Anatomic Structures of the Bovine Mammary Gland
Cattle have a single udder that is composed of four independent mammary glands that are referred to as quarters. Suspensory ligaments attach the udder to the pelvic bone and abdominal muscles of the ventral body wall.
The four quarters are anatomically independent with no direct physical connections,44 but researchers have demonstrated mild inflammatory responses in apparently healthy quarters adjacent to quarters experiencing clinical mastitis indicative of some interdependence.45 Each mammary gland consists of a teat with a single opening, small cisterns (also called sinuses) in the teat and base of the gland, a system of ducts, and secretory tissue. The secretory tissue is organized into lobes that are composed of lobules containing hundreds of microscopic alveoli lined by mammary epithelial cells that synthesize and secrete milk.5 Nutrients required for milk synthesis are delivered to each mammary gland by the circulatory system and transported into epithelial cells or directly into the alveolar lumen. Milk components, including casein, lactose, and fat, are synthesized within epithelial cells and secreted into the alveolar lumen, where they combine with other constituents to form milk. Production of milk is continuous, and during the interval between milkings about 20% of the milk accumulates in the larger spaces of the udder such as the teat cistern, gland cistern, and large ducts (cisternal fraction). However, about 80% of the milk remains fixed by capillary forces within the alveoli (alveolar fraction), and removal of this fraction is dependent on achieving successful milk ejection (milk letdown).4Milk letdown occurs in response to tactile stimulation of teats, which triggers a neurohormonal reflex, causing the pituitary gland to release oxytocin into the bloodstream.46 Milk is ejected from the alveoli into the cisterns when the surrounding myoepithelial cells contract after binding oxytocin.
Milk flows through the teat orifice after resistance of the teat canal (also called the streak canal) is overcome due to the pressure of milk in the teat, suckling by offspring, or the process of milking. Activation of the sympathetic nervous system during periods of stress or excitement can inhibit the release of oxytocin from the pituitary gland or the binding of oxytocin to myoepithelial cells, thus preventing milk ejection.47The teat canal and surrounding musculoelastic tissue are the primary physical barrier to microbial invasion and also prevent leakage of milk between milkings.48,49 The teat canal is a narrow, longitudinally folded cylinder lined with stratified squamous epithelium. The tortuous shape of the canal provides physical protection against infection, as well as keratin, which lines the canal and is produced by the epithelial cells. Lack of keratin in the teat canal greatly increases the risk of IMI because keratin physically plugs the teat canal, traps invading microbes, and contains bacteriocidal fatty acids and proteins.50-52 Hyperkeratosis of the teat end; trauma-associated abrasion of the teat orifice; or teat damage from ineffective pulsation, excessive milking vacuum, or poorly fitting teat cup liners can also increase the risk of IMI.53-55
Because of the importance of the teat in mammary gland defense, dairy producers should routinely monitor teat condition and use a combination of genetic selection and management practices to promote teat health and sound conformation of the udder. However, it must be emphasized that cows housed on wet or soiled bedding, or udders and teats exposed to excessive water during milking preparation, are at high risk for IMI, despite good teat end health.56-58 In addition, lack of aseptic preparation of teat ends before cannula insertion or use of multiple-dose drug preparations that are not intended for intramammary infusion can result in outbreaks of infection
59 caused by opportunistic pathogens.59
Immunologic Responses to Intramammary Infection
Reducing pathogen exposure is the foundation of prevention of infectious disease, including mastitis.
When exposure to pathogens is sufficient to traverse the teat canal and multiply in the milk, the ensuing response on the part of the host immune system initiates inflammation of the mammary gland, which is recognized as subclinical or clinical mastitis. If the innate and acquired immune systems effectively eliminate the invading microbes, the mastitis will be mild and transient. However, when defense mechanisms are compromised (e.g., during the periparturient period, periods of heat stress, or after transportation and commingling of animals in the herd) or when the pathogen expresses virulence factors that resist phagocytosis or intracellular destruction, severe or chronic mastitis may develop. The intensity of the inflammatory response determines whether mastitis is subclinical or clinical.With subclinical mastitis, the inflammatory process does not result in visible abnormalities in the milk, mammary gland, or cow, although leukocytosis and other soluble changes in milk occur. With clinical mastitis, milk from the affected quarter is visibly abnormal, the gland may undergo marked inflammation, and the cow may exhibit signs of systemic illness. Although mastitis can result from trauma, the vast majority of mastitis is caused by IMI via the teat canal. As stated earlier, limiting exposure to pathogens at the teat end remains the primary means to prevent infection. Mastitis occurs in all herds, but herds that manage pathogen exposure will endure less mastitis.
MAMMARY INFLAMMATION. If invading pathogens are able to overcome the teat canal barrier and gain entry into the gland, a series of innate host defenses help to limit bacterial growth. Because bovine mastitis is essentially a disease of bacterial and occasional mycotic or algal pathogens, the primary effectors of mammary immunity are polymorphonuclear neutrophils (PMNs). However, as in other tissues, these phagocytes rely on a complex system of soluble and cellular mediators that recognize pathogen presence and subsequently recruit and activate phagocytes to eliminate the microbes, termed the innate immune system.
Soluble factors include complement, lactoferrin, and acute phase proteins (such as mannose-binding lectin and C-reactive protein). Lactoferrin is an iron-binding glycoprotein produced by mammary epithelial cells and found in PMN granules.60 By sequestering iron, lactoferrin prevents multiplication of iron-dependent microorganisms, such as coliform bacteria.61 Mannose-binding lectin and C-reactive protein will bind to molecules specific to pathogen cell walls and help to activate the complement cascade. Innate host defenses differentiate host tissue from pathogens by recognizing molecules that are universal to many microbes but not present on host cells. These pathogen-associated molecular patterns (PAMPs) are recognized and selectively bound by serum proteins and receptors in host cells, which are called pathogen recognition receptors (PRRs).62Toll-like receptors (TLRs) are an important class of PRRs that reside both on cell surfaces and within host cells. These receptors invoke gene expression and release of inflammatory cytokines (e.g., interleukins, tumor necrosis factor [TNF]-α) from the host cells and act as the critical link between recognition of foreign agents and initiation of the host response.62,63 Examples of specific pathogen targets bound by TLRs include peptidoglycan and lipoteichoic acid of gram-positive bacteria (TLR-2), gram-negative lipopolysaccharide (LPS; TLR-4), bacterial flagellin (TLR-5), and bacterial DNA as CpG oligonucleotides (TLR-9).64 Much of the local inflammation and systemic signs (when they occur) associated with mastitis are attributable to this response. Numerous cell types, including epithelial and endothelial cells, macrophages, and other phagocytic cells possess TLRs, which can contribute to an inflammatory response.65 The inflammatory response is further modulated by activation of eicosanoid (arachidonic acid) metabolites such as prostaglandins and leukotrienes, as well as by endocrine and adipose mediators.66,67
Milk from healthy mammary glands contains a low concentration of leukocytes.
However, when microbial ligands (molecules) are detected by TLRs on macrophages and epithelial cells, these cells release proinflammatory cytokines, such as interleukin 1 (IL-1), IL-6, and TNF-α.62 These mediators cause vasodilation and expression of adhesion molecules on endothelial cells, which in turn trigger an influx of neutrophils.64 Circulating blood neutrophils must adhere to the vascular endothelium before migrating into the milk. Vascular neutrophils loosely bind and roll along the endothelium, using a surface adhesion molecule (CD62L or L-selectin).68 L-selectin is highly expressed on the surface of normal circulating neutrophils, whereas similar molecules—E-selectin (CD62E) and P-selectin (CD62P)—are expressed on vascular endothelial cells at the site of inflammation.68 Neutrophil CD62L is responsible for making initial contact between fast-flowing blood neutrophils and the vascular wall, which rapidly slows neutrophil movement in small postcapillary vessels, whereas endothelial CD62E and CD62P direct slow-moving neutrophils to the specific site of inflammation. Additional proinflammatory mediators, such as IL-8, leukotriene B4, and complement component 5a, serve as neutrophil chemotactic agents and promote expression of surface receptors on endothelial cells.68 This in turn causes neutrophils to express the β2-integrin adhesion complex CD11b/CD18. The CD11b/CD18 complex firmly anchors neutrophils to vascular and intercellular adhesion molecules.68-70 Once bound, neutrophils migrate between the endothelial and mammary epithelial cells into the milk (diapedesis), traveling along a chemotactic gradient to the site of infection.68 During infection, neutrophils often exceed concentrations of 1 million cells/mL. Both the speed of recruitment and the extent of neutrophil influx into milk influence microbial clearance and are critical determinants of infection outcome.64,71-73 Thus, compared to noninfected quarters, milk from infected quarters reflects the recruitment of PMNs and lymphocytes to eliminate the infection after initial signaling by antigen processing cells such as macrophages, and chemotactic modulators from endothelial and antigen processing cells. This leads to higher absolute numbers of neutrophils, lymphocytes, and macrophages but greater proportions of PMNs and lymphocytes relative to macrophages.74,75During migration and under the influence of proinflamma- tory cytokines (e.g., TNF-α and interferon gamma [IFN-γ], IL-17), neutrophils are further activated to become focused killer cells.68 In this way, the leukocytes arrive at the infection site ready to recognize, phagocytose, and kill infecting pathogens. Phagocytosis is accomplished through a variety of specialized receptors on the surface of neutrophils, which are generally upregulated in response to the proinflammatory signals received during diapedesis and chemotaxis. Expression of CD14 receptors enables neutrophils to bind bacterial LPS in the presence of LPS-binding protein (LBP); this binding facilitates nonopsonic phagocytosis of gram-negative bacteria.76 At the same time, soluble CD14 is shed into the milk, where it neutralizes free LPS and binds to epithelial cells, enhancing chemoattractant release.73 The most important neutrophil receptor for opsonic phagocytosis is the Fc receptor, which binds the Fc region of immunoglobulins (Ig), particularly IgG2 and IgM, enabling the phagocytosis of antibody-coated pathogens.77,78 Complement component 3b is also opsonic for bovine neutrophils.69
Receptor binding stimulates the neutrophil to extend pseudopods and engulf the adhered pathogen into a phagosome. The phagosome fuses with cytoplasmic secretory granules to form an intracellular vesicle (phagolysosome), where degranulation and microbial killing take place. Neutrophil granules contain cationic peptides called defensins, which have broad-spectrum antibacterial and antifungal activities.60,64 These proteins, along with lactoferrin, and hydrolytic enzymes contribute to oxygen-independent killing of engulfed pathogens.
To effectively respond to pathogen invasion, the lactating mammary gland of ruminants must overcome numerous deficits. The number of somatic cells (which in lactating dairy cattle are primarily leukocytes) in milk is typically less than 100,000 cells/mL in uninfected glands, which is approximately 100-fold lower than in blood. Concentrations of factors such as lactoferrin and complement are also lower in milk relative to plasma.79 In addition, the ability of circulating macrophages in milk to re-enter tissue for antigen presentation remains speculative. As compared to blood, macrophages and neutrophils in milk have decreased phagocytic ability.80,81 Neutrophils engulf fat globules and casein, which reduces subsequent pseudopod formation as well as intracellular killing capacity.73 Host-adapted pathogens (such as S. aureus) often have numerous virulence factors to help evade recognition and phagocytosis, which further compromises the ability of host defenses to eliminate the infection.
As mentioned earlier, postpartum cows may be particularly susceptible to infections compared to cows later in lactation. CD4+ (T-helper) lymphocytes are predominantly Th2 (downregulation of inflammation) compared with Th1 (proinflam- matory) within 3 days after calving. This alters the repertoire of cytokines produced and thus the effectiveness of phagocytic cells such as PMNs and macrophages.82
Specific Immune Responses. If a pathogen survives innate host defenses, the specific (also called acquired) immune system is triggered. This branch of the immune system recognizes specific antigens of pathogens, and if repeated exposure occurs, an immunologic “memory” initiates a faster and more intense response with longer duration. As with neutrophils, mammary macrophages ingest and kill microorganisms. In addition to initiating inflammation and killing pathogens, macrophages and dendritic cells play a key role in processing and presenting antigen to T lymphocytes. Antigen presentation is critical to activate T-helper cells (CD4+), thus enabling secretion of cytokines, activation of B lymphocytes, as well as cytotoxic (CD8+), suppressor, and memory functions.83 To date, the role of the CD4+ (helper), CD8+ (cytotoxic and suppressor), and CD17 (phagocyte agonist) subsets in bovine mammary immunity are best understood.84 B lymphocytes also are critical for antigen processing and serve as precursors to immunoglobulin-producing plasma cells.
Concentrations of immunoglobulin are low in normal milk but rise in response to IMI. This increase is partly a result of local Ig production but mostly from increased permeability of vascular endothelial cells and mammary epithelial cell tight junctions, which allows an influx of plasma Ig (and other plasma proteins) into milk. Influx of opsonizing Ig (IgM and IgG2) enables .
In addition, elevated serum NEFAs lead to undesirable expression of proinflammatory mediators, adhesion molecules, and reactive oxygen species.67 The altered plasma fatty acid profile in cattle during periods of negative energy balance also changes the phospholipid content in membranes of endothelial and mononuclear cells.96 Fatty acids from membrane phospholipids are the precursors for numerous arachidonic acid metabolites, the control of which is critical for the magnitude of inflammatory responses.
Oxidative stress resulting from high energy use by high- producing dairy cows further increases the expression of oxylipid (peroxylipid and other metabolites of polyunsaturated fats) pathways. Especially because of the dynamics of lipid metabolism in dairy cows around parturition, profound changes in the composition and concentration of oxylipids systemically and in the mammary gland may be responsible for dysfunctional inflammatory responses.66 The key to modulating this potential problem is to manage the feeding of cows to mitigate excessive body condition loss, especially in transition cows.
Antioxidant supplementation has also been demonstrated to play an important role in mammary immune function. Antioxidants such as vitamin E, selenoenzymes, and zinc-dependent superoxide dismutase protect cells and membranes from oxidative damage that could result from expression of reactive oxygen species during phagocyte killing.97 Phagocytosis and killing is reduced in neutrophils collected from selenium-deficient cows as compared to those collected from selenium-supplemented cows.98 In addition, selenium deficiency resulted in more severe outcomes in cows with coliform mastitis.99 In one herd, vitamin E supplementation above National Research Council (NRC) guidelines (4000 IU∕day) to dry cows in the last 14 days before calving was reported to decrease clinical mastitis in the first 7 days of lactation.100 However, in a larger field study in five herds, feeding dry cows 3000 IU∕day of vitamin E (which also exceeds NRC recommendations) was found to increase both subclinical and clinical mastitis in the first 3 months after lactation.101 In this field study, antioxidant status in cows fed high levels of vitamin E varied, which may reflect the dependency of plasma α-tocopherol concentrations on lipid mobilization, which itself varies with individual cow energy balance.102 Thus while antioxidant supplementation is critical for mammary health, there is no compelling evidence to feed selenium, vitamin E, or any other nutrient of this type above NRC guidelines.
Recently, recombinant bovine granulocyte colony-stimulating factor covalently bound to polyethylene glycol (PEG rbG-CSF) administered twice subcutaneously to cows before and within 24 hours after calving was found to increase circulating numbers of PMNs. This induced neutrophilia is hypothesized to permit a more robust and timely response for cows that may be prone to postpartum infectious diseases, including mastitis.103 In a subsequent field trial conducted in four dairies in diverse geographic locations in the United States, administration of two doses of PEG rbG-CSF to cows before and after calving reduced the overall clinical mastitis rate during the first 30 days after calving by 35% compared to control cows.104 However, the magnitude of the decrease in clinical mastitis varied by herd. This technology may help transition cows to overcome PMN-related immune dysfunction, although other pathways of postpartum immune dysfunction may not be affected by this approach. In addition, clinical benefits will rely on the incidence of postpartum clinical mastitis in a herd and should not be used as a substitute for other sound transition cow management practices.