Mastitis Case Studies
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.
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).
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.
Antigen: A foreign substance that stimulates a specific immune response. Generally a protein or polysaccharide.
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.
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
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.
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.
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.
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.
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.
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.
Lysosomes contain many anti-microbial components.
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.
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:
Mastitis Case Studies