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Immune Response to Toxic Staphylococcal Cellular Products and Efficacy of Mastitis Therapy Treatment
Immune Response to Toxic Staphylococcal Cellular Products and Efficacy of Mastitis Therapy Treatment J. D. SKEAN 1 and W. W. OVERCAST
Department of Dairying, Tennessee Agricultural Experiment Station, Knoxville Abstract
attempted one to three weeks later. Plommet (12) claimed to have cured bovine mastitis by injecting a- and fl-antitoxins and staphylococcal cells into the udder. Cures occurred in about 50% of the incidents treated, including some that had not yielded to therapy with several antibiotics. Derbyshire (4) suggested that intramammary vaccination be investigated more thoroughly. Presumably, stimulating the formation of protective antibody directly in the udder where it is needed to combat mastitis would be more efficacious than systemic vaccination, since it is known that staphylococcal mastitis may occur in cows having high serum titers of antibody. Our studies were made to determine the humoral immune response to toxic staphylococcal cellulax products introduced into the cow's udder and the efficacy of serum therapy in treatment of staphylococcal mastitis.
Experimental staphylococcal mastitis was inducible in both nonvaccinated and vaccinated cows by intramammary infusion of sterile inflanunatory agent containing cultural products from a bovine strain of Staphylococcus. Infusion of 10 ml of inflammatory agent containing 10 international units of a-hemolysin provoked an inflammatory response, with overt symptoms closely resembling those of clinical mastitis. Vaccinated and nonvaceinated cows appeared equally susceptible to the experimental mastitis. This mastitis elicited a systemic immune response in nonvaccinated cows. Neither equivalent nor multiequivalent infusions of homologous bovine antiserum lessened the severity of experimentally induced mastitis in vaccinated and nonvaccinated cows. This was true when the antiserum was infused immediately after the inflammatory agent or six hours later. Moreover, the antiserum alone sometimes provoked an inflammatory response.
Materials and Methods
The increased use of antimicrobial agents has been accompanied by a shift in the general etiology of bovine mastitis from streptococcal to staphylococcal (9). Since none of the available antimierobial agents eradicates staphylococci from the cow's udder, various workers have studied immunological control of staphylococcal mastitis, but the results have not been entirely satisfactory (2, 5, 6, 13, 17). The belief of some investigators that systemic vaccination does not effect adequate proteetion against staphylococcal mastitis has led to experimentation with local vaccination, in which the vaccine is introduced through the teat directly into the mammary gland. Pillet et al. (11) reported that a vaccine containing a- and fl-toxoids and staphylococcal cells introduced into the teat duct protected sheep against experimental intramammary infection Received for publication December 15, 1967. 1 Present address: Department of Biology, Western Kentucky University, Bowling Green. 47
Eight pairs of cows were used. They were paired according to breed, age, and stage of lactation, insofar as possible. One cow of each p a i r had been vaccinated with a staphylococcal vaccine (14). Two types of experiments were quadruplicated. I n one type, mastitis was induced in both cows of each p a i r by infusing different levels of inflammatory agents into different mammary quarters. I n the other type, mastitis was induced in both cows by infusing them with antiserum. The inflammatory agent used to induce mastitis was a saline suspension of toxic staphylococcal culture products prepared as described previously (14) and sterilized by filtration. The cultural products were of the same lot used to make the vaccine given to the vaccinated cows. The a-hemolytic potency [international units (IU) per milliliter] of the inflammatory agent never exceeded the serum titer of antistaphylococcal a-hemolysin for the vaccinated cow given the inflannnatory agent. The antitoxic potencies of the sera were determined by the method of the National Institutes of Health (10), as modified (14), except that the mixture of one international unit of laboratory hemolysin and serum was
48
SKEAN AND OVERCAST
let stand for 20 rain at room temperature before the indicator rabbit cells were added and the antitoxic potencies were recorded as serum titers in international units of antistaphylococcal a-hemolysin. The laboratory standard hemolysin was prepared as needed from lyophilized cultural products of bovine staphylococcal Strain 89. One international unit of laboratory standard hemolysin was the amount of cultural products having a toxic potency of one hemolytic test dose. For each serum, the end point was obtained from among a series of dilutions exhibiting relative protective potencies ranging from complete to none. For sera having 10 IU/milliliter or fewer, the titrations were done in one-IU increments. For more potent sera the titrations were done in two-IU increments and the end points interpolated, if necessary. The antiserum used for each pair was obtained from the blood of the vaccinated cow, diluted with saline solution to the desired titer of antistaphylococcal a-hemolysin, and sterilized by filtration. The titer of the antiserum equaled or exceeded the a-hemolytic potency of the companion inflammatory agent. Within one hour after the afternoon milking, a cannula was aseptically passed through the teat into the gland cistern, the residual milk expressed, and the gland infused with a 10-ml volume of the test material. Serum titers of antistaphylococcal a-hemolysin were determined for each cow at two- or three-day intervals during a 22-day period, beginning two days before the intramammary infusions were given. During this same period, the leucocyte count and titer of antistaphylococcal a-hemoysin were determined for samples of foremilk aseptically obtained from each mammary quarter. These determinations were made daily for the first five days of the period, and thereafter at two- or three-day intervals. The numbers of leucocytes in the milk were estimated by the direct microscopic method. The titers of antistaphylococcal a-hemolysin and serum were determined and recorded, except before estimating end points the intact rabbit cells were sedimented by centrifugation. Results
The data pertaining to artificially induced staphylococcal mastitis in nonvaccinated and vaccinated dairy cows presented in Tables 1 and 2 are representative of the eight pairs of cows. These data show that an intramammary infusion of toxic staphylococcal cultural products containing as few as 10 I U of staphyJ. DAIRY SCIENCE VOL. 52, NO. I
lococcal a-hemolysin provoked an inflammatory response, with overt symptoms resembling very closely those of clinical mastitis; that is, the infused gland produced abnormal milk containing many leucocytes. I n vaccinated cows, the intralnammary or milk titer of antistaphylococcal a-hemolysin was generally much lower than the extramammary or serum titer. The leucocyte counts of the mastitic milks indicate that the vaccinated cows with evaluated serum titers of antistaphyloccoccal a-hemolysin were as susceptible to experimental inastitis as were nonvaecinated cows with very low serum titers. The appearance of the milks did not indicate any diference in the severity of experimental mastitis suffered by vaccinated and nonvaccinated cows. But the leucocyte counts of the mastitic milks indicate that in two experiments as typified in Table 1, the vaccinated cow sustained a milder incident than did the nonvacciuated cow, and that in two experiments as typified in Table 2 the nonva~cinated cow sustained a milder incident than did the vaccinated cow. I n each nonvaccinated cow sustaining experimental mastiffs, there developed an elevated serum titer of antistaphylococcal a-hemolysin. This indicates that the hemolysin traversed the mammary barrier and induced a systemic immune response. Table 3 contains data pertaining to artificially induced staphylococcal mastitis and serum therapy for one pair of cows. These data are representative of and similar to those from three other pairs. Again, in these experiments, neither the appearance nor the leucocyte counts of the mastitic milks indicated that vaccinated cows were more resistant to experimental mastitis than were nonvaeeinated cows. Intramammary infusion of homologous bovine antiserum subsequent to intramammary infusion of toxic staphylococcal cultural products did not lessen the severity of induced mastitis. This was indicated by the appearance and leucocyte counts of mastitic milks produced by the infused glands. Moreover, even though the antiserum was prepared only from the blood of the vaccinated cow in each experiment and was treated minimally before use, it alone twice provoked an inflammatory response in the recipient gland, as indicated by a large increase in numbers of leucocytes in the milk produced by these glands. Discussion
Transient clinical mastitis was readily induced in both vaccinated and nonvaccinated
TABLE 1. E x p e r i m e n t a l staphylococcal m a s t i t i s in Cows T-86 and T-73.
Day
1 2 3f 4 5 8 10 12 15 17 19 22
o
Cow and treatment ~ LR T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V
Leucocytes in milk from quarter °
Deseriptio~ of milk from quarter b LF
SPE SPE Clots Clots
]~F
SPE SPE Clots Clots
RR
SPE SPE Clots Clots
LR
LF
2.2 2.2 4.6 0.99 4.6 1.4 3.3 1.5 7.2 1.9 7.1 2.0 5.3 0.87 3.0 2.9 4.2 1.6 3.8 2.6 4.9 1.6 5.5 4.0
( X 10-~/ml) 0.036 0.47 0.68 0.38 0.016 0.74 0.45 0.43 0.052 0.66 0.42 0.4 TNTC TNTC TNTC TNTC TNTC 16.0 9.0 1.2 4.3 4.2 1.7 1.2 0.83 4.6 0.68 0.36 0.66 3.6 3.8 2.8 2.2 6.7 4.2 0.69 1.1 3.7 2.6 0.99 1.1 7.1 0.8 0.52 0.78 5.6 4.6 2.8
R.F
Milk titer f r o m q u a r t e r `~'° RR
LR
LF
RF
RR
Serum titer ~ 0.5 56
(IU/ml) 0.94 0.12 1.2 0.24 1.1 0.12 TNTC TNTC 18.0 12.0 4.9 1.5 5.1 0.31 2.3 0.83 1.8 0.38 1.4 0.38 1.6 0.24 1.6 0.3
1
1
1
1
1
1
1
1 0.1 42
1
1
1
1
1
1
1
1
1
0.1 30 0.5 26 6 30 10 15 12 30 12 22 16 28 11 20
5~
NV, nonvaccinated ; V, vaccinated. b No data, n o r m a l ; S P E , sero-purulent exudate. e TNTC, too n u m e r o u s to count. No d a t a indicates titers of less t h a n one. e Titers of antistaphyloc0ccal a-hemolysin. Day of i n t r a m a m m a r y i n f u s i o n s : LR, 10 ml of saline solution; L F , 10 I U of a-hemolysin in 10 ml of saline solution; RF, 20 I U of a-hemolysin in 10 5111 of saline solution ; and R~R, 40 I U of ~-hemolysin in 10 ml of saline solution.
¢D
TABLE 2. E x p e r i m e n t a l staphylococcal m a s t i t i s in Cows 219 and 184.
Day 1 2 3r 4 5 8 10 12 15 17 19 22
Cow and t r e a t m e n t a LR 219NV 184V 219NV 184V 219NV 184V 219]NV 184V 219NV 184V 219NV 184V 219NV 184V 219NV 184V 219NV 184V 219NV 184V 219NV 184V 219NV 184V
Clots SPE SPE Clots
Description of milk from quarter b LF
t~F
Leucocytes in milk from quarter c ~R
LR
LF
t~F
0.33 4.0 2.5 3.9 0.85 4.9 0.26 3.3 0.47 4.7 0.28 3.4 21.0 4.0 18.2 3.4 TNTC 4.7 TNTC 3.6 TNTC 4.2 4.2 3.2
( X lO~/ml) 0.73 0.12 0.14 0.19 0.76 0.14 0.36 0.16 0.88 0.14 0.16 0.16 15.0 12.0 7.2 14.0 9.0 10.0 7.9 15.0 0.66 0.74 1.4 1.4 0.8 0.36 4.0 0.76 1.7 2.0 0.87 1.3 0.64 0.74 1.5 1.8 0.62 0.35 0.57 1.0 0.22 0.66 0.85 0.61 0.24 0.62 0.71 0.85
Milk titer f r o m q u a r t e r `~'e RR
LR
LF
RF
RR
Serum titer e
(IU/ml) 0.69 TNTC 0.4 TNTC 0.48 TNTC 18.0 TNTC 11.0 TNTC 0.71 TNTC 0.61 TNTC 2.4 TNTC 0.61 TNTC 1.2 TNTC 0.64 TNTC 1.5 TNTC
0.25 26 0.25 28 1 1
1
1 1
1 0.25 29 0.25 29 0.25 26 1 28 5 26 6 26 4 24 2 22
a NV, nonvaccinated; V, vaccinated. b No data, norma3; S P E , sero-purulent exudate. c TNTC, too n u m e r o u s to count. d No data indicates titers of less t h a n one. Titers of antistaphy]oeoccal a-hemolysin. D a y of i n t r a m a m m a r y i n f u s i o n s : LR, 10 ml of saline solution; L F , 10 I U of a-hemolysin in 10 ml of saline solution; RF, 20 I U of a-hemolysin in 10 ml of saline solution; and RR, 40 I U of a-hemolysin in 10 ml of saline solution.
TABLE 3. E x p e r i m e n t a l staphylococcal m a s t i t i s and serum t h e r a p y in Cows 241 and 239. Leucocytes in milk from quarter ~
Description of milk from quarter b Day 1 2 3~ 4 5 8 10 12 15 17 19 22
Cow and t r e a t m e n t a LR 241NV 239V 241NV 239V 241NV 239V 241NV 239V 241NV 239V 241NV 239V 241NV 239V 241NV 239V 241NV 239V 241NV 239V 241NV 239V 241NV 239V
LF
Clots SPE Clots Clots Flakes
RF
Clots Clots Flal~es
Flakes
ttR
Clots SPE SPE Clots Flakes
LR 0.59 0.66 0.3 1.8 0.64 0.82 9.6 0.69 12.0 0.19 14.0 5.4 10.0 2.2 6.7 2.6 4.8 1.7 11.0 1.2 13.0 2.9 12.0 2.0
LF
~F
( X 10-"/ml) 0.14 0.26 0.73 0.5 0.17 0.19 0.87 0.92 0.69 0.17 0.48 0.54 TNTC TNTC 22.0 14.0 17.0 TNTC 10.0 TNTC 1.9 2.1 2.9 4.1 0.73 0.76 1.8 1.4 0.54 0.48 2.2 1.3 0.17 0.23 1.0 0.68 0.59 0.73 1.0 0.61 0.24 0.28 2.4 1.3 0.38 0.33 1.7 0.95
Milk titer f r o m q u a r t e r a'e tlR
LR
LF
RF
RR
Serum titer e
(IU/ml) 0.45 0.33 0.01 0.71 0.01 0.36 TNTC 16.0 TNTC 16.0 1.8 3.4 0.57 2.0 0.49 1.5 0.31 1.5 0.59 1.0 0.24 1.2 0.16 0.88
0.1 29 0.1 30 1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
0.1 24 0.5 19 6 22 13 22 13 22 25 22 24 20 22 2O
" NV, nonvaccinated; V, vaccinated. b No data, n o r m a l ; S P E , sero-purulent exudate. " TNTC, too n u m e r o u s to count. d NO data indicates titers of less than one. e Titers of antistaphylococcal a-hemolysin. Day of i n t r a m a m m a r y infusions: LR, 80 I U of anti-a-hemolysin in 10 m l of s e r u m ; L F , 40 I U of a-hemolysin in 10 ml of saline solution; R F , 40 I U of a-hemolysin in 10 ml of saline solution + 80 I U of anti-a-hemolysin in 10 ml of serunl given immediately; RR, same as R F except serum given 6 hr later.
O
52
S K E A N AND OVERCAST
cows by intramammary infusion of staphylococcal cultural products containing known quantities of a-hemolysin. The appearance and leucocyte counts of resulting mastitic milks did not indicate that vaccination stinmlated any protection against staphylococcal cultural products in the udder, despite elevated serum titers of antistaphylococcal ahemolysin in the vaccinated cows. a-Hemolysin has been shown not to be a major contributor to the virulence of coagulase-positive staphylococci in man (7), bu~ coagulase-negative staphylococci that produce a-hemolysin are associated with acute bovine mastitis (8). There is experimental evidence that immunity to a-hemolysin is important in specific resistance to staphylococcal mastitis (4). The pathogenic role of a-hemolysin in bovine mastitis is not clear, but results from this study show that the hemolysin is deleterious to the mammary epithelium to the extent that it is able to cross this barrier and elicit the production of specific antibody. Presumably, this tissuedegrading activity could contribute to pathogenesis by the organism producing it. The cultural products used to induce mastitis contained unknown amounts of staphylococcal fl-hemolysin, fl-ttemolysin seems to be a distinctive property of bovine staphylococci isolated from incidents of mastitis, but it does not seem to be a strong irritant in the bovine mammary gland (15). However, fl-hemolysin does have a possible pathogenic role in mastitis of multiple etiology. Many strains of bovine staphylococci that are apparently nonpathogenic, or produee minute amounts of fl-hemolysin in vitro, are very hemolytic when cultured in vitro with strains of streptococci in the Lancefield Group B (18). Derbyshire (3) demonstrated that some irritation of the mammary epithelium was neeessary before circulating antitoxin could pass into the milk, although a very mild irritant was sufficient. In these experiments the titers of antistaphylococcal a-hemolysin in milks from the insulted glands of vaccinated cows did not indicate that much inflow of circulating antibodies had occurred, even in cows having reIatively high serum titers (Tables 1, 2, 3). The milk titers were generally low and occurred sporadically. There have been no reports concerning the therapeutic use of antiserum against mastitis (4). I n our study attempts to test the efficacy of homologous bovine antiserum in preventing experimentally induced staphylococcal mastitis i~dicated that the antiserum was inefJ. DAIRY SCIENCE X~OL. 52, NO. 1
fective in preventing an inflammatory response. The numbers of leucocytes in the milk indicated that sterile bovine antiserum alone sometimes elicited an inflammatory response upon intramammary infusion. Such a response occurred in a nonvaccinated cow (Table 3) and in the vaccinated cow of another pair. References
(1) Blobel, H., and Y. Katsube. 1964. Effects of experimentally induced ]eucocytosis in bovine mammary glands upon infections with Staphylococcus aureus, Streptococcus agalactiae and Aerobacter aerogenes. Amer. J. Vet. Res., 25: 1085. (2) Brown, R. W., H. G. Blobel, W. D. Pounden, O. W. Schahn, L. W. Slanetz, and G. R. Spencer. 1963. Current concepts of bovine mastitis. National Mastltis Council, Inc., Hinsdale, Illinois. (3) Derbyshire, J. B. 1960. Studies in immunity to experimental staphylococcal mastitis in the goat and cow. g. Comp. Pathol. Therap., 70 : 222. (4) Derbyshire, g. B. 1962. Immunity in bovine mastitis. Vet. Bull., 32: I. (5) Derbyshire, J. B., and S. J. Edwards. 1963. A field trial of a staphylococcal cell-toxoid vaccine in the control of staphylococcal mastitis in cows. Vet. Record, 75: 1208. (6) Drury, A. R. 1962. Diagnosis and Treatment. I n A. R.. Drury, D. L. Murray, and G. W. Reed, ads. p. 14-22. Mastitis Control in Michigan ~erds. Michigan State Univ., Ext. Bull. 344. (7) Elek, S. D., 1959. Staphylococcus pyogenes m~d Its Relation to Disease. E. and S. Livingstone Ltd., London. (8) Joshi, I-I. D., and A. •. Drury. 1962. Pathogenicity of staphylococci associated with the bovine udder. Michigan State Univ. Yet., 23: 40. (9) Murray, D. L. 1962. Introduction. I n A. R. Drury, D. L. Murray, and G. W. Reed, ads. p. 3. Mastitis Control in Michigan Herds. Michigan State Univ., Ext. Bull. 344. (10) National Institutes of Health. 1938. Tentative staphylococcus toxoid requirements. Washington, D.C. (11) Pillet, J., O. Girard, t[. Dutheil, and B. Orta. 1959. Recherches sur ]'Immunisation de la t~rebls et Yaccination locale. Ann. Inst. Pasteur, 96: 591. (12) Plommet, M. 1960. Essais de Traitment de la Mammite staphylococcique de la ~'ache par Vaccination locale. Ann. Inst. Pasteur, 99 : 618. (13) Schu]tze, W. D., J. W. Smith, V. C. Womack, and M. A. Norcross. 1963. Observations on the efficacy of antistaphylococcal
BOVINE
vaccination in the control of mastit~s in a dairy cattle herd. J. Dairy Sci., 46: 625. (14) Skean, J. D., and W. W. Overcast. 1968. The efficacy of different adjuvants used with a staphylococcal vaccine in dairy cows. J. Dairy Sci., 51:1239. (15) Slanetz, L. W., and C. H. Bartley. 1953. The diagnosis of staphylococcal mastitis with special reference to the characteristics of mastitis staphylococci. J. Infectious Diseases, 92: 139.
MASTITIS
53
(16) Slanetz, L. W., C. I-I. Baxtley, and F. E. Allen. 1963. Vaccination of dairy cattle against staphylococcic mastltis. Amer. J . Vet. Res., 24: 923. (17) Slanetz, L. W., C. It. Bartley, and F. E. Allen. 1965. Evaluation of cell-toxoid vancines for the vaccination of dairy cattle against staphylococcic mastitis. Amer. J. Vet. Res., 26: 688. (18) Zemelman, R., and L. Longeri. 1965. Characterization of staphylococci isolated from raw milk. Appl. Microblol., 13: 167.
J. DAIRY SCIENCE VOL, 52, NO. I
Department of Dairying, Tennessee Agricultural Experiment Station, Knoxville Abstract
attempted one to three weeks later. Plommet (12) claimed to have cured bovine mastitis by injecting a- and fl-antitoxins and staphylococcal cells into the udder. Cures occurred in about 50% of the incidents treated, including some that had not yielded to therapy with several antibiotics. Derbyshire (4) suggested that intramammary vaccination be investigated more thoroughly. Presumably, stimulating the formation of protective antibody directly in the udder where it is needed to combat mastitis would be more efficacious than systemic vaccination, since it is known that staphylococcal mastitis may occur in cows having high serum titers of antibody. Our studies were made to determine the humoral immune response to toxic staphylococcal cellulax products introduced into the cow's udder and the efficacy of serum therapy in treatment of staphylococcal mastitis.
Experimental staphylococcal mastitis was inducible in both nonvaccinated and vaccinated cows by intramammary infusion of sterile inflanunatory agent containing cultural products from a bovine strain of Staphylococcus. Infusion of 10 ml of inflammatory agent containing 10 international units of a-hemolysin provoked an inflammatory response, with overt symptoms closely resembling those of clinical mastitis. Vaccinated and nonvaceinated cows appeared equally susceptible to the experimental mastitis. This mastitis elicited a systemic immune response in nonvaccinated cows. Neither equivalent nor multiequivalent infusions of homologous bovine antiserum lessened the severity of experimentally induced mastitis in vaccinated and nonvaccinated cows. This was true when the antiserum was infused immediately after the inflammatory agent or six hours later. Moreover, the antiserum alone sometimes provoked an inflammatory response.
Materials and Methods
The increased use of antimicrobial agents has been accompanied by a shift in the general etiology of bovine mastitis from streptococcal to staphylococcal (9). Since none of the available antimierobial agents eradicates staphylococci from the cow's udder, various workers have studied immunological control of staphylococcal mastitis, but the results have not been entirely satisfactory (2, 5, 6, 13, 17). The belief of some investigators that systemic vaccination does not effect adequate proteetion against staphylococcal mastitis has led to experimentation with local vaccination, in which the vaccine is introduced through the teat directly into the mammary gland. Pillet et al. (11) reported that a vaccine containing a- and fl-toxoids and staphylococcal cells introduced into the teat duct protected sheep against experimental intramammary infection Received for publication December 15, 1967. 1 Present address: Department of Biology, Western Kentucky University, Bowling Green. 47
Eight pairs of cows were used. They were paired according to breed, age, and stage of lactation, insofar as possible. One cow of each p a i r had been vaccinated with a staphylococcal vaccine (14). Two types of experiments were quadruplicated. I n one type, mastitis was induced in both cows of each p a i r by infusing different levels of inflammatory agents into different mammary quarters. I n the other type, mastitis was induced in both cows by infusing them with antiserum. The inflammatory agent used to induce mastitis was a saline suspension of toxic staphylococcal culture products prepared as described previously (14) and sterilized by filtration. The cultural products were of the same lot used to make the vaccine given to the vaccinated cows. The a-hemolytic potency [international units (IU) per milliliter] of the inflammatory agent never exceeded the serum titer of antistaphylococcal a-hemolysin for the vaccinated cow given the inflannnatory agent. The antitoxic potencies of the sera were determined by the method of the National Institutes of Health (10), as modified (14), except that the mixture of one international unit of laboratory hemolysin and serum was
48
SKEAN AND OVERCAST
let stand for 20 rain at room temperature before the indicator rabbit cells were added and the antitoxic potencies were recorded as serum titers in international units of antistaphylococcal a-hemolysin. The laboratory standard hemolysin was prepared as needed from lyophilized cultural products of bovine staphylococcal Strain 89. One international unit of laboratory standard hemolysin was the amount of cultural products having a toxic potency of one hemolytic test dose. For each serum, the end point was obtained from among a series of dilutions exhibiting relative protective potencies ranging from complete to none. For sera having 10 IU/milliliter or fewer, the titrations were done in one-IU increments. For more potent sera the titrations were done in two-IU increments and the end points interpolated, if necessary. The antiserum used for each pair was obtained from the blood of the vaccinated cow, diluted with saline solution to the desired titer of antistaphylococcal a-hemolysin, and sterilized by filtration. The titer of the antiserum equaled or exceeded the a-hemolytic potency of the companion inflammatory agent. Within one hour after the afternoon milking, a cannula was aseptically passed through the teat into the gland cistern, the residual milk expressed, and the gland infused with a 10-ml volume of the test material. Serum titers of antistaphylococcal a-hemolysin were determined for each cow at two- or three-day intervals during a 22-day period, beginning two days before the intramammary infusions were given. During this same period, the leucocyte count and titer of antistaphylococcal a-hemoysin were determined for samples of foremilk aseptically obtained from each mammary quarter. These determinations were made daily for the first five days of the period, and thereafter at two- or three-day intervals. The numbers of leucocytes in the milk were estimated by the direct microscopic method. The titers of antistaphylococcal a-hemolysin and serum were determined and recorded, except before estimating end points the intact rabbit cells were sedimented by centrifugation. Results
The data pertaining to artificially induced staphylococcal mastitis in nonvaccinated and vaccinated dairy cows presented in Tables 1 and 2 are representative of the eight pairs of cows. These data show that an intramammary infusion of toxic staphylococcal cultural products containing as few as 10 I U of staphyJ. DAIRY SCIENCE VOL. 52, NO. I
lococcal a-hemolysin provoked an inflammatory response, with overt symptoms resembling very closely those of clinical mastitis; that is, the infused gland produced abnormal milk containing many leucocytes. I n vaccinated cows, the intralnammary or milk titer of antistaphylococcal a-hemolysin was generally much lower than the extramammary or serum titer. The leucocyte counts of the mastitic milks indicate that the vaccinated cows with evaluated serum titers of antistaphyloccoccal a-hemolysin were as susceptible to experimental inastitis as were nonvaecinated cows with very low serum titers. The appearance of the milks did not indicate any diference in the severity of experimental mastitis suffered by vaccinated and nonvaccinated cows. But the leucocyte counts of the mastitic milks indicate that in two experiments as typified in Table 1, the vaccinated cow sustained a milder incident than did the nonvacciuated cow, and that in two experiments as typified in Table 2 the nonva~cinated cow sustained a milder incident than did the vaccinated cow. I n each nonvaccinated cow sustaining experimental mastiffs, there developed an elevated serum titer of antistaphylococcal a-hemolysin. This indicates that the hemolysin traversed the mammary barrier and induced a systemic immune response. Table 3 contains data pertaining to artificially induced staphylococcal mastitis and serum therapy for one pair of cows. These data are representative of and similar to those from three other pairs. Again, in these experiments, neither the appearance nor the leucocyte counts of the mastitic milks indicated that vaccinated cows were more resistant to experimental mastitis than were nonvaeeinated cows. Intramammary infusion of homologous bovine antiserum subsequent to intramammary infusion of toxic staphylococcal cultural products did not lessen the severity of induced mastitis. This was indicated by the appearance and leucocyte counts of mastitic milks produced by the infused glands. Moreover, even though the antiserum was prepared only from the blood of the vaccinated cow in each experiment and was treated minimally before use, it alone twice provoked an inflammatory response in the recipient gland, as indicated by a large increase in numbers of leucocytes in the milk produced by these glands. Discussion
Transient clinical mastitis was readily induced in both vaccinated and nonvaccinated
TABLE 1. E x p e r i m e n t a l staphylococcal m a s t i t i s in Cows T-86 and T-73.
Day
1 2 3f 4 5 8 10 12 15 17 19 22
o
Cow and treatment ~ LR T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V T-86NV T-73V
Leucocytes in milk from quarter °
Deseriptio~ of milk from quarter b LF
SPE SPE Clots Clots
]~F
SPE SPE Clots Clots
RR
SPE SPE Clots Clots
LR
LF
2.2 2.2 4.6 0.99 4.6 1.4 3.3 1.5 7.2 1.9 7.1 2.0 5.3 0.87 3.0 2.9 4.2 1.6 3.8 2.6 4.9 1.6 5.5 4.0
( X 10-~/ml) 0.036 0.47 0.68 0.38 0.016 0.74 0.45 0.43 0.052 0.66 0.42 0.4 TNTC TNTC TNTC TNTC TNTC 16.0 9.0 1.2 4.3 4.2 1.7 1.2 0.83 4.6 0.68 0.36 0.66 3.6 3.8 2.8 2.2 6.7 4.2 0.69 1.1 3.7 2.6 0.99 1.1 7.1 0.8 0.52 0.78 5.6 4.6 2.8
R.F
Milk titer f r o m q u a r t e r `~'° RR
LR
LF
RF
RR
Serum titer ~ 0.5 56
(IU/ml) 0.94 0.12 1.2 0.24 1.1 0.12 TNTC TNTC 18.0 12.0 4.9 1.5 5.1 0.31 2.3 0.83 1.8 0.38 1.4 0.38 1.6 0.24 1.6 0.3
1
1
1
1
1
1
1
1 0.1 42
1
1
1
1
1
1
1
1
1
0.1 30 0.5 26 6 30 10 15 12 30 12 22 16 28 11 20
5~
NV, nonvaccinated ; V, vaccinated. b No data, n o r m a l ; S P E , sero-purulent exudate. e TNTC, too n u m e r o u s to count. No d a t a indicates titers of less t h a n one. e Titers of antistaphyloc0ccal a-hemolysin. Day of i n t r a m a m m a r y i n f u s i o n s : LR, 10 ml of saline solution; L F , 10 I U of a-hemolysin in 10 ml of saline solution; RF, 20 I U of a-hemolysin in 10 5111 of saline solution ; and R~R, 40 I U of ~-hemolysin in 10 ml of saline solution.
¢D
TABLE 2. E x p e r i m e n t a l staphylococcal m a s t i t i s in Cows 219 and 184.
Day 1 2 3r 4 5 8 10 12 15 17 19 22
Cow and t r e a t m e n t a LR 219NV 184V 219NV 184V 219NV 184V 219]NV 184V 219NV 184V 219NV 184V 219NV 184V 219NV 184V 219NV 184V 219NV 184V 219NV 184V 219NV 184V
Clots SPE SPE Clots
Description of milk from quarter b LF
t~F
Leucocytes in milk from quarter c ~R
LR
LF
t~F
0.33 4.0 2.5 3.9 0.85 4.9 0.26 3.3 0.47 4.7 0.28 3.4 21.0 4.0 18.2 3.4 TNTC 4.7 TNTC 3.6 TNTC 4.2 4.2 3.2
( X lO~/ml) 0.73 0.12 0.14 0.19 0.76 0.14 0.36 0.16 0.88 0.14 0.16 0.16 15.0 12.0 7.2 14.0 9.0 10.0 7.9 15.0 0.66 0.74 1.4 1.4 0.8 0.36 4.0 0.76 1.7 2.0 0.87 1.3 0.64 0.74 1.5 1.8 0.62 0.35 0.57 1.0 0.22 0.66 0.85 0.61 0.24 0.62 0.71 0.85
Milk titer f r o m q u a r t e r `~'e RR
LR
LF
RF
RR
Serum titer e
(IU/ml) 0.69 TNTC 0.4 TNTC 0.48 TNTC 18.0 TNTC 11.0 TNTC 0.71 TNTC 0.61 TNTC 2.4 TNTC 0.61 TNTC 1.2 TNTC 0.64 TNTC 1.5 TNTC
0.25 26 0.25 28 1 1
1
1 1
1 0.25 29 0.25 29 0.25 26 1 28 5 26 6 26 4 24 2 22
a NV, nonvaccinated; V, vaccinated. b No data, norma3; S P E , sero-purulent exudate. c TNTC, too n u m e r o u s to count. d No data indicates titers of less t h a n one. Titers of antistaphy]oeoccal a-hemolysin. D a y of i n t r a m a m m a r y i n f u s i o n s : LR, 10 ml of saline solution; L F , 10 I U of a-hemolysin in 10 ml of saline solution; RF, 20 I U of a-hemolysin in 10 ml of saline solution; and RR, 40 I U of a-hemolysin in 10 ml of saline solution.
TABLE 3. E x p e r i m e n t a l staphylococcal m a s t i t i s and serum t h e r a p y in Cows 241 and 239. Leucocytes in milk from quarter ~
Description of milk from quarter b Day 1 2 3~ 4 5 8 10 12 15 17 19 22
Cow and t r e a t m e n t a LR 241NV 239V 241NV 239V 241NV 239V 241NV 239V 241NV 239V 241NV 239V 241NV 239V 241NV 239V 241NV 239V 241NV 239V 241NV 239V 241NV 239V
LF
Clots SPE Clots Clots Flakes
RF
Clots Clots Flal~es
Flakes
ttR
Clots SPE SPE Clots Flakes
LR 0.59 0.66 0.3 1.8 0.64 0.82 9.6 0.69 12.0 0.19 14.0 5.4 10.0 2.2 6.7 2.6 4.8 1.7 11.0 1.2 13.0 2.9 12.0 2.0
LF
~F
( X 10-"/ml) 0.14 0.26 0.73 0.5 0.17 0.19 0.87 0.92 0.69 0.17 0.48 0.54 TNTC TNTC 22.0 14.0 17.0 TNTC 10.0 TNTC 1.9 2.1 2.9 4.1 0.73 0.76 1.8 1.4 0.54 0.48 2.2 1.3 0.17 0.23 1.0 0.68 0.59 0.73 1.0 0.61 0.24 0.28 2.4 1.3 0.38 0.33 1.7 0.95
Milk titer f r o m q u a r t e r a'e tlR
LR
LF
RF
RR
Serum titer e
(IU/ml) 0.45 0.33 0.01 0.71 0.01 0.36 TNTC 16.0 TNTC 16.0 1.8 3.4 0.57 2.0 0.49 1.5 0.31 1.5 0.59 1.0 0.24 1.2 0.16 0.88
0.1 29 0.1 30 1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
0.1 24 0.5 19 6 22 13 22 13 22 25 22 24 20 22 2O
" NV, nonvaccinated; V, vaccinated. b No data, n o r m a l ; S P E , sero-purulent exudate. " TNTC, too n u m e r o u s to count. d NO data indicates titers of less than one. e Titers of antistaphylococcal a-hemolysin. Day of i n t r a m a m m a r y infusions: LR, 80 I U of anti-a-hemolysin in 10 m l of s e r u m ; L F , 40 I U of a-hemolysin in 10 ml of saline solution; R F , 40 I U of a-hemolysin in 10 ml of saline solution + 80 I U of anti-a-hemolysin in 10 ml of serunl given immediately; RR, same as R F except serum given 6 hr later.
O
52
S K E A N AND OVERCAST
cows by intramammary infusion of staphylococcal cultural products containing known quantities of a-hemolysin. The appearance and leucocyte counts of resulting mastitic milks did not indicate that vaccination stinmlated any protection against staphylococcal cultural products in the udder, despite elevated serum titers of antistaphylococcal ahemolysin in the vaccinated cows. a-Hemolysin has been shown not to be a major contributor to the virulence of coagulase-positive staphylococci in man (7), bu~ coagulase-negative staphylococci that produce a-hemolysin are associated with acute bovine mastitis (8). There is experimental evidence that immunity to a-hemolysin is important in specific resistance to staphylococcal mastitis (4). The pathogenic role of a-hemolysin in bovine mastitis is not clear, but results from this study show that the hemolysin is deleterious to the mammary epithelium to the extent that it is able to cross this barrier and elicit the production of specific antibody. Presumably, this tissuedegrading activity could contribute to pathogenesis by the organism producing it. The cultural products used to induce mastitis contained unknown amounts of staphylococcal fl-hemolysin, fl-ttemolysin seems to be a distinctive property of bovine staphylococci isolated from incidents of mastitis, but it does not seem to be a strong irritant in the bovine mammary gland (15). However, fl-hemolysin does have a possible pathogenic role in mastitis of multiple etiology. Many strains of bovine staphylococci that are apparently nonpathogenic, or produee minute amounts of fl-hemolysin in vitro, are very hemolytic when cultured in vitro with strains of streptococci in the Lancefield Group B (18). Derbyshire (3) demonstrated that some irritation of the mammary epithelium was neeessary before circulating antitoxin could pass into the milk, although a very mild irritant was sufficient. In these experiments the titers of antistaphylococcal a-hemolysin in milks from the insulted glands of vaccinated cows did not indicate that much inflow of circulating antibodies had occurred, even in cows having reIatively high serum titers (Tables 1, 2, 3). The milk titers were generally low and occurred sporadically. There have been no reports concerning the therapeutic use of antiserum against mastitis (4). I n our study attempts to test the efficacy of homologous bovine antiserum in preventing experimentally induced staphylococcal mastitis i~dicated that the antiserum was inefJ. DAIRY SCIENCE X~OL. 52, NO. 1
fective in preventing an inflammatory response. The numbers of leucocytes in the milk indicated that sterile bovine antiserum alone sometimes elicited an inflammatory response upon intramammary infusion. Such a response occurred in a nonvaccinated cow (Table 3) and in the vaccinated cow of another pair. References
(1) Blobel, H., and Y. Katsube. 1964. Effects of experimentally induced ]eucocytosis in bovine mammary glands upon infections with Staphylococcus aureus, Streptococcus agalactiae and Aerobacter aerogenes. Amer. J. Vet. Res., 25: 1085. (2) Brown, R. W., H. G. Blobel, W. D. Pounden, O. W. Schahn, L. W. Slanetz, and G. R. Spencer. 1963. Current concepts of bovine mastitis. National Mastltis Council, Inc., Hinsdale, Illinois. (3) Derbyshire, J. B. 1960. Studies in immunity to experimental staphylococcal mastitis in the goat and cow. g. Comp. Pathol. Therap., 70 : 222. (4) Derbyshire, g. B. 1962. Immunity in bovine mastitis. Vet. Bull., 32: I. (5) Derbyshire, J. B., and S. J. Edwards. 1963. A field trial of a staphylococcal cell-toxoid vaccine in the control of staphylococcal mastitis in cows. Vet. Record, 75: 1208. (6) Drury, A. R. 1962. Diagnosis and Treatment. I n A. R.. Drury, D. L. Murray, and G. W. Reed, ads. p. 14-22. Mastitis Control in Michigan ~erds. Michigan State Univ., Ext. Bull. 344. (7) Elek, S. D., 1959. Staphylococcus pyogenes m~d Its Relation to Disease. E. and S. Livingstone Ltd., London. (8) Joshi, I-I. D., and A. •. Drury. 1962. Pathogenicity of staphylococci associated with the bovine udder. Michigan State Univ. Yet., 23: 40. (9) Murray, D. L. 1962. Introduction. I n A. R. Drury, D. L. Murray, and G. W. Reed, ads. p. 3. Mastitis Control in Michigan Herds. Michigan State Univ., Ext. Bull. 344. (10) National Institutes of Health. 1938. Tentative staphylococcus toxoid requirements. Washington, D.C. (11) Pillet, J., O. Girard, t[. Dutheil, and B. Orta. 1959. Recherches sur ]'Immunisation de la t~rebls et Yaccination locale. Ann. Inst. Pasteur, 96: 591. (12) Plommet, M. 1960. Essais de Traitment de la Mammite staphylococcique de la ~'ache par Vaccination locale. Ann. Inst. Pasteur, 99 : 618. (13) Schu]tze, W. D., J. W. Smith, V. C. Womack, and M. A. Norcross. 1963. Observations on the efficacy of antistaphylococcal
BOVINE
vaccination in the control of mastit~s in a dairy cattle herd. J. Dairy Sci., 46: 625. (14) Skean, J. D., and W. W. Overcast. 1968. The efficacy of different adjuvants used with a staphylococcal vaccine in dairy cows. J. Dairy Sci., 51:1239. (15) Slanetz, L. W., and C. H. Bartley. 1953. The diagnosis of staphylococcal mastitis with special reference to the characteristics of mastitis staphylococci. J. Infectious Diseases, 92: 139.
MASTITIS
53
(16) Slanetz, L. W., C. I-I. Baxtley, and F. E. Allen. 1963. Vaccination of dairy cattle against staphylococcic mastltis. Amer. J . Vet. Res., 24: 923. (17) Slanetz, L. W., C. It. Bartley, and F. E. Allen. 1965. Evaluation of cell-toxoid vancines for the vaccination of dairy cattle against staphylococcic mastitis. Amer. J. Vet. Res., 26: 688. (18) Zemelman, R., and L. Longeri. 1965. Characterization of staphylococci isolated from raw milk. Appl. Microblol., 13: 167.
J. DAIRY SCIENCE VOL, 52, NO. I