J. S. Hogan
Department of Animal Sciences
OARDC/The Ohio State University
Wooster, OH 44691.
The theory that bovine nutrition and resistance to mastitis are interrelated has been demonstrated by a number of different models. The primary interest in recent years has been refined to the effects of micronutrient deficiencies on different mammary health parameters. As evidenced by the brilliant work presented by the noted scientists in this symposium in honor of Dr. A.L. Moxon, Se and vitamin E are integral components the host defenses against infectious diseases. The concerted efforts of nutritionists, immunologists and veterinarians to define optimum dietary concentrations of Se and vitamin E have led to an universal improvement in bovine mammary health and milk quality.
ßIncidence and severity of mastitis are related to vitamin E and Se status of a dairy herd. Dietary vitamin E and Se supplementation of dairy cows reduces the rate and duration of clinical mastitis. The first controlled studies on the effects of vitamin E and Se supplementation on clinical mastitis was reported by Smith et al. (1984; 1985) and thoroughly outlined by Dr. Weiss in this symposium. These pioneering trials led to a series of field investigations and surveys that substantiated vitamin E and selenium deficiencies were common on dairy farms around the world and related to mammary health.
Erskine et al. (1987) reported a negative correlation between the percentage of quarters infected with major pathogens and mean herd GSH-Px activity in whole blood. Serum a-tocopherol did not differ between high and low SCC herds (Erskine et al., 1987). Atroshi et al. (1986) reported Finnish cows with clinical mastitis had lower erythrocyte GSH-Px activity and lower milk and plasma a-tocopherol concentrations than uninfected herd mates. In contrast, Ropstad et al. (1987) reported that Norwegian herds with high Se status had higher frequencies of treatments for clinical mastitis and higher milk SCC than herds with lower Se status. In a survey of Ohio herds, vitamin E and Se were related to rate of clinical mastitis and bulk tank milk SCC (Weiss et al., 1990). High serum Se concentrations were associated with reduced rates of clinical mastitis and low bulk tank milk SCC. Concentration of Se in serum was correlated positively to concentration of Se in the diet until cows consumed more than 5 mg/d of Se. Above this value, serum Se was independent of Se intake. Concentration of vitamin E in the diet was negatively correlated with rate of clinical mastitis. Vitamin E intake was correlated positively with plasma a-tocopherol concentrations, but vitamin E intake had a greater effect on serum a-tocopherol values in dry cows than in lactating cows.
Dietary supplementation of mammals with vitamin E and Se is important to maintain host defense mechanisms, including antibody production, cell proliferation, cytokine production, prostaglandin metabolism and neutrophil function (reviewed; Smith, 1986). The greatest body of information concerning the bovine host defense deals with neutrophil function. Neutrophils are considered to be a primary defense mechanism against bacterial infections in mammals. The importance of neutrophils in host defense against bovine intramammary infections (IMI) is well-documented (Craven and Williams, 1985). Incidence and severity of clinical signs associated with IMI depend on responsiveness of neutrophils. Therefore, herd management practices that result in optimal vitamin E and Se status of dairy cows also optimize neutrophil responses and increase resistance to IMI.
The earliest trials to determine effects of vitamin E and Se on neutrophil function were performed on species other than the cow. Neutrophils that were from either vitamin E-deficient or Se-deficient mice (Heinzerling et al., 1974) and humans (Baehner et al., 1977; Boxer, 1986; Boxer et al., 1979) had impaired bactericidal activities. The respiratory burst by neutrophils is characterized by marked changes in oxygen metabolism that result in increased production of superoxide and hydrogen peroxide (Baboir, 1984). Although neutrophil-generated oxygen metabolites are necessary in antimicrobial defense mechanisms, these free radicals also can damage the neutrophil and surrounding tissues. Vitamin E and GSH-Px both are cellular antioxidants that protect against the cytotoxic capabilities of oxygen metabolites. Vitamin E protects at the membrane, whereas GSH-Px activity is in the cytosol. Glutathione peroxidase converts hydrogen peroxide to water and lipid hydroperoxides to the corresponding alcohol. Vitamin E inhibits autoxidation of polyunsaturated fatty acids in neutrophil membranes (Baehner et al., 1977; Baker and Cohen, 1983). Vitamin E is localized in cellular membranes in close proximity to the mixed function oxidase enzymes that initiate the production of free radicals.
Benefits of Se supplementation on speed of neutrophil response to mammary irritation was tested by experimental challenge of quarters with mastitis pathogens. Erskine et al. (1989) infused Escherichia coli into mammary quarters of cows fed diets supplemented with Se (.14 ppm of Se) or unsupplemented (.04 ppm of Se). Selenium-supplemented cows had more rapid SCC response following challenge, maintained lower bacterial colony forming units per milliliter of milk, eliminated IMI more rapidly and had less severe clinical signs than did unsupplemented cows. Selenium status of cows also had an effect on the ability of milk neutrophils to kill mastitis pathogens in vitro (Grasso et al., 1990). Neutrophils collected from cows fed Se-supplemented diets had increased intracellular kill of bacteria, enhanced viability and reduced extracellular hydrogen peroxide concentration compared with neutrophils harvested from milk of cows fed Se-deficient diets. Intracellular kill of bacteria also was greater in blood-derived neutrophils isolated from cows supplemented with parenteral Se than in neutrophils from cows without supplemental Se (Gyang et al, 1984). Ability of neutrophils to phagocytize bacteria was independent of Se (Grasso et al., 1990; Gyang et al., 1984; Hogan et al., 1990).
Vitamin E supplementation of diets increased intracellular kill of Staphylococcus aureus and E. coli by bovine blood neutrophils but had no effect on phagocytic index (Hogan et al., 1990). These results differed from those in which dietary supplementation of vitamin E increased phagocytosis and decreased intracellular kill by human neutrophils (Boxer, 1986). The bovine neutrophil responses to vitamin E were comparable with bovine neutrophil responses to supplementation of Se (Boyne and Arthur, 1979; 1981). Bovine plasma concentrations of greater than 3.5 g/ml of a-tocopherol are considered to be adequate as evidenced by the relationship between intracellular kill of bacteria by neutrophils and plasma vitamin E concentrations (Weiss et al., 1992).
The effects of vitamin E and Se supplementation on intracellular kill of bacteria by neutrophils are not additive. Supplementation with both vitamin E and Se did not result in greater intracellular kill of bacteria by blood neutrophils than did supplementation with either one of the nutrients alone. However, vitamin E and GSH-Px have sparing effects on the requirements for one another relative to intracellular kill of bacteria (Hogan et al., 1990). The protection afforded cellular membranes by vitamin E may spare the requirement for GSH-Px by oxidizing free radicals at the membrane, thereby preventing leakage of free radicals into the cytosol and maintaining intracellular kill capacity of the cell. Conversely, GSH-Px activity in the cytosol may have spared the requirement for vitamin E in the membranes. Boxer et al. (1979) reported a similar sparing effect of vitamin E for protecting GSH-Px-deficient human neutrophils against oxidative damage.
The importance of vitamin E in mammary health can be exemplified by monitoring a-tocopherol concentrations in milk during mastitis. The primary source of a-tocopherol in milk is the fat globule membrane. However, during mastitis a shift apparently occurs toward sources of a-tocopherol other than milk fat (Barrett et al., 1997; Hogan et al., 1995). Neutrophils in milk are a rich source of vitamin E, averaging 17 ng of a-tocopherol/106 cells. Neutrophils are the principal line of defense for the mammary gland once bacteria have penetrated the teat canal. Acute inflammation in the mammary gland results in acute phase response, including recruitment of neutrophils into damaged tissue. a-tocopherol from somatic cells for 10% of total a-tocopherol in milk from uninfected glands and 25% in mastitis glands. Therefore, neutrophils normally contribute to the concentration of a-tocopherol in milk in healthy glands, and this contribution increases during inflammation.
The recommended dietary and blood concentrations of vitamin E and Se as discussed by Dr. Weiss in the next paper relate to maintenance of host defenses to protect against infections. Optimal blood concentrations of antioxidants may be greater during periods of stress. One such period of stress appears to be parturition. Plasma vitamin E concentrations in dairy cows are normally lowest when rates of IMI are highest and when neutrophil functions are depressed during the periparturient period (Kehrli et al., 1989). The decrease in plasma a-tocopherol during the periparturient period is related to changes in consumption of vitamin E and to decreased transport capacity for the vitamin in plasma. Concentration of plasma a-tocopherol typically decreases 7 to 10 days prior to calving and remains low during the first couple of weeks of lactation, even when dietary vitamin E offered to cows is constant throughout this period. Administration of vitamin E to late gestation cows other than in feed was tested as a means of preventing a drop in plasma concentrations of vitamin E. Parenteral administration of vitamin E successfully elevated a-tocopherol concentrations in plasma and neutrophils during late gestation and early lactation periods. Cows injected with vitamin E had greater plasma a-tocopherol concentrations during the periparturient period than did cows injected with placebo. Neutrophils from cows injected with vitamin E had greater intracellular kill of bacteria at calving than did neutrophils from placebo-injected cows. Neither phagocytic index nor percentage of neutrophils phagocytizing differed between vitamin E-injected and placebo-injected cows. Dietary vitamin E during the dry period had less effect on neutrophil function at calving than did parenteral vitamin E.
Impaired neutrophil function in cows during the periparturient period has been thoroughly documented (Guidry et al., 1976). Impairment of neutrophil microbicidal mechanisms and membrane-associated activities of chemokinesis and ingestion were reported in cows during the first week after calving (Kehrli et al., 1989). Specifically, neutrophil functions associated with the oxidative burst of metabolism were altered. These neutrophil impairments were similar to depressed neutrophil activity in vitamin E-deficient animals (Hogan et al., 1990). Cows that received vitamin E injections maintained intracellular kill by blood neutrophils and neutrophil concentrations of vitamin E (Hogan et al., 1992). Subcutaneous injections of vitamin E prior to calving negated the suppression of in vitro intracellular kill of E. coli by neutrophils at calving. Intracellular kill by blood neutrophils from treated cows was constant from calving through the first month of lactation. Neutrophils from cows injected with the placebo had depressed intracellular kill at calving compared with their values at 2 and 4 weeks postpartum.
A known physiological parameter that influences neutrophil function in vivo is plasma cortisol concentrations. Changes in neutrophil activity at calving are preceded by increased corticosteroid in blood (Guidry et al., 1976). These responses can be induced experimentally by injections of exogenous ACTH that increase blood cortisol and decrease antibacterial activity of blood-derived neutrophils. Nockels (1991) reported that injection of ACTH also reduced vitamin E content in neutrophils. Chan et al. (1989) reported that during infection and other periods of inflammation, an increased destruction of tocopherol in phagocytes can be expected, and treatment of the animal with high doses of vitamin E will have a beneficial effect. These data imply that parenteral vitamin E may temper the adverse effects of increased cortisol on neutrophil vitamin E content. Concentration of vitamin E in neutrophils was related linearly to killing ability of neutrophils at parturition (Weiss et al., 1994). In summary, parenteral vitamin E prior to calving successfully elevated plasma and neutrophil a-tocopherol concentrations and maintained intracellular kill of bacteria by neutrophils when dietary supplementation did not maintain these parameters.
Immunological responses to specific mastitis pathogens can be heightened by vaccination. Vitamin E has been shown to have immunoenhancing properties when incorporated into vaccines. The addition of all-rac-a-tocopherol acetate into vaccine adjuvants enhanced immune responses in poultry, sheep and mice compared with activity following use of conventional adjuvants. Positive attributes ascribed to vitamin E in adjuvant systems include detoxification of reactive oxygen radicals generated at the sites of injection during antigen processing and presentation by immune cells (Tengerdy, 1989).
Vitamin E was evaluated as an adjuvant in an Escherichia coli J5 mastitis vaccine (Hogan et al., 1993). The use of oil in water adjuvants can result in lumps or abscesses at the site of injection (Tengerdy et al., 1983). Adjuvants containing a mixture of oil in water and vitamin E successfully vaccinated animals without creating lumps or abscesses at the injection site. Replacement of one-half of the oil in water with vitamin E reduced the swelling at E. coli J5 injection sites but did not eliminate the occurrence of lumps detected by palpation (Hogan et al., 1993). Vitamin E alone was not an effective adjuvant. Serum and milk IgG titers in cows vaccinated with the vitamin E adjuvant vaccine did not differ from those in cows vaccinated with the placebo adjuvant. Milk IgG titers were greater in cows vaccinated with the oil in water plus vitamin E adjuvant than those of cows vaccinated with the other adjuvant formulations. Serum IgG titers to E. coli J5 did not differ between cows vaccinated with the bacterin containing oil in water adjuvant and those vaccinated with the oil in water plus vitamin E bacterin. Results using E. coli J5 vaccine suggested that an adjuvant mixture of vitamin E and oil in water is promising, but the immunoenhancing properties were considerably less than in other species, as reported by Tengerdy et al. (1989).
Vitamin E and GSH-Px are both cellular antioxidants that protect against the cytotoxic capabilities of oxygen metabolites produced by neutrophils in response to bacterial IMI. Deficiencies in either of these micronutrients resulted in impaired bactericidal activity of neutrophils and increased mastitis. Dietary levels should maintain basal tissue concentrations adequate to defend against oxidation by free radicals produced during normal physiological events. Parenteral vitamin E and Se can augment dietary supplementation.
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