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Mastitis Case Studies

Disease Resistance Mechanisms


This section discusses the immunological functions and roles in the mammary gland and the disease resistance factors in the mammary gland.


Immunological Functions and Roles in the Mammary Gland

The immunological and protective functions of the mammary gland come into play both in terms of protection of the neonate and protection of the mammary gland.

  • Selective transport of IgG1 during colostrum formation and during early involution. This is relatively specific to the mammary gland compared to other epithelial and mucosal tissues.
  • Selective transport of secretory immunoglobulins (IgA, IgM) during colostrum formation and lactation. Similar mechanisms exist in other epithelial and mucosal tissues.
  • Involvement of leukocytes and serum-derived factors in protection from and response to infection.

Disease Resistance Factors in the Mammary Gland

This section discusses mammary gland's Physical Barriers, Cellular Immunity, Humoral Immunity, and Phagocytosis. Immunity includes all physiological mechanisms allowing the body to identify and neutralize foreign or abnormal bodies. The immune system is comprised of Cellular immunity (leukocytes; white blood cells) and Humoral immunity (soluble immune components such as immunoglobulins and complement).

Physical Barriers

The Teat and Streak Canal are the gland's first line of defense. Length and diameter of streak canal can influence susceptibility to entry by bacteria. The keratin lining in streak canal contains factors that seem to be bacteriostatic (see Hogan et al., 1988).

Teat Shape: Has a moderate to high heritability. Pointed or rounded teat ends seem to have the best resistance to IMI. Flat or inverted teat ends are least resistant. Funnel shaped teats are more resistant than cylindrical teats.

Streak Canal : Is the primary line of defense. The streak canal extends from teat orifice to the squamocolumnar junction with teat cistern.. The streak canal is lined with stratified squamous epithelium. The canal has several defense mechanisms, such as the physical closing of the entryway for bacteria into the gland and the formation of a keratin plug to prevent entry of bacteria.

Teat Sphincter : This is situated in the teat wall at the distal 2 mm of canal. It has no antibacterial activity, but by contracting and shutting off the streak canal it causes a physical obstruction to bacteria. Some cows have leaky sphincters (poor tone) and may be more susceptible to IMI.

Keratin : Is a "meshlike" substance, formed from desquamated epithelial cells + fatty acids + cationic proteins. It functions as a physical obstruction to bacteria and by the adsorption of bacteria (up to 1 million). The keratin lining is desquamation during milking which removes bacteria in the streak canal. The keratin's fatty acids are bactericidal and bacteriostatic and it has proteins which bind to and cause lysis of gram positive bacterial cell walls. However, certain bacteria can survive and grow in keratin. The thicker keratin provides more resistance to IMI. Fatty acid composition of the keratin is heritable.

Lauric, myristic, and palmitoleic acids are associated with resistance to IMI, while stearic, oleic, and linoleic acids are associated with susceptibility to IMI.

Canal Diameter : The lumen of the canal is folded and becomes dessicated between milkings. Peristalsis of the smooth muscle lining the canal pushes bacteria out. The canal becomes dilated during milking (8.6 mm long, 1.2 mm dia.), and stays dilated and has no peristalsis for 2 to 4 hours after milking. A temporary engorgement of teat end tissues with fluid (blood, extracellular fluid) occurs after milking. It takes time after milking for the fluid to leave the teat end. Short wide canals milk out faster but have less resistance to mastitis. The length and diameter of the canal varies with age, which can affect resistance to mastitis.

Furstenberg's Rosette : Is situated at the internal end of the streak canal. It has a protective leukocyte population which are thought to leave he teat wall and enter the cistern via Furstenberg's Rosette. It contains bactericidal cationic proteins (eg. ubiquitin).

Intramammary infusion reduces the natural defense mechanisms of the streak canal by dilating the streak canal, scraping off or removing the keratin (this takes 2 to 4 weeks to recover), and pushing organisms into teat cistern. One solution to this problem is partial insertion of needle into the teat end, resulting in higher treatment efficacy. This results in deposition of the antibiotic into the streak canal.

Bacteria may escape the natural defense mechanisms by direct inoculation into teat cistern via intramammary infusion, by multiplication of bacterial colonies along the streak canal (especially after milking), or by propulsion into teat by vacuum fluctuations at the teat end during milking. Once past the physical barrier of the streak canal, invading pathogens are confronted with the immune system. In most tissues, the immune system usually overcomes the bacteria. However, in the mammary gland a number of factors can compromise the effectiveness of the immune components, as indicated above.


Humoral Immunity

Antigen: A foreign substance that stimulates a specific immune response. Generally a protein or polysaccharide.
Antibody: A special protein capable of combining with a specific antigen. Synthesized by B lymphocytes and plasma cells. Antibodies are the class of proteins called immunoglobulins.

Immunoglobulins

  • Classes of immunoglobulins (antibodies) include - IgG, IgM, IgA, IgD, IgE
  • Antibodies function to to form antibody-antigen complexes, which -
- direct the enhancement of phagocytosis (opsonization or recognition),
- direct the neutralization of the antigen (such as toxins, viruses),
- cause activation of complement system.

Complement

Complement is also part of the Humoral Immune System. Complement is composed of a series of blood proteins which interact as an enzyme cascade and function as a component of the acute inflammatory response. There are 11 complement components. They can function in concert with immunoglobulins and leukocytes or independently of immunoglobulins. The net result of complement activation is lysis of the target cell. Complement is low in bovine milk - dependent on the stage of lactation and pathological status of the gland.

Cellular Immunity

The immune cells include leukocytes (white blood cells) and derivatives of leukocytes that reside in tissues. These are the effector cells of the immune system. All of these cells originate in bone marrow.

Types of Leukocytes

  • Granulocytes -
Neutrophils (polymorphonuclear neutrophils, PMN, these have segmented nuclei), basophils, and eosinophils. All have granules which contain hydrolytic enzymes and other antibacterial and cell lysing components. Granulocytes are phagocytic, that is they ingest and destroy foreign material. During mastitis or involution, the PMN are the first cells to enter the tissue. They are considered the "second line of defense" in the mammary gland.

  • Lymphocytes -
There are two general types. B cells are involved in antibody production (produce the humoral immune components). T cells are involved in cell mediated immunity (killer cells, helper cells, etc.). The specific roles of B and T lymphocytes in the mammary gland are largely unknown. However, plasma cells in the tissue are B cells that reside in the tissue and locally secrete immunoglobulin.

  • Monocytes/Macrophages -
Both are mononucleated (not a segmented nucleus like the PMN). Both are phagocytic. Monocytes are the form found in the blood. Once monocytes leave the blood and enter the tissue they are called macrophages. Macrophages are important in initiating both the humoral and cellular immune responses, as well as in phagocytosis of foreign cells and debris.
Milk leukocytes.
Milk leukocytes.
PMN from milk. Note the segmented nucleus in the cell circled. Leukocytes from milk. M = marophage; P = PMN; L = lymphocyte.

In the mammary gland less than 2% of total milk somatic cells are epithelial cells. Others are leukocytes. PMN predominate during early stages of inflammation or involution. May account for greater than 90% of total milk somatic cells then. Macrophages and lymphocytes enter the tissue later and predominate after a few days. Macrophages present an antigen to T lymphocytes to initiate humoral and cellular immunity responses.

  • Specific Immunity -
Lymphocytes are responsible for specific immunity. Specific immunity requires prior exposure to antigen, recognition of that antigen again and a response by the lymphocytes. Specific immunity includes both humoral immunity and cell-mediated immunity.

  • Nonspecific Immunity -
Mediated by granulocytes and macrophages. Nonspecific immunity does not require prior exposure to antigen. It is generally nonselective, that is PMN and macrophages can ingest most anything, and will try to. Nonspecific immunity is particularly important in the initial exposure to bacteria.


Phagocytosis

Phagocytosis is a complex process by which phagocytes (neutrophils, macrophages) move into the tissue (chemotaxis), recognize foreign material, ingest the material, and destroy/digest the material. Phagocytosis is the major host mechanism for eliminating foreign material.

Chemotaxis

  • All leukocytes are mobile.
  • Directed cellular migration along a concentration gradient of a chemoattractant. Migrate toward highest concentration. Chemoattractants bind to receptors on the PMN membrane, induce changes in the PMN cytoskeleton, and direct movement of PMNs along concentration gradient of the chemoattractant into gland.
  • Numerous chemotactic stimulants have been identified, including lymphokines, C5a, and toxins and peptides derived from bacteria.
  • PMNs enter the gland by diapedesis between epithelial cells. Entrance is facilitated by capillary permeability.
  • Chemotaxis is primarily responsible for bringing PMN into contact with the bacteria. It is critical for initiating the recognition phase.

Recognition

  • Although phagocytes will ingest most things, ingestion can be facilitated if the phagocyte specifically recognizes a substance as foreign.
  • Opsonization - immunoglobulins (the Fab end of the Ig) and/or complement (particularly C3b) can bind to the antigen or foreign body. This is called opsonization. The other end of the immunoglobulin then can bind to specific Fc receptors on the phagocyte's surface, completing the recognition process. Therefore, the antibody molecule acts as a linker between the antigen and the phagocyte. IgG1 is the most prevalent antibody in milk, but is not an effective opsonin for PMNs. IgG2 is opsonic.
  • Staphylococcus aureus resists recognition. Protein A on its surface binds the Fc portion of IgG, preventing opsonization, and coating the bacterium with host antibody which is not recognized as foreign.
  • Complement bound to a foreign substance also can bind to specific complement receptors on the phagocyte's surface.
  • IgA is produced primarily in the mammary gland. It can agglutinate bacteria, and directly binds and neutralizes toxins without binding to PMN.

Ingestion

  • Pseudopods are formed by out-pocketing of the phagocyte's surface membrane, resulting in engulfment of the foreign material. The pseudopods join together resulting in formation of an intracellular vacuole containing the foreign body. (the organism surrounded by cell membrane). This vacuole is called a phagosome.
  • As the phagosome is formed or immediately after formation of the phagosome, cytoplasmic lysosomes fuse with the phagosome and release their contents into the phagosome vacuole. This now is called a phagolysosome. Intracellular killing occurs in the phagolysosome.
  • The process of fusion of lysosomes to the phagosome and release of their contents is called degranulation. Phagocytes are said to degranulate during phagocytosis.
  • If the phagosome has not completely formed before the lysosomes start fusing with it, as most often is the case, then the hydrolytic contents of the lysosomes released into the phagosome can also leak out of the cell. Much of the tissue damage that occurs during inflammation is caused by this release of lysosomal enzymes and other hydrolytic components from the phagocytes.

Digestion and Killing

There are two fundamental systems for killing and digesting phagocytized foreign material, and an oxygen-independent system. There is a great deal of flexibility and redundancy in each system.

Oxygen-Dependent System

  • Concurrent with ingestion there is a major burst of oxidative metabolism. There is increased oxygen consumption, increased production of hydrogen peroxide and superoxide anion, and increased glucose oxidation via hexose monophosphate shunt.
  • This results in production of several microbicidal oxidizing agents in the phagolysosomes, including superoxide anion, hydroxyl radical, singlet oxygen, and hydrogen peroxide.
  • These will oxidize lipids, such as in the bacterial membranes causing lysis of the bacteria, and will oxidize or cross-link protein destroying their function.
  • The primary enzyme involved in catalyzing the oxidation of foreign materials in the phagocytes is myeloperoxidase, which is contained in the lysosomes. Superoxide dismutase is also involved.

Oxygen-Independent System

Lysosomes contain many anti-microbial components.

  • Acids are formed in the phagosomes, this directly kills acid-sensitive bacteria, but also provides an acidic environment for the lysosomal hydrolases (their pH optimum is ~4.5).
  • Lactoferrin (LF) is an iron-binding protein and maintains its iron-binding capacity even at acid pH. LF is synthesized in secretory epithelial cells, it is found in milk, but it also is found in secondary granules of PMNs. It binds iron needed for growth of coliform bacteria. LF is inhibited by citrate. Citrate binds iron and makes it available for bacteria. Citrate is low during mastitis. LF activity is enhanced by bicarbonate. Bicarbonate in milk is increased during mastitis.

During phagocytosis, secondary granules migrate to cell membrane and release lactoferrin to the outside of the cell. LF binds iron and is again internalized. The LF-iron complex inhibits hydroxyl radical formation. LF increases PMN adhesiveness, keeping them in the inflamed site. LF may play a role in normal functioning of lymphocytes, PMNs, and macrophages.

  • Hydrolytic enzymes - lysozyme, glycosidases
  • Cationic proteins, including low molecular weight antimicrobial proteins.
  • Lysozyme hydrolyzes B1-4 glycoside linkages in gram positive cell walls resulting in cell death. It is absent in cow mammary gland, but is an important antimicrobial protein milks of humans and horses.
  • Lactoperoxidase is produced by the secretory epithelial cells. It reacts with thiocyanate (from green plants in the diet) and hydrogen peroxide (synthesized by streptococci). The oxidative reaction disrupts streptococcal membrane and results in cell death. Lactoperoxidase is not effective against other bacteria unless hydrogen peroxide is added.

Endotoxin-Induced Mastitis

E. coli endotoxin (LPS or lipopolysaccharide) is a toxin produced by coliform bacteria. Exposure of a tissue to endotoxin results in a rapid inflammation. To study mammary responses to inflammation (mastitis) investigators often infuse endotoxin into the mammary gland (into the teat and gland cisterns). This results in a sterile intramammary inflammation. Changes in the tissue and milk composition are similar to those seen when bacteria actually invade the gland. For references using this technique, see:

  • Lengemann, F. W. and M. Pitzrick. 1986. Effects of endotoxin on mammary secretion of alctating cows. J. Dairy Sci. 69:1250.
  • Lohuis, J. A. C. M., W. Van Leeuwen, J. H. M. Verheijden, A. Brand, and A. S. J. P. A. M. Van Miert. 1989. Effect of steroidal anti-inflammatory drugs on Escherichia coli endotoxin-induced mastitis in the cow. J. Dairy Sci. 72:241.
  • Jackson, J. A., D. E. Shuster, W. J. Silvia, and R. J. Harmon. 1990. Physiological responses to intramammary or intravenous treatment with endotoxin in lactating dairy cows. J. Dairy Sci. 73:627.
  • Shuster, D. E., R. J. Harmon, J. A. Jackson, and R. W. Hemken. 1991. Endotoxin mastitis in cows milked four times daily. J. Dairy Sci. 74:1527.
  • McFadden, T. B., R. M. Akers, and A. V. Capuco. 1988. Relationship of milk proteins in blood with somatic cell counts in milk of dairy cattle. J. Dairy Sci. 71:826.
  • Shuster, D. E., R. J. Harmon, J. A. Jackson, and R. W. Hemken. 1991. Reduced lactational performance following intravenous endotoxin administration to dairy cows. J. Dairy Sci. 74:3407.

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