Introduction
Selenium (Se) is important in the nutrition of dairy cattle because of its association with reproductive performance and mastitis which continues to present a major threat to dairy herds globally. This is especially true for high yielding cows. In spite of improvement in management practices and dry cow therapy, mastitis remains a worldwide problem of prime economic importance to dairy producers (National Mastitis Council, 2005). Se is also important because of the growing public concern over food safety and milk quality especially in populations which are marginal in their dietary intake of Se. Bacterial infection and growth in the udder is the main cause of bovine mastitis (Burvenich et al., Reference Burvenich, Paape, Hill, Guidry, Miller, Heyneman, Kremer and Brand1994). Mastitis pathogens and residues of drugs (mainly penicillin and other beta-lactam antibiotics) used to treat mastitis are significant concerns. Furthermore, the producer suffers economic losses as a result of decreased quantity and quality of milk and increased veterinary costs.
The prevalence of mastitis in dairy herds and concerns about contamination of milk have stimulated a search for alternative treatment and control strategies. The innate and acquired immune responses evoked as a result of various pathogenic stimuli which cause mastitis have been studied by numerous research groups. As the neutrophils have been found to play a key role in combating mastitis pathogens, immunomodulation to strengthen the innate immune responses through genetic traits selection for enhanced neutrophil function has been proposed (Rainard and Riollet, Reference Rainard and Riollet2006). Besides genetic selection, which is a long-term strategy, more efficient nutritional modulation is another option. Research over several decades has shown that vitamins (A, E and β-carotenes) and micronutrients (Se, copper and zinc) affect the host immune system. Amongst these, Se has shown great promise in terms of effect on the neutrophil function, mammary gland health, mastitis, and economic losses.
Se performs its biological role through the genetically encoded selenocysteine residue of selenoproteins. It is now known that Se can affect three broad areas of cellular function: antioxidant activities, thyroid hormone metabolism, and the regulation of redox-active protein activity (McKenzie et al., Reference McKenzie, Arthur and Beckett2002). Out of 30–50 known selenoproteins (Köhrle et al., Reference Köhrle, Brigelius-Flohe, August Böck, Gärtner, Meyer and Flohe2000) at least 12 have been relatively well characterized as having wide ranging implications in immune function, malignancy and viral pathogenesis. The best-known selenoenzyme with respect to dairy cattle nutrition is glutathione peroxidase (GSH-Px). Indeed, it is an essential component of the cellular antioxidant defense mechanism which removes potentially damaging lipid hydro-peroxides and hydrogen peroxides and protects the immune cells from oxidative stress-induced damage. However, enhanced immune function of the component cells can not all be directly attributed to protection against peroxides. It is proposed that selenoenzymes may influence eicosanoid metabolism to modulate inflammation and chemotactically active compounds (Arthur et al., Reference Arthur, McKenzie and Beckett2003).
The role of Se in the immune function of the bovine mammary gland can be ascribed to its highly coordinated components. Interactions of Se with neutrophils are most important. Bactericidal activities of neutrophils are dependent on the production of free radicals, which can unequivocally result in harmful effects on the neutrophils themselves. Cytosolic GSH-Px helps to detoxify these detrimental metabolites. Neutrophils from Se-deficient mice, rats and cattle are able to ingest pathogens in vitro but are less efficient in killing them than are neutrophils from Se-sufficient animals (Arthur et al., Reference Arthur, McKenzie and Beckett2003). Improved Se status of cows is associated with increased adhesion and intracellular killing of bacteria (Cebra et al., Reference Cebra, Heidel, Crisman and Stang2003). There is a difference in the percentage of Escherichia coli killed by blood neutrophils from cows supplemented with Se-yeast or sodium selenite at 0.3 ppm in the diet (P<0.05) compared with cows fed with a diet without the Se supplement. Neutrophils from the Se-yeast fed group exhibited a little higher value than did neutrophils from the group fed sodium selenite (Weiss and Hogan, Reference Weiss and Hogan2005). Unspecified antibacterial activity against Staphylococcus aureus has been reported in milk whey from cows fed 4 mg supplemental Se as Se-yeast in their daily rations (Malbe et al., Reference Malbe, Attila and Atroshi2006).
Studies with the mouse have shown that Se is involved in the up-regulation of T-cell high affinity IL-2 cytokine receptors, which provides a vehicle for enhanced T-cell responses (Roy et al., Reference Roy, Kiremidjian-Schumacher, Wishe, Cohen and Stotzky1994). In dairy cows, it has been noted that intra-mammary infusion of IL-2 resulted in a cure rate of approximately 60% of mammary glands infected with S. aureus (Erskine et al., Reference Erskine, Eberhart, Grasso and Scholz1989). In fact, Se can partially reverse the age-related decrease in immunity by increasing the responsiveness to IL-2. Blood lymphocyte proliferation and chemotaxin production by milk macrophages can be improved by Se supplementation (Ndiweni and Finch, Reference Ndiweni and Finch1995, Reference Ndiweni and Finch1996). Negative correlations (P<0.05) have been reported between somatic cell count (SCC) and Se status of the animal (Hemingway Reference Hemingway1999; Kommisrud et al., Reference Kommisrud, Osteras and Vatn2005; Mukerjee, Reference Mukherjee2008).
It is evident that the health and immune function of the bovine mammary gland can be improved following Se supplementation of deficient animals. It has been found that plateau effects in Se status obtained after supplementation with organic and inorganic sources are quite different. A level of 600 ng Se ml−1 in the blood of dairy cows fed with selenized yeast at a dietary concentration of 6.25 mg kg−1 dry matter (DM) has been described as the maximum threshold level for the incorporation of Se in serum protein (Palacios et al., Reference Palacios, Encinar, Bertin and Lobinski2005) which is far higher than the plasma Se plateau level of 90 ng ml−1 reported earlier for cows supplemented with Se-yeast at a dietary level of 0.3 mg kg−1 DM (Ortman and Pehrson, Reference Ortman and Pehrson1999). Although the phenomenon of non-specific incorporation of selenomethionine in proteins may be involved, it has been noted that only about one-third of total selenium is in the form of selenomethione in milk from cows fed with Se-yeast as supplement (Juniper et al., Reference Juniper, Phipps, Jones and Bertin2006). Whether the improved Se status in animals supplemented with high doses of Se-yeast could be completely translated in terms of health benefits is debatable. Some of the most recent reports (Pavalata et al., Reference Pavlata, Prasek, Filipek and Pechova2004; Guyot et al., Reference Guyot, Spring, Andrieu and Rollin2007) with high supplemental doses of Se from both organic and inorganic sources have described the potential being translated in terms of disease resistance and immunoglobulin (Ig) production. Further research may help to delineate the effects of high doses of Se-yeast on the immune function and health of the dairy animals as limited data are available. Hence, this review article aims to integrate the information available on the topic of Se and its relationship with the immune function and health of the bovine mammary gland.
Mammary gland immune system – interactions with Se
The immune response is characterized by heterogeneity of reactive cells and their products, having specificity for the response and memory following subsequent antigen exposures. The bovine mammary gland produces colostrum, which is rich in antibodies that can protect the newborn from infectious agents (Sordillo et al., Reference Sordillo, Shafer-Weaver and DeRosa1997). The bovine mammary gland is itself protected by a variety of defense mechanisms, which can be separated into two distinct categories: innate immunity and adaptive immunity, each having sensing and effectors arms (Rainard and Riollet, Reference Rainard and Riollet2006). The innate and acquired immune systems interact closely in an attempt to provide protection against pathogens (Sordillo et al., Reference Sordillo, Shafer-Weaver and DeRosa1997; Burvenich et al., Reference Burvenich, Van Merris, Mehrzad, Diez-Fraile and Duchateau2003). The acquired immune response uses many innate immune effector mechanisms to eliminate micro-organisms and its action frequently increases innate antimicrobial activity (Oviedo-Boyso et al., Reference Oviedo-Boyso, Valdez-Alarcon, Cajero-Juarez, Ochoa-Zarzosa, Lopez-Meza, Braro-Patino and Baizabal Aguirre2007). The efficacy of the adaptive immune response rests in its specificity, memory of the immune cells and also, to some extent, on the immune stimulus, which is augmented by repeated exposure to the antigen. On the other hand, innate immunity is non-antigen-specific, exists prior to the encounter with the pathogens, and is related to the processes of acute and chronic inflammation and sepsis (Finlay and Hancock, Reference Finlay and Hancock2004).
Physical barriers
The first lines of defense against foreign molecules and invading pathogenic micro-organisms are the natural physical barriers of the body. Mastitis can occur when bacteria gain entrance into the mammary gland via the teat canal. The teat end contains sphincter muscles that maintain tight closure between milkings and hinder bacterial penetration. Increased patency of these muscles is directly related to an increased incidence of mastitis (Murphy and Stuart, Reference Murphy and Stuart1953; Myllys et al., Reference Myllys, Honkanen-Buzalski, Virtanen, Pyorala and Muller1994). The teat canal is lined with keratin, which is crucial to the maintenance of the barrier function of the teat and removal of the keratin correlates with increased susceptibility to bacterial invasion and colonization (Capuco et al., Reference Capuco, Mein, Nickerson, Jack, Wood, Bright, Aschenbrenner, Miller and Bitman1994; Sordillo and Steichner, Reference Sordillo and Streicher2002). Teat keratin is a waxy material derived from stratified squamous epithelium that traps invading bacteria and also exhibits bactericidal properties (Hibbitt et al., Reference Hibbitt, Cole and Reiter1969; Craven, Reference Craven1985). Esterified and non-esterified fatty acids (myristic, palmitoleic and linoleic) function as bacteriostatic agents, and are associated with keratin of the teat canal (Miller et al., Reference Miller, Bitman, Bright, Wood and Capuco1992). More recently, it has been noted that certain cationic proteins associated with keratin can bind to pathogenic micro-organisms, thus increasing their susceptibility to osmolarity changes leading to the lysis and death of the invading pathogens (Paulrud, Reference Paulrud2005). As a result of the efficacy of the teat canal barrier, the intra-mammary lumen is an aseptic chamber to which the aseptic character of normal milk can be attributed. Thus, the teat canal is an important barrier against intra-mammary infections.
There may be a role for Se in teat canal keratin function as it has been found that in mammalian spermatozoa phospholipid hydroperoxide GSH-Px (PHGSH-Px), a selenoprotein, is functionally associated with the cross-linking of the structural elements of the cytoskeleton via the oxidation of high sulfur keratin-associated proteins (Maiorino et al., Reference Maiorino, Roveri, Benazzi, Bosello, Mauri, Toppo, Tosatto and Ursini2005). There is no direct evidence of the association of Se with the bovine mammary gland teat canal.
Cellular factors
Bacteria and other pathogens, upon entry into the body tissues, are only able to cause disease by overcoming the body's natural cellular defense mechanism. A pivotal role is played by different types of cells in combating the pathogens. Cellular factors of the bovine mammary gland immune system come from two main types: the mammary epithelial cells (MECs) and the immune cells comprising macrophages, neutrophils, natural killer (NK) and dendritic cells. Collectively these constitute the somatic cells of the milk.
MECs were previously considered to be the major cell type in milk (Schalm et al., Reference Schalm, Carroll and Jain1971). However, later study confirmed that MECs are rarely found in the milk and the major cell type of the tissue and secretion of the bovine mammary gland is the macrophages (McDonald and Anderson, Reference McDonald and Anderson1981). The presence of sub- and intra-epithelial leukocytes, and the repertoire and distribution of sensor receptors on MECs makes the immune system of the mammary gland peculiar, resembling the urinary tract system and differing from the intestine (Rainard and Riollet, Reference Rainard and Riollet2006). MECs express mRNA for TLR 2, 4 and 9 and β-defensin 5, thus contributing positively towards the sensing of pathogens (Goldammer et al., Reference Goldammer, Zerbe, Molenaar, Schuberth, Brunner, Kata and Seyfert2004). Adhesion of bacteria and the interaction of bacterial toxins with the epithelial cells has been reported to induce the synthesis of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and IL-8 (Rainard, Reference Rainard2003).
Phagocyte responses
Much of the uptake of foreign antigens is performed by macrophages, neutrophils and NK cells in the mammary gland. During the defense of the mammary gland against bacterial infection, tissue and milk macrophages recognize the invading pathogen and initiate the inflammatory response by releasing pro-inflammatory cytokines (TNFα- and IL-1β), that induce neutrophil recruitment to the mammary gland (Banermann et al., Reference Bannerman, Paape, Goff, Kimura, Lippolis and Hope2004).
Macrophages are the major cell type in milk, secretions of the involuted udder, and mammary tissue (Jensen and Eberhart, Reference Jensen and Eberhart1981; McDonald and Anderson, Reference McDonald and Anderson1981). Although macrophages can ingest common mastitis pathogens, they are less active phagocytes than are milk neutrophils. Furthermore, both milk cell types are less efficient than their blood counterparts (Mullan et al., Reference Mullan, Carter and Nguyen1985). In addition to phagocytic activity, macrophages also play a role in antigen presentation (Politis et al., Reference Politis, Zhao, McBride and Burton1992), and are responsible for the removal of neutrophils following the elimination of bacterial pathogens. The functional capabilities of mammary macrophages decrease markedly during peri-parturient periods and this alteration has been linked to an increased mastitis incidence (Waller, Reference Waller2000; Sordillo and Streicher, Reference Sordillo and Streicher2002). Apart from the stress associated with parturition and the start of lactation, the underlying mechanism of the peri-parturient immunuosuppression is still unclear.
Ndiweni and Finch (Reference Ndiweni and Finch1995) worked with bovine mammary gland macrophages obtained from cows fed with a Se adequate diet. They investigated the effect of various doses of vitamin E, sodium selenite and combination of both on cellular functions in vitro. Sodium selenite supplementation in vitro from 1 nM to 10 μM to S. aureus-stimulated macrophages enhanced the production of chemotactic factors significantly (P<0.0033). Similar effects were recorded with vitamin E supplementation in the range from 5 ng ml−1 to 50 μg ml−1. There were no synergistic effects of both nutrients. Concentrations of Se above 0.1 mM depressed chemotaxin production. It was suggested that the stimulatory effect of Se might be attributed to its role as cofactor of LTB4 synthase or hydrase, as peritoneal macrophages from rats fed with selenium-deficient diets are not able to produce a respiratory burst reaction and as a result their antimicrobial function is compromized (Parnham et al., Reference Parnham, Bittner and Winkelmann1983).
Neutrophil numbers in normal milk from healthy bovine mammary gland are too low for efficient phagocytosis (Leijh et al., Reference Leijh, van den Barselaar, van Zwet, Dubbeldeman-Rempt and van Furth1979). Pro-inflammatory cytokines released by macrophages and MECs activate the expression of cellular adhesion molecules by endothelial cells that cause the binding and subsequent migration of blood neutrophils from blood to the site of infection, or in the milk where they are further localized. Following bacterial entry into the mammary gland, neutrophils are the first cells that are recruited into the milk and represent the predominant cell type. Neutrophil recruitment from the circulation to the site of infection is essential in the defense of the mammary gland against invading bacteria. The promptness of the recruitment and the number of recruited neutrophils, which vary in intensity according to pathogen type and the cow, determines the outcome of the infection.
Neutrophil concentrations increase rapidly between 3 and 12 h post-challenge and can reach more than 107 ml−1 in milk following Escherichia coli infusion in the mammary gland, whereas in the case of a S. aureus challenge, the recruitment is delayed (between 24 and 48 h) and remains below 106 ml−1 (Riollet et al., Reference Riollet, Rainard and Poutrel2000; Rainard and Riollet, Reference Rainard and Riollet2006). Recruited neutrophils at the site of infection phagocytose bacteria and produce reactive oxygen species, low molecular weight antibacterial peptides and defensins, which eliminate a wide variety of pathogens (Mehrzahd et al., Reference Mehrzad, Duchateau, Pyorala and Burvenich2002; Sordillo and Streicher, Reference Sordillo and Streicher2002; Paape et al., Reference Paape, Bannerman, Zhao and Lee2003). The increase in the concentration of milk neutrophil is, in fact, the origin of high SCC during mastitis and this is the reason why their presence is inversely correlated with the risk of intramammary infections (Burton and Erskine, Reference Burton and Erskine2003).
The most important and widely investigated association between Se and the immune function in dairy cows is the effect of this micronutrient on neutrophil function. Neutrophils perform their microbe killing function by producing super-oxide derived radicals. This type of process is a balance between sufficient radical production for microbial killing and the system that protects the neutrophils themselves from these radicals. This balance is attributed to the cytosolic GSH-Px activity within the neutrophils, which is impaired in Se deficiency, which permits neutrophils to be self-destroyed. The earliest evidence regarding the effect of Se on neutrophil function was reported by Boyne and Arthur (Reference Boyne and Arthur1979). In that study, it was noted that the ability of neutrophils to phagocytize Candida albicans cells was not different (P<0.05) between Se-deficient and Se-supplemented calves receiving 0.1 mg of dietary Se day−1. However, the number of neutrophils with the ability to kill phagocytosed C. albicans cells was about three times less for Se-deficient animals having undetectable levels of blood GSH-Px activity. On the other hand, both phagocytosis (P<0.05) and killing (P<0.01) of S. aureus by blood PMN leukocytes were higher (P<0.05) when the dairy cows received between 10 and 17 mg Se day−1, along with an additional 350–1000 mg vitamin E day−1 for a period of 16 days (Gyang et al., Reference Gyang, Stevens, Olson, Tsitsamis and Usenik1984). However, phagocytosis by neutrophils from cattle supplemented with selenite or selenate at low levels (2 mg day−1 or 0.2 mg/kg−1 DM, respectively) was not different from that of neutrophils from unsupplemented cows.
Direct and indirect measures of bacterial killing were higher (P<0.05) in neutrophils isolated from Se-supplemented cattle as compared with those from unsupplemented cows (Grasso et al., Reference Grasso, Scholz, Erskine and Eberhart1990; Hogan et al., Reference Hogan, Smith, Weiss, Todhunter and Schockey1990). In a survey conducted by Cebra et al. (Reference Cebra, Heidel, Crisman and Stang2003), it was observed that higher blood Se levels (>300 ng ml−1) were associated with enhanced neutrophil adhesion and intracellular kill by the neutrophils obtained from post-parturient cows. With PMN cells isolated from the blood of Se-adequate cows, it was found that in vitro supplementation of Se (10 μM) had greater stimulatory effect (129%) on their random migration than did vitamin E (71%) and, at the highest concentration of selenite used (1 mM), random migration of PMN was inhibited (Ndiweni and Finch, Reference Ndiweni and Finch1996). On the other hand, vitamin E enhanced phagocytosis of S. aureus to a greater extent than did sodium selenite after a 2 h incubation period. Both nutrients were not significantly different in their ability to stimulate PMN cells to produce superoxides. Enhanced recruitment of neutrophils at the site of infection in Se-supplemented cows has also been reported previously (Ali-Vehmas et al., Reference Ali-Vehmas, Vikerpuur, Fang and Sandholm1997).
Organic and inorganic sources of Se at 0.3 mg kg−1 DM intake have been compared for their effect on the function of neutrophils obtained from the blood of lactating cows (Weiss and Hogan, Reference Weiss and Hogan2005). There were no significant differences regarding either the ability of neutrophils to phagocytose bacteria or the percentage of E. coli that were killed, although there was a slight increase in the percentage kill for the Se-yeast group. These observations agree with those of Malbe et al. (Reference Malbe, Klaassen, Fang, Myllys, Vikerpuur, Nyholm, Sankari, Suoranta and Sandholm1995) regarding the effect of Se source on bovine neutrophil phagocytosis of S. aureus. A plausible explanation for this effect might be the non-specific pooling of selenomethionine from organic Se sources into tissue proteins instead of methionine and the presence of 0.2% sulfur in the diets. However, it is difficult to interpret such data as a negative control was not included. More recently, Mukherjee (Reference Mukherjee2008) has reported an improvement (P<0.05) in phagocytosis of S. aureus by milk neutrophils obtained from mastitic riverine buffaloes that had been injected with a Se/vitamin E preparation containing sodium selenite and had been treated with enrofloxacin.
Lymphocyte responses
Long-term cellular specific immunity is a function of both antigen-presenting cells and lymphocytes, which are the only cells of the immune system that recognize antigens by membrane receptors specific to invading pathogens. If the invading pathogens survive the activities of macrophages and neutrophils, T and B lymphocytes and monocytes become the predominant cell type. Leitner et al. (Reference Leitner, Eligulashvily, Krifucks, Perl and Saran2003) observed that lymphocytes were the most common infiltrating cell type within the two-layer epithelium lining the teat cistern; monocytes and macrophages were present in lower number. Nevertheless, neutrophils remain most important in chronic mastitis (Rainard and Riollet, Reference Rainard and Riollet2006). T lymphocytes are classified into two main groups: Tαβ includes CD4+ (helpers), CD8+ (suppressors) and Tγδ. In healthy mammary glands, CD8+ lymphocytes are the prevailing type, whereas in mastitis-infected mammary glands, CD4+ cells are predominantly activated by the formation of a molecular complex between the major histocompatability complex class II (MHC II) and antigens presented by B lymphocytes and macrophages (Park et al., Reference Park, Joo, Park, Moon, Kim and Kwon2004). Through their ability to secrete certain cytokines, CD4+ cells help B lymphocytes to proliferate and secrete antibodies. CD4+ cells are mainly found in the inter-alveolar tissue of the mammary gland whereas CD8+ cells surround the alveoli (Leitner et al., Reference Leitner, Eligulashvily, Krifucks, Perl and Saran2003).
In contrast with the milk, cells obtained from blood exhibit a higher ratio of CD4+ to CD8+ cells; however, the functional significance of this elevated frequency has not been clearly established. CD8+ cells may be either cytotoxic or suppressor type. Post-partum they are mainly of the cytotoxic type, whereas during mid- and late-lactation they are of the suppressor type (Sordillo et al., Reference Sordillo, Shafer-Weaver and DeRosa1997). Cytotoxic T cells recognize and eliminate altered self-cells via antigen presentation in conjunction with MHC I molecules. They act as the scavengers of old and damaged secretory cells and their secretions are related to the susceptibility of the bovine mammary gland to infections (Oviedo-Boyso et al., Reference Oviedo-Boyso, Valdez-Alarcon, Cajero-Juarez, Ochoa-Zarzosa, Lopez-Meza, Braro-Patino and Baizabal Aguirre2007). Although Tγδ cells are not well characterized, they are associated with the epithelial surface where they destroy damaged epithelial cells (Yamaguchi et al., Reference Yamaguchi, Hiratsuka, Asai, Kai and Kumagai1999).
NK cells, B cells and dendritic cells are also parts of the bovine mammary gland immune system. NK cells are large granular lymphocytes that have cytotoxic activity independent of MHC, through antibody-dependent cell-mediated cytotoxicity. In contrast with neutrophils and macrophages, they are critical to the removal of intracellular pathogens. Bovine NK-like cells (CD2+, CD3− and T Lymphocytes), express bactericidal activity against S. aureus upon stimulation with IL-2 in a non-specific manner (Sordillo et al., Reference Sordillo, Kendall, Corl and Cross2005). These cells destroy both Gram-positive and Gram-negative bacteria and are fundamental to the prevention of bovine mammary gland infections (Sordillo and Streicher, Reference Sordillo and Streicher2002). The primary role of B lymphocytes is to produce antibodies against invading pathogens. In doing so, they utilize their cell-surface receptors to recognize specific pathogens and process the antigens. Processed antigens are thus presented to T helper cells, which secrete cytokine IL-2 which, in turn, induces the proliferation and differentiation of B lymphocytes into either plasma cells that produce antibodies or memory cells. Not much is known about the density and role of dendritic cells in the bovine mammary gland immune system. Normally they are associated with antigen presentation.
It has been suggested that Se and vitamin E deficiencies affect T lymphocytes to a greater extent than B lymphocytes (Larsen, Reference Larsen1993). This was suggested to be the result of higher levels of polyunsaturated fatty acids in T lymphocytes and associated with higher membrane fluidity. Se and vitamin E deficiencies may affect both the maturation of specific lymphocyte subpopulations and proliferative capabilities of peripheral lymphocytes (Surai, Reference Surai2006). In an experiment with dairy cows fed with either basal diet (~0.05 mg Se/kg DM) or a diet supplemented with sodium selenite (~0.20 mg Se kg−1 DM), it was noted that con A stimulated lymphocyte proliferation was significantly higher in the Se-supplemented group (Cao et al., Reference Cao, Weaver, Reddy and Sordillo2002). Similar findings have been reported when bovine peripheral blood lymphocytes were supplemented with sodium selenite in vitro from 1 nM to 10 μM concentrations (Ndiweni and Finch, Reference Ndiweni and Finch1995).
Se supplementation or deficiency in mice altered the kinetics of IL-2 receptor expression (Roy et al., Reference Roy, Kiremidjian-Schumacher, Wishe, Cohen and Stotzky1994). Supplementation in vitro or in vivo resulted in an earlier expression of high affinity IL-2 receptors, whereas Se deficiency resulted in a delayed expression of receptors. This may explain the stimulatory role of Se in the enhanced T cell function. In healthy aged humans, Se supplementation (400 μg day−1, for 6 months) enhanced NK cell cytotoxicity over pre-treatment levels by 58% (Wood et al., Reference Wood, Beckham, Yosioka, Darban and Watson2000). There is no information on the effect of Se on NK cell and dendritic cell function in dairy cows. It is interesting to note that enhanced immune cell function resulted from Se supplementation levels which are higher than normally recommended.
Soluble factors
Soluble factors of the bovine mammary gland immune system are made up of various proteins that include complement proteins, cytokines and Igs. Each class performs its physiologically defined function with a high level of specificity.
The bovine complement system is a collection of proteins that is present in serum and milk, and has an important role in the defense of the mammary gland. Complement proteins are predominantly produced by hepatocytes, though they are also produced by monocytes and macrophages in different tissues. In the presence of antibodies they lyse invading pathogens. Complement component C3b binds the antibody bacteria complex for efficient phagocytosis by neutrophils and macrophages (Paape et al., Reference Paape, Bannerman, Zhao and Lee2003), whereas C5a stimulates the recruitment of neutrophils, which augments their phagocytic and bactericidal activities (Rainard and Pourtel, Reference Rainard and Poutrel2000).
Cytokines are produced by both immune and non-immune cells and are essential in almost all aspects of host defense. They regulate the activities of cells involved in the immune function. A variety of cytokines such as IL-1β, IL-2, IL-6, IL-8, IL-12, colony stimulating factor (CSF), interferon gamma (IFN-γ) and TNF-α have been detected in healthy and infected bovine mammary glands (Sordillo and Streicher, Reference Sordillo and Streicher2002; Alluwaimi, Reference Alluwaimi2004). TNF-α is the main cytokine produced by macrophages, neutrophils and epithelial cells during the early stage of infection and participate in the neutrophil chemotactic activity (Persson et al., Reference Persson, Colditz, Lun and Östensson2003). IFN-γ is produced by CD4+ and CD8+ lymphocytes and NK cells in response to mitogenic and antigenic stimuli. IFN-γ functions in activating the acquired immune response and phagocytic activity of neutrophils and is important in viral infections (Strichman and Samuel, Reference Strichman and Samuel2001). IL-1β is produced by monocytes, macrophages and epithelial cells. During the inflammatory response, IL-1β regulates the expression of adhesion molecules and neutrophil chemotaxis in E. coli infections (Yamanak et al., Reference Yamanaka, Hisaeda, Hagiwara, Kirisawa and Iwai2000). IL-2, produced by CD4+ lymphocytes, regulates the acquired immune response by stimulating the growth and differentiation of B lymphocytes and the activation of NK and T cells. Alterations in IL-2 production cause a decrease in the mammary gland immune response capacity, which facilitates mastitis (Sordillo et al., Reference Sordillo, Campos and Babiuk1991; Sordillo and Streicher, Reference Sordillo and Streicher2002).
Igs are synthesized by plasma cells that are differentiated from B lymphocytes upon activation by IL-2. In milk, Igs are either synthesized locally or originate from blood (Sordillo and Nickerson, Reference Sordillo and Nickerson1988). The role of antibodies in the natural defense mechanisms of the udder is to opsonize bacterial pathogens, thereby aiding the neutrophils and macrophages in phagocytosis.
Four classes of Ig are known to influence mammary gland defense against bacteria causing mastitis: IgG1, IgG2, IgA and IgM. Each of these classes differs in physiochemical and biological properties (Gershwin et al., Reference Gershwin, Krakowka and Olsen1995). The concentration of each Ig in the mammary secretion varies with the stage of lactation, increasing during dry periods and approaching peak concentrations during colostrogenesis (Sordillo and Nickerson, Reference Sordillo and Nickerson1988). The largest part of the opsonic antibodies in adult serum and milk of cows is IgM (Willam and Hill, Reference Williams and Hill1982; Hill et al., Reference Hill, Heneghan and Williams1983). The presence of IgM in cows, without a previous history of mastitis, suggests that they are mainly autoantibodies directed against self-antigens and are poly-reactive in nature (Rainard and Riollet, Reference Rainard and Riollet2006). In this regard, the cow is not different from humans or rodents, who also have these types of antibodies in their blood (Saini et al., Reference Saini, Allore, Jacobs and Kaushik1999).
Non-specific proteins such as lactoferrin, lysozyme, transferrin, xanthine oxidase and the lactoperoxidase system, exhibit bacteriostatic and bactericidal activities against common mastitis pathogens. In normal function, various components of the innate and adaptive immune systems are coordinated to provide protection to animals against invasion by pathogens.
Improved Se status of animals results in enhanced Ig titer in colostrum from cows receiving a high dose of Se, administered by intramuscular injection pre-partum (Pavlata et al., Reference Pavlata, Prasek, Filipek and Pechova2004). Earlier studies reported lower (P<0.05) concentrations of IgG and IgM in plasma and colostrum of beef cows and calves fed with a free-choice salt/mineral mixture containing 20 ppm Se as sodium selenite, compared with the cows and calves fed with a salt mixture containing 60 ppm Se in the form of Se-yeast compound, or 120 ppm sodium selenite. The source of Se affects only the IgM concentration of plasma with higher concentrations (P<0.05) when cows are fed a Se-yeast supplement (Awadeh et al., Reference Awadeh, Abdelrehman, Kincaid and Fineley1998).
It was recommended that consideration should be given to the concentrations of T3 (thyroid hormone) and IgG while determining the nutritional requirement of cattle for Se. Swecker et al. (Reference Swecker, Eversole, Thatcher, Blodgett, Schurig and Meldrum1989) also confirmed higher concentrations of colostral IgG in beef cows fed with higher Se levels in free choice mineral mixtures. Enhanced proliferation of B lymphocytes in cell cultures containing 100 ng ml−1 Se suggests a mechanism for the increased IgM production (Stabel et al., Reference Stabel, Reinhardt and Nonnecke1991). Contradictory observations of a non-effect (P<0.05) of Se on the Igs have also been reported (Lacetera et al., Reference Lacetera, Bernabucci, Ronchi and Nardone1996; Leyan et al., Reference Leyan, Wittwer, Contreras and Kruze2004). It is noteworthy that positive effects have been observed only when higher Se doses were used. Depression in several leukocyte function parameters, including the forced antibody response, was noted in pre-parturient beef cows consuming 6 ppm or 12 ppm Se as sodium selenite from their diets (Yaeger et al., Reference Yaeger, Neiger, Holler, Fraser, Hurley and Palmer1998).
Studies on the interactions of Se with the immune system of the mammary gland are summarized in Table 1. There is little data on the effect of Se on cytokines and other soluble factors in dairy cows available.
The effects of Se on the immune responses of dairy cattle
a Se=selenium; b C=controlled experiment; c DM=dry matter.
Se status of cows and udder health
The nutritional status of the animal is related to its overall health and its capacity to combat disease. The nutritionally modulated improvement of the immune system should culminate in increased resistance to disease. Research on micronutrients and their immunoregulatory role regarding udder health and bovine mastitis, has focused mainly on Se, vitamin A, vitamin E, β-carotene, copper and zinc. Among these, Se has been the most characterized trace element affecting bovine mammary gland health through its role in cell function.
Having been recognized as a dietary essential, Se is being routinely supplemented in the rations of farm animals. In the United States, 0.1 mg Se kg−1 DM is recommended for ruminant rations to correct symptoms of a Se deficiency. However, owing to the beneficial effects of the additional Se supplementation, the recommendation was increased to a level of 0.3 mg kg−1 DM (NRC, 2001). The German Society for Nutritional Physiology (GfE) has recommended that Se intake levels for dairy cattle should range from 0.15 to 0.2 mg kg−1 DM (GfE, 2001) whereas the recommendations by the British authorities are 0.1 mg kg−1 DM (MAFF, Reference Haresign and Cole1983). Supplementation of this nutrient to dairy animals can be one of the best options, not only to protect the animal from disease threats, but also to raise the selenium level in milk and subsequently transfer this essential element to the human population, many of whom are marginal deficient in selenium.
Biomarkers for Se status
The scientific controversy regarding the identification of the best biomarker for Se status assessment is still unresolved. In dairy cows, several approaches have been followed to assess the status of the herd or the individual animal. These approaches include the direct estimation of Se in whole blood, serum or plasma, milk and others tissues of interest; and indirect measures such as the intra- and extra-cellular activities of the Se containing enzyme, GSH-Px in whole blood, serum or plasma. A number of studies have shown that Se or GSH-Px activity represents the short-term Se status, while parameters for the whole blood or erythrocytes reflect the long-term Se status. The reference range of serum Se level of 70–100 ng ml−1 was determined by Stowe and Herdt (Reference Stowe and Herdt1992). This value has been described as an adequate level. Earlier reports (Maus et al., Reference Maus, Martz, Belyea and Weiss1980; deToledo and Perry, Reference deToledo and Perry1985) suggested that an adequate Se level in blood serum should be in the range of 40–120 ng ml−1. Variations in these findings may be the result of dietary concentration and nutritional management practices. Gerlof (Reference Gerloff1992), on review of the data from various research groups, considered the value of 70–100 ng ml−1 for serum Se as a consensus of opinion regarding the adequacy of Se, particularly when the dietary source is inorganic selenate or selenite.
With the discovery that GSH-Px has selenocysteine as its essential component, the activity of this enzyme has been regarded as the pertinent parameter for the assessment of Se status. Although numerous studies have associated the activity of GSH-Px with the Se status of the animal because of a linear response of GSH-Px activity with Se supplementation, GSH-Px activity as the parameter of Se status assessment has been criticized (Stowe and Herdt, Reference Stowe and Herdt1992). Inconsistency of units used in expressing the enzyme concentrations, difficulty in ensuring the proper storage conditions of samples, enzyme concentrations that reach a plateau, whereas serum Se concentrations continue to rise and delayed response to supplementation and different cellular and extra cellular forms are all points which need to be taken into account when considering GSH-Px activity as a criterion for Se status of the animal. On the other hand, the relationship between GSH-Px and health is better explained than between plasma Se concentration and health. Awadeh et al. (Reference Awadeh, Abdelrehman, Kincaid and Fineley1998) showed that only one-third of total Se intake is incorporated into GSH-Px, and that GSH-Px activity is largely confined to the erythrocytes.
Milk Se concentrations can potentially be used as a simple parameter for the Se status assessment of dairy herds. In a study conducted with large dairy herds over several seasons, a sigmoid relationship with an adjusted R 2 value of 0.92 (P<0.0001) was observed between the bulk tank milk Se and mean serum Se values (Wichtel et al., Reference Wichtel, Keefe, Van Leeuwen, Spangler, McNiven and Ogilvie2004). A plateau effect was noted in serum Se concentrations when milk concentrations exceeded 20 μg l−1. On the basis of the relationship observed tentative reference values for bulk tank milk Se have been generated. Milk Se concentrations less than 9.6 ng ml−1 are considered to indicate a deficiency, while a value of 21.8 ng Se ml−1 appears to represent an adequate Se supply. The value 15.7 ng ml−1 is the median between the marginal range of the low and high categories. However, it is notable that the source of the Se has not been considered while making the bulk tank milk Se an accurate measure of the herd Se status. Many studies have reported that milk Se concentrations were significantly higher when diets were supplemented with Se-yeast as compared with sodium selenite at the same level (Ortman and Pehrson, Reference Ortman and Pehrson1999; Muniz Naviero et al., Reference Muniz-Naveiro, Dominguez-Gonzalez, Bermejo-Barrera, De Juan, Bermudez, Pereiras, Santamaria, Lede, Puente, Gomez and Bermejo-Barrera2005; Juniper et al., Reference Juniper, Phipps, Jones and Bertin2006). Positive correlations, irrespective of the source of Se supplementation, of 0.59, 0.64 and 0.68 have been observed between the cows' milk and their calves erythrocytes GSH-Px activity, whole blood, and plasma Se concentrations, respectively (Perhson et al., Reference Pehrson, Orman, Madjid and Trafikowska1999). A cautious estimate of the herd Se status can be made by bulk tank milk Se concentrations, keeping the source of Se supplementation in mind.
The source and dietary level of the nutrient are important in determining the nutritional status of the animal. Different supplements of Se are categorized on the basis of organic and inorganic forms. Sodium selenite and sodium selenate are common inorganic forms, whereas the organic form of Se is produced from the yeast Saccharomyces cerevisiae, with almost 90% of the total Se represented by selenomethionine (Muniz-Naveiro et al., Reference Muniz-Naveiro, Dominguez-Gonzalez, Bermejo-Barrera, De Juan, Bermudez, Pereiras, Santamaria, Lede, Puente, Gomez and Bermejo-Barrera2005). As far as the bioavailability of Se from organic versus inorganic sources is concerned, whole blood Se concentration, GSH-Px activity and milk Se concentration in dairy cattle increase more efficiently after dietary Se supplementation using organic sources compared with inorganic ones (Malbe et al., Reference Malbe, Klaassen, Fang, Myllys, Vikerpuur, Nyholm, Sankari, Suoranta and Sandholm1995; Ortman and Pehrson, Reference Ortman and Pehrson1999; Awadeh et al., Reference Awadeh, Abdelrehman, Kincaid and Fineley1998; Knowels et al., Reference Knowles, Grace, Wurms and Lee1999; Gunter et al., Reference Gunter, Beck and Phillips2003). However, Se yeast and selenite follow a similar pattern of distribution among serum proteins (Awadeh et al., Reference Awadeh, Abdelrehman, Kincaid and Fineley1998). Cattle fed with Se yeast have a higher percentage of Se in whole blood (average 20%), milk (average 90%) and increased activity of GSH-Px (16%) compared with cattle fed with inorganic Se (Weiss, Reference Weiss2005). Previously, Knowles et al. (Reference Knowles, Grace, Wurms and Lee1999) had reported no difference in the blood GSH-Px activity between cows fed with Selenite and those fed with a Se-yeast compound, provided the cows consumed 4 mg day−1 of supplemental Se (approximately 0.2 ppm). However, when cows were fed 2 mg day−1, the GSH-Px activity was 50% higher than when Se yeast was used as source of dietary Se. Comparative increases in milk and blood Se levels after supplementing the diet of cows with a Se-yeast source have been largely attributed to non-specific incorporation of selenomethionine from the diet into the tissue proteins (Weiss, Reference Weiss2005). However, Juniper et al. (Reference Juniper, Phipps, Jones and Bertin2006), after conducting a study with Se supplementation in the range of 0.27–0.4 ppm from a selenium-containing yeast source, reported that only 25–33% of total milk Se increase could be attributed to selenomethionine and that there are other selenoproteins in milk, which might play a role as an antioxidant.
Interactions between the Se status of dairy cows and the udder defense system have been explored. Parameters of milk somatic cells and microbial counts, incidence and duration of clinical mastitis cases in dairy herds, and controlled experiments with or without the experimental challenge of pathogenic microbes, have been the prime focus in this area. With the advent of Se yeast products on the market, research is now focusing on safety and comparative efficacies. Based on the information cited above, it can be inferred that the Se status of the animal is directly correlated with dietary level and source, and organic Se sources tend to be comparatively more efficient in maintaining the Se status of the animal than are inorganic sources.
SCC and Se status
The SCC of milk is used as a benchmark parameter to estimate udder health and consequently milk quality. Cell concentration of the milk varies widely as a function of the lactation cycle. In healthy udder conditions, very few leukocytes should migrate into milk during full lactation. At cessation of milking, the SCC might increase owing to the intense physiological changes occurring in the udder. Milk from a healthy bovine udder should contain very few somatic cells (<20 000 ml−1), and whenever the SCC rises above 20 000 ml−1, there has been histological evidence of inflammation in the udder (Schalm et al., Reference Schalm, Carroll and Jain1971). Rainard and Riollet (Reference Rainard and Riollet2006) reported that the SCC in most uninfected and uninflamed quarters is considerably less than 100 000 ml−1, with a low portion of neutrophils which can increase up to 40% near the drying off period. Somatic cell concentrations increase to reach 2–5×106 ml−1 during the first 7–10 days of the dry period. They then remain stabilized in the range of 1–3×106 ml−1 After parturition, the SCC decreases to 105 ml−1 in the first 7–10 days after calving (Jensen and Eberhart, Reference Jensen and Eberhart1981; McDonald and Anderson, Reference McDonald and Anderson1981).
Higher SCC values in milk reflect a diseased udder making the milk less valuable. It is evident (Table 2) that milk SCC is negatively correlated to the Se status of the animal. It was reported that the cow's udder is more prone to infection if GSH-Px activity in the blood is below 3.3 μkat g−1 of hemoglobin (Malbe et al., Reference Malbe, Klaassen, Kaartinen, Attila and Atroshi2003). Lack of GSH-Px activity causes oxidative damage to soft tissue, thus making the udder more vulnerable to mastitis pathogens. Consequently, infiltration of neutrophils in the udder tissue will cause the SCC to rise to higher levels. The effective role of neutrophils in combating the microbial threat is also dependent on GSH-Px activity. Enhanced viability and vitality of neutrophils in response to optimum GSH-Px activity could be a plausible explanation for the low SCC in the milk of cows having improved Se status and consequent enhanced GSH-Px activity.
The effects of Se bovine udder health and susceptibility to mastitis
a Se=selenium; bC=controlled experiment; cSCC=somatic cell count; d GSH-Px=glutathione peroxidase; e DM=dry matter.
Few studies failed to find a correlation (Grace et al., Reference Grace, Knowles and Lee1997; Wichtel et al., Reference Wichtel, Keefe, Van Leeuwen, Spangler, McNiven and Ogilvie2004) between the Se status of cows and disease susceptibility. This has been attributed to the fact that the data involved the results of surveys conducted with herds having different management practices. Marginal bulk tank milk Se levels of (0.018 μg ml−1), and corresponding marginal serum Se levels, could have been the reason why Wichtel et al. (Reference Wichtel, Keefe, Van Leeuwen, Spangler, McNiven and Ogilvie2004) did not find any substantial relationships between bulk tank milk Se levels and the general parameters used to assess udder health.
Mastitis susceptibility and Se status
Low Se status is linked to increased susceptibility of dairy cows to intramammary infections (Table 2). Marked reduction (up to 60%) in infected mammary gland quarters has been observed in dairy cows after Se supplementation for a period of 8 weeks at 0.2 ppm dietary level (Malbe et al., Reference Malbe, Klaassen, Fang, Myllys, Vikerpuur, Nyholm, Sankari, Suoranta and Sandholm1995; Ali-Vehmas et al., Reference Ali-Vehmas, Vikerpuur, Fang and Sandholm1997). Duration of clinical mastitis was reduced by 46% in cows supplemented with Se and by 62% in cows supplemented with Se and vitamin E (Smith et al., Reference Smith, Harrison, Hancock, Todhunter and Conrad1984).
Supplementation with Se and/or vitamin E at levels far above those required for growth and normal physiological function can result in the improvement of various components of the immune system and general animal health (Surai, Reference Surai2006). This is particularly important for cows infected with pathogens. In an experiment described by Hemingway (Reference Hemingway1999), 14 of 36 cows receiving intramammary antibiotic infusions at drying off needed extra treatment in the subsequent lactation, whereas only 5 of 36 cows which received additionally 4 mg Se at drying off needed such treatment. Udder health benefits have been attributed to antibacterial activities against S. aureus in milk whey protein (Ali-Vehmas et al., Reference Ali-Vehmas, Vikerpuur, Fang and Sandholm1997; Malbe et al., Reference Malbe, Attila and Atroshi2006). The underlying mechanism of this antibacterial activity is not well understood. However, it was proposed that impaired microbial growth rate in the whey fraction exhibiting high GSH-Px activity may account for the results. The absence of both glutathione and GSH-Px in bovine milk has been reported (Stagsted, Reference Stagsted2006). Therefore, further generation of more reactive radical oxygen species by phagocytes or the presence of other selenoproteins in milk may account for the results obtained. It can be concluded that Se may affect mastitis susceptibility of the mammary gland by improving the phagocyte recruitment to the infected quarters, increasing their vitality and inducing unspecified antibacterial activity in milk whey against various pathogens.
Conclusion
Survey findings and controlled studies with or without experimental challenge indicate a role for Se in the immune function and improvement in bovine mammary gland health. Although Se status has been noted to markedly increase as a result of the supplementation with Se yeast as compared with inorganic sources; whether this increase is completely translated in terms of health benefits to the animal is not clear. Most recent findings have confirmed that Se levels higher than those considered adequate can potentially enhance the natural defense mechanisms of the bovine mammary gland at maximum, especially the humoral responses.
There are limited studies on the clinical aspects of the health of the bovine mammary gland, as affected by organic versus inorganic Se sources, or a combination of the two sources of Se. Moreover, there are many gaps in our knowledge of the interactions of Se with the immune function of the bovine mammary gland. Neutrophil function has been the major point of focus of the research. Other aspects of the immune response, notably, the activity of NK cells as affected by Se supplementation in combating both Gram-positive and Gram-negative mastitis pathogens, has not been studied. Furthermore, certain cytokines and MECs and lymphocyte proliferation response have great implications for mammary gland health and mastitis control. More work is required to delineate these interactions. It is proposed that dietary Se recommendations can be increased up to 0.6 mg kg−1 DM to enhance its benefits while remaining far below the toxic level.
Acknowledgments
We acknowledge the support to S. Salman provided by the Deutscher Akademischer Austausch Dienst (DAAD) and the Higher Education Commission (HEC) of Pakistan.
