saline containing .1% oyster glycogen into two mammary quarters resulted in a milk leukocytosis of 17 x 109 PMN leukocytes/infused quarter (Table 3) 135
136
PAAPE ET AL.
TABLE 1. Comparative blood leukocyte counts for different species, a
Species
Total leukocyte count (cells/mm 3 )
Mature PMN leukocytes (%)
Total no. mature PMN leukocytes (cells/mm 3 )
Man Dog Cat Horse Pig Cow
7,780 11,500 12.500 9,000 16,000 8,000
47 61 60 52 29 25
3,600 7,000 7,500 4,700 5,500 2,000
aschalm et al. (47).
(37). Cytological e x a m i n a t i o n of these milk PMN l e u k o c y t e s indicated that 98% o f the cells were mature. The total and differential circulating l e u k o c y t e counts before and after infusion indicate that the ceils do n o t shift to the i m m a t u r e f o r m in circulating b l o o d (Tables 3 and 4) (40). These results suggest that the num-
TABLE 2. Storage pools of PMN leukocytes in the bovine.
Storage pool
Total no. mature PMN leukocytes X 109
Circulating Marginal Reserve (bone marrow) a Total available
101.0 68.2 ? over 169.2
a11.9% of leukocytes in bone marrow are mature PMN. Schalm and Lasmanis (48).
ber of mature PMN l e u k o c y t e s in the circulating and marginal pools is large enough to mobilize mature PMN l e u k o c y t e s into milk. Alternatively, infusion of .5 mg of Escbericbia coli e n d o t o x i n , a p o t e n t c h e m o t a c t i c agent, results in an excess of 53 X 106 PMN l e u k o c y t e s / ml of milk, depletes the three storage pools of m a t u r e PMN l e u k o c y t e s and causes release of i m m a t u r e PMN l e u k o e y t e s f r o m bone m a r r o w (Table 5) (39). Despite this capacity to mobilize tremendous numbers of mature and i m m a t u r e PMN l e u k o c y t e s f r o m three sources, the bovine m a m m a r y gland is highly susceptible to mastitis (27). N u m e r o u s PMN l e u k o c y t e s along with viable mastitis pathogens f r e q u e n t l y are shed f r o m infected quarters. Somatic cell counts (90% PMN leukocytes) in subclinically infected quarters averaged 700,000 cells/ml of milk; counts in clinically i n f e c t e d quarters average several million cellshnl of milk (90% PMN leuk o c y t e s ) (Table 6) (38). Despite these high leuk o c y t e counts, cows m a y remain subclinically
TABLE 3. Number of PMN leukocytes in primary and residual milk following infusion of saline containing oyster glycogen, a Milk fraction Parameter measured
Primary
Residual
Volume of milk (ml) Concentration of somatic cells (× 106/ml in milk) Total no. of somatic cells X 109 PMN leukocytes, % Immature PMN leukocytes, %
780 9.0 7.0 91.4 1.0
540 19.4 10.0 92.2 2.0
apaape et al. (37). Journal of Dairy Science Vol. 62, No. 1, 1979
SYMPOSIUM: BOVINE MAST1TIS
13 7
TABLE 4. Total and differential circulating leukocyte count before and after intramammary infusion of saline containing .1% oyster glycogen,a Parameter measured (per mm 3 in blood)
Before infusion
After infusion
Total circulating leukocytes
7,106
6,754
3,503 295
3,911 335
2,533 0 40 638
2,079 40 0 379
Differential circulating leukocytes Lymphocytes Monocytes Neutrophils Segmented Band Juvenile Eosinophils aEach value represents the mean from 3 animals.
infected in the same quarter for several lactations, and antibiotics still are required to cure clinically infected animals. This paper explores reasons why the PMN leukocytes, which greatly outnumber the infectious organisms in milk, are unable to eliminate effectively mastitis pathogens from the mammary gland.
C Y T O L O G Y OF THE B O V I N E PMN L E U K O C Y T E
The fine structure of a bovine PMN leukocyte isolated from blood is illustrated in Fig. 1. The ultrastructure of this cell is similar to that for other species (5). The cell is bounded by a plasma membrane which has receptors that bind immunogtobulin molecules or certain components of complement (53). Binding of immunoglobulins (primarily IgG series) and corn-
plement (C3b) allows the PMN leukocytes to recognize bacteria that have become opsonized or coated with immunoglobulins or complement. A prominent multilobed nucleus is the most conspicuous organelle in the cytoplasm. It has been suggested (14) that the multilobed shape of this organelle allows the PMN leukocyte to move through small openings so that cells easily can enter tissue to perform their specialized role of phagocytosis. In addition to the nucleus, two distinct types of cytoplasmic granules are azurophilic or primary granules and specific or secondary granules. Both types of granules contain bactericidal substances that enable PMN leukocytes to kill and digest bacteria. Finally islets of glycogen appear in the cytoplasm. During phagocytosis, glycogen is a source of glucose that flows through the hexose monophosphate shunt and produces NADPH. Hydrogen peroxide, an end product of NADPH
TABLE 5. Leukocyte response following intramammary infusion of .5 mg of Escbericbia coli endotoxin,a Hours relative to infusion
Somatic cells
Juvenile
0 8 18
(× 106/ mlin milk) .057 37.261 53.776
0 406 1,287
Blood PMN leukocytes Band
Mature
(per mm 3 in blood) 61 379 804
2,389 662 1,544
apaape et al. (39). Journal of Dairy Science Vol. 62, No. 1, 1979
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SYMPOSIUM: BOVINE MASTITIS and N A D H oxidation, is an i m p o r t a n t c o m p o n ent of the antimicrobial system in the cell.
TABLE 6. Milk somatic cell counts and infectious status of the mammary gland, a
GLUCOSE-6 -PHOSPHATE
F
V0s.
I-.2°qr,,
Infectious status Uninfected
6-PHOSPHOGLUCONAT£
[ t
FizO
02
RZBULOSE 5-PHOSPHATE RIB(]SE 5 -PHOSPHATE
139
Somatic cells/ml milk
Corynebacterium boris infected
40,000 150,000
Pathogen infected Subclinical Clinical
700,000 millions
apaape, M. J., and W. D. Schultze (38).
F o r a review of this system, the reader is referred to Sbarra et al. (47). PHAGOCYTOSIS BY B O V I N E PMN L E U K O C Y T E
Phagocytosis can be divided into several stages. The first stage consists of c o n t a c t between the PMN l e u k o c y t e and foreign particle and adherence of the particle to the surface of the l e u k o c y t e (Fig. 2). This stage is f o l l o w e d by the m o v e m e n t of pseudopods around the particle (Fig. 3 and 4). A f t e r the particle is surr o u n d e d by pseudopods, it is pulled into the interior of the cell as a p h a g o s o m e that is limited by a m e m b r a n e (Fig. 5). Inside the cell, cytoplasmic granules migrate toward and fuse their m e m b r a n e s with that of the p h a g o s o m e to f o r m the phagolysosome (Fig. 6). The particle subsequently is digested in the p h a g o l y s o s o m e where catabolic products are prevented f r o m leaking out into the cytoplasm. As a result, the cell is p r o t e c t e d f r o m self destruction. Phagocytosis of bacteria by l e u k o c y t e s in milk has been observed in vitro by several inves-
tigators. However, only a few compare the phagocytic ability of PMN l e u k o c y t e s of milk with that of PMN l e u k o c y t e s of blood. Wisniowski et al. (60) r e p o r t e d that PMN leukocytes isolated f r o m milk were p o o r e r phagocytes than PMN l e u k o c y t e s isolated f r o m blood. This observation was later c o n f i r m e d by K e n t and N e w b o u l d (18), whose microscopic e x a m i n a t i o n of internalized or adhered staphylococci indicated that 44% of the PMN leukocytes of blood c o n t a i n e d 1.7 S. aureus whereas only 30% of the PMN l e u k o c y t e s of milk contained 1.07 S. aureus. More recently, Russell and Reiter (46) r e p o r t e d that the n u m b e r of staphylococci killed by PMN l e u k o c y t e s isolated f r o m milk was significantly less than the n u m b e r killed by PMN l e u k o c y t e s isolated f r o m blood. T h e y attributed this reduced bactericidal activity to a deficiency by PMN leukocytes of milk to p h a g o c y t o s e and to kill intracellularly. Several suggestions have been proposed to
FIG. 1. Transmission electron micrograph of a polymorphonuclear (PMN) leukocyte isolated from bovine blood. The cell, which is limited by the plasma membrane, contains a lobulated nucleus (N 1 to N 4), specific (S) and azurophitic (A) granules, and aggregations of glycogen (G). The azurophilic granules are stained more intensely than the surrounding cytoplasm because the leukocyte was incubated with diaminobenzidine (DAB) (8) before postfixation with osmium tetroxide. As a result of this procedure, an electron dense deposit, indicative of peroxidase activity, formed in the granules. × 22,000. FIG. 2. Scanning electron micrograph of a PMN leukocyte that had been incubated with yeast cells for 10 min. Pseudopods (arrows) of the leukocyte (L) have made contact with a single yeast (Y) cell. X9,000. FIG. 3. Scanning electron micrograph of a leukocyte and several yeast cells. The plasma membrane (arrows) of the leukocyte (L) has begun to surround one of the yeast cells (Y). X 8,000. FIG. 4. Scanning electron micrograph of a yeast cell (Y) that is partly engulfed by advancing pseudopod membranes of a leukocyte. X 11,000. FIG. 5. Scanning electron micrograph of a PMN leukocyte that was incubated with yeast cells for 60 rain. The leukocyte (L), which was cryofractured after incubation, appears to contain three yeast (Y) cells that are internalized in phagocytic vacuoles. X 9,000. Journal of Dairy Science Vol. 62, No. 1, 1979
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TABLE 7. Percentage of milk PMN leukocytes isolated from milk containing fat globules, a PMN containing fat globules (%) Fat globules/PMN (no.)
TABLE 8. Effect of fat globules on phagocytosis, a staphylococcus a~re~s
68 2.1
aMean of 10 cows.
explain the reduced phagocytic and bacterial properties of PMN leukocytes of milk. Naidu and N e w b o u l d (24) f o u n d that PMN l e u k o c y t e s of milk c o n t a i n e d 38% less glycogen than PMN leukocytes of blood. Adding glucose to media containing PMN leukocytes of milk and staphylococci apparently increases phagocytosis (26). Wisniowski et al. (60) suggested that deficiency of opsonins in milk could be a factor contributing to the reduced phagocytosis by PMN leukocytes. To test this hypothesis, N e w b o u l d (26) added i m m u n e serum to skimmed milk and observed an increase in the percentage of PMN l e u k o c y t e s that ingested staphylococci. However, Russell and Reiter (46) failed to stimulate phagocytosis of staphylococci by PMN leukocytes of milk after adding normal bovine serum to skimmed milk. As a result, they concluded that the reduced phagocytosis of PMN leukocytes of milk was n o t due to a deficiency of opsonins in milk. To clarify this a p p a r e n t discrepancy, Guidry et al. (12) prepared w h e y f r o m cows that were i m m u n i z e d with heat kiIled S. a u r e u s . When PMN l e u k o c y t e s of milk and S. a u r e u s were incubated in vitro in this whey, phagocytosis increased by 62%. Absorption of the i m m u n e w h e y with h o m o l o g o u s S. a u r e u s r e m o v e d the opsonizing effects of immunization.
Studied material
phagocytosed (%)
Serum Whole milk Skimmed milk Serum plus cream Skimmed milk plus cream
80 44 74 56 63
aEach value represents the mean of 11 cows. Paape et al. (33).
The reduced phagocytic ability of PMN l e u k o c y t e s of milk also has been attributed to interference from c o m p o n e n t s in the milk (33, 45). Light microscopic observations of PMN l e u k o c y t e s of milk, which were isolated after infusion of a sterile irritant into the m a m m a r y gland, indicated that 68% of the PMN leukocytes contained on the average 2.1 phagocytosed fat globules (Table 7) (59). The presence of fat globules in PMN l e u k o c y t e s of milk has been c o n f i r m e d subsequently by a positive staining reaction with Oil Red 0 (Fig. 7 and 8). Fat globules also have been identified in PMN l e u k o c y t e s of milk by transmission electron m i c r o s c o p e (TEM) (2, 15, 41, 44) (Fig. 9). Studies of in vitro phagocytosis indicate that the fat globules in milk exert an inhibitory effect on phagocytosis (Table 8) (33). Phagocytosis decreases to 44% w h e n S, a u r e u s and PMN leukocytes o f blood are incubated in
FIG. 6. Part of a PMN leukocyte isolated from milk and incubated in vitro with S t a p b y l o c o c c u s aureus for 60 rain. The leukocyte contains two bacterial cells (SA). The cell that lies to the right has been engulfed recently by a pseudopod assembly. On the left, a bacterium, which appears to be digested partially, is internalized in a phagolysosome that is surrounded by specific granules (S). X 28,000. FIG. 7 and 8. Light micrographs of PMN leukocytes that were isolated from milk. In Fig. 7, the cells, which were stained with hematoxylin, show clear circular areas (arrows). When similar cells were stained with Oil Red 0 and counterstained with hematoxylin, as in Fig. 8, no clear areas were present. These results indicate that the clear areas in Fig. 7 represent phagocytosed fat globules. × 3,500. FIG. 9. PMN leukocyte containing four fat globules (FG), which was isolated from milk. The leukocyte had been incubated in diaminobenzidine to localize peroxidase activity as described for Fig. 1. As a result of this procedure, positive reaction products were observed in the azurophilic granules (A) and around the periphery of the fat globules (arrows). × 16,000. FIG. 10. Portion of a PMN leukocyte isolated from milk. The cell contains numerous phagosomes (P) that appear to be filled with casein micelles. × 20,000. Journal of Dairy Science Vol. 62, No. 1, 1979
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TABLE 9. Phagocytosis and intracellular killing of whole milk and skimmed milk. a
S t a p b y l o c o c c u s aureus
Total surviving S.
by PMN leukocytes incubated in
aureus c
Intracellular Studied material b
Arithmetic mean (CFU X 104)
Whole milk Skimmed milk
.50
.14
Extracellular Log mean d
Arithmetic mean
Log mean d
(%)
(CFU)
(CFU X 106)
(%)
(CFU)
5.2
5.51 e
1.4
5.10 f
2.03 .35
21.7 3.1
5.97 e 5.43 f
aRepresents the mean from 8 separate determinations run in duplicate. Paape et al. (32). bStudied material (.8 ml) was in a total volume of 2 ml with PBS and incubated for 60 rain at 37 C. CTotal number of S. aureus and PMN leukocytes (0 h) was 10.7 × 106 CFU and 100 × 106 PMN leukocytes. dMeans in a column with no superscript letter in common differ from each other (P
