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The effect of local and parenteral vaccination on the response of the guinea pig mammary gland to staphylococcal challenge
Journal of Reproductive Immunology, 6 (1984) 365-376 Elsevier
365
JRI 00320
The effect of local and parenteral vaccination on the response of the guinea pig mammary gland to staphylococcal challenge * Brian J. Nonnecke 1 and Stanislaw P. Targowski 2 I Department of Veterinary Preventive Medicine, College of Veterinary Medicine, Ohio State University, Columbus, OH 43210 and e National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 70, Ames, IA 50010, U.S.A. (Accepted for publication 21 March 1984)
Preparturient guinea pigs vaccinated locally (orally and intramammarily) or parenterally (intradermally) with killed Staphylococcus aureus (KS), were challenged intramammarily (IMM) with KS or viable S. aureus during the next lactation. The number and types of leukocytes emigrating into the milk were determined before and after IMM challenge. The milk leukocytosis after challenge with KS was the greatest in the intradermally (1D) vaccinated animals, moderate in the IMM vaccinated animals, and insignificant in the unvaccinated or orally vaccinated animals. The polymorphonuclear (PMN) leukocyte predominated in the milk of the IMM and ID vaccinated animals during the initial 30 h after challenge with KS. Later (30-102 h postchaUenge), the mononuclear leukocyte (macrophage and lymphocytes) was the major cell-type. No significant changes in the number or types of leukocytes occurred in the milk of the unvaccinated or orally vaccinated animals after challenge. Intramammary challenge with viable S. aureus caused a large increase in the number of leukocytes in the milk of all animals. The milk leukocytosis occurred more rapidly in locally vaccinated guinea pigs than unvaccinated or ID vaccinated guinea pigs. The PMN leukocyte predominated in the milk of all animals during the period of maximum response. The incidence and severity of staphylococcal mastitis were less in guinea pigs vaccinated locally than ID vaccinated or unvaccinated animals. Key words: staphylococcal challenge, guinea pig
Introduction
The humoral and cellular components of the immune system play an important role in the response of the mammary gland to infection (Newby et al., 1982; Watson, 1982). In domestic animals and humans, severe or chronic mastitis has frequently been attributed to Staphylococcus aureus (Newbould, 1974). Enhancement of the immunity of the mammary gland to staphylococcal infection by vaccination has been the subject of many investigations. However, these studies have not defined a
* No endorsements are implied herein. 0165-0378/84/$03.00 © 1984 Elsevier Science Publishers B.V.
366
procedure with proven efficacy. Vaccination by the local route, compared with the parenteral route, resulted in an increased concentration of specific antibody in milk of ewes (Lascelles and McDowell, 1974). In other studies, the effect of local and parenteral vaccination was measured by the magnitude of the milk leukocytosis after IMM challenge (DeCueninck, 1979; Targowski and Berman, 1975; Targowski and Nonnecke, 1982; Coldwitz and Watson, 1982). In locally vaccinated animals, the leukocytic response of the mammary gland to the same dose of antigen was more rapid and greater than in unvaccinated animals. Since the polymorphonuclear leukocyte predominated during the milk leukocytosis, it is conceivable that protection against invading bacteria may be associated with the magnitude of this response. In the present study, the effect of vaccination route (local versus parenteral) on the magnitude of leukocytic response of the mammary gland to challenge with staphylococcal antigens and the degree of protection provided against challenge with live staphylococci was investigated.
Materials and Methods
Animals Hartley strain female guinea pigs (originally obtained from Dr. D.T. Berman, University of Wisconsin, Madison, WI) were employed. The animals were vaccinated during their first gestation period, and challenged in the subsequent lactation. Vaccination procedure Pregnant guinea pigs were vaccinated with a formalin-killed (0.5% v / v formaldehyde), logarithmic-stage culture of Staphylococcus aureus, strain Smith, ATCC 13709. The killed staphylococci (KS) were washed twice in phosphate-buffered saline (PBS), resuspended in pyrogen-free PBS, and subjected to mild sonication. The concentration of KS in the vaccine and challenge preparations was determined using
TABLE 1 Vaccination procedure Vaccination route
No. of killed staphylococci
Administration schedule
Intramammary a
1.0-5.0 x 109 in 50 txl of PBS 1.0-5.0 x 109 in 1 ml of PBS 1.0-5.0 x 109 in 50 pl of PBS
Days 60 and 70 of pregnancy
Oral b Intradermal
a Injected into the glandular duct of the left gland. Intragastric administration by stomach tube.
Daily, from day 60 of pregnancy to day 5 of lactation Day 60 and 70 of pregnancy
367
a Petroff-Hausser bacteria counter (C.A. Hausser and Son, Philadelphia, PA). The pregnant animals were divided into the treatment groups described in Table 1. I M M challenge with KS
Five unvaccinated, 10 intramammarly (IMM) vaccinated, 6 orally vaccinated, and 6 intradermally (ID) vaccinated guinea pigs were IMM challenged with KS on the sixth day of their lactation. Both mammary glands of each animal were injected intraductally with 1.0-5.0 x 10 4 KS (suspended in 50 #1 of pyrogen-free PBS) using a 1 cc tuberculin syringe fitted with a 30 gauge needle. 1 M M challenge with viable S. aureus
Seventeen unvaccinated guinea pigs, 13 IMM vaccinated, 15 orally vaccinated, and 15 ID vaccinated guinea pigs were IMM challenged with viable S. aureus, ATCC 13709, on the sixth day of their lactation. Both mammary glands of each animal were injected intraductally with approximately 128 + 30 colony-forming units (CFU) of S. aureus (harvested during logarithmic stage of growth, washed in PBS, and suspended in 50 /11 of pyrogen-free PBS). The number of S. aureus administered was determined by triplicate plating of 100/~1 of fluid from the syringe on 5% bovine blood agar immediately after IMM challenge. Milk collection
Milk (0.5-1.0 ml) was expressed asceptically from each gland 48 and 24 h before, and 6, 16, 23, 30, 54, 78, and 102 h after IMM challenge with KS or viable S. aureus. Isolation of bacteria from milk and/or the total number of leukocytes in the milk exceeded 5.0 x 105/ml were criteria for the determination of mastitis. Animals with mastitis at 48 h and/or 24 h before IMM challenge with staphylococci were excluded from the study. Milk cell enumeration and differentiation
A smear from each milk sample was stained using the Levowitz-Weber modification of the Newman-Lambert stain (Schalm et al., 1971) for total cell count determinations. A second smear, for differential cell counts, was stained using the Wright-Leishman procedure (Schalm, 1965). At least 200 cells per smear were categorized as either polymorphonuclear (PMN) leukocytes, macrophages, or lymphocytes. Epithelial cells, typically less than 1% of cells counted, were excluded from the differential cell count. Eosinophils and basophils, rarely observed, were included with the PMN leukocytes. Milk samples with fewer than 105 leukocytes per ml were centrifuged using an Eppendorf centrifuge (Model 5413, Brinkman Inst. Inc., Westburg, NY). The cell pellet was resuspended in PBS (20-50/~1) providing an adequate cell concentration for leukocyte differentiation. Bacteriology
One-hundred/~1 of each asceptically collected milk sample was briefly sonicated (to release intracellular bacteria and reduce clumping) and plated on blood (5% v/v) agar. The antibiotic susceptibility patterns and cultural characteristics of the
368
staphylococcal isolates were used to confirm infection by S. aureus, ATCC 13709. Antibiotics used for this purpose were ampicilin (10/~g), chloramphenicol (30/~g), cloxacillin (1 /~g), gentamicin (10 #g), and streptomycin (10 t~g). Isolation of one or more C F U (equivalent to >~ 10 organisms/ml) of S. aureus, ATCC 13709, after 18 h of incubation at 37°C, indicated infection by this organism. Cutaneous delayed-type hypersensitivity Guinea pigs challenged IMM with KS on day 6 of lactation were also challenged I D on the l l t h day postpartum with 104 KS (diluted from vaccine) suspended in 50 /xl of pyrogen-free PBS. The minimum and maximum diameters (in mm) of the cutaneous responses were measured 24, 48, 72, and 96 h after dermal challenge. Statistical analyses Statistical significance was evaluated using a 2-tailed Student's t-test.
Results
Clinical observations The unvaccinated and locally vaccinated guinea pigs did not develop clinical mastitis after I M M challenge with KS. However, the ID vaccinated guinea pigs often developed mastitis characterized by decreased milk secretion and abnormal milk from 6 to 23 h after challenge. The severity of mastitis was consistently greater in animals challenged with viable S. aureus than those challenged with KS. The unvaccinated and I D vaccinated animals more frequently had acute and persistent mastitis than the locally vaccinated guinea pigs. The incidence or severity of mastitis in the left or right glands of the I M M vaccinated animals did not differ markedly; even then, only the left gland of these animals was vaccinated. Effect of I M M challenge with K S The number of leukocytes in the milk from the left and right m a m m a r y gland of I M M and I D vaccinated animals increased significantly ( P < 0.05) between 6 and 30 h after I M M challenge with KS (Table 2). The magnitude of this leukocytosis was significantly greater in the ID vaccinated than the I M M vaccinated animals. In contrast, challenge with KS did not produce a significant increase in the number of milk leukocytes in the milk of the unvaccinated and orally vaccinated animals (Table 2). The difference in the number of leukocytes infiltrating the milk of the left and right gland in all animals was insignificant. The P M N leukocyte was the predominant cell-type in the milk of ID and IMM vaccinated animals during the initial 30 h after challenge with KS (Fig. lc, d). Between 30 and 102 h after challenge and prior to challenge, the mononuclear leukocyte (macrophage and lymphocytes) predominated in the milk of these animals. The mononuclear leukocyte was the major cell-type in the milk of the unvaccinated and orally vaccinated guinea pigs for the entire experimental period (Fig. la, b). Effect of I M M challenge with viable S. aureus To simplify data interpretation, the leukocyte counts of milk samples from the
2.70+0.50
2.52_+0.40 1.48_+0.19 3.30_+0.20 1.80_+0.13
2.80_+0.10 2.44_+0.11
2.13-+0.26
2.36_+0.26 2.38+0.60
2.49_+0.24 2.81_+0.35
2.28_+0.21 2.80_+0.24 67.80_+17.45
Right Left
Right
Left
Right
Left
Right
49.50_+17.45
1.52-+ 0.14
4.09_+ 1 . 0 9
5.87_+ 0.28
2.49_+ 0.42 5.94_+ 0.26
2.24+ 0.39
S. aureus.
66.10_+14.05
13.15_+ 1 . 6 2
1.98-+ 0 . 3 7
3.38_+ 0 . 7 4
8.77_+ 0 . 2 6
2.53_+ 0 . 5 1 8.56_+ 0.26
3.13± 0.42
+ 16
a Each gland was challenged intramammarily with 1.0-5.0 × 104 killed b Number of animals in each group.
3.37-+0.16
1.82+0.30
+ 6
Left
- 24
Unvaccinated (n=5) b lntramammarilyvaccinated (n =10) Orally vaccinated (n = 8) Intradermally vaccin(n = 6)
- 48
M e a n ( _+ S E M ) no. o f l e u k o c y t e s ( x 1 0 5 / m l ) - t i m e (h)
Gland
Animal groups
Mean number of leukocytes in milk collected from guinea pigs challenged with killed
TABLE 2 a
+ 30
2.06_+0.20
+ 54
1.46_+0.20
+ 78
1.31_+0.53
+ 102
2.11-+0.14
16.99+4.27
9.39-+2.13
3.95_+0.29 9.11_+2.25
3.87+0.49
3.06_+0.42 4.68+0.68
9.54_+0.40 4.49_+0.30
2.27-+0.27
2.45_+0.33
1.27-+0.18
1.48-+0.16
2.62_+0.12
1.96_+0.20
1.88_+0.12
1.97-+0.44
1.26-+0.20
2.42_+0.12
2.64_+0.15
2.12_+0.20
1.22-+0.18
2.34_+0.80
2.49-+0.17
2.36_+0.53 2.00_+0.49 1.72_+0.52 1.37_+0.29 1.10_+0.51 11.92_+0.61 6.42_+0.57 3.29_+0.29 2.42_+0.11 2.14+0.73
2.96_+0.27 3.03+0.32
+ 23
S. aureus
370
left and right glands of animals challenged with viable S. aureus were pooled at each sampling period, since there were no significant differences in the number of leukocytes in milk from the left and right glands of animals within a treatment group (Table 3). Intramammary challenge with live S. aureus versus KS resulted in a greater number of leukocytes in the milk of all animals during the postchallenge period. At 6 h after infection with S. aureus, the magnitude of the milk leukocytosis was significantly (P < 0.05) greater in the locally (IMM and orally) vaccinated guinea pigs than in the unvaccinated or ID vaccinated guinea pigs. However, for the period between 23 to 102 h after challenge, the number of leukocytes in the milk from the ID vaccinated and unvaccinated animals exceeded the number in the milk from the locally vaccinated animals. The number of various leukocytes present in the milk of the experimentally infected guinea pigs is depicted in Fig. 2a, b. During the initial 23 h after challenge, the PMN leukocyte was the predominant cell type in the secretion of all the animals. At 6 h after challenge, the number of PMN leukocytes was greater in the milk from
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~t~
..,ago °
o -48
-24
+6
~,
.16 +23 +30 +54 +78 +102 -48 TiME (h)
-24
+6
+16 +23 +30 -1-54 ÷78 +102 TIME (h)
d 42
C
i 08462 o
-48
i -24
I +6
÷ ll6
t i +23 +30 TiME (5)
i
+54
i
t
+T8 +i02
-48
-24
+6
÷i6
4"23 +30 TIME (h)
+54
+78 .',-102
Fig. 1. Leukocytes in the milk from the left gland of guinea pigs IMM challenged with KS. Each point represents the mean number of PMN leukocytes (e e), macrophages (O O), or lymphocytes (A. . . . . . A), from (a) unvaccinated, (b) orally vaccinated, (c) ID vaccinated, and (d) IMM vaccinated animals.
2.38_+0.11
3.41 -+ 0.12
2.27 + 0.07
2.75 _+0.12
2.17 + 0.10
66.29 _+21.99
14.88 -+ 1.98
28.27-+7.51
+ 23
56.35 _+10.42
14.52 _+1.18
24.41_+7.51
a Mean derived from pooled leukocyte counts from left and right mammary glands. b Number of animals in each group.
13.29 _+1.30
23.78 -+ 2.06
28.01+3.37
19.50_+2.14
55.19_+7.48
3.23_+0.18
11.41_+1.12
+ 16
Unvaccinated (n = 17) b Intramammarily vaccinated (n =13) Orally vaccinated (n =15) lntradermally (n =15)
+6
2.02_+0.10
- 48
1.63_+0.18
groups
- 24
Mean ( -+ SEM) number of leukocytes ( x 105/ml) - time (h)
Animal
Mean number a of leukocytes in milk collected from guinea pigs challenged with live S. a u r e u s
TABLE 3
+ 30
66.16 _+7.19
26.01 -+ 2.46
40.10-+7.47
98.39_+13.34
+ 54
79.87 -+ 14.41
9.98 _+1.76
26.88_+5.30
65.25_+10.56
+ 78
28.52 _+4.07
6.78 ± 0.40
28.56_+6.52
42.34+6.32
+ 102
24.55 ± 3.71
8.77 -+ 0.90
21.99-+4.82
25.37-+3.22
372
locally vaccinated animals than ID vaccinated or unvaccinated animals. However, at 16 h after challenge, the concentration of PMN leukocytes in the milk of the latter animals increased markedly. The macrophage comprised a greater proportion of the leukocyte population in the milk of animals challenged with S. aureus than animals challenged with KS. Isolation of S. aureus from the milk of experimentally infected guinea pigs Staphylococci were isolated in markedly greater numbers from the infected milk from the I D vaccinated and unvaccinated animals than the infected milk from locally vaccinated animals after I M M inoculation with S. aureus. The frequency of isolation of staphylococci from the milk samples of animals challenged with live staphylococci is illustrated in Fig. 3. The locally vaccinated animals, compared with those unvaccinated or I D vaccinated, had consistently fewer infected glands at each postchallenge sampling period. Staphylococci were isolated from 52 and 54% of the
50
o
b
40
i, ° =-"
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0
i
-48
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-24
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+23 +30 +54 TIME(h}
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41
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+16
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,
,
,
-48 -24 +6 +16 +23 +30 +54 +78 ÷102 -48 -24 +6
TIME(h)
+16 +23 +30 +54 +78 +102
TIME(h)
Fig. 2. Leukocytes in the milk from guinea pigs IMM challenged with live S . a u r e u s . Each point represents the mean number of PMN leukocytes ( e e), macrophages (O ©), or lymphocytes (A . . . . . . A) from (a) unvaccinated, (b) ID vaccinated, (c) IMN vaccinated, and (d) orally vaccinated animals. Mean derived from average of left and right gland cell counts.
373 TABLE 4 Cutaneous response of guinea pigs intradermally challenged with killed
S. a u r e u s a
Animal groups
Mean ( + SD) diameters of the cutaneous response - time (h) +24
+48
+72
+96
Unvaccinated (n = 5) lntramammarily vaccinated n = 10) Orally vaccinated (n = 6) Intradermally vaccinated (n = 6)
2.4_+0.8 ~'
0.0 b
0.0 b
0.0 b
5.1 + 0.8 c
3.8 _+0.8 ~
2.2 _+1.3 c
0.3 _+0.8 b
3.6 + 1.9 bx
2.2 _+1.1 c
1.4 +_0.5 c
0.0 b
9.3 +_1.6 d
5.5 _+0.9 d
4.6 +_0.5 d
3.3 _+1.3 ~
Challenged with 10 4 killed staphylococci in 50/~1 of PBS. hx,d Significant difference (P < 0.01) at specific times after challenge. a
milk samples from the unvaccinated and ID vaccinated animals, respectively, and f r o m 23 a n d 17% o f m i l k s a m p l e s f r o m t h e I M M a n d o r a l l y v a c c i n a t e d a n i m a l s , respectively, during the entire postchallenge period. None of the ID vaccinated or u n v a c c i n a t e d animals were free of infection during the entire p o s t c h a l l e n g e period. I n c o n t r a s t , 23 (3 o f 13) a n d 46% (7 o f 15) o f t h e I M M a n d o r a l l y v a c c i n a t e d animals, respectively, were free of infection. D u r i n g the entire p o s t c h a l l e n g e period,
I00 9O 80
70
30 20
~"
I0
"
~'t"
0
I
I
I
t
I
I
I
23 30 54 78 102 TIME (h) Fig. 3. Percentage of milk samples from individual glands infected with S. a u r e u s at various time intervals after IMM challenge from unvaccinated (O O), IMM vaccinated (e O), orally vaccinated (A. . . . . . A), and ID vaccinated (zx. . . . . . zx) animals. 0
6
16
374
88 (15 of 17) and 80% (12 of 15) of the unvaccinated and ID vaccinated animals had both glands infected at least once, as compared to 31 (5 of 13) and 7% (1 of 15) of the IMM and orally vaccinated animals, respectively. Dermal response of cutaneous challenge with K S
The ID vaccinated guinea pigs had significantly (P < 0.05) larger and more persistent dermal lesions than the unvaccinated or locally vaccinated guinea pigs after cutaneous challenge with KS (Table 4). The lesions in the locally vaccinated animals were significantly greater than those of the unvaccinated controls during the 72 h period after challenge.
Discussion
In the present study, the magnitude of the leukocytosis in the milk of unvaccinated and vaccinated animals after IMM challenge with staphylococci differed significantly (P < 0.05). The number of leukocytes in the milk of ID or IMM vaccinated animals challenged with KS and in the milk of all animals (unvaccinated and vaccinated) challenged with viable S. aureus was significantly (P < 0.05) greater than the diurnal fluctuations in the number of leukocytes in normal cavian milk (Targowski and Nonnecke, 1982). In vaccinated ruminants, a significant increase in the number of milk leukocytes has been observed after intramammary challenge with antigens (Targowski and Berman, 1975; DeCueninck, 1979). The moderate, but significant, milk leukocytosis after challenge with KS was associated with a moderate dermal reaction to the cutaneous injection of KS in the IMM vaccinated animals. In the ID vaccinated animals, a large leukocytosis after IMM challenge with KS was correlated with a large dermal reaction to the cutaneous injection of KS. The responses of the unvaccinated animals to the cutaneous and IMM injections of KS were insignificant. These observations indicate that the magnitude of the milk leukocytosis and dermal response of animals in each group were correlated. Although the magnitude of these responses was related, their cellular composition was different. The PMN leukocyte was the major cell-type in the milk of the ID and IMM vaccinated animals from 23 h to 30 h after IMM challenge with KS. In contrast, the cellular infiltrate in dermal lesions of animals sensitized parenterally and challenged ID with staphylococci consisted primarily of mononuclear leukocytes at 24 h postchaUenge (Johnson et al. 1961). The unresponsiveness of the orally vaccinated guinea pigs to IMM challenge with KS might indicate a failure of the vaccination procedure. However, the orally vaccinated animals had weak, but significant (P < 0.05) dermal responses to the injection of KS and a significant increase in the number of leukocytes in their milk after the second IMM challenge in the same lactation (data not shown). Furthermore, these guinea pigs were more protected against experimental infection with S. aureus than the ID or unvaccinated animals. The protection induced by oral vaccination may have been due to the migration of sensitized lymphocytes from the intestinal lymphoid tissue to the mammary gland. Numerous studies have shown
375 that both T and B lymphocytes from the mesenteric lymph nodes or Peyer's patches home to the mammary gland tissue or enter the secretion in late pregnancy and during lactation (McDermott and Beinenstock, 1970; Manning and Parmely, 1980; Head and Seeling, 1983). The increased protection against staphylococcal infection provided by local (IMM and oral) vaccination compared with no vaccination and ID vaccination, may be due to the greater number of PMN leukocytes in the milk of these animals 6 h after challenge. Approximately 84% of the milk samples from the locally vaccinated animals were free of staphylococci at 6 h after challenge, compared to only 50% of the milk samples from unvaccinated and ID vaccinated animals. A milk leukocytosis of similar magnitude did not occur in the latter animals until 16 to 23 h after challenge, and failed to eliminate staphylococci from the mammary glands during the postchallenge period. In ewes, vaccination by the IMM infusion of killed staphylococci also enhanced the initial neutrophil influx in milk, compared to unvaccinated animals, resulting in greater protection of the gland against experimental infection with S. aureus (Colditz and Watson, 1982). Infusion of KS into the glands of locally vaccinated guinea pigs caused a delayed migration of leukocytes into the milk, whereas infusion of live staphylococci caused an immediate increase in the number of milk leukocytes. In contrast, the milk leukocytosis in animals vaccinated ID was rapid after infusion of KS, and delayed after the infusion of live staphylococci. The mechanism responsible for this difference cannot be determined on the basis of the data presented. However, it is conceivable that antigens elaborated by the live staphylococci interacted with the sensitized lymphocytes in the gland of the locally vaccinated animals triggering the rapid leukocytosis. Antibody might not have been involved in mediating the leukocytosis, since no agglutinating antibody was detected in the secretion (data not shown). In conclusion, local vaccination provided the greatest protection against IMM challenge with live staphylococci, even though the dermal and milk leukocytic responses were moderate after challenge with KS. In contrast, ID vaccination induced large dermal reactions and milk leukocytic responses to KS, but did not protect the mammary gland against challenge with live staphylococci. It seems that ID vaccination of animals inudced primarily delayed-type hypersensitivity without mammary protection.
References Colditz, I.G. and Watson, D.L. (1982) Effect of immunizationon the early influx of neutrophils during staphylococcalmastitis in ewes. Res. Vet. Sci. 33, 146-151. DeCueninck, B.J. (1979) Immune-mediatedinflammationin the lumen of the bovine mammary gland. Int. Arch. AllergyAppl. Immunol.59, 394-402. Head, J. and Seeling, L.L., Jr. (1983) Autoradiographicanalysis of lymphocytemigration into the mammaryepitheliumand milk of lactatingfemalerats. J. Reprod. Immunol.5, 61-72. Jain, N.C. (1976) Neutrophilleukocytesand inflammationof the bovine mammarygland.Theriogenology 6, 153-173.
376 Johnson, J.E., Cluff, L.E. and Goshi, K. (1961) Studies on the pathogenesis of staphylococcal infection. I. The effect of repeated skin infections. J. Exp. Med. 113, 235-247. Lascelles, A.K. and McDowell, G.S. (1974) Localized humoral immunity in particular reference to ruminants. Transplant. Rev. 19, 170-208. Manning, L.S. and Parmely, M.J. (1980) Cellular determinants of mammary cell-mediated immunity in the rat. I. The migration of radioisotopically labelled T-lymphocytes. J. lmmunol. 125, 2508-2514. McDermott, M.R. and Beinenstock, J. (1979) Evidence for a common mucosal immunologic system. I. Migration of B lymphoblasts into intestinal, respiratory, and genital areas. J. I mmunol. 122, 1892-1898. Newbould, F.H.S. (1974) Microbial diseases of the mammary gland. In Lactation: A Comprehensive Treatise (Larson, B.L. and Smith, V.R., eds.), pp. 237-269. Academic Press, New York. Newby, T.J., Stokes, C.R. and Bourne, B.J. (1982) Immunological activities of milk. Vet. Immunol. Immunopathol. 3, 67-94. Schalm, O.W., Carroll, E.J. and Jain, N.C. (1971) Bovine Mastitis, Lea and Febiger, Philadelphia. Targowski, S.P. and Berman, D.T. (1975) Leukocytic response of bovine mammary gland to injection of killed cells and cell walls of Staphylococcus aureus. Am. J. Vet. Res. 36~ 1561-1565. Targowski, S.P. and Nonnecke, B.J. (1982) Cell-mediated immune response of the mammary gland in guinea pigs. I. Effect of antigen injection into vaccinated and unvaccinated glands. Am. J. Reprod. Immunol. 2, 29-38. Watson, D.L. (1980) Immunological functions of the mammary gland and its secretion - comparative review. Aust. J. Biol. Sci. 33, 403-422. Watson, D.L. (1981) Immunologically-specific resistance to infection with particular reference to staphylococcal mastitis. Adv. Exp. Med. Biol. 137, 579-590.
365
JRI 00320
The effect of local and parenteral vaccination on the response of the guinea pig mammary gland to staphylococcal challenge * Brian J. Nonnecke 1 and Stanislaw P. Targowski 2 I Department of Veterinary Preventive Medicine, College of Veterinary Medicine, Ohio State University, Columbus, OH 43210 and e National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 70, Ames, IA 50010, U.S.A. (Accepted for publication 21 March 1984)
Preparturient guinea pigs vaccinated locally (orally and intramammarily) or parenterally (intradermally) with killed Staphylococcus aureus (KS), were challenged intramammarily (IMM) with KS or viable S. aureus during the next lactation. The number and types of leukocytes emigrating into the milk were determined before and after IMM challenge. The milk leukocytosis after challenge with KS was the greatest in the intradermally (1D) vaccinated animals, moderate in the IMM vaccinated animals, and insignificant in the unvaccinated or orally vaccinated animals. The polymorphonuclear (PMN) leukocyte predominated in the milk of the IMM and ID vaccinated animals during the initial 30 h after challenge with KS. Later (30-102 h postchaUenge), the mononuclear leukocyte (macrophage and lymphocytes) was the major cell-type. No significant changes in the number or types of leukocytes occurred in the milk of the unvaccinated or orally vaccinated animals after challenge. Intramammary challenge with viable S. aureus caused a large increase in the number of leukocytes in the milk of all animals. The milk leukocytosis occurred more rapidly in locally vaccinated guinea pigs than unvaccinated or ID vaccinated guinea pigs. The PMN leukocyte predominated in the milk of all animals during the period of maximum response. The incidence and severity of staphylococcal mastitis were less in guinea pigs vaccinated locally than ID vaccinated or unvaccinated animals. Key words: staphylococcal challenge, guinea pig
Introduction
The humoral and cellular components of the immune system play an important role in the response of the mammary gland to infection (Newby et al., 1982; Watson, 1982). In domestic animals and humans, severe or chronic mastitis has frequently been attributed to Staphylococcus aureus (Newbould, 1974). Enhancement of the immunity of the mammary gland to staphylococcal infection by vaccination has been the subject of many investigations. However, these studies have not defined a
* No endorsements are implied herein. 0165-0378/84/$03.00 © 1984 Elsevier Science Publishers B.V.
366
procedure with proven efficacy. Vaccination by the local route, compared with the parenteral route, resulted in an increased concentration of specific antibody in milk of ewes (Lascelles and McDowell, 1974). In other studies, the effect of local and parenteral vaccination was measured by the magnitude of the milk leukocytosis after IMM challenge (DeCueninck, 1979; Targowski and Berman, 1975; Targowski and Nonnecke, 1982; Coldwitz and Watson, 1982). In locally vaccinated animals, the leukocytic response of the mammary gland to the same dose of antigen was more rapid and greater than in unvaccinated animals. Since the polymorphonuclear leukocyte predominated during the milk leukocytosis, it is conceivable that protection against invading bacteria may be associated with the magnitude of this response. In the present study, the effect of vaccination route (local versus parenteral) on the magnitude of leukocytic response of the mammary gland to challenge with staphylococcal antigens and the degree of protection provided against challenge with live staphylococci was investigated.
Materials and Methods
Animals Hartley strain female guinea pigs (originally obtained from Dr. D.T. Berman, University of Wisconsin, Madison, WI) were employed. The animals were vaccinated during their first gestation period, and challenged in the subsequent lactation. Vaccination procedure Pregnant guinea pigs were vaccinated with a formalin-killed (0.5% v / v formaldehyde), logarithmic-stage culture of Staphylococcus aureus, strain Smith, ATCC 13709. The killed staphylococci (KS) were washed twice in phosphate-buffered saline (PBS), resuspended in pyrogen-free PBS, and subjected to mild sonication. The concentration of KS in the vaccine and challenge preparations was determined using
TABLE 1 Vaccination procedure Vaccination route
No. of killed staphylococci
Administration schedule
Intramammary a
1.0-5.0 x 109 in 50 txl of PBS 1.0-5.0 x 109 in 1 ml of PBS 1.0-5.0 x 109 in 50 pl of PBS
Days 60 and 70 of pregnancy
Oral b Intradermal
a Injected into the glandular duct of the left gland. Intragastric administration by stomach tube.
Daily, from day 60 of pregnancy to day 5 of lactation Day 60 and 70 of pregnancy
367
a Petroff-Hausser bacteria counter (C.A. Hausser and Son, Philadelphia, PA). The pregnant animals were divided into the treatment groups described in Table 1. I M M challenge with KS
Five unvaccinated, 10 intramammarly (IMM) vaccinated, 6 orally vaccinated, and 6 intradermally (ID) vaccinated guinea pigs were IMM challenged with KS on the sixth day of their lactation. Both mammary glands of each animal were injected intraductally with 1.0-5.0 x 10 4 KS (suspended in 50 #1 of pyrogen-free PBS) using a 1 cc tuberculin syringe fitted with a 30 gauge needle. 1 M M challenge with viable S. aureus
Seventeen unvaccinated guinea pigs, 13 IMM vaccinated, 15 orally vaccinated, and 15 ID vaccinated guinea pigs were IMM challenged with viable S. aureus, ATCC 13709, on the sixth day of their lactation. Both mammary glands of each animal were injected intraductally with approximately 128 + 30 colony-forming units (CFU) of S. aureus (harvested during logarithmic stage of growth, washed in PBS, and suspended in 50 /11 of pyrogen-free PBS). The number of S. aureus administered was determined by triplicate plating of 100/~1 of fluid from the syringe on 5% bovine blood agar immediately after IMM challenge. Milk collection
Milk (0.5-1.0 ml) was expressed asceptically from each gland 48 and 24 h before, and 6, 16, 23, 30, 54, 78, and 102 h after IMM challenge with KS or viable S. aureus. Isolation of bacteria from milk and/or the total number of leukocytes in the milk exceeded 5.0 x 105/ml were criteria for the determination of mastitis. Animals with mastitis at 48 h and/or 24 h before IMM challenge with staphylococci were excluded from the study. Milk cell enumeration and differentiation
A smear from each milk sample was stained using the Levowitz-Weber modification of the Newman-Lambert stain (Schalm et al., 1971) for total cell count determinations. A second smear, for differential cell counts, was stained using the Wright-Leishman procedure (Schalm, 1965). At least 200 cells per smear were categorized as either polymorphonuclear (PMN) leukocytes, macrophages, or lymphocytes. Epithelial cells, typically less than 1% of cells counted, were excluded from the differential cell count. Eosinophils and basophils, rarely observed, were included with the PMN leukocytes. Milk samples with fewer than 105 leukocytes per ml were centrifuged using an Eppendorf centrifuge (Model 5413, Brinkman Inst. Inc., Westburg, NY). The cell pellet was resuspended in PBS (20-50/~1) providing an adequate cell concentration for leukocyte differentiation. Bacteriology
One-hundred/~1 of each asceptically collected milk sample was briefly sonicated (to release intracellular bacteria and reduce clumping) and plated on blood (5% v/v) agar. The antibiotic susceptibility patterns and cultural characteristics of the
368
staphylococcal isolates were used to confirm infection by S. aureus, ATCC 13709. Antibiotics used for this purpose were ampicilin (10/~g), chloramphenicol (30/~g), cloxacillin (1 /~g), gentamicin (10 #g), and streptomycin (10 t~g). Isolation of one or more C F U (equivalent to >~ 10 organisms/ml) of S. aureus, ATCC 13709, after 18 h of incubation at 37°C, indicated infection by this organism. Cutaneous delayed-type hypersensitivity Guinea pigs challenged IMM with KS on day 6 of lactation were also challenged I D on the l l t h day postpartum with 104 KS (diluted from vaccine) suspended in 50 /xl of pyrogen-free PBS. The minimum and maximum diameters (in mm) of the cutaneous responses were measured 24, 48, 72, and 96 h after dermal challenge. Statistical analyses Statistical significance was evaluated using a 2-tailed Student's t-test.
Results
Clinical observations The unvaccinated and locally vaccinated guinea pigs did not develop clinical mastitis after I M M challenge with KS. However, the ID vaccinated guinea pigs often developed mastitis characterized by decreased milk secretion and abnormal milk from 6 to 23 h after challenge. The severity of mastitis was consistently greater in animals challenged with viable S. aureus than those challenged with KS. The unvaccinated and I D vaccinated animals more frequently had acute and persistent mastitis than the locally vaccinated guinea pigs. The incidence or severity of mastitis in the left or right glands of the I M M vaccinated animals did not differ markedly; even then, only the left gland of these animals was vaccinated. Effect of I M M challenge with K S The number of leukocytes in the milk from the left and right m a m m a r y gland of I M M and I D vaccinated animals increased significantly ( P < 0.05) between 6 and 30 h after I M M challenge with KS (Table 2). The magnitude of this leukocytosis was significantly greater in the ID vaccinated than the I M M vaccinated animals. In contrast, challenge with KS did not produce a significant increase in the number of milk leukocytes in the milk of the unvaccinated and orally vaccinated animals (Table 2). The difference in the number of leukocytes infiltrating the milk of the left and right gland in all animals was insignificant. The P M N leukocyte was the predominant cell-type in the milk of ID and IMM vaccinated animals during the initial 30 h after challenge with KS (Fig. lc, d). Between 30 and 102 h after challenge and prior to challenge, the mononuclear leukocyte (macrophage and lymphocytes) predominated in the milk of these animals. The mononuclear leukocyte was the major cell-type in the milk of the unvaccinated and orally vaccinated guinea pigs for the entire experimental period (Fig. la, b). Effect of I M M challenge with viable S. aureus To simplify data interpretation, the leukocyte counts of milk samples from the
2.70+0.50
2.52_+0.40 1.48_+0.19 3.30_+0.20 1.80_+0.13
2.80_+0.10 2.44_+0.11
2.13-+0.26
2.36_+0.26 2.38+0.60
2.49_+0.24 2.81_+0.35
2.28_+0.21 2.80_+0.24 67.80_+17.45
Right Left
Right
Left
Right
Left
Right
49.50_+17.45
1.52-+ 0.14
4.09_+ 1 . 0 9
5.87_+ 0.28
2.49_+ 0.42 5.94_+ 0.26
2.24+ 0.39
S. aureus.
66.10_+14.05
13.15_+ 1 . 6 2
1.98-+ 0 . 3 7
3.38_+ 0 . 7 4
8.77_+ 0 . 2 6
2.53_+ 0 . 5 1 8.56_+ 0.26
3.13± 0.42
+ 16
a Each gland was challenged intramammarily with 1.0-5.0 × 104 killed b Number of animals in each group.
3.37-+0.16
1.82+0.30
+ 6
Left
- 24
Unvaccinated (n=5) b lntramammarilyvaccinated (n =10) Orally vaccinated (n = 8) Intradermally vaccin(n = 6)
- 48
M e a n ( _+ S E M ) no. o f l e u k o c y t e s ( x 1 0 5 / m l ) - t i m e (h)
Gland
Animal groups
Mean number of leukocytes in milk collected from guinea pigs challenged with killed
TABLE 2 a
+ 30
2.06_+0.20
+ 54
1.46_+0.20
+ 78
1.31_+0.53
+ 102
2.11-+0.14
16.99+4.27
9.39-+2.13
3.95_+0.29 9.11_+2.25
3.87+0.49
3.06_+0.42 4.68+0.68
9.54_+0.40 4.49_+0.30
2.27-+0.27
2.45_+0.33
1.27-+0.18
1.48-+0.16
2.62_+0.12
1.96_+0.20
1.88_+0.12
1.97-+0.44
1.26-+0.20
2.42_+0.12
2.64_+0.15
2.12_+0.20
1.22-+0.18
2.34_+0.80
2.49-+0.17
2.36_+0.53 2.00_+0.49 1.72_+0.52 1.37_+0.29 1.10_+0.51 11.92_+0.61 6.42_+0.57 3.29_+0.29 2.42_+0.11 2.14+0.73
2.96_+0.27 3.03+0.32
+ 23
S. aureus
370
left and right glands of animals challenged with viable S. aureus were pooled at each sampling period, since there were no significant differences in the number of leukocytes in milk from the left and right glands of animals within a treatment group (Table 3). Intramammary challenge with live S. aureus versus KS resulted in a greater number of leukocytes in the milk of all animals during the postchallenge period. At 6 h after infection with S. aureus, the magnitude of the milk leukocytosis was significantly (P < 0.05) greater in the locally (IMM and orally) vaccinated guinea pigs than in the unvaccinated or ID vaccinated guinea pigs. However, for the period between 23 to 102 h after challenge, the number of leukocytes in the milk from the ID vaccinated and unvaccinated animals exceeded the number in the milk from the locally vaccinated animals. The number of various leukocytes present in the milk of the experimentally infected guinea pigs is depicted in Fig. 2a, b. During the initial 23 h after challenge, the PMN leukocyte was the predominant cell type in the secretion of all the animals. At 6 h after challenge, the number of PMN leukocytes was greater in the milk from
0
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0
1
,,,.
/ ].0
"
,,
~
,,
-
f
,. .,,
\7, ~
~t~
..,ago °
o -48
-24
+6
~,
.16 +23 +30 +54 +78 +102 -48 TiME (h)
-24
+6
+16 +23 +30 -1-54 ÷78 +102 TIME (h)
d 42
C
i 08462 o
-48
i -24
I +6
÷ ll6
t i +23 +30 TiME (5)
i
+54
i
t
+T8 +i02
-48
-24
+6
÷i6
4"23 +30 TIME (h)
+54
+78 .',-102
Fig. 1. Leukocytes in the milk from the left gland of guinea pigs IMM challenged with KS. Each point represents the mean number of PMN leukocytes (e e), macrophages (O O), or lymphocytes (A. . . . . . A), from (a) unvaccinated, (b) orally vaccinated, (c) ID vaccinated, and (d) IMM vaccinated animals.
2.38_+0.11
3.41 -+ 0.12
2.27 + 0.07
2.75 _+0.12
2.17 + 0.10
66.29 _+21.99
14.88 -+ 1.98
28.27-+7.51
+ 23
56.35 _+10.42
14.52 _+1.18
24.41_+7.51
a Mean derived from pooled leukocyte counts from left and right mammary glands. b Number of animals in each group.
13.29 _+1.30
23.78 -+ 2.06
28.01+3.37
19.50_+2.14
55.19_+7.48
3.23_+0.18
11.41_+1.12
+ 16
Unvaccinated (n = 17) b Intramammarily vaccinated (n =13) Orally vaccinated (n =15) lntradermally (n =15)
+6
2.02_+0.10
- 48
1.63_+0.18
groups
- 24
Mean ( -+ SEM) number of leukocytes ( x 105/ml) - time (h)
Animal
Mean number a of leukocytes in milk collected from guinea pigs challenged with live S. a u r e u s
TABLE 3
+ 30
66.16 _+7.19
26.01 -+ 2.46
40.10-+7.47
98.39_+13.34
+ 54
79.87 -+ 14.41
9.98 _+1.76
26.88_+5.30
65.25_+10.56
+ 78
28.52 _+4.07
6.78 ± 0.40
28.56_+6.52
42.34+6.32
+ 102
24.55 ± 3.71
8.77 -+ 0.90
21.99-+4.82
25.37-+3.22
372
locally vaccinated animals than ID vaccinated or unvaccinated animals. However, at 16 h after challenge, the concentration of PMN leukocytes in the milk of the latter animals increased markedly. The macrophage comprised a greater proportion of the leukocyte population in the milk of animals challenged with S. aureus than animals challenged with KS. Isolation of S. aureus from the milk of experimentally infected guinea pigs Staphylococci were isolated in markedly greater numbers from the infected milk from the I D vaccinated and unvaccinated animals than the infected milk from locally vaccinated animals after I M M inoculation with S. aureus. The frequency of isolation of staphylococci from the milk samples of animals challenged with live staphylococci is illustrated in Fig. 3. The locally vaccinated animals, compared with those unvaccinated or I D vaccinated, had consistently fewer infected glands at each postchallenge sampling period. Staphylococci were isolated from 52 and 54% of the
50
o
b
40
i, ° =-"
y/f/r...........~
0
i
-48
_
-24
+6
+23 +30 +54 TIME(h}
+78 +102
+i02
2olc
,61
/\
41
I, . ; . . . . . .
~ o,'~ , ~'
+16
/A\
,
." ,
"."" ,
,
,
,
-48 -24 +6 +16 +23 +30 +54 +78 ÷102 -48 -24 +6
TIME(h)
+16 +23 +30 +54 +78 +102
TIME(h)
Fig. 2. Leukocytes in the milk from guinea pigs IMM challenged with live S . a u r e u s . Each point represents the mean number of PMN leukocytes ( e e), macrophages (O ©), or lymphocytes (A . . . . . . A) from (a) unvaccinated, (b) ID vaccinated, (c) IMN vaccinated, and (d) orally vaccinated animals. Mean derived from average of left and right gland cell counts.
373 TABLE 4 Cutaneous response of guinea pigs intradermally challenged with killed
S. a u r e u s a
Animal groups
Mean ( + SD) diameters of the cutaneous response - time (h) +24
+48
+72
+96
Unvaccinated (n = 5) lntramammarily vaccinated n = 10) Orally vaccinated (n = 6) Intradermally vaccinated (n = 6)
2.4_+0.8 ~'
0.0 b
0.0 b
0.0 b
5.1 + 0.8 c
3.8 _+0.8 ~
2.2 _+1.3 c
0.3 _+0.8 b
3.6 + 1.9 bx
2.2 _+1.1 c
1.4 +_0.5 c
0.0 b
9.3 +_1.6 d
5.5 _+0.9 d
4.6 +_0.5 d
3.3 _+1.3 ~
Challenged with 10 4 killed staphylococci in 50/~1 of PBS. hx,d Significant difference (P < 0.01) at specific times after challenge. a
milk samples from the unvaccinated and ID vaccinated animals, respectively, and f r o m 23 a n d 17% o f m i l k s a m p l e s f r o m t h e I M M a n d o r a l l y v a c c i n a t e d a n i m a l s , respectively, during the entire postchallenge period. None of the ID vaccinated or u n v a c c i n a t e d animals were free of infection during the entire p o s t c h a l l e n g e period. I n c o n t r a s t , 23 (3 o f 13) a n d 46% (7 o f 15) o f t h e I M M a n d o r a l l y v a c c i n a t e d animals, respectively, were free of infection. D u r i n g the entire p o s t c h a l l e n g e period,
I00 9O 80
70
30 20
~"
I0
"
~'t"
0
I
I
I
t
I
I
I
23 30 54 78 102 TIME (h) Fig. 3. Percentage of milk samples from individual glands infected with S. a u r e u s at various time intervals after IMM challenge from unvaccinated (O O), IMM vaccinated (e O), orally vaccinated (A. . . . . . A), and ID vaccinated (zx. . . . . . zx) animals. 0
6
16
374
88 (15 of 17) and 80% (12 of 15) of the unvaccinated and ID vaccinated animals had both glands infected at least once, as compared to 31 (5 of 13) and 7% (1 of 15) of the IMM and orally vaccinated animals, respectively. Dermal response of cutaneous challenge with K S
The ID vaccinated guinea pigs had significantly (P < 0.05) larger and more persistent dermal lesions than the unvaccinated or locally vaccinated guinea pigs after cutaneous challenge with KS (Table 4). The lesions in the locally vaccinated animals were significantly greater than those of the unvaccinated controls during the 72 h period after challenge.
Discussion
In the present study, the magnitude of the leukocytosis in the milk of unvaccinated and vaccinated animals after IMM challenge with staphylococci differed significantly (P < 0.05). The number of leukocytes in the milk of ID or IMM vaccinated animals challenged with KS and in the milk of all animals (unvaccinated and vaccinated) challenged with viable S. aureus was significantly (P < 0.05) greater than the diurnal fluctuations in the number of leukocytes in normal cavian milk (Targowski and Nonnecke, 1982). In vaccinated ruminants, a significant increase in the number of milk leukocytes has been observed after intramammary challenge with antigens (Targowski and Berman, 1975; DeCueninck, 1979). The moderate, but significant, milk leukocytosis after challenge with KS was associated with a moderate dermal reaction to the cutaneous injection of KS in the IMM vaccinated animals. In the ID vaccinated animals, a large leukocytosis after IMM challenge with KS was correlated with a large dermal reaction to the cutaneous injection of KS. The responses of the unvaccinated animals to the cutaneous and IMM injections of KS were insignificant. These observations indicate that the magnitude of the milk leukocytosis and dermal response of animals in each group were correlated. Although the magnitude of these responses was related, their cellular composition was different. The PMN leukocyte was the major cell-type in the milk of the ID and IMM vaccinated animals from 23 h to 30 h after IMM challenge with KS. In contrast, the cellular infiltrate in dermal lesions of animals sensitized parenterally and challenged ID with staphylococci consisted primarily of mononuclear leukocytes at 24 h postchaUenge (Johnson et al. 1961). The unresponsiveness of the orally vaccinated guinea pigs to IMM challenge with KS might indicate a failure of the vaccination procedure. However, the orally vaccinated animals had weak, but significant (P < 0.05) dermal responses to the injection of KS and a significant increase in the number of leukocytes in their milk after the second IMM challenge in the same lactation (data not shown). Furthermore, these guinea pigs were more protected against experimental infection with S. aureus than the ID or unvaccinated animals. The protection induced by oral vaccination may have been due to the migration of sensitized lymphocytes from the intestinal lymphoid tissue to the mammary gland. Numerous studies have shown
375 that both T and B lymphocytes from the mesenteric lymph nodes or Peyer's patches home to the mammary gland tissue or enter the secretion in late pregnancy and during lactation (McDermott and Beinenstock, 1970; Manning and Parmely, 1980; Head and Seeling, 1983). The increased protection against staphylococcal infection provided by local (IMM and oral) vaccination compared with no vaccination and ID vaccination, may be due to the greater number of PMN leukocytes in the milk of these animals 6 h after challenge. Approximately 84% of the milk samples from the locally vaccinated animals were free of staphylococci at 6 h after challenge, compared to only 50% of the milk samples from unvaccinated and ID vaccinated animals. A milk leukocytosis of similar magnitude did not occur in the latter animals until 16 to 23 h after challenge, and failed to eliminate staphylococci from the mammary glands during the postchallenge period. In ewes, vaccination by the IMM infusion of killed staphylococci also enhanced the initial neutrophil influx in milk, compared to unvaccinated animals, resulting in greater protection of the gland against experimental infection with S. aureus (Colditz and Watson, 1982). Infusion of KS into the glands of locally vaccinated guinea pigs caused a delayed migration of leukocytes into the milk, whereas infusion of live staphylococci caused an immediate increase in the number of milk leukocytes. In contrast, the milk leukocytosis in animals vaccinated ID was rapid after infusion of KS, and delayed after the infusion of live staphylococci. The mechanism responsible for this difference cannot be determined on the basis of the data presented. However, it is conceivable that antigens elaborated by the live staphylococci interacted with the sensitized lymphocytes in the gland of the locally vaccinated animals triggering the rapid leukocytosis. Antibody might not have been involved in mediating the leukocytosis, since no agglutinating antibody was detected in the secretion (data not shown). In conclusion, local vaccination provided the greatest protection against IMM challenge with live staphylococci, even though the dermal and milk leukocytic responses were moderate after challenge with KS. In contrast, ID vaccination induced large dermal reactions and milk leukocytic responses to KS, but did not protect the mammary gland against challenge with live staphylococci. It seems that ID vaccination of animals inudced primarily delayed-type hypersensitivity without mammary protection.
References Colditz, I.G. and Watson, D.L. (1982) Effect of immunizationon the early influx of neutrophils during staphylococcalmastitis in ewes. Res. Vet. Sci. 33, 146-151. DeCueninck, B.J. (1979) Immune-mediatedinflammationin the lumen of the bovine mammary gland. Int. Arch. AllergyAppl. Immunol.59, 394-402. Head, J. and Seeling, L.L., Jr. (1983) Autoradiographicanalysis of lymphocytemigration into the mammaryepitheliumand milk of lactatingfemalerats. J. Reprod. Immunol.5, 61-72. Jain, N.C. (1976) Neutrophilleukocytesand inflammationof the bovine mammarygland.Theriogenology 6, 153-173.
376 Johnson, J.E., Cluff, L.E. and Goshi, K. (1961) Studies on the pathogenesis of staphylococcal infection. I. The effect of repeated skin infections. J. Exp. Med. 113, 235-247. Lascelles, A.K. and McDowell, G.S. (1974) Localized humoral immunity in particular reference to ruminants. Transplant. Rev. 19, 170-208. Manning, L.S. and Parmely, M.J. (1980) Cellular determinants of mammary cell-mediated immunity in the rat. I. The migration of radioisotopically labelled T-lymphocytes. J. lmmunol. 125, 2508-2514. McDermott, M.R. and Beinenstock, J. (1979) Evidence for a common mucosal immunologic system. I. Migration of B lymphoblasts into intestinal, respiratory, and genital areas. J. I mmunol. 122, 1892-1898. Newbould, F.H.S. (1974) Microbial diseases of the mammary gland. In Lactation: A Comprehensive Treatise (Larson, B.L. and Smith, V.R., eds.), pp. 237-269. Academic Press, New York. Newby, T.J., Stokes, C.R. and Bourne, B.J. (1982) Immunological activities of milk. Vet. Immunol. Immunopathol. 3, 67-94. Schalm, O.W., Carroll, E.J. and Jain, N.C. (1971) Bovine Mastitis, Lea and Febiger, Philadelphia. Targowski, S.P. and Berman, D.T. (1975) Leukocytic response of bovine mammary gland to injection of killed cells and cell walls of Staphylococcus aureus. Am. J. Vet. Res. 36~ 1561-1565. Targowski, S.P. and Nonnecke, B.J. (1982) Cell-mediated immune response of the mammary gland in guinea pigs. I. Effect of antigen injection into vaccinated and unvaccinated glands. Am. J. Reprod. Immunol. 2, 29-38. Watson, D.L. (1980) Immunological functions of the mammary gland and its secretion - comparative review. Aust. J. Biol. Sci. 33, 403-422. Watson, D.L. (1981) Immunologically-specific resistance to infection with particular reference to staphylococcal mastitis. Adv. Exp. Med. Biol. 137, 579-590.