Immune Response in the Mammary Gland1

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MASTITIS

Immune Response in the Mammary Gland 1 N. L. NORCROSS Department of Large Animal Medicine, Obstetrics and Surgery New York State Veterinary College, Corr}ell University, Ithaca, New York ]4850 Abstract

1. Three classes of immunoglobnlins have been identified in lacteal secretions. IgG1 a p p e a r s to be s e l e c t i v e l y t r a n s f e r r e d through the glandular epithelium into the udder. Presumably any method of immunization that stimulates circulating antibodies of the IgG1 class will be beneficial, resulting in IgG1 antibodies in the milk. 2. I g A class immunoglobulins are also found in the milk, however, in lower concentration than IgG1 immnnoglobulins. I g A is produced locally in the gland since an increase is seen in antigen-infused quarters over noninfused quarters. A factor that might be important is that probably the total amount of any immunoglobulin in lacteal secretions will not be increased by systemic immunization whereas local imnmnization does increase the amount of IgA. 3. The method and route of immunization is possibly very important. Intramuscular, subcutaneous, or other routes that result in systemic stimulation of antibodies, to be effective, must for the most p a r t stimulate class IgG 1 immunoglobulins. Hammer, Kickhofen, and ttenning (7) have recently published that there is a quantitative shift of antibody activity from IgGx to I g G 2 between the first and third courses of immunization of cattle (7). These IgG2 antibodies would be of little value for mammary gland immunity insofar as antibodies in the milk are concerned. However, since the tissues of the mammary gland are involved in a mastitis infection, specific antibodies of all immunoglobulin classes may be beneficial. Imnmnization by inoculation of a Streptovoccus agalactiae, type I I preparation into the area of the supramammary lymph node stimulates protective antibodies in the milk. These antibodies have been shown to be of IgG class principally, with less IgA. Finally, by intramammary infusion of antigen into one or more quarters

of a nonlactating gland, an increase in specific antibodies of only the I g A class was noted. 4. Progress in the determination of the imnmnogenic substance necessary to stimulate resistance to staphylococcal mastitis has been slow. Serological typing of staphylococci is not available; however, on the basis of phage typing, most mastiffs staphylococci may fall into a dozen or so types (]4). Serological typing and the immunogenic response of the group B streptococci are much further advanced. The immunology of the group C and group E streptococci is also in a primitive state. I t probably is not necessary to separate and p u r i f y each immunogen that is required in a preparation; however, it is necessary that inactivated bacteria of each different antigenic type be present in a bacterin in order to stimulate all the various antibodies necessary. One can speculate that a combination of immunization routes that result in the stimulation of several of the classes of immunoglobulins might produce sufficient protective antibodies in the milk and in the mammary gland tissues to increase resistance. Local stimulation would increase specific I g A while systemic stimulation would result in specific IgGl, IgG2 and IgM. Local stimulation would presumably have to occur in the nonlaetating gland with a nonirritating or nontissue damaging antigen. The antigen may be a bacterin consisting of a combination of each of the types of staphylococci and streptococci and be inactivated with formalin or some other inaetivant. The baeterin should also contain staphylococcal toxoids. Introduction

This review characterizes bovine immunity as it pertains to the mammary gland and includes a discussion of the antibodies in the gland as well as methods of their stimulation. I n addition a description of the antigens that may be 1 Presented at the Sixty-sixth Annual Meeting important in increasing resistance to mastiffs is of the American Dairy Science Association, Mich- given. There are already, in the literature, sevigan State University, East Lansing, June 22, eral reviews on this subject (2, 5, 15) and only 1971. recent developments will be included here. Two 1880

sY~eosruM recent texts are also available that include much information pertinent to this subject (14, 19).

Immunoglobulins The term antibody has been replaced in newer immunological literature by a more descriptive word--immunoglobulin. These are a family of proteins in the body fluids that have the p r o p erty of combining with antigen and, in the situation where the antigen is a pathogen, sometimes inactivating it and producing a state of immunity. Butler (2) recently reviewed bovine immunoglobulins indicating that there have been, so far, three classes identified, IgG, IgA, and IgM. The greatest concentration is in class IgG which contains two subclasses, IgG1 and IgG2. The classes of immunoglobulins can be distinguished from each other by physical measurements and biological characteristics. IgM is the largest, with a molecular weight of about 1,000,000. I g A is intermediate, and the IgG class is smallest with a molecular weight of 150,000 to 165,000. I g A has often been termed "secretory I g A " because of its high concentration in most body secretions. An exception to this statement is bovine milk which is, of course, a body secretion, and IgG1 appears to be the class of immunoglobulin found in the highest concentration (17). The predominant immunoglobulin in colostrum, in concentration, is also IgG1, and most researchers speak of only traces of IgG2 in all lacteal secretions (10). Bovine colostrum and milk also contain both IgM and I g A (6). Table i tabulates the concentration of gamma globulin (total immunoglobulin) in the various lacteal secretions. These were published by Dixon, Weigle, and Vazquez about 10 years ago and may be on the high side; how-

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ever, they were measuring casein-free colostrum and whey, and the table does indicate the vast differences in gamma globulin content of lacteal secretions (6). Table 2 is a division of these immunoglobulins in classes emphasizing again the generally high concentration of immunoglobulins in the colostrum and also the much lower IgG2 (11). I n terms of each of the iummnoglobulin classes the quantitation may be somewhat inaccurate since the range of concentration varies greatly among individual animals. Colostrum, very rich in immunoglobulins, has been the subject of many of the immunological analyses; however, this period represents at most 2 or 3 days. The emphasis in a discussion of mammary gland should probably be placed on the postcolostral period of lactation since it represents the longest period of mastitis susceptibility. Specificity. While the presence of immunoglobulins in the mammary gland is indicative of a potential for immune reactivity, it is the induction of specific immunoglobulins or antibodies against the pathogens that infect the mammary gland that is of practical importance to the researcher interested in mastitis. Presumably it is necessary to have protective immunoglobulins in the gland itself and probably in the milk to attain a state of heightened resistance. The term "heightened resistance" is employed because there is some doubt that complete immunity or refractoriness to mammary gland infection is possible. There are methods of measuring the concentration of specific immunoglobulins against mastitis pathogens in the milk, but methods of determining the immune status of the gland and the cow are difficult to evaluate. Direct challenge of ira-

TABLE 1. Gamma globulin concentration in lacteal secretions, a Dry secretions

Colostrum

Gamma globulin (mg/ml)

183

100

Gamma globulin, p e r cent of total protein

95

78

Early milk 1.8 17

Midmilk 0.5 7

a Modified from (3) TABLE 2. Concentration of bovine immunoglobulins in various body fluids. Source

IgG1

[gG2

IgM

IgA

Serum Colostrum Milk

(mg/ml) 10.5 a 75 a .3-.4

(mg/ml) 7.5 a 1.9 ~ .03-.04

(mg/ml) 2.5 3 -5 .05- .08

(mg/ml) .5 1 -6 .05- .1

a F r o m Reference (7). J.

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munized cows would appear to be the most satisfactory method of measuring resistance, but in practice it is difficult to determine the dose of challenge organisms necessary to measure "heightened resistance." I f complete refractoriness was obtained, the challenge dose of organisms might not be as critical and this method of evaluation might be applicable. The expense of adequate numbers of cows is also an important factor. Where a degree of resistance must be dealt with it is probably more accurate in determining this level to immunize several herds and depend on natural exposure to evaluate the immunogen. Induction into udder. The induction in the cow and in the mammary gland of specific immunoglobulins against mastitis pathogens has been reported by numerous workers, many indieating moderate success (15). They can be divided into two approaches on the basis of the source of imnmnoglobulins: 1) transfer through the secretory epithelium from the general blood circulation, and 2) by local production in or near the gland. I n nlany studies the antigen was inoculated intramuscularly or subcutaneously in a manner to initiate systemic production of immunoglobulins, i.e., antibodies in the circulating blood. Presumably then, these immunoglobulins would be carried to the mammary gland tissue and then be transferred through the secretory epithelium. From the previous discussion much of these immunoglobulins in the lacteal secretions would be mainly in class IgG 1. A second route of inoculation of antigen, which might be considered intermediate between systemic and local stimulation, is the area of the supramammary lymph node that drains the area of the mammary gland. This route has been reported to result in antibodies in the mammary gland in nmch greater concentration and earlier than occurs with immunization via

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intramusculax or subcutaneous routes. Table 3 contains the results of one such experiment where the intramuscular, prescapular lymph node, and supramammary lymph nodes were the sites of immunization. The results indicate that all three routes produced antibodies in the serum but the latter route was most successful in the production of milk antibodies. The specific antibodies from the milk of Cow 2 were isolated and concentrated. This was accomplished by preparing an immunoadsorbent of polymerized Streptococcus agalactiae. Antibodies in the milk adhered to the immunoadsorbent and, after other milk proteins were washed away, were eluted by lowered p i t of 2.3. Table 4 shows the results of a mouse protection test indicating that the antibodies were removed in the eluate. The eluate, after being concentrated, contained S. apalactiae antibodies in all three immunoglobulin classes. Figure 1 is an immunodiffusion slide containing concentrated milk antibodies in the center surrounded by antisera to IgM, IgA, IgG, and bovine serum. This experiment was useful in demonstrating that the supramammary lymph node area was an effective route to stimulate antibodies in the mammary gland. The third method of immunization that has received much attention is the infusion of antigen directly into the mammary quarters via the teat canal. Many early workers studied this route and were generally suecessful in stimulating a local immune response (9, 18). Most success has been noted when the antigen was infused during the dry period, a practice that results not only in heightened antigenic stimulation but also less irritation to delicate udder tissue. This latter problem, i.e., tissue irritation and damage, has been a limiting factor in the use of the intramammary infusion route. Much of the more recent work using this method has been done with sheep (16). Ewes immunized in

TABLE 3. Comparison of antibody level in serum and milk produced by three routes of inoculation of antigen,a Cow

Route of inoculation

Serum

Antibodyb milk

1 2

Supramammary lymph node

10 4 10 6

101 10 2

3 4

Intramuscular

10 6 10 6

3 3

5 6

Preseapular lymph node

10 6 10 5

3 3

a Using passive mouse protection test. b Expressed as number of organisms in LDso. J'. DAIRY SCIEI~CE VOL. 54, NO. 312

Control 3

s~POSlU~ T~BLE 4. Passive mouse protection test indicating removal of specific Streptococcus agalactiae antibodies from milk by passage through an immunoadsorbent column. Number of organisms in challenge Route

135

18

2

Mira

4/5a

0/5

0/5

5/5

3/5

5/5

3/5 1/5 4/5

1/5 2/5 0/5 2/5

Milk through : Colmnn Wash Eluate Control

4/5 5/5

aNumber of dead, 4/5 ~ 4 out of 5. one side of the udder exhibited higher antibodies in the colostrum of infused quarters over noninfused quarters. This higher antibody continued over the 10 weeks of lactation. A large p a r t of the antibody in the antigen-infused quarters has been identified as I g A (13). Presumably systemic immunization will result in increased antibodies in the IgG 1 class in the udder whereas intramammary or local immunization will increase antibodies of the I g A class. In a similar experiment in the bovine the right rear quarter was infused with a S. agalactiae antigen during the dry period. The concentration of I g A in each quarter was followed as was the presence of antibodies against the same antigen. Table 5 shows that Quarter 3 (right hind) contained higher IgA, and antibodies

G

A

FIG. 1. Immunodiffusion reaction with concentrated eluate containing milk antibodies in middle well surrounded by wells filled with rabbit antisera against IgM, IgA, IgG, and bovine serum.

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TA~L~ 5. Concentration of I g A in the secretions of a cow infused with antigen into the right rear quarter, a Quarter Source

Left front

Left rear

Right Right rear front

Colostrum Milk, 1 Week Milk, 1 Month

- 1.17 .059 .052

(mg/ml)-1.26 1.32 .049 .265 .047 .073

1.27 .052 .050

a Immunized during dry period. were higher in this quarter than the other three nonimmunized quarters. The estimation of antibody against the bovine streptococci is more difficult to determine than the actual amount of immunoglobulin i n t h e secretions. However, the general phenomenon o£ the selective stimulation of antibodies in infused quarters in the bovine has been well established for many years (5). Effective Antigens

I n progressing to the relationship between immune reaction in the mammary gland and immunity to bovine mastitis, one must first determine the antigens that effectively stimulate antibodies against these pathogens. Progress in this area has been slow because of the large number of different species of bacteria that cause mastitis and, even within the species that have been studied most thoroughly, the effective antigen among the many other proteins and polysaccharides of the bacterium has not been elucidated. Immunity in the mammary gland has been studied in large p a r t with antigens that stimulate antibodies that are easy to measure (Brucella abortus, Salmonella antigens, etc.) and often not antibodies effective against normal mastitis pathogens. Many of these latter are not good antigens in that they stimulate antibodies poorly, procedures for their measurement are somewhat insensitive, and the effectiveness in increasing resistance has n o t been established. Causative Organisms Most mastitis is caused by infection of the mammary gland with hemolytic Staphylococcus or Streptococcus. There are many species and types of each of these genera and each must be represented in any vaccine that is to have wide spectrum effects. No estimate is available of the number of antigenically different strains of hemolytic staphylococci that cause bovine mastitis. There appear to be several cellular J. DAIRY SCIEI~'OE VOL. 54, NO. 12

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and extracellular antigens associated with the staphylococci that produce beneficial antibodies in immunization procedures. One example would be the toxins that have great lytic effects and these can be toxoided by formalin or other procedures to produce good antigens. Antibodies against these toxoids are antitoxins, and they are effective in combating the systemic effects of staphylococcal mammary gland infections (4). These antigens are not type or strain specific and would give beneficial effects against many different staphylococcal infections. They will not, however, eliminate a staphylococcal infection nor protect against subsequent infections but only cause them to be less severe. I n addition, there are substances in the cell wall including a mucopeptide and certain ribitol teichoic acids that have protective capacity as immunizing agents (20). The general conclusion that may be gathered from reports of vaccination against staphylococcal mastitis is that baeterimtoxoid preparations stimulate the production of antibodies to several substances associated with virulence. There is significant resistance to mammary infections with homologous strains, i.e. the same strain as used to make the bacterin; however, the resistance to heterologous strains is considerably less (1). The degree of protection against heterologous strains was probably attributable to the antitoxins because of their capability in neutralizing toxins which are responsible for much tissue damage. For the streptococci that cause mastitis, there is a somewhat analogous situation; however, a little more is known of some of the antigenic factors that might be important. S. agalactiae is the most prevalent pathogen; it is the only species belonging to Lancefield's group B. I t is primarily a pathogen of the bovine mammary gland but has been found increasingly often in humans, principally in the adult upper respiratoo" tract and urogenital tract, and also in newborn infants (8, 12). There are four type specific polysaccharide antigens that will stimulate protective antibodies and two type specific antigens of a protein nature about which less is known. Presumably each type would have to be represented in an antigenic preparation to stimulate cross protection. The type specific polysaccharides are not highly immunogenic, and several inoculations are required for a minimal response. Table 6 contains the results of a passive mouse protection test with antiserum from a cow immunized with a bacterin containing only S. agalactiae, type I I antigens. The mice were challenged with five types of live S. agalactiae, J'. DAIRY SCIENCE VOL. 64, NO. 12

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TABLE 6. Passive mouse protection test using antisermn from immunized cow. a Serum

Challenge type

LD50

Immuneu Normal Immune Normal Immune Normal Immune Normal Immune Normal

Ia

10-7 10-s 10-s 10.9 10.6 10.6 104 10-9

Ib Ii II III

1 0 -5

10-~

a Bacterin contained only S. agalactiae, Type I I antigens. b 21 Days post-immunization. TABLE 7. Passive mouse protection test on antiserum from cow immunized with a polyvalent bacterin,a Serum

Challenge type

LD~0

Immunev Normal Immune Normal Immune Normal Immune Normal Immune Normal

Ia

10-5 10 s 10-5 10-9 104 10-~ 10-~ 10-9 104 10.5

Ib Ii II III

a Bacterin contained each of the 5 type specific antigens. 24 Days post-immunization. and results clearly indicate the type specificity of the antibodies. Table 7 is the same experiment with antiserum from a cow that was immunized with a bacterin containing each of the type specific antigens. I n each case the mice receiving immune serum were more resistant than those receiving normal serum. Streptococcus uberis is another udder pathogen that has received little attention from immunoIogists. I t belongs to Lancefield's group E and is less prevalent than S. agalactiae. Several serological types have been identified, the im: munological importance of which awaits fur: tiler study (15). Streptococcus dysgalactiae is a Lancefield's group C species that is responsible for a significant number of mastitis infections. Three

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t y p e specific a n t i g e n s h a v e been identified; however, t h e i r i m m u n o g e n i e p r o p e r t i e s i n t h e bovine have n o t been established (]5, 21). (12) References (1) Blobel, H~ans, and David T. ]3erman. 1962. Vaccination of dairy cattle against staphylococcic mastitis. Amer. J. Vet. Res., 23: 7. (2) Butler, J o h n E. 1969. Bovine immunoglobulins: A review. J. Dairy Sci., 52: 1895. (3) Davidson, Inn. 1961. A set of bacteriophages for typing bovine staphyloeocei. Res. Vet. Sei., 2: 396. (4) Derbyshire, J. ]3. 1960. Studies in immunity to experimental staphylococcal mastitis in the goat and cow. J. Comp. Pathol. Therapeutics, 70 : 222. (5) Derbyshire, J. ]3. 1962. Immunity ir~ bovine mastitis. Vet. Bull., 32: 1. (6) Dixon, F r a n k J., William O. Weigle, and Jacinto J. Vazquez. 1961. Metabolism and mammary secretion of serum proteins in the cow. J. Lab. Invest., 10:216. (7) Hammer, D. K., I~. Kiekhofen, a n d G. Henning. 1970. Studies on the formation and association characteristics of IgG class antibodies in bovine serum and colostrum against protein antigens. J. Immunol., 104 : 1016. (8) Jelinkova, J., M. Neubauer, and J. Duben. 1970. Group B streptococci in human pathology. Zentralbl. Bakteriol., I. Abt. Orig., 214 : 450. (9) Kerr, W. R., J. K. L. Pearson, and J. E. :~. Rankin. 1959. The bovine udder and its agglutinins. British Vet. J., 115: 1. (10) Klaus, G. G. B., A. Bennett, and E. W. Jones. 1968. A quantitative study of the transfer of colostral immunoglobulins to the newborn calf. Immunology, 16:293. (11) Maeh, Jean-Pierre, and Jean-Jacques Pahud.

(13)

(14)

(15)

(16)

(17)

(18)

(20)

(21)

1971. Secretory IgA, a m a j o r immunoglobulin in most bovine external secretions. J. Immunol., 106: 552. MacKnight, J o h n F., Patrieia J. Ellis, l~aren A. Jensen, and B a r b a r a Franz. 1969. Group B streptococci in neonatal deaths. App. Microbiol., 17: 926. McDowell, G. H., and A. K. Lascelles. 1969. Local production of antibody by ovine mammary glands infused with salmonella flagellar antigens. Australian J. Exp. Biol. Med. Sei., 47: 669. MeKenzie, H u g h A., ed. 1970. Milk Proteins. Vol. 1. Academic Press. New York City. Norcross, 1V. L., and D. M. Stark. 1970. Immunity to mastitis. A review. J. Dairy Sci., 53: 387. 0utteridge, P. M., and Lascelles, A. K. 1967. Local immunity in the lactating mammary gland following the infusion of staphylococcal toxoids. Res. Vet. Sci., 8: 313. Pierce, A. E., and A. Feinstein. 1965. Biophysical and immunological studies on bovine immune globulins with evidence for selective transport within the mammary gland from maternal plasma to colostrum. Immunology, 8: 106. Porterfield, I. D., W. E. Petersen, and Berry Campbell. 1959. Antibody response of the bovine udder. Sci. J. Ser. Minn. Agr. Exp. Sta., 3939: 1. Singleton, L., G. W. Boss, R. A. Stedman, and K. V. Chanter. 1967. Immunisation with staphylococcal cell walls against mastitis. J. Comp. PathoL, 77: 279. Stark, D. M., and N. L. Norcross. 1971. Isolation and characterization of two virulent antigens of Streptococcus dysgalactiae. Amer. J. Vet. Res., 32:485.

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