Abbreviation Key: E.coli - Escherichia coli; IFN- Interferon; Ig – Immunoglobulin; IL- Inter-leukin; IMI - Intra-Mammary Infection; IMM- Intramammary; Lf –Lactoferrin; LPS- Lipopolysaccharide; MCO- Mastitis-causing Organism; MG- Mammary Gland; NK- Natural killer; PMN – Neutrophils/PolymorphonuclearNeutrophils; SCC - Somatic Cell Count; Se- Selenium; Str. – Streptococcus; S. – Staphylococcus; Vit- Vitamin.
Key words: mastitis, non-specific immunity, specific immunity, defence mechanisms, mammary gland, vaccination
Introduction
Consistent production of high-quality milk is a goal of every dairy producer. The benefits of high-quality milk include longer shelf life and increased cheese yield. These factors ultimately increase consumer usage of dairy products. Preventing mammary gland (MG) infections is one of the cornerstones in the production of high-quality milk. In addition to providing a suitable environment, correct milking procedures, and maintenance of milking equipment, a sound nutrition program is critical for the production of high-quality milk.
Mastitis is an inflammation of the mammary gland that mainly develops in response to intramammary infection (IMI) by mastitis-causing organisms (MCO) (Bannerman et al. 2004) and is a major cause of economic losses (Riollet et al. 2000).
Whether or not udder infection occurs depends on the interaction of host, MCO and environmental factors (Hurley et al 2002). To induce inflammation, the MCO must first pass the teat canal to enter the gland, survive the microbiostatic and microbiocidal mechanisms of the MG and then multiply. Recovery from mastitis occurs with elimination of the MCO and resolution of the elicited inflammatory response (Paape et al 2003). The highest infection rates occur during the early dry and peri-partum periods, correlating with involution and colostogenesis (Godden et al. 2003). This may be related to decreased effectiveness of defence mechanisms, such as impaired function of neutrophils (PMN) after parturition (Hoeben et al. 1997).
Bovine mastitis results in decreased milk production and compositional changes that vary with the intensity and duration of infection (Harmon 1994) and can be of three types, i.e., (i) clinical or (ii) subclinical and (iii) chronic with eventual sporadic clinical episodes. S.aureus and E.coli account for the majority of clinical mastitis cases in cattle (Bannerman et al. 2004). Striking differences exist between the courses of bovine intramammary infection caused by S. aureus and E. coli. E.coli can cause severe mastitis, during which death or extensive damage to mammary tissues may. If the cow survives, the clinical episode is followed by a spontaneous microbial cure. In contrast, S.aureus infection often starts with an acute phase and generally becomes chronic and subclinical (Riollet et al. 2000) with infection persisting for the life of the animal (Bannerman et al. 2004).
The MG is protected from the MCO by a variety of defence mechanisms, which can be separated, into two distinct systems: non-specific (innate) and specific (adaptive) immunity (Sordillo et al. 1997). Non-specific factors of the udder defence mechanism are not enhanced by repeated exposure to the same insult, and they react with many MCO. They offer a first line of defence when a MCO invades, and are frequently able to resist the invasion of a number of potentially infectious organisms. The specific immunity is activated by a specific organism or strain, which facilitates selective elimination. Frequently this system, once developed, remembers the invading MCO or strain, which can be resisted by the specific immunity factors on a later occasion (Hibbitt et al 1995). During MCO challenge all factors work synergistically. In this paper they are explained individually for simplicity.
There are at least three phases of host defence to overcome MCO (Adams and Templeton 1998). The first defence mechanism is the skin and epithelial barrier. The skin and epithelium provide a physical barrier blocking entry of potential MCO, but the epithelium also provides cell surface receptors for attachment of some organisms, which are the first step in the establishment of many IMI. The second phase of host defence is non-specific, or innate, immune mechanisms. Cells involved in non-specific immunity include neutrophils, macrophages and natural killer (NK) cells (Adams and Templeton 1998) which phagocytose MCO. Humoral mediators of non-specific host defences include the Lactoferrin (Lf), Lactoperoxydase, Lysozyme, Cytokines, complement system and the interferon system. The third phase of host defence is specific, or adaptive, immunity (Adams and Templeton 1998).
One means of mastitis control is to increase the MG resistance to new IMI. Two main approaches can be used to increase the resistance of the udder. The first is to enhance the non-specific factors effective against the MCO. This will require both management and pharmacological procedures. The second involves enhancing the specific immunity to the MCO but this is difficult as there are a great variety of causative organisms or strains (Kenny et al 1995).
Defence mechanisms of the udder
Non-specific factors (Innate immunity)
The non-specific immune factors consist of four reactive barriers (Deitemeyer 2003), that are discussed bellow. They are generally non-selective and the predominant means of defence during the early stages of a new IMI (Sordillo et al. 1997). Non-specific factors do not require prior exposure to a MCO and represent the natural disease resistance factors (Bannerman et al. 2004; Rupp and Boichard 2003). On the most global scale, resistance could be defined as the ability to avoid any infection or the quick recovery from an infection (Rupp and Boichard 2003). The inherent capability of the innate immune system to respond to a vast number of MCO is mediated by its ability to recognise highly conserved motifs shared by diverse organisms, such as microbial cell wall components, lipopolysaccharide (LPS), peptidoglycan and lipoteichoic acid (Bannerman et al. 2004). Non-specific factors work in close collaboration with specific immunity by priming lymphocytes to generate specific humoral or cell-mediated responses.
Anatomical or physical barriers of the udder
These are the skin, ducts and their epithelium, closing mechanisms of the teat orifice and a flushing action. To maintain the integrity of the anatomic barriers, a cow needs to be protected from trauma because damaged area provides a site for MCO growth and access to unprotected tissue.
1. Skin - The teat skin is covered with a normal squamous cell epithelium and is hairless. The main function of the udder skin is protective one. It covers the underlying tissues, protects them from injury and extremes of temperatures. There are no sweat glands or sebaceous glands found on the teats of the cow. Because of the lack of glands teat skin is very sensitive to drying off. Damaged skin tissue, such as stepped on or injured teats, frostbite, teat ends lesions and sun burned or chapped teats provide excellent opportunities for MCO, such S.aureus, to attach and colonise sites on the teat.
2. Teat (streak) canal – is considered to be the first line of defence against invading MCO (Paape and Capuco 1997; Sordillo et al. 1997). The teat canal provides a mechanical barrier to invading MCO and the efficiency of this barrier depends upon the efficiency of the teat sphincter, which consists of smooth sphincter muscles that maintains tight closure between milkings. Generally it is accepted that there is an increased risk of mastitis if the cows have poor muscle tone. The teat canal is dilated during milking and for 2-4 hours after milking (Hibbitt et al 1995). Muscle tone varies considerably between individual. The length and diameter of teat canal varies with age. When this is coupled with the closing muscle tone it can influence susceptibility to entry by MCO.
When the MCO pass the teat orifice and colonise the teat canal then the next defence factor is the presence of antimicrobial substances within the canal. The keratin is white wax-like material that is derived from the surface stratified squamous epithelium cells of the teat canal. It is formed from desquamated epithelial cells, fatty acids and cationic proteins derived form the blood and produced locally. It functions as a physical obstruction and adsorption of the MCO that may enter the teat canal. Within the keratin antimicrobial substances have been identified. Some of the keratin’s fatty acids are microbiostatic- myristic, palmitoleic and linoleic (Hibbitt et al 1995; Sordillo et al. 1997). The cationic proteins bind to and cause lysis of gram-positive organisms’ cell walls interfering with the maintenance of their cell osmolarity (Sordillo et al. 1997). Approximately 40% of the keratin lining in teat canals of Holstein dairy cattle is removed during the milking process, thus requiring continuous regeneration.A major factor allowing the invasion of MCO into the MG during the dry period may be that there is often a significant delay in the formation of a complete keratin plug in the teat canal (Godden et al. 2003)
3. Flushing effect. The frequent flushing action during milking has an important role in the protection against infection. It helps to wash out MCO which colonise the epithelium of the ducts and teat orifice (Nickerson 1985). Due to the continual sloughing of the epithelial cells they are flushed out during the milking together with the MCO which are adhered to them. Increased new infection rate of quarters at drying off has been partially attributed to the cessation of milking and consequent failure to flush-out the organisms (Hibbitt et al 1995).
If the MCO pass through the canal and enter the milk in the teat sinus, a third system operates by the mobilisation of the PMN situated at the Furstenberg’s rosette. The Furstenberg’s Rosette is situated at the internal end of the teat canal that has a protective leukocyte population and contains antimicrobial cationic proteins.
4. Teat and udder shape - One of the inherited characteristics, which may affect susceptibility to mastitis, is the teat and udder shape.Funnel shaped teats are generally more resistant than cylindrical teats. Udder depth and udder attachment generally is also important with higher and more tightly attached udders associated to lower somatic cell counts (SCC) and less clinical mastitis (Rupp and Boichard 2003).The shape of the teat end may also be important. Cows with pointed teat ends appear to have smaller diameter teat canals and those with inverted, funnel-shaped ends or recessed plate-like end appear to be more susceptible to infection.
Phagocytic barriers of the mammary gland
All phagocytic cells have the common ability to adhere to extracellular material and microbes, ingest them, and finally kill and digest them. Ingestion is done through contact, recognition and adherence to the organisms’ cell walls (opsonisation). Complement’s and immunoglobulins’ roles in coating organisms are critical to allow phagocytes to adhere to them, so they can be ingested (Paape et al 1977).The final result of phagocytosis should be complete destruction of the MCO.
An acute inflammatory reaction is crucial in the defence of mammary tissue against invading MCO. Leukocytes, especially polymorpho-neutrophil leukocytes (PMN), are the major contributors to this mechanism of natural defence, and their migration to the site of infection is determinant for the outcome of the infection. Neutrophil migration is elicited by inflammatory mediators which are produced in the infected tissue by cells responding to microbial toxins or metabolites. The array of known inflammatory mediators is vast and includes complement fragments, arachidonic acid metabolites, vasoactive amines, and cytokines. Cytokines can also enhance the bactericidal activity of phagocytes (Riollet et al. 2000).
Killing and digestion is achieved by oxidation involving myeloperoxidase and hydrogen peroxide or through the oxygen-independent digestion by the enzymes present in lysosomes. Most gram-negative organisms are killed by oxidation, but gram-positive organisms resist oxidative killing by generating catalase and they are killed by the oxygen independent digestion (Sordillo et al. 1997).
Once complement components and immunoglobulins (Ig) bind to receptors on the PMN surface, the cell becomes activated and generation of the oxidative burst is initiated (Paape et al 2003). This response is called the “respiratory burst” (Paape et al 2003). During the respiratory burst there is increased oxygen consumption and metabolism within the cell and increased production of the peroxides necessary to kill the MCO. These are, however, potentially dangerous to the cell and surrounding tissue (Paape et al 2003; Smith et al. 1997). Accumulation of hydrogen peroxide in a PMN reduces its ability to kill MCO intracellularly (Paape et al 2003; Smith et al. 1997).
Important parameters for the outcome of an IMI are the antimicrobial efficiency of PMN and the invading organisms’ antiphagocytic and cytotoxic properties (Rainard and Riollet 2003). Many MCO avoid recognition and in that way phagocytosis. For example: slime and protein A producing strains of S.aureus are more resistant to phagocytosis and intracellular killing by bovine PMN compared to non-slime producing strains. The slime layer prevents binding of immunoglobulins to the cell membrane of S.aureus, increasing the antimicrobial resistance and enhancing MCO virulence. The toxins (alpha and beta) appear to play a major role in the staphylococcal virulence (Cucurella et al. 2004; Paape et al 2003; Riollet et al. 2000). It is believed that S.aureus induces early PMN apoptosis. An apoptoic PMN may serve as a vehicle by which the S.aureus could enter macrophages without stimulating microbicidal activities, while simultaneously being provided with a protective barrier against exogenous host defence mechanisms and/or antimicrobials (Bayles et al. 1998).
Leukocytes - The phagocytic cells include leukocytes and tissue derivatives of leukocytes, such as macrophages. There are three main types of leukocytes: granulocytes, lymphocytes and monocytes/macrophages. Leukocytes are involved in both non-specific and specific immunity of the MG. The activities of resident and newly recruited leukocytes are very important when new MCO passes from the environment into the MG. If the organisms overcome the leukocytes then the occurrence of a new IMI is common. In the MG the PMN, NK and macrophages are important phagocytes.
Granulocytes are represented by neutrophils (polymorphonuclear neutrophils-PMN), basophils and eosinophils. The most important in udder defence are the PMN, which are considered to be a primary cellular defence mechanism to MCO that penetrate the physical barrier of the teat canal (Hogan et al. 1993; Paape and Capuco 1997; Paape et al 2003). They are considered the “second line of defence” in the mammary gland (Nickerson 1985). PMN are phagocytic cells dealing with foreign material, such as MCO, small particles of cellular debris or accumulated milk components. During mastitis or involution, the PMN are the first cells to enter the MG alveoli (Kelly et al. 2000). The PMN work rapidly but only for short periods in the early stages of IMI. They are short-lived and have no ability to present antigen to the adaptive immune system. Their main role is to sacrifice themselves and neutralise the MCO before the infection spreads. PMN mobilisation can occur within one hour of infection and within four hours large numbers can be presented in the milk. PMN predominate during early stages of inflammation or involution and may account for greater than 90% of total milk somatic cells at those times (Sordillo et al. 1997). Their attraction to the inflammatory site is a corner-stone of the host defence against invading MCO (Rainard and Riollet 2003) associated with the incidence and severity of clinical mastitis (Hogan et al. 1993). Depletion of neutrophils results in a dramatic increase in susceptibility to IMI and with severe clinical forms (Paape and Capuco 1997; Rainard and Riollet 2003). On the contrary, improved recruitment of the PMN through immunisation reduces the severity of mastitis and enhances the occurrence of microbial cure (Rainard and Riollet 2003).
MCO release metabolic by-products, potent toxins, or cell-wall components as they colonise and grow in the MG (Sordillo et al. 1997); these serve as chemoatractants for PMNs and activate epithelial cells in the MG to secrete cytokynes. There are four main cell types involved in the secretion of chemotactic factors for PMN: epithelial cells, macrophages, lymphocytes (Rainard and Riollet 2003) and residual PMN. Other chemotactic factors are likely to be generated in milk by the activation of complement (Rainard and Riollet 2003). In response to chemotactic stimuli the PMN migrate (diapedesis) from blood into the milk. Current knowledge about PMN diapedesis from blood into milk is marginal. The time of transit of PMNs from the circulation to the milk is about 2 h after infusion of the teat cistern with endotoxin. This represents the time required for the migratory signal to develop, plus the time for a PMN to adhere and migrate through the endothelium, subepithelial matrix, basement membrane and mammary epithelium into the milk (Paape and Capuco 1997; Paape et al. 2003; Rainard and Riollet 2003).
However, the intense recruitment of PMN is not sufficient per se to cope with the MCO. To exert their antimicrobial potential, PMN require opsonins to recognise the MCO, antitoxins to counter MCO’s toxins, and a proper state of activation, provided by the cytokines and chemokines associated with the inflammatory and immune responses (Rainard and Riollet 2003). Stimulated mobilisation of PMN without opsonins and antitoxins would probably result in exacerbation of the severity of the disease, as it apparently occurs with S.uberis or mycoplasmal mastitis (Rainard and Riollet 2003).
Several explanations can be put forward to explain why the PMN, despite outnumbers MCO in milk, are frequently unable to eliminate them. One of the reasons is that the phagocytic capacity of PMN is reduced in the MG (Hoeben et al. 1997; Rainard and Riollet 2003), because of the ingestion of milk particles (Sordillo et al. 1997)and a deficiency in phagocytosis-promoting antibodies (opsonins) in milk (Kenny et al 1995; Rainard and Riollet 2003) or due to a lack of compound that favours their function, such as glucose (Rainard and Riollet 2003). Diapedesis utilises the energy reserves of a PMN which are needed for efficient phagocytosis and killing of invading MCO (Paape et al. 2003; Rainard and Riollet 2003). Intracellular killing of phagocytosed MCO is inhibited by milk components because of loss of lysosomes that fuse with phagosomes containing fat and casein instead of phagosomes containing MCO (Paape and Capuco 1997; Paape et al. 2003). Consequently, diapedesis of PMN across mammary epithelium results in decreased phagocytosis and oxidative burst activity. Casein exerts an inhibitory effect on both phagocytosis and microbial activity of PMN due to interfering with the oxygen radical generating pathway and depressing the oxydative burst (Rainard and Riollet 2003). Phagocytic killing by PMN is optimal when the MCO are opsonised with immunoglobulins (Ig) or Ig and complement (Rainard and Riollet 2003). Finally, virulence factors of MCO may reduce the efficiency of phagocytic killing by PMN. Encapsulated organisms are usually more resistant to phagocytosis than are un-encapsulated (Rainard and Riollet 2003). Apart from antiphagocytic surface material, MCO may target PMN with toxins which interfere with the phagocytic process. Leukotoxins, which are exotoxins produced by many S.aureus strains isolated from mastitis, are able to kill bovine PMN (Rainard and Riollet 2003) or induce early apoptosis (Bayles et al. 1998) and consequently are likely to reduce the efficiency of the phagocytic defence during mastitis. As a result, large numbers of PMN are needed to prevent the new IMI. However, the number of PMN storage pools that respond to an irritation in the MG depends on severity of the irritation and strength of the chemotactic agent (Paape et al. 2003).
In the course of phagocytosing and destroying invading pathogens, PMN release chemicals that not only kill the MCO but also cause injury to the delicate lining of the MG (Paape et al 2003; Rainard and Riollet 2003). One mechanism by which this may occur is through release of toxic oxygen radicals and proteases, which are both antimicrobial and cytotoxic to MG tissue. Prolonged irritation to the mammary secretory epithelium results in scarring and leads to a permanent decrease in milk production due to reduced numbers of secretory cells. Rapid elimination of PMN by macrophages following MCO neutralisation is essential to minimising inflammatory-derived injury to the host (Paape et al 2003). Also, the life span of PMN is limited by the onset of apoptosis (Paape et al 2003). Apoptosis is a form of cell death morphologically characterised by chromatin condensation, nuclear fragmentation, cell shrinkage and blebbing of the plasma membrane. The end result of apoptosis is fragmentation of PMN into small membrane-bound bodies that are quickly cleared by other phagocytotic cells. In contrast, necrotic cell death is characterised by cell swelling and lysis. The loss of membrane integrity of necrotic cells is accompanied by release of cellular contents that injure neighboring cells and trigger an inflammatory response (Paape et al 2003). The significance of this process is that it prevents disintegration in vivo, and therefore release of toxic products of phagocytosis and minimize the damage to mammary tissue (Paape and Capuco 1997).
Milking removes compromised PMN, which are replaced by healthy cells, thus enhancing the defence against MCO. This phenomenon could partially explain the reduced incidence of clinical mastitis for cows milked four times a day compared to cows milked two times a day (Paape and Capuco 1997).
Clearly, the speed with which PMN can be mobilised following MCO invasion and the efficiency of intracellular kill are critical to the protection of the MG from infection. Vitamin E (Vit E) and selenium (Se) play essential roles in these events and dietary deficiencies of either leads to impaired PMN function and increased incidence of IMI in dairy cows (Smith et al. 1997). Vit E and GSH-Px both are cellular antioxidants that protect against the cytotoxic effect of oxygen metabolites produced during phagocytosis. Vitamin E protects the membrane, whereas GSH-Px the cytosol (Hogan et al. 1993). A lack of Se also lowers the production of leucotrienes of polymorphonuclear leukocytes and thus lowers chemotaxis of neutrophils (Jukola et al. 1996).
Mastitis is commonly treated with IMM administration of antimicrobials. These antimicrobials may affect body defences directly as well as indirectly via changes in MCO (e.g., changes in the microbial cell membrane and inhibition of protein synthesis). Efficient removal of the MCO requires both sensitivity to the drug and an effective immune system (Hoeben et al. 1997). The oxidative burst activity of bovine PMN can be altered after IMM treatment. Cloxacillin has no effect, enrofloxacin increases PMN activity, whereas neomycin, lincomycin, dihydrostreptomycin, doxycycline, oxytetracycline, danofloxacin, penicillin, ceftiofur, spiramycin, erythromycin and chloramphenicol reduce the oxidative burst activity of bovine PMN (Hoeben et al. 1997; Paape et al 2003). Because the functional activity of bovine PMN in milk are already impaired, use of antimicrobials that will not cause further impairment of PMN function becomes more critical in the ability to clear invading MCO from the MG. Corticosteroids show no adverse effect on oxidative burst activity of bovine PMN at therapeutic concentrations (Paape et al 2003).
There is variation in the phagocytic properties of PMN among cows. The variation may explain some of the differences in susceptibility to new IMI that exist between animals (Paape and Capuco 1997; Paape et al. 1977; Paape et al. 2003)
Monocytes/Macrophages - Once monocytes leave blood and enter tissue they mature and become macrophages. Both are phagocytic. Monocytes/macrophages start acting later with extended phagocytic activity compared to PMN. Macrophages are important in initiating both the humoral and cellular adaptive immune responses, as well as in phagocytosis of foreign cells and debris. They are the predominant cells found in the secretions and tissues of a healthy involuted or the milk and tissues of the lactating MG (Hibbitt et al 1995). These cells are long-lived, and play several roles, from clearing the infection to repairing tissue (Kenny et al 1995). The macrophages are often in contact with MCO in the very first phases of infection. After phagocytosis of an MCO, the macrophages are able to secrete factors which are chemotactic for PMN (Rainard and Riollet 2003). The capacity to secrete a whole range of inflammatory mediators, cytokines or components derived from arachidonic acid, confers to the macrophages a significant role in the defence of the MG (Rainard and Riollet 2003). Several cytokines are secreted by stimulated macrophages in particular IL-1b, TNF-a, IL-8 and GM-CSF (Rainard and Riollet 2003). The stage of lactation is relevant to their activity: IL-8 and GM-CSF are active in the dry period, but not in a gland during lactation, whereas IL-1b and TNF-a are active whatever the physiological stage (Rainard and Riollet 2003). Macrophages quickly engulf and phagocytose apoptotic PMN, thereby minimising the release of PMN granular contents that are damaging to the secretory tissue (Paape et al 2003).The phagocytic rate of macrophages can be increased substantially in the presence of opsonic antibodies for specific MCO (Hibbitt et al 1995). Macrophages can phagocytose S.aureus but a percentage survives intra-cellularly (Kenny et al 1995).
Soluble barriers of the mammary gland
The soluble defence factors of the MG are associated with the cellular factors. They also can be divided into non-specific and specific factors. The mammary gland contains non-specific microbiostatic components that work independently and in concert with antibodies (immunoglobulins- Ig) and cellular factors. These factors include lactoferrin, lactoperoxydase system, lysozyme, complement and cytokines.
Lactoferrin (Lf) is an iron-binding protein produced by epithelial cells and leukocytes. Lactoferrin has many proposed biological functions, including antimicrobial/ antiinflammatory activities, participation in local secretory immune systems in synergism with some Ig’s and other protective proteins, it is an iron-binding antioxidant protein in tissues, and possibly promotes the growth of some animal cells such as lymphocytes. Its iron binding properties prevents the growth of MCO, which have iron requirements, such as staphylococci and coliforms (Sordillo et al. 1997). Hydrolysis of Lf also releases a peptide which has strong antimicrobial activity. Citrate can abolish the antimicrobial activity of the Lf (Hibbitt et al 1995) probably by chelating iron and making it available to the MCO.
The concentration of citrate in milk is high and Lf is low at an average concentration of about 0.2 grams/litre during lactation. During the dry period concentration of citrate in the MG secretion is low and Lf is high (Green et al 2002; Hibbitt et al 1995; Smith et al 1977). Thus it is considered that the role of Lf in the defence of the MG is more important in the involuted gland than during lactation, especially against coliforms (Sordillo et al. 1997). Some organisms, such as Str. agalactiae may be able to utilise Lf as an iron source by binding it to surface receptors (Hibbitt et al 1995; Sordillo et al. 1997).
Lactoperoxidase is an enzyme, which in the presence of hydrogen peroxide and thiocyanate form the lactoperoxidase system. This system is microbiostatic for some gram-positive organisms such as S. aureus and Streptococci and microbicidal for gram-negative organisms, including E. coli (Hibbitt et al 1995). Hydrogen peroxide and thiocyanate are naturally distributed in animal and human tissues, although they are generally in very low concentrations. For normal function of lactoperoxidase the concentration of oxygen is important. The low oxygen tension of the MG can inhibit the production of peroxides, thus limiting the effectiveness of the peroxidase system against the MCO. The antimicrobial property of the lactoperoxidase system is based upon inhibition of vital bacterial metabolic enzymes due to oxidation by hypothiocyanate. Bovine milk contains concentrations of about 0.03 grams/liter. In bovine colostrum, the lactoperoxidase content is very low, but increases rapidly after 4 to 5 days postpartum.
Lysozyme is a microbicidal protein that is present in low concentration in bovine milk and functions by destroying peptidoglycan, a gluco-peptide in the organisms’ cell wall (Nickerson 1985). There are two types of lysozyme. One type is found in the egg-white of hens and is known as chicken-type or c-lysozyme. The other type is found in the white of geese eggs and is known as goose type or g-lysozyme. Cow’s milk may contain both c- and g-lysozymes. Lysozyme possesses antimicrobial activity against a number of MCO. This enzyme usually functions in association with Lf or Ig A. Lysozyme is effective against E.coli in concert with Ig A. In addition, lysozyme can limit the migration of PMN into damaged tissue and might function as an anti-inflammatory agent. It probably offers little protection against new IMI, but its concentrations are increased during mastitis.
The complement complex is composed of at least 29 distinct plasma and membrane proteins, with antimicrobial, opsonic and phlogistic functions (Rainard 2003). It is one of the most intriguing complexes produced by the immune system. Complement proteins assemble in a cascade process (Deitemeyer 2003), called complement activation, and adhere to the MCO surface. The activation of the system results in a sequence of biochemical reactions in which one component activates another component in a cascade fashion. Three activation systems have been described: the classical pathway, the alternative pathway, and the lectin pathway of activation (Rainard 2003). The net result of complement activation is lysis of the MCO, promoting opsonisation, activation of the inflammatory processes or immune clearance of the antibody-complement complexes in the liver or spleen either directly or through cooperation with phagocytic cells. The complement system can influence the immune response and constitute a bridge between innate and acquired immunity (Rainard 2003). The opsonisation with fragments of the components C3 and C4 makes them prone to ingestion by phagocytic cells possessing receptors for these opsonins. The regulation of the inflammatory response by the component fragments C3a and C5a, which induce histamine release from mast cells and basophils, vasodilatation, increased vascular permeability, and the recruitment of phagocytes at sites of inflammation, are also important functions of the complement system (Rainard 2003). The concentration of complement is generally low in bovine milk and is dependent on the stage of lactation and pathological status of the gland. The concentration of complement is highest during the colostrum period, and in inflamed glands (Hibbitt et al 1995; Kenny et al 1995; Rainard 2003; Sordillo et al. 1997). Therefore, because of its intermittent presence in the milk, complement is thought to play only a minor antimicrobial role in the MG. However, the alternative complement pathway will be activated by the presence of many gram-negative organisms, such as some strains of E. coli (Hibbitt et al 1995; Sordillo et al. 1997) and kill them. The complement enhancing the function of phagocytic cells and the immune response is of greater consequence than the complement-mediated lysis (Rainard 2003).
Cytokines are peptides with potent biological activity at extremely low concentrations, which are produced by immune and non-immune cells locally in various tissues including the MG (Sordillo et al. 1991). Their function is complex (Sordillo 1995). Cytokines play an important role of the host defence by regulating the activity of cells that participate in all aspects of specific and non-specific immunity (Hibbitt et al 1995; Sordillo et al. 1991). The cell-regulating activity of individual cytokines can interact with other cytokines in a synergistic, additive, or antagonistic fashion on multiple cellular targets (Sordillo et al. 1991). Because of their extreme potency, elevated levels of certain cytokines can be detrimental to the host (Hibbitt et al 1995; Sordillo et al. 1997). Cytokines play an important role in stimulating the recruitment of cells into the udder following invasion by a MCO. They are important mediators of inflammation and play a role in B lymphocyte growth and differentiation (Sordillo et al. 1997); they are also involved in the activation of NK cells.
Colostrum has a lower concentration of cytokines and diminished immune cell function therefore increasing the susceptibility to new IMI (Sordillo et al. 1991). Significant quantities of inflammatory inducing cytokines (IL-1b, TNF-a, IL-6 and IL-8) are found in milk in experimental or natural E.coli mastitis. Staphylococci, however do not release as many irritants and the intensity of inflammation is more modest (Rainard and Riollet 2003). The low levels of C5a and the absence of cytokines in milk from S. aureus-infected cows, compared to the high levels found in milk from E. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk after S. aureus inoculation in the lactating bovine MG could contribute to the establishment of chronic mastitis. This should be considered during the design of preventive or curative strategies against chronic mastitis (Riollet et al. 2000).
Interferons (IFN) are a group of closely related proteins with two major classes. Class I IFN consist of three closely related types: IFN-g, IFN-b, and IFN-v. The IFN-a and IFN-b are produced by a variety of cell types in response to several inducers, including viral infections, bacterial products, and tumor cells. In the bovine, the IFN-v genes code for proteins produced by the early embryonic trophoblast, and these are referred to as IFN-t. The second class of IFN consists of a single protein, IFN-a, which is unrelated to the class I. IFN has been found to exhibit a variety of immunomodulatory properties to many aspects of the immune system (Sordillo et al. 1997).
Inflammatory barriers of the mammary gland
Inflammatory barriers are the bodies responses to infection including: vasodilation, increased capillary permeability and phagocyte emigration (Deitemeyer 2003). Acute inflammation is the response of tissue to injury or infection (Erskine and Bartlett 1993). The acute inflammatory reaction is primarily a manifestation of non-specific immunity. It starts when the luminal compartment of the udder is invaded by MCO, even if these organisms have never caused infection before in that animal (Rainard and Riollet 2003). The inflammatory cascade begins with the release of signal molecules from the damaged tissue or detection of foreign material (Deitemeyer 2003) from the MCO after phagocytosis by the PMNs or macrophages. The most likely signal molecules are the cytokines. Humoral factors and cytokines released in this process mediate systemic events, collectively termed the acute phase response (Erskine and Bartlett 1993). A key component of the host innate immune response to infection is the upregulation of cytokine production. Two well-described proinflammatory cytokines, tumor necrosis factor alpha (TNF-a) and interleukin 1b (IL-1b), mediate the inflammatory response at both the local and systemic levels. Locally, these cytokines induce vascular endothelial adhesion molecule expression, thereby promoting neutrophil transendothelial migration (diapedesis) to the site of infection. Systemically, TNF-a and IL-1b are potent inducers of fever and the acute-phase response. Induction of the acute-phase response results in increased hepatic synthesis of proteins, such as LPS-binding protein and C-reactive protein, which facilitate host detection of MCO-wall products and complement activation, respectively. Neutrophil recruitment to the site of infection is further mediated by the upregulation of the chemoattractant IL-8. IL-12 contributes to the innate immune response by stimulating the production of gamma interferon (IFN-g), an activator of PMN and macrophages. Finally, resolution of the inflammatory process is mediated by the upregulation of IL-10, which downregulates pro-inflammatory cytokine production (Bannerman et al 2004; Deitemeyer 2003; Erskine and Bartlett 1993; Sordillo et al. 1997). Several other chemical mediators are involved: C-reactive protein, histamine, kinin peptides, lipopolysaccharides, enzymes etc (Erskine and Bartlett 1993).
Table 1. Defence mechanisms of the udder
|
Defence mechanisms
of the udder |
Non-specific (Innate)
factors |
Anatomical (physical) barriers |
Skin |
|
Ductal system epithelium |
|
Teat sphincter |
|
Peristalsis of the teat canal |
|
Keratin |
|
Furstenbergs’ rosete |
|
Phagocytic barriers |
Neutrophils |
|
Monocytes/Macrophages |
|
Soluble barriers |
Lactoferrin |
|
Lactoperoxidase system |
|
Lysozyme |
|
Complement |
|
Cytokines |
|
Inflammatory barriers |
Vasodilatation |
|
Capillary permeability |
|
Phagocyte emigration |
|
Specific
(Adaptive) factors |
Cell-mediated immunity |
Macrophages |
|
Lymphocytes |
|
Humoral immunity |
IgG1 |
|
IgG2 |
|
IgM |
|
IgA |
Specific factors of the udder’s defence mechanism (adaptive or acquired immunity)
The specific factors of an udder’s defence mechanism recognise, process and clear antigens through cell-mediated or humoral immunity. It produces a specific memory guide for defence against the subsequent attacks by the same MCO (Rainard and Riollet 2003) or strain, resulting in a humoral or cellular structure that is capable of recognising the antigen and eliminating it form the MG (Losnedahl et al. 1996). These specific responses can be more intense and more efficient than the unspecific reactions (Losnedahl et al. 1996; Rainard and Riollet 2003).
Cell-mediated immunity
The effective specific cell-mediated immunity involves two types of leukocytes: macrophages (as antigen-presenting cells) and lymphocytes (as executive cells – producing humoral factors or sensitised cells).
Macrophages in addition to their role in the early non-specific defences (which is discussed previously) also play a key role in antigen processing and presentation (Sordillo et al. 1997). Antigens from the ingested MCO are processed within the macrophages and appear on the membrane in association with the major histocompatibility complex (Sordillo et al. 1997)that are required by the executive cells (lymphocytes) for the recognition of the antigens.
Lymphocytes are the only cells of the immune system that recognise antigens through membrane receptors which are specific for each antigen (MCO or strain).Lymphocytes consist of T and B cells together with a population of lymphoid cells, which do not consistently carry markers of either T or B cells, the so-called null or NK cells (Hibbitt et al 1995; Sordillo et al. 1997). The specific roles of B and T lymphocytes in the MG are complex. B-lymphocytes are involved in antibody production and T-lymphocytes are involved in cell-mediated immunity (Hurley et al 2002). There is a variety of T-lymphocytes with different activities. T-helper lymphocytes produce cytokines and play an important role in activating B-lymphocytes, other T-lymphocytes, macrophages and various other cells that participate in the immune response. T-cytotoxic lymphocytes are involved in recognition and elimination of specific cells in conjunction with other immunity factors. There is also a group of T-lymphocytes that are probably involved in the protection of the epithelial cells, etc. Plasma cells in the mammary tissue are B cells that reside locally and secrete immunoglobulins. Under certain conditions, differentiation of B lymphocytes can be directly stimulated by an antigen, such as lipopolysacharide from coliforms.
Humoral immunity
The Ig classes contained in milk differ in concentrations depending on the stage of lactation and the health of the gland (Hibbitt et al 1995). Antibodies in the MG can be produced locally or are selectively transported from the serum (Hibbitt et al 1995). Immunoglobulins act as soluble or humoral effectors of specific immune responses. The purpose of Ig is to perform one of two primary functions, either deactivation or elimination of a MCO. Their mechanisms of action in milk include prevention of adherence to epithelial cells, neutralisation of the elaborated toxins, opsonisation and direct lysis of the MCO. The key role of the humoral immunity of the udder is based on the fact that most of the MCO are extra-cellular. Immunoglobulins in the bovine colostrum are important in the transfer of passive immunity from the cow to the calf.
There are four main types of Ig involved in MG’s humoral immunity; these are IgG1, IgG2, IgA, and IgM (Burvenich et al 1995; Hibbitt et al 1995; Hurley et al 2002). IgG1, IgG2 and IgM are probably main opsonins for bovine milk PMN (Sordillo et al. 1997). They are also involved in the complement adherence processes. IgM is a very effective opsonin (Hibbitt et al 1995), but due to the transient nature after immunisation compared with IgG2, effectiveness of IgM as a protective opsonin is limited. IgA does not bind component complex or opsonise MCO. Instead, IgA appears to contribute to prevention of adherence to the epithelial lining and organism colonisation, agglutination and toxin neutralisation (Nickerson 1985; Salmon 2003; Sordillo et al. 1997).
IgG1 is transported preformed from the blood. Colostrum has a markedly higher concentration of IgG1 than serum. IgA and IgM are produced mainly by the antigen-activated plasma cells (B-lymphocytes) that reside in the mammary tissue (Hibbitt et al 1995).
In healthy glands the concentration of Ig is low during lactation, but slowly increases during the dry period, with peak concentrations during colostrogenesis (Losnedahl et al. 1996; Sordillo et al. 1997). High concentrations of Ig are also present during inflammation (Hibbitt et al 1995; Hurley et al 2002; Sordillo et al. 1997).
The total antimicrobial effect in milk is greater than the sum of the individual contributions of Ig and non-Ig defence proteins and antimicrobial peptides. This is most likely due, at least in part, to their synergy.
There is an increased cellular influx in milk in the hours following an infection when the cows have been immunised beforehand with a particular MCO. This is true of infections by mycoplasma, staphylococci, streptococci or E.coli (Rainard and Riollet 2003). This reaction, which is induced by systemic immunisation, is a local manifestation of a general phenomenon of sensitisation to an antigen.
Due to the importance of the Ig for the local defence of the MG, many research workers are focusing on enhancing of their concentrations in milk and sera (Hibbitt et al 1995; Sordillo et al. 1997). |