Vaccination
Against Mastitis 1 R. W. MELLENBERGER Department of Dairy Science Michigan State University East Lansing 48824
ABSTRACT
toxoid vaccines is a potential means of increasing a cow's resistance to bacterial invasion of the udder. However, the variety of bacterial species involved in mastitis makes immunization a complex and questionable practice. For example, there are undetermined numbers of staphylococcal strains and 70-plus capsular varieties of Klebsiella. Thus, different strains of pathogenic bacteria often do not respond to an individual vaccine (17, 33).
Immunization of lactating dairy cows with bacterin-toxoid vaccines is a potential method of increasing a cow's resistance to bacterial invasion of the udder. Challenge experiments with mice, guinea pigs, goats, and dairy cows indicate that vaccination during lactation and during the nonlactating state will decrease the rate of new infections by Streptococcus agalactiae and Staphylococcus aureus and lessen the severity of clinical attacks. In contrast, field studies with commercial dairy herds have shown that immunization with bacterin-toxoid vaccines does not decrease the new infection rate caused by streptococcal and staphylococcal bacteria. Commercial vaccines have limited value against clinical mastitis since an autogenous vaccine is essential. Therefore, vaccination against mastitis in the lactating dairy cow is of limited value because 1) new infections are not prevented; 2) protection against severe clinical attacks requires an autogenous vaccine and repeated injections; 3) cost of vaccines is high. Further research is necessary on antigens, route of administration, and other factors to make vaccination a feasible management tool.
Vaccination--Definition and Theory
Vaccination against mastitis is defined as injection of a suspension of sensitized, attenuated, or killed bacteria into the body or udder to induce immunity against the same species of bacteria or their toxins. Vaccines developed from bacteria cultured from the cows to be inoculated are autogenous vaccines whereas a vaccine made from any other virulent strain or strains of the same bacterial species is a stock vaccine. In response to vaccination, a cow should develop an antibody titer in the blood and milk against particular bacterial strains or their toxins. Also, successful vaccination or immunization should prevent a majority of new infections caused by the bacterial strains for which the vaccine was intended. Vaccination Against Streptococcus agalactiae
Mastitis, despite much research, remains a costly disease in milk-producing animals. Practically and economically a prevention program offers the most viable solution to the mastitis problem. Despite prevention programs, mastitis caused by Staphylococcus aureus, Streptococcus nonagalactiae, and Gram negative Coliform species remains difficult to treat and control. Immunization of cows with bacterin-
Received January 12, 1977. 1Michigan Agricultural Experiment Station Journal Article No. 7917.
Streptococcus agalactiae (S. agalactiae) remains one of the most prevalent mastitis-causing organisms. In recent years intramammary therapy with penicillin and synthetic penicillins have been used to eradicate S. agalactiae from closed dairy herds. Prior to the use of antibiotics, vaccination was considered a potential means to prevent mammary infections caused by S. agalactiae. Holman et al. (11), Pattison (23) and Pattison and Holman (24) vaccinated mice, guinea pigs, and goats with whole cell preparations of several strains of S. agalactiae. These authors reported a strain-specific protection was provided by various routes of vaccina-
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OUR INDUSTRY TODAY tion against intraperitoneal or intramammary S. agalactiae challenges. In addition, Norcross and Stark (19) reported that dairy cows injected with whole S. agalactiae cells were resistant to an intramammary challenge of 2 to 5 S. agalactiae colony forming units. In contrast Howell et al. (12) reported no effect of vaccination on the new infection rate of S. agalactiae during a challenge experiment with dairy cows. Howell et al. (12) injected 22 cows subcutaneously with a series of six 10-ml doses of S. agalactiae strain A 100. Following an intramammary challenge with three concentrations of S. agalactiae strain A 100, the 22 vaccinated cows had lower neutrophil counts and a decreased duration of infection as compared with 22 unvaccinated cows, but vaccination did not affect the new infection rate of S. agalactiae. Field experiments utilizing S. agalactiae vaccines to prevent or control mastitis in the dairy cow contradict laboratory challenge experiments. Bracewell and Pattison (2) and Derbyshire (6) found no significant differences in the occurrence of new infections or clinical cases of mastitis between dairy cows vaccinated against S. agalactiae and nonvaccinated control cows. Bracewell and Pattison (2) concluded that vaccination was not a useful means of controlling S. agalactiae infections in commercial dairy herds. Apparently, the interaction of cow and environment has a major influence on the effectiveness of S. agalactiae vaccines. Vaccination Against Staphylococcus aureus
Since S t a p h y l o c o c c u s aureus (S. aureus) responds poorly to antibiotic therapy (28), research has been extensive on the vaccination of cattle, goats, and sheep with S. aureus toxin and bacterin-toxid preparations. Staphylococci possess many strain specific antigens which are either soluble or bound to the cell wall. Thus, S. aureus strains are potentially controllable with immunization procedures. Generally researchers have administered two or three intramuscular or subcutaneous injections of antigen to cows at 2- to 4-wk intervals followed by booster injections 6 to 12 mo later (1, 32, 33). Regardless of the type of vaccine, dose administered, or injection schedule, there is a wide variation in animal response which is partly due to the titer of serum antibodies at
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the start of the vaccination procedure. Bacterintoxoids stimulate the highest S. aureus antitoxin titers which reach a maximum within 2 to 3 wk after the first injection and then decline to prevaccination titers within 2 to 4 mo (1, 34). In addition, Pearson (25) reported that 6-mo booster injections could not maintain serum antibody titers against S. aureus toxins. Despite the increase in serum antibody titers, milk whey contains less than 2% as much staphylococcal antitoxin as blood serum since degree of permeability between mammary tissue and blood vessels determines the quantity of milk antitoxin. The antitoxin titer of milk does not increase following a subcutaneous or intramuscular injection of S. aureus antigen because an irritation or bacterial invasion of mammary epithelium is required before substantial quantities of circulating antibodies pass into mammary tissue or milk during lactation (5, 9, 14). Thus, S. aureus vaccines injected subcutaneously or intramuscularly conceivably could reduce the rate and severity of clinical and acute mastitis, but the new infection rate would remain the same (7, 25). Recent research by Tarjowski and Berman (38) indicates, however, that cows parentally immunized against S. aureus show a marked leukocytosis in response to a second intramammary challenge with killed whole S. aureus bacteria or cell wall material. The leukocytosis may indicate a cell-mediated immunity as a result of the vaccination. However, Tarjowski and Berman (38) also observed that the leukoeytosis actually may have increased S. aureus multiplication due to mammary tissue damage from the immune inflammatory response. Laboratory and field research data with parentally administered S. aureus bacterintoxoid vaccines have been controversial. Early work by Slanetz et al. (32, 33) indicated that intramuscular injections of S. aureus vaccine administered to lactating cows prevented an increase in the new infection rate while acute infections were reduced 50% as compared with nonvaecinated cows. Derbyshire (5) and Blobel and Berman (1) confirmed that vaccination decreased the clinical rate of S. aureus, but the new infection rate in these trials was not different from control cows. In contrast, Pearson (25) reported that vaccination against S. aureus did not prevent or control S. aureus infections. During the various field studies with Journal of Dairy Science Vol. 60, No. 6
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S. aureus vaccines, heterologous vaccines pro-
vided little or no protection against specific strains of S, aureus in cows or goats (5, 7, 31). Derbyshire (5) suggested that immunity against S. aureus produced in the cow's udder was a local effect since vaccinated udder halves showed an increased leukocytosis to S. aureus challenge while no resistance was apparent in nonvaccinated udder halves. McDowell and Lascelles (16), McDowell and Watson (17) and Outteridge et al. (20, 22) utilized the local immunity theory when they vaccinated ewes against S. aureus during the nonlactating or dry period. Vaccination during the dry period increases the potential transfer of antitoxins from blood serum to mammary tissue since cellular permeability in the udder increases as parturition approaches (6). Colostrum from vaccinated cows or ewes will contain antitoxin titers equal to or in excess of titers in blood. However, the antitoxin titer in the milk decreases as the transition from colostrum to milk occurs. Data on dry period immunization against S. aureus indicate that autogenous S, aureus bacterin-toxoid vaccines decrease the severity of clinical attacks but not the incidence of new S. aureus infections. Outteridge and Lascelles (20) immunized ewes with a polyvalent celltoxoid during the nonlactating period and found that vaccinated ewes produced 18% more milk than controls during the first 4 days following an intramammary challenge with 10 6 S. aureus cells 3 wk postpartum. McDowell and Watson (17) administered (intramuscular and intramammary injections) an autogenous S, aureus bacterin-toxoid vaccine to ewes during the dry period. The ewes were challenged with an intramammary injection of 106 S. aureus cells 30 days after parturition. Most ewes (immunized, nonimmunized) developed severe clinical mastitis. However, immunized ewes had lower body temperatures, less udder inflammation, and higher milk production compared with controls. Schultze et al. (30) found no difference in the new infection rate between vaccinated and nonvaccinated cows from a commercial S. aureus vaccine 2.
2Jcn. Sal Lab, Kansas City, MO. Journal of Dairy Science Vol. 60, No. 6
Vaccination Against Streptococcus nonagalactiae, Gram Negative Coliform Species, and Mvcoplasmas
An immunization program for these organisms could be beneficial as their response to antibiotic therapy is variable and eradication is not practical because they exist in many areas of the environment. Smith et al. (36) reported that the goat mammary gland had an increased resistance to Streptococcus dysgalactiae infections following immunization with an autogenous bacterin. Stark and Norcross (37) indicated no cross reactivity between antibodies against several strains of S. dysgalactiae. Coliform infections comprise only 1 to 2% of the total mammary gland infections. This low rate could be partially due to the fact that most cows have significant natural antibody to Gram negative species which has been stimulated by constant exposure to the endotoxic antigen of these organisms. Schalm et al. (27) suggested that these normally occurring titers may play a significant role during the early stages of a coliform infection to control unlimited growth of the organism provided the antibody or antitoxin could cross from the serum to milk. Schalm et al. (28) found that serum from cows vaccinated with Aerobacter aerogenes possessed greater bactericidal activity for the organism than did serum of control cows although hemagglutinating antibodies were not found following experimental udder infections with the organism. Dairymen faced with acute outbreaks of coliform mastiffs (18) have used autogenous bacterins to decrease the rate of acute infections but not the new infection rate (8). Perhaps the best solution is to control the new infection rate of Gram negative coliform species and S t r e p t o c o c c u s nonagatactiae species through sanitation and hygiene. Mycoplasma organisms have been responsible for major mastiffs problems in California (27) and Europe (10). Gourlay et al. (9) reported that cows inoculated (intramammary) with virulent strains of Mycoplasma dispar were resistant to a M. dispar challenge 3 to 4 wk later. Challenge of quarters that had been inoculated previously with virulent M. dispar resulted in a massive polymorphonuclear response similar to the finding of Tarjowski and Berman (38). Work by Carroll et al. (4) with Mycoplasma agalactiae var. boris points out the short duration of antigenic stimulation. The
OUR INDUSTRY TODAY injection of three strains of M. agalactiae var. bovis from mastitic cows into rabbits resulted in a significant indirect hemagglutination titer. However, despite continued intravenous antigen administration, the hemagglutination titers decreased sharply after 4 to 6 wk, and bacterial inhibiting antibodies were not found. Vaccines-Route of Administration
Immunization via subcutaneous or intramuscular injections is transitory, and gland irritation associated with subclinical infections is n o t sufficient to permit transfer of substantial quantities of antibodies from blood to milk (14). Subcutaneous and intramuscular injections of vaccine increased the blood serum antibody titer (2, 32, 34), but Pearson (25) states "that there is no constant relationship between blood titer and staphylococcal udder infection, neither does it appear from our results that such a titer stimulated by an udder infection gives any protection whatsoever against further bacterial invasion of the udder tissue". Intramammary immunization is a possibility in the dry or lactating cow, but severe inflammation problems caused by the vaccine (13, 17, 19) plus the short antibody titer life (2 to 6 wk of lactation) limit the usefulness of intramammary immunization. Commercial Vaccination Products
One commercially available product is a whey protein antibody s. This product is designed to be injected subcutaneously during the dry period and early lactation to aid in the prevention and treatment of specific bacteria or to aid in the reduction of California Mastitis Test scores caused by the bacteria stated on the label. Derbyshire and Edwards (7), MacKenzie et al. (14) and Reiter and Bramley (26) indicated that serum antibodies cannot cross the mammary epithelial cell barrier during lactation until an irritation such as clinical mastitis is present. Therefore, from a theoretical viewpoint the subcutaneous injection of
3 lmpro Products, Inc., Waukon, IA or Eitzen, MN. 4 Somato-Staph; Anchor Laboratories, Inc., Indianapolis, IN. SJen-Sal. Labs, Kansas City, MO.
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whey protein antibody during the dry period or during lactation should not be effective in the prevention of mastitis. Work by Smith et al. (35) and Marx (15) indicated a nonsignificant increase in the number of new S. agalactiae and S. aureus infections following the use of the whey protein antibody. Schellpfeffer and Hunter (29) determined that the whey antibody preparation contained only minute quantities of gamma globulin. A second commercial vaccine 4 contains five phage types of S. aureus as a lysed preparation. Recommended vaccination procedure is to inject the vaccine intramuscularly twice with 15 days during lactation. Work by Williams et al. (39) indicated that this commercial vaccine reduced the number of new S. a u r e u s infections. However, serum antibodies do not cross the mammary cell barrier during lactation until tissue damage occurs, so it is improbable that this commercial stock vaccine could prevent S. aureus infections. A third commercial vaccine s against mastitis has not prevented mammary gland infections or altered the clinical rate (15, 30). Cost of the three commercial vaccines ranges from $5 to $10 or more per injection. Booster injections are recommended each 6 to 12 mo with the original injection schemes remaining the same each year because of the lack of a true immunization (25). Commercial vaccines, therefore, have not been beneficial in the control of mastitis. A true long-lasting immunization against any mastitis-causing organism cannot be achieved with vaccines available. It is possible that immunization in dry period with an autogenous bacterin-toxoid may have some benefit against the severity of S. a u r e u s and Gram negative coliform infections during parturition and early lactation. However, this is a transitory protection and actually masks the effort to find the real cause of the problem. SUMMARY
Bruford et al. (3) stated that "in any bacterial disease the possibility of conferring an active immunity by vacciantion has to be considered. The difficulties with mastitis have been, firstly, the many different organisms associated with the disease, and secondly, the fact that infection of the gland, so long as it Journal
of Dairy Science Vol. 60, No. 6
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r e m a i n s in t h e m a m m a r y d u c t s a n d alveoli, is s e p a r a t e d f r o m h u m o r a l a n t i b o d i e s . It is only w h e n t h e r e is a c t u a l p e n e t r a t i o n o f t h e m u c o u s m e m b r a n e as a result o f i n f e c t i o n or i r r i t a t i o n , t h a t h u m o r a l a n t i b o d i e s are able t o c o m e in c o n t a c t w i t h t h e o r g a n i s m s t h e y are m e a n t to c o m b a t . T h u s , v a c c i n a t i o n w o u l d a p p e a r to b e capable o f p r o t e c t i n g o n l y against clinical f o r m s o f m a s t i t i s " . In general, a n t i b a c t e r i a l f a c t o r s n o r m a l l y in m i l k have little i n f l u e n c e o n t h e course o f a m a s t i t i s i n f e c t i o n , a n d a n y beneficial e f f e c t p r o b a b l y is o v e r s h a d o w e d by t h e cellular r e s p o n s e to i n f e c t i o n (6). V a c c i n a t i o n against m a s t i t i s m a y b e c o m e practical in t h e f u t u r e . However, m o r e research is n e e d e d o n antigens, t h e i r specificity a n d l o n g e v i t y (19). D a i r y m e n are c o n f u s e d t h a t v a c c i n a t i o n against Brucellosis, Leptospirosis, a n d o t h e r diseases successfully p r e v e n t s these diseases f o r m a n y m o n t h s o r years. D a i r y m e n n e e d to have t h e same assurance with m a s t i t i s v a c c i n a t i o n b e f o r e it is a r e c o m m e n d e d procedure. S c h a l m et al. (27) s u m m a r i z e d v a c c i n a t i o n against m a s t i t i s as follows: 1) Large a n d r e p e a t e d i n j e c t i o n s of v a c c i n e are r e q u i r e d t o p r o d u c e high s e r u m antib o d y titers, a n d these titers are o n l y m a i n t a i n e d for a f e w m o n t h s . Vaccines are strain specific a n d d o n o t p r o t e c t a d e q u a t e l y against h e t e r o l o g o u s strains. 2) A n i n v a d i n g o r g a n i s m w o u l d have to m u l t i p l y in t h e g l a n d in sufficient n u m bers t o i n i t i a t e an i n f l a m m a t o r y r e s p o n s e n e c e s s a r y t o a l l o w s e r u m a n t i b o d i e s to cross t h e cell b a r r i e r b e t w e e n b l o o d a n d milk. T h e r e f o r e , v a c c i n a t i o n does n o t p r o t e c t t h e u d d e r against initial i n f e c t i o n . 3) O n l y u n d e r e x p e r i m e n t a l c o n d i t i o n s , as o p p o s e d t o n a t u r a l f a r m c o n d i t i o n s , have vaccines i n c r e a s e d a c o w ' s r e s i s t a n c e t o m a s t i t i s ( l o w e r clinical rate). 4) A n t i b o d y titers to bacterial t o x i n s increase in m i l k at t h e t i m e of p a r t u r i t i o n . However, t h e i n c i d e n c e of m a s t i t i s is high at p a r t u r i t i o n , especially cows w i t h chronic udder infections. REFERENCES
1 Blobel, H., and D. T. Berman. 1962. Vaccination of dairy cattle against staphylococci mastitis. Amer. J. Vet. Res. 23:7. Journal of Dairy Science Vol. 60, No. 6
2 Bracewell, C. D., and I. H. Pattison. 1958. Experimental streptococcal mastltis. XII. Rarther immunological studies in the cow. J. Comp. Path. Therap. 68:121. 3 Bruford, J. W., R. M. Loosmore, P. S. Blackburn, D. L. Hughes, J. K. L. Pearson, O. Ulvarov, J. B. White, and C. D. Wilson. 1965. Controlling bovine mastitis. Vet. Rec. 77:612. 4 Carroll, E. J., M. Rollins, and D. E. Jasper. 1976. The immune response of rabbits to 3 strains of Mycoplasma agalactiae var. boris isolated from mastitic bovine udders. Cornel[ Vet. 66:143. 5 Derbyshire, J. B. 1961. Further immunological studies in experimental staphylococcal mastitis. J. Comp. Path. Therap. 71:146. 6 Derbyshire, J. B. 1962. Immunity in bovine mastiffs. Vet. Bull. 32:1. 7 Derbyshire, ]. B., and S. J. Edwards. 1963. A field trial of a staphylococcal cell-toxoid vaccine in the control of staphylococcal mastitis in cows. Vet. Rec. 73:1208. 8 Gibson, C. 1975. Personal communication. 9 Gourlay, R. M., C. J. Howard, and J. Brownlie. 1975. Localized immunity in experimental bovine mastitis caused by Mycoplasma dispar. Infect. ~mmun. 12:947. 10 Gourlay, R. N., E. J. Stott, J. Espinasse, and C. Barle. 1974. Isolation of Mycoplasma agalactiae var. boris and infectious bovine rhinotracheitis virus from an outbreak of masdtis in France. Vet. Rec. 93:534. 11 Holman, H. H., I. H. Pattison, and W. S. Gordon. 1952. Studies on experimental streptococcal mastitis. VII. Immunological studies in goats. J. Comp. Path. Therap. 63:196. t 2 Howell, D. G., I. M. Smith, H. H. Holman, and I. H. Pattison. 1956. Experimental streptococcal mastitis. Vi. Immunological studies in the cow. J. Comp. Path. Therap. 66:49. 13 Kerr, W. R., J. K. L. Pearson, and J. E. F. Rankin. 1959. The bovine udder and its agglutinins. Brit. Vet. J. 115:105. 14 MacKenzie, D. D. S., P. M. Outteridge, and A. K. Lascelles. 1966. Aust. J. Exp. Biol. Med. Sci. 44:181. 15 Marx, G. D. 1976. Mastitis and milking management studies. Minnesota Dairy Rep. 16 McDowell, G. H., and A. K. Lascelles. 1971. Local immunization of ewes with staphylococcal cell and cell-toxoid vaccines. Res. Vet. Sci. 12:258. 17 McDowell, G. H., and D. L. Watson. 1974. Immunity to experimental staphylococcal mastitis: Comparison of local and systemic immunization. Aust. Vet. J. 50:533. 18 Newman, L. E., and J. J. Kowalski. 1973. Fresh sawdust bedding-a possible source of Klebsiella organisms. Arner. J. Vet. Res. 34:979. 19 Norcross, N. L., and D. M. Stark. 1969. Role of immunization in mastiffs control. J. Dairy Sci. 52:714. 20 Outteridge, P. M., and A. K. Lascelles. 1967. Local immunity in the lactating mammary gland following the infusion of staphylococcal toxoids. Res. Vet. Sci. 8:313.
O U R INDUSTRY T O D A Y 21 Outteridge, P. M., J. D. Rock, and A. K. Lascelles. 1965. The i m m u n e response of the m a m m a r y gland and regional l y m p h node following antigenic stimulation. Aust. J. Exp. Biol. Med. Sci. 43:265. 22 Outteridge, P. M., R. C. Williams, and A. K. Lascelles. 1968. Local i m m u n i t y in the m a m m a r y gland following the infusion of a staphylococcal cell-toxoid vaccine. Res. Vet. Sci. 9:416. 23 Pattison, I. H. 1948. Immunological studies with Group-B streptococci. J. Path. Bact. 60;219. 24 Pattison, I. H., and H. H. Holman. 1953. Experimental streptococcal mastitis. VIII. Further i m m u nological studies in goats. J. Comp. Path. Therap. 63:304. 25 Pearson, J. K. L. 1959. A u t o g e n o u s toxoid vaccine in the prophylaxis of staphylococcal mastiffs in cattle. J. Dairy Res. 26:9. 26 Reiter, B., and A. J. Bramley. 1975. Defense m e c h a n i s m s of t h e udder and their relevance to mastitis control. Paper presented at the International Dairy Federation meeting at NIRD, Shinfield, Reading, England. April, 1975. 27 Schalm, O. W., E. J. Carroll, and N. C. Jain. 1971. Bovine mastitis. Lea and Febiger, Philadelphia, PA. 28 Schalm, O. W., E. J. Carroll, and J. Lasmanis. 1964. The leukocyte barrier and serologic investigations of experimental coliform (Aerobacter aerogenes) mastitis in cattle. Amer. J. Vet. Res. 25:90. 29 Schellpfeffer, D. A., and A. G. Hunter. 1971. Characterization of Impro., a w h e y antibody preparation. J. Dairy Sci. 54:798. (Abstr.). 30 Schultze, W. D., J. W. Smith, V. C. W o m m a c k , a n d M. A. Norcross. 1963. Observations on the efficacy of antistaphylococcal vaccination in the control of mastitis in a dairy cattle herd. J. Dairy Sci. 46:625. (Abstr.).
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31 Singleton, L., G. W. Ross, R. A. Stedman, and K. V. Chanter. 1967. I m m u n i z a t i o n with staphylococcal cell walls against mastitis. J. Comp. Path. Therap. 77:279. 32 Slanetz, L. W., C. H. Bartley, and F. E. Allen. 1959. The immunization of dairy cattle against staphylococcal mastitis. J. Amer. Vet. Med. Ass. 134:155. 33 Slanetz, L. W., C. H. Bartley, and F. E. Alien. 1963. Vaccination of dairy cattle against staphylococcic mastitis. Amer. J. Vet. Res. 24:923. 34 Slanetz, L. W., C. H. Bartley, and F. E. Allen. 1965. Evaluation of cell-toxoid vaccines for the vaccination of dairy cattle against staphylococcic mastiffs. Amer. J. Vet. Res. 26:688. 35 Smith, J. W., C. A. Kiddy, R. D. Plowman, W. D. Schultze, and N. W. Hooven, Jr. 1970. Whey antibody preparation: Effects of p r e p a r t u m injection on milk production in dairy cows. Amer. J. Vet. Res. 31:1485. 36 Smith, I. M., I. H. Pattison, and H. H. Holman. 1954. The increased resistance of t h e udder o f goats to infection with Streptococcus dysgalactiae strain 419, after vaccination with the same strain. J. Comp. Path. Therap. 64:206. 37 Stark, D. M., and N. L. Norcross. 1969. A passive hemagglutination technique for detection of Streptococcus dysgalactiae antibody. Amer. J. Vet. Res. 30:127. 38 Tarjowski, S. P., and D. T. Berman. 1975. Leukocytic response of bovine m a m m a r y gland to injection of killed cells and cell walls of Staphylococcus aureus. Amer. J. Vet. Res. 36:1561. 39 Williams, J. M., H. J. Mayerhofer, and R. W. Brown. 1966. Clinical evaluation of a Staphylococcus aureus bacterin (polyvalent somatic antigen). Vet. Med./SAC 61: 789.
Journal of Dairy Science Vol. 60, No. 6
Against Mastitis 1 R. W. MELLENBERGER Department of Dairy Science Michigan State University East Lansing 48824
ABSTRACT
toxoid vaccines is a potential means of increasing a cow's resistance to bacterial invasion of the udder. However, the variety of bacterial species involved in mastitis makes immunization a complex and questionable practice. For example, there are undetermined numbers of staphylococcal strains and 70-plus capsular varieties of Klebsiella. Thus, different strains of pathogenic bacteria often do not respond to an individual vaccine (17, 33).
Immunization of lactating dairy cows with bacterin-toxoid vaccines is a potential method of increasing a cow's resistance to bacterial invasion of the udder. Challenge experiments with mice, guinea pigs, goats, and dairy cows indicate that vaccination during lactation and during the nonlactating state will decrease the rate of new infections by Streptococcus agalactiae and Staphylococcus aureus and lessen the severity of clinical attacks. In contrast, field studies with commercial dairy herds have shown that immunization with bacterin-toxoid vaccines does not decrease the new infection rate caused by streptococcal and staphylococcal bacteria. Commercial vaccines have limited value against clinical mastitis since an autogenous vaccine is essential. Therefore, vaccination against mastitis in the lactating dairy cow is of limited value because 1) new infections are not prevented; 2) protection against severe clinical attacks requires an autogenous vaccine and repeated injections; 3) cost of vaccines is high. Further research is necessary on antigens, route of administration, and other factors to make vaccination a feasible management tool.
Vaccination--Definition and Theory
Vaccination against mastitis is defined as injection of a suspension of sensitized, attenuated, or killed bacteria into the body or udder to induce immunity against the same species of bacteria or their toxins. Vaccines developed from bacteria cultured from the cows to be inoculated are autogenous vaccines whereas a vaccine made from any other virulent strain or strains of the same bacterial species is a stock vaccine. In response to vaccination, a cow should develop an antibody titer in the blood and milk against particular bacterial strains or their toxins. Also, successful vaccination or immunization should prevent a majority of new infections caused by the bacterial strains for which the vaccine was intended. Vaccination Against Streptococcus agalactiae
Mastitis, despite much research, remains a costly disease in milk-producing animals. Practically and economically a prevention program offers the most viable solution to the mastitis problem. Despite prevention programs, mastitis caused by Staphylococcus aureus, Streptococcus nonagalactiae, and Gram negative Coliform species remains difficult to treat and control. Immunization of cows with bacterin-
Received January 12, 1977. 1Michigan Agricultural Experiment Station Journal Article No. 7917.
Streptococcus agalactiae (S. agalactiae) remains one of the most prevalent mastitis-causing organisms. In recent years intramammary therapy with penicillin and synthetic penicillins have been used to eradicate S. agalactiae from closed dairy herds. Prior to the use of antibiotics, vaccination was considered a potential means to prevent mammary infections caused by S. agalactiae. Holman et al. (11), Pattison (23) and Pattison and Holman (24) vaccinated mice, guinea pigs, and goats with whole cell preparations of several strains of S. agalactiae. These authors reported a strain-specific protection was provided by various routes of vaccina-
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OUR INDUSTRY TODAY tion against intraperitoneal or intramammary S. agalactiae challenges. In addition, Norcross and Stark (19) reported that dairy cows injected with whole S. agalactiae cells were resistant to an intramammary challenge of 2 to 5 S. agalactiae colony forming units. In contrast Howell et al. (12) reported no effect of vaccination on the new infection rate of S. agalactiae during a challenge experiment with dairy cows. Howell et al. (12) injected 22 cows subcutaneously with a series of six 10-ml doses of S. agalactiae strain A 100. Following an intramammary challenge with three concentrations of S. agalactiae strain A 100, the 22 vaccinated cows had lower neutrophil counts and a decreased duration of infection as compared with 22 unvaccinated cows, but vaccination did not affect the new infection rate of S. agalactiae. Field experiments utilizing S. agalactiae vaccines to prevent or control mastitis in the dairy cow contradict laboratory challenge experiments. Bracewell and Pattison (2) and Derbyshire (6) found no significant differences in the occurrence of new infections or clinical cases of mastitis between dairy cows vaccinated against S. agalactiae and nonvaccinated control cows. Bracewell and Pattison (2) concluded that vaccination was not a useful means of controlling S. agalactiae infections in commercial dairy herds. Apparently, the interaction of cow and environment has a major influence on the effectiveness of S. agalactiae vaccines. Vaccination Against Staphylococcus aureus
Since S t a p h y l o c o c c u s aureus (S. aureus) responds poorly to antibiotic therapy (28), research has been extensive on the vaccination of cattle, goats, and sheep with S. aureus toxin and bacterin-toxid preparations. Staphylococci possess many strain specific antigens which are either soluble or bound to the cell wall. Thus, S. aureus strains are potentially controllable with immunization procedures. Generally researchers have administered two or three intramuscular or subcutaneous injections of antigen to cows at 2- to 4-wk intervals followed by booster injections 6 to 12 mo later (1, 32, 33). Regardless of the type of vaccine, dose administered, or injection schedule, there is a wide variation in animal response which is partly due to the titer of serum antibodies at
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the start of the vaccination procedure. Bacterintoxoids stimulate the highest S. aureus antitoxin titers which reach a maximum within 2 to 3 wk after the first injection and then decline to prevaccination titers within 2 to 4 mo (1, 34). In addition, Pearson (25) reported that 6-mo booster injections could not maintain serum antibody titers against S. aureus toxins. Despite the increase in serum antibody titers, milk whey contains less than 2% as much staphylococcal antitoxin as blood serum since degree of permeability between mammary tissue and blood vessels determines the quantity of milk antitoxin. The antitoxin titer of milk does not increase following a subcutaneous or intramuscular injection of S. aureus antigen because an irritation or bacterial invasion of mammary epithelium is required before substantial quantities of circulating antibodies pass into mammary tissue or milk during lactation (5, 9, 14). Thus, S. aureus vaccines injected subcutaneously or intramuscularly conceivably could reduce the rate and severity of clinical and acute mastitis, but the new infection rate would remain the same (7, 25). Recent research by Tarjowski and Berman (38) indicates, however, that cows parentally immunized against S. aureus show a marked leukocytosis in response to a second intramammary challenge with killed whole S. aureus bacteria or cell wall material. The leukocytosis may indicate a cell-mediated immunity as a result of the vaccination. However, Tarjowski and Berman (38) also observed that the leukoeytosis actually may have increased S. aureus multiplication due to mammary tissue damage from the immune inflammatory response. Laboratory and field research data with parentally administered S. aureus bacterintoxoid vaccines have been controversial. Early work by Slanetz et al. (32, 33) indicated that intramuscular injections of S. aureus vaccine administered to lactating cows prevented an increase in the new infection rate while acute infections were reduced 50% as compared with nonvaecinated cows. Derbyshire (5) and Blobel and Berman (1) confirmed that vaccination decreased the clinical rate of S. aureus, but the new infection rate in these trials was not different from control cows. In contrast, Pearson (25) reported that vaccination against S. aureus did not prevent or control S. aureus infections. During the various field studies with Journal of Dairy Science Vol. 60, No. 6
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S. aureus vaccines, heterologous vaccines pro-
vided little or no protection against specific strains of S, aureus in cows or goats (5, 7, 31). Derbyshire (5) suggested that immunity against S. aureus produced in the cow's udder was a local effect since vaccinated udder halves showed an increased leukocytosis to S. aureus challenge while no resistance was apparent in nonvaccinated udder halves. McDowell and Lascelles (16), McDowell and Watson (17) and Outteridge et al. (20, 22) utilized the local immunity theory when they vaccinated ewes against S. aureus during the nonlactating or dry period. Vaccination during the dry period increases the potential transfer of antitoxins from blood serum to mammary tissue since cellular permeability in the udder increases as parturition approaches (6). Colostrum from vaccinated cows or ewes will contain antitoxin titers equal to or in excess of titers in blood. However, the antitoxin titer in the milk decreases as the transition from colostrum to milk occurs. Data on dry period immunization against S. aureus indicate that autogenous S, aureus bacterin-toxoid vaccines decrease the severity of clinical attacks but not the incidence of new S. aureus infections. Outteridge and Lascelles (20) immunized ewes with a polyvalent celltoxoid during the nonlactating period and found that vaccinated ewes produced 18% more milk than controls during the first 4 days following an intramammary challenge with 10 6 S. aureus cells 3 wk postpartum. McDowell and Watson (17) administered (intramuscular and intramammary injections) an autogenous S, aureus bacterin-toxoid vaccine to ewes during the dry period. The ewes were challenged with an intramammary injection of 106 S. aureus cells 30 days after parturition. Most ewes (immunized, nonimmunized) developed severe clinical mastitis. However, immunized ewes had lower body temperatures, less udder inflammation, and higher milk production compared with controls. Schultze et al. (30) found no difference in the new infection rate between vaccinated and nonvaccinated cows from a commercial S. aureus vaccine 2.
2Jcn. Sal Lab, Kansas City, MO. Journal of Dairy Science Vol. 60, No. 6
Vaccination Against Streptococcus nonagalactiae, Gram Negative Coliform Species, and Mvcoplasmas
An immunization program for these organisms could be beneficial as their response to antibiotic therapy is variable and eradication is not practical because they exist in many areas of the environment. Smith et al. (36) reported that the goat mammary gland had an increased resistance to Streptococcus dysgalactiae infections following immunization with an autogenous bacterin. Stark and Norcross (37) indicated no cross reactivity between antibodies against several strains of S. dysgalactiae. Coliform infections comprise only 1 to 2% of the total mammary gland infections. This low rate could be partially due to the fact that most cows have significant natural antibody to Gram negative species which has been stimulated by constant exposure to the endotoxic antigen of these organisms. Schalm et al. (27) suggested that these normally occurring titers may play a significant role during the early stages of a coliform infection to control unlimited growth of the organism provided the antibody or antitoxin could cross from the serum to milk. Schalm et al. (28) found that serum from cows vaccinated with Aerobacter aerogenes possessed greater bactericidal activity for the organism than did serum of control cows although hemagglutinating antibodies were not found following experimental udder infections with the organism. Dairymen faced with acute outbreaks of coliform mastiffs (18) have used autogenous bacterins to decrease the rate of acute infections but not the new infection rate (8). Perhaps the best solution is to control the new infection rate of Gram negative coliform species and S t r e p t o c o c c u s nonagatactiae species through sanitation and hygiene. Mycoplasma organisms have been responsible for major mastiffs problems in California (27) and Europe (10). Gourlay et al. (9) reported that cows inoculated (intramammary) with virulent strains of Mycoplasma dispar were resistant to a M. dispar challenge 3 to 4 wk later. Challenge of quarters that had been inoculated previously with virulent M. dispar resulted in a massive polymorphonuclear response similar to the finding of Tarjowski and Berman (38). Work by Carroll et al. (4) with Mycoplasma agalactiae var. boris points out the short duration of antigenic stimulation. The
OUR INDUSTRY TODAY injection of three strains of M. agalactiae var. bovis from mastitic cows into rabbits resulted in a significant indirect hemagglutination titer. However, despite continued intravenous antigen administration, the hemagglutination titers decreased sharply after 4 to 6 wk, and bacterial inhibiting antibodies were not found. Vaccines-Route of Administration
Immunization via subcutaneous or intramuscular injections is transitory, and gland irritation associated with subclinical infections is n o t sufficient to permit transfer of substantial quantities of antibodies from blood to milk (14). Subcutaneous and intramuscular injections of vaccine increased the blood serum antibody titer (2, 32, 34), but Pearson (25) states "that there is no constant relationship between blood titer and staphylococcal udder infection, neither does it appear from our results that such a titer stimulated by an udder infection gives any protection whatsoever against further bacterial invasion of the udder tissue". Intramammary immunization is a possibility in the dry or lactating cow, but severe inflammation problems caused by the vaccine (13, 17, 19) plus the short antibody titer life (2 to 6 wk of lactation) limit the usefulness of intramammary immunization. Commercial Vaccination Products
One commercially available product is a whey protein antibody s. This product is designed to be injected subcutaneously during the dry period and early lactation to aid in the prevention and treatment of specific bacteria or to aid in the reduction of California Mastitis Test scores caused by the bacteria stated on the label. Derbyshire and Edwards (7), MacKenzie et al. (14) and Reiter and Bramley (26) indicated that serum antibodies cannot cross the mammary epithelial cell barrier during lactation until an irritation such as clinical mastitis is present. Therefore, from a theoretical viewpoint the subcutaneous injection of
3 lmpro Products, Inc., Waukon, IA or Eitzen, MN. 4 Somato-Staph; Anchor Laboratories, Inc., Indianapolis, IN. SJen-Sal. Labs, Kansas City, MO.
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whey protein antibody during the dry period or during lactation should not be effective in the prevention of mastitis. Work by Smith et al. (35) and Marx (15) indicated a nonsignificant increase in the number of new S. agalactiae and S. aureus infections following the use of the whey protein antibody. Schellpfeffer and Hunter (29) determined that the whey antibody preparation contained only minute quantities of gamma globulin. A second commercial vaccine 4 contains five phage types of S. aureus as a lysed preparation. Recommended vaccination procedure is to inject the vaccine intramuscularly twice with 15 days during lactation. Work by Williams et al. (39) indicated that this commercial vaccine reduced the number of new S. a u r e u s infections. However, serum antibodies do not cross the mammary cell barrier during lactation until tissue damage occurs, so it is improbable that this commercial stock vaccine could prevent S. aureus infections. A third commercial vaccine s against mastitis has not prevented mammary gland infections or altered the clinical rate (15, 30). Cost of the three commercial vaccines ranges from $5 to $10 or more per injection. Booster injections are recommended each 6 to 12 mo with the original injection schemes remaining the same each year because of the lack of a true immunization (25). Commercial vaccines, therefore, have not been beneficial in the control of mastitis. A true long-lasting immunization against any mastitis-causing organism cannot be achieved with vaccines available. It is possible that immunization in dry period with an autogenous bacterin-toxoid may have some benefit against the severity of S. a u r e u s and Gram negative coliform infections during parturition and early lactation. However, this is a transitory protection and actually masks the effort to find the real cause of the problem. SUMMARY
Bruford et al. (3) stated that "in any bacterial disease the possibility of conferring an active immunity by vacciantion has to be considered. The difficulties with mastitis have been, firstly, the many different organisms associated with the disease, and secondly, the fact that infection of the gland, so long as it Journal
of Dairy Science Vol. 60, No. 6
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r e m a i n s in t h e m a m m a r y d u c t s a n d alveoli, is s e p a r a t e d f r o m h u m o r a l a n t i b o d i e s . It is only w h e n t h e r e is a c t u a l p e n e t r a t i o n o f t h e m u c o u s m e m b r a n e as a result o f i n f e c t i o n or i r r i t a t i o n , t h a t h u m o r a l a n t i b o d i e s are able t o c o m e in c o n t a c t w i t h t h e o r g a n i s m s t h e y are m e a n t to c o m b a t . T h u s , v a c c i n a t i o n w o u l d a p p e a r to b e capable o f p r o t e c t i n g o n l y against clinical f o r m s o f m a s t i t i s " . In general, a n t i b a c t e r i a l f a c t o r s n o r m a l l y in m i l k have little i n f l u e n c e o n t h e course o f a m a s t i t i s i n f e c t i o n , a n d a n y beneficial e f f e c t p r o b a b l y is o v e r s h a d o w e d by t h e cellular r e s p o n s e to i n f e c t i o n (6). V a c c i n a t i o n against m a s t i t i s m a y b e c o m e practical in t h e f u t u r e . However, m o r e research is n e e d e d o n antigens, t h e i r specificity a n d l o n g e v i t y (19). D a i r y m e n are c o n f u s e d t h a t v a c c i n a t i o n against Brucellosis, Leptospirosis, a n d o t h e r diseases successfully p r e v e n t s these diseases f o r m a n y m o n t h s o r years. D a i r y m e n n e e d to have t h e same assurance with m a s t i t i s v a c c i n a t i o n b e f o r e it is a r e c o m m e n d e d procedure. S c h a l m et al. (27) s u m m a r i z e d v a c c i n a t i o n against m a s t i t i s as follows: 1) Large a n d r e p e a t e d i n j e c t i o n s of v a c c i n e are r e q u i r e d t o p r o d u c e high s e r u m antib o d y titers, a n d these titers are o n l y m a i n t a i n e d for a f e w m o n t h s . Vaccines are strain specific a n d d o n o t p r o t e c t a d e q u a t e l y against h e t e r o l o g o u s strains. 2) A n i n v a d i n g o r g a n i s m w o u l d have to m u l t i p l y in t h e g l a n d in sufficient n u m bers t o i n i t i a t e an i n f l a m m a t o r y r e s p o n s e n e c e s s a r y t o a l l o w s e r u m a n t i b o d i e s to cross t h e cell b a r r i e r b e t w e e n b l o o d a n d milk. T h e r e f o r e , v a c c i n a t i o n does n o t p r o t e c t t h e u d d e r against initial i n f e c t i o n . 3) O n l y u n d e r e x p e r i m e n t a l c o n d i t i o n s , as o p p o s e d t o n a t u r a l f a r m c o n d i t i o n s , have vaccines i n c r e a s e d a c o w ' s r e s i s t a n c e t o m a s t i t i s ( l o w e r clinical rate). 4) A n t i b o d y titers to bacterial t o x i n s increase in m i l k at t h e t i m e of p a r t u r i t i o n . However, t h e i n c i d e n c e of m a s t i t i s is high at p a r t u r i t i o n , especially cows w i t h chronic udder infections. REFERENCES
1 Blobel, H., and D. T. Berman. 1962. Vaccination of dairy cattle against staphylococci mastitis. Amer. J. Vet. Res. 23:7. Journal of Dairy Science Vol. 60, No. 6
2 Bracewell, C. D., and I. H. Pattison. 1958. Experimental streptococcal mastltis. XII. Rarther immunological studies in the cow. J. Comp. Path. Therap. 68:121. 3 Bruford, J. W., R. M. Loosmore, P. S. Blackburn, D. L. Hughes, J. K. L. Pearson, O. Ulvarov, J. B. White, and C. D. Wilson. 1965. Controlling bovine mastitis. Vet. Rec. 77:612. 4 Carroll, E. J., M. Rollins, and D. E. Jasper. 1976. The immune response of rabbits to 3 strains of Mycoplasma agalactiae var. boris isolated from mastitic bovine udders. Cornel[ Vet. 66:143. 5 Derbyshire, J. B. 1961. Further immunological studies in experimental staphylococcal mastitis. J. Comp. Path. Therap. 71:146. 6 Derbyshire, J. B. 1962. Immunity in bovine mastiffs. Vet. Bull. 32:1. 7 Derbyshire, ]. B., and S. J. Edwards. 1963. A field trial of a staphylococcal cell-toxoid vaccine in the control of staphylococcal mastitis in cows. Vet. Rec. 73:1208. 8 Gibson, C. 1975. Personal communication. 9 Gourlay, R. M., C. J. Howard, and J. Brownlie. 1975. Localized immunity in experimental bovine mastitis caused by Mycoplasma dispar. Infect. ~mmun. 12:947. 10 Gourlay, R. N., E. J. Stott, J. Espinasse, and C. Barle. 1974. Isolation of Mycoplasma agalactiae var. boris and infectious bovine rhinotracheitis virus from an outbreak of masdtis in France. Vet. Rec. 93:534. 11 Holman, H. H., I. H. Pattison, and W. S. Gordon. 1952. Studies on experimental streptococcal mastitis. VII. Immunological studies in goats. J. Comp. Path. Therap. 63:196. t 2 Howell, D. G., I. M. Smith, H. H. Holman, and I. H. Pattison. 1956. Experimental streptococcal mastitis. Vi. Immunological studies in the cow. J. Comp. Path. Therap. 66:49. 13 Kerr, W. R., J. K. L. Pearson, and J. E. F. Rankin. 1959. The bovine udder and its agglutinins. Brit. Vet. J. 115:105. 14 MacKenzie, D. D. S., P. M. Outteridge, and A. K. Lascelles. 1966. Aust. J. Exp. Biol. Med. Sci. 44:181. 15 Marx, G. D. 1976. Mastitis and milking management studies. Minnesota Dairy Rep. 16 McDowell, G. H., and A. K. Lascelles. 1971. Local immunization of ewes with staphylococcal cell and cell-toxoid vaccines. Res. Vet. Sci. 12:258. 17 McDowell, G. H., and D. L. Watson. 1974. Immunity to experimental staphylococcal mastitis: Comparison of local and systemic immunization. Aust. Vet. J. 50:533. 18 Newman, L. E., and J. J. Kowalski. 1973. Fresh sawdust bedding-a possible source of Klebsiella organisms. Arner. J. Vet. Res. 34:979. 19 Norcross, N. L., and D. M. Stark. 1969. Role of immunization in mastiffs control. J. Dairy Sci. 52:714. 20 Outteridge, P. M., and A. K. Lascelles. 1967. Local immunity in the lactating mammary gland following the infusion of staphylococcal toxoids. Res. Vet. Sci. 8:313.
O U R INDUSTRY T O D A Y 21 Outteridge, P. M., J. D. Rock, and A. K. Lascelles. 1965. The i m m u n e response of the m a m m a r y gland and regional l y m p h node following antigenic stimulation. Aust. J. Exp. Biol. Med. Sci. 43:265. 22 Outteridge, P. M., R. C. Williams, and A. K. Lascelles. 1968. Local i m m u n i t y in the m a m m a r y gland following the infusion of a staphylococcal cell-toxoid vaccine. Res. Vet. Sci. 9:416. 23 Pattison, I. H. 1948. Immunological studies with Group-B streptococci. J. Path. Bact. 60;219. 24 Pattison, I. H., and H. H. Holman. 1953. Experimental streptococcal mastitis. VIII. Further i m m u nological studies in goats. J. Comp. Path. Therap. 63:304. 25 Pearson, J. K. L. 1959. A u t o g e n o u s toxoid vaccine in the prophylaxis of staphylococcal mastiffs in cattle. J. Dairy Res. 26:9. 26 Reiter, B., and A. J. Bramley. 1975. Defense m e c h a n i s m s of t h e udder and their relevance to mastitis control. Paper presented at the International Dairy Federation meeting at NIRD, Shinfield, Reading, England. April, 1975. 27 Schalm, O. W., E. J. Carroll, and N. C. Jain. 1971. Bovine mastitis. Lea and Febiger, Philadelphia, PA. 28 Schalm, O. W., E. J. Carroll, and J. Lasmanis. 1964. The leukocyte barrier and serologic investigations of experimental coliform (Aerobacter aerogenes) mastitis in cattle. Amer. J. Vet. Res. 25:90. 29 Schellpfeffer, D. A., and A. G. Hunter. 1971. Characterization of Impro., a w h e y antibody preparation. J. Dairy Sci. 54:798. (Abstr.). 30 Schultze, W. D., J. W. Smith, V. C. W o m m a c k , a n d M. A. Norcross. 1963. Observations on the efficacy of antistaphylococcal vaccination in the control of mastitis in a dairy cattle herd. J. Dairy Sci. 46:625. (Abstr.).
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31 Singleton, L., G. W. Ross, R. A. Stedman, and K. V. Chanter. 1967. I m m u n i z a t i o n with staphylococcal cell walls against mastitis. J. Comp. Path. Therap. 77:279. 32 Slanetz, L. W., C. H. Bartley, and F. E. Allen. 1959. The immunization of dairy cattle against staphylococcal mastitis. J. Amer. Vet. Med. Ass. 134:155. 33 Slanetz, L. W., C. H. Bartley, and F. E. Alien. 1963. Vaccination of dairy cattle against staphylococcic mastitis. Amer. J. Vet. Res. 24:923. 34 Slanetz, L. W., C. H. Bartley, and F. E. Allen. 1965. Evaluation of cell-toxoid vaccines for the vaccination of dairy cattle against staphylococcic mastiffs. Amer. J. Vet. Res. 26:688. 35 Smith, J. W., C. A. Kiddy, R. D. Plowman, W. D. Schultze, and N. W. Hooven, Jr. 1970. Whey antibody preparation: Effects of p r e p a r t u m injection on milk production in dairy cows. Amer. J. Vet. Res. 31:1485. 36 Smith, I. M., I. H. Pattison, and H. H. Holman. 1954. The increased resistance of t h e udder o f goats to infection with Streptococcus dysgalactiae strain 419, after vaccination with the same strain. J. Comp. Path. Therap. 64:206. 37 Stark, D. M., and N. L. Norcross. 1969. A passive hemagglutination technique for detection of Streptococcus dysgalactiae antibody. Amer. J. Vet. Res. 30:127. 38 Tarjowski, S. P., and D. T. Berman. 1975. Leukocytic response of bovine m a m m a r y gland to injection of killed cells and cell walls of Staphylococcus aureus. Amer. J. Vet. Res. 36:1561. 39 Williams, J. M., H. J. Mayerhofer, and R. W. Brown. 1966. Clinical evaluation of a Staphylococcus aureus bacterin (polyvalent somatic antigen). Vet. Med./SAC 61: 789.
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