Effect of dietary Se depletion, different dietary Se sources, and supranutritional Se-supplementation dosages on immune responses in healthy and FR-affected ewes
The immune system has two functional divisions: innate and adaptive immunity. Both divisions involve various blood-borne factors (e.g., complement, antibodies, and cytokines) and cells (e.g., neutrophils, lymphocytes, and macrophages). Neutrophils are the most numerous and important cellular component of innate immunity. Their primary functions are phagocytosis and destruction of microorganisms. They serve as the body’s first line of defenses against invading microorganisms. Phagocytosed bacteria are rapidly killed by proteolytic enzymes (e.g., myeloperoxidase), antimicrobial proteins, and ROS when membrane-bound granules fuse with phagocytic vesicles. To assess innate immunity of neutrophils, an ex vivo
biologic assay was performed using E. coli
and measuring percent bacterial killing. Neutrophils from healthy sheep not receiving Se supplementation demonstrated higher percent bacterial killing compared with neutrophils from FR-affected sheep not receiving Se supplementation, consistent with our results published previously for another flock 
. In the current study we were able to show that Se supplementation, regardless of source or dosage, restored neutrophil bacterial killing in FR-affected ewes back to percentages consistent with Se-supplemented or non-supplemented healthy ewes. The bacterial killing percentage for neutrophils tended to be greater in FR-affected ewes receiving Se-yeast compared with Na-selenite. We saw no clear benefit from supranutritional Se dosages in neutrophil bacterial killing.
In a companion paper 
, we reported on neutrophil-related gene expression profiles from these ewes and showed a U-shaped relationship with supranutritional Se-yeast supplementation and Se depletion both enhancing gene expression of L-Selectin (L-Sel), interleukin 8 receptor (IL-8R), and toll-like receptor 4 (TLR4). All three are essential for bacterial recognition and neutrophil migration, phagocytosis, and killing. In addition, expression of selenoprotein S (SEPS1) and glutathione peroxidase 4 (GPx4), which are both involved in controlling inflammation, was increased for both with supranutritional Se-yeast supplementation and Se depletion 
. When we correlated (nonparametric spearman correlation) neutrophil bacterial killing activity in this study with the previously reported neutrophil gene expression profiles for ewes receiving 0, 4.9, 14.7, and 24.5 mg Se/wk, significant negative correlations were observed for GPx4 (r
0.01) and SEPS1 (r
0.01). Both genes act as anti-inflammatory agents: GPx4 promotes cell survival and blocks eicosanoid synthesis, including cyclooxygenase (COX) II 
, and SEPS1 protects immune cells from apoptosis and decreases the release of the proinflammatory cytokines IL-6 and TNF-α 
. Others have shown in mice that moderate selenium deficiency down-regulates inflammation-related genes and reduces myeloperoxidase and lysozyme activities in Se-restricted leukocytes 
. Myeloperoxidase is expressed in neutrophils and monocytes and generates ROS that are important for antimicrobial and cytotoxic effects. Thus, Se-supplementation may restore optimal neutrophil bacteria killing in the presence of FR disease, but healthy ewes may have different requirements for dietary Se.
Neutrophil bacterial killing also decreased linearly in ewes with age. Ewes six years and older (43%) had similar killing percentages as FR-affected ewes receiving no Se supplement (40%). Se supplementation delayed the age-associated decline in neutrophil killing ability, as ewes receiving Se had similar killing percentages as non supplemented ewes that were one age category younger. We have previously shown in aged Beagle dogs that older dogs have a significant decrease in neutrophil bacterial killing and, in addition, have lower levels of mRNA for neutrophil-related gene expression compared with younger dogs, including mRNA for myeloperoxidase 
, which may contribute to increased morbidity and mortality with aging. These results are consistent with our finding in this study that older ewes had a higher FR severity and prevalence. Neutrophils from older humans have also been shown to have less phagocytic ability than those from younger adults, and the respiratory burst was altered in neutrophils from aged participants 
Tests used to assess adaptive immunity include measuring an antibody titer in response to sensitization/immunization (humoral immune response). The ewe is injected with a novel protein (e.g., KLH) that elicits an immune response. Following sensitization, antibody titers to KLH are measured. Consistent with our results in another flock 
and results for bacterial killing by neutrophils in the current study, healthy ewes receiving no Se supplementation had higher KLH antibody titers compared with FR affected ewes receiving no Se supplementation. In the current study, we were also able to show that Se supplementation, regardless of source, restored KLH antibody titers in FR-affected ewes back to titers consistent with Se-supplemented and non-supplemented healthy ewes. Others have shown that Se supplementation increases antibody production in Se deficient sheep (reviewed in 
). Our results suggest that Se supplementation may also improve antibody titers in response to a novel antigen in Se-replete yet FR-affected sheep, which is consistent with results we demonstrated in Se-replete adult beef cattle 
and weaned beef calves (paper under review).
We did see a disadvantage of supranutritional Se treatment with Na-selenite (24.5 mg/wk dosage) in that KLH titers in FR-affected ewes receiving this Se source and dosage remained suppressed. We also observed at this Na-selenite dosage a higher propensity for FR lesions at several time points. Furthermore, WB-Se concentrations did not increase from the 14.7 mg/wk to the 24.5 mg/wk Na-selenite. In a companion paper, we reported a lower transfer of IgG from ewe colostrum to lamb serum if ewes received 24.5 versus 4.9 mg Se/wk as Na-selenite. 
. These results suggest that Na-selenite may have potentially deleterious effects at higher dosages. In support, comparative toxicosis studies in sheep showed that oxidative effects were greater for Na-selenite than equivalent amounts of SeMet 
, which is the main selenocompound in Se-yeast 
. Thus, the efficacy of supranutritional treatment with Na-selenite at 5 times the maximal FDA-permitted level requires further study.
The DTH test, which is also known as a type IV hypersensitivity reaction, is another test used to assess the adaptive immune response. This test provides a general measure of CMI. Professional antigen presenting cells, e.g., dendritic cells, present antigen to T lymphocytes. This results in antigen specific activation of T lymphocytes in local tissues. Inflammatory cytokines produced by these stimulated T lymphocytes cause other mononuclear cells (lymphocytes and macrophages) to migrate to the area and proliferate. To perform this test, foreign antigen is injected under the epidermis of the skin. The immune system responds to this antigen by producing a small raised wheal that can be measured 24 to 96 h after injection. The larger and thicker the wheal, the greater is the DTH response.
In our study, FR-affected sheep demonstrated suppressed CMI at 24 h after intradermal KLH challenge, consistent with our results in another flock 
. In the current study, this response was significant using ear wheal diameter measurements, and although numerically true for ear thickness measurements, the latter results were not significant. We reported previously in another flock that FR-affected ewes with WB-Se concentrations above 250 ng/mL at the time of the DTH assay had greater ear thickness and ear wheal diameter responses than FR-affected ewes with WB-Se concentrations below 250 ng/mL 
. In the current study we saw no effect of Se source and dosage on DTH responses. The DTH test may be too insensitive under field conditions to detect a difference in CMI with Se supplementation. It is well known that large variation exists in immune function measures, even among healthy animals. For example, differences in genetics, age, diet, body condition scores, stress, levels of exercise, and infectious disease history are important contributors to observed variation 
, such that demonstrating a consistent improvement in immune function with Se supplementation is challenging. In addition, differences in DTH methodology, with variable injection sites, response times, and measurement techniques may account for differences between studies. Nonetheless, finding once again an attenuated T-lymphocyte response in FR-affected sheep is important, and could be the result of decreased activation, migration, proliferation, or a combination of these effects. Se supplementation alone may or may not (current study) be sufficient to improve the DTH response under FR-disease conditions.
We also assessed the 30-min skin test response following intradermal KLH challenge in healthy and FR-affected sheep to determine if results differed for the type I hypersensitivity reaction normally induced by histamine and inflammatory cytokines. The KLH antigen stimulates inflammatory cytokine production. We found that FR-affected ewes had attenuated 30 min ear-thickness responses to KLH intradermal injection compared with healthy ewes, consistent with results of a previous study 
. The KLH response was not influenced by source of Se treatment. Footrot bacterial infection could suppress the Type I hypersensitivity response by affecting the release of histamine, or virulence factors such as leukotoxin, endotoxin, haemolysin, haemagglutinin, and adhesion.
Histamine normally increases capillary permeability and relaxes vascular smooth muscle, allowing edema fluid accumulation. Influx of proinflammatory cytokines triggers production of ROS. When produced in excess, ROS are important mediators of cell and tissue injury (reviewed in Murr et al. 
). As a component of the glutathione peroxidase family of enzymes, Se contributes to the reduction of hydroperoxides in cells. Glutathione peroxidase reduces ROS to less reactive metabolites, decreasing oxidant stress. Because Se is involved in redox reactions, and immune activation is usually associated with increased production of ROS by cells of the immune system, we hypothesized that ewes receiving supranutritional Se supplementation might have suppressed 30 min skin reactions compared with ewes receiving less Se supplementation. . We observed, however, a U-shaped relationship between Se dosage and the 30-min skin test responses to KLH, similar to what we reported in a companion paper for Se-yeast dosage and neutrophil gene expression 
. Ewes receiving either no Se supplementation or supranutitional Se supplementation both had accentuated rather than suppressed 30-min skin test responses to KLH compared with ewes receiving the maximum FDA-allowed levels. A U-shaped relationship between Se status and human health has been postulated in a review by Rayman 
, whereby supplemental Se intake may benefit people with low Se status (or in our case under certain disease conditions such as FR), but cautions that those with adequate to high Se status might be affected adversely and should not take Se supplements. Decreased inflammation and inflammation-dependent plasma cell tumors have been reported in Se-deficient mice 
. Both Se deficiency and high levels of Se have been reported to decrease the incidence and progression of liver tumors in transgenic mice prone to liver cancer 
. A vigorous 30-min skin response to a novel antigen, induced by histamine and proinflammatory gene products, may be necessary for a successful defense against FR-causing bacteria, as FR-affected sheep had a lower 30-min skin test response than healthy sheep in this and another flock 
. Our results suggest that optimal 30 min skin test responses vary depending on the underlying disease condition and the Se supplementation rate.
Effect of dietary Se sources and supranutritional Se-supplementation on whole-blood and serum-Se concentrations and FR morbidity in healthy and FR-affected ewes
In the current study, ewes affected with FR at baseline had lower WB- and serum-Se concentrations compared with healthy ewes, although mean concentrations were within the normal reference interval for adult sheep. [The normal reference interval for Se in WB of adult sheep > 700 days of age at the Michigan State University diagnostic laboratory is 150 to 500 ng/mL (T. Herdt, personal communication)]. This finding is consistent with our previously published observations in another flock 
. In a companion paper, we reported that WB-Se and serum-Se concentrations increased linearly with supranutritional Se-yeast supplementation 
. However, WB-Se concentrations in ewes receiving supranutritional Na-selenite supplementation reached a plateau similar to concentrations attained in ewes receiving 4.9 mg/wk of Se-yeast 
. In the current analysis, we showed that ewe FR status does not affect Se-supplementation induced increases in WB- or serum-Se concentrations irrespective of Se-source and Se-dosage. Thus, it is more likely that Se intake was lower in ewes with FR compared with healthy ewes at baseline because sheep affected with FR are less mobile and, therefore, unable or unwilling to consume as much Se-containing mineral supplement as healthy sheep 
than the alternative hypothesis that Se requirements are higher in the presence of an infectious disease like FR because more Se is required for removal of reactive oxygen species (ROS) associated with inflammation 
We previously reported in another sheep flock that parenteral Se-supplementation in conjunction with routine control practices accelerated recovery from FR in sheep 
. In the current study, even though we were able to raise WB- and serum-Se concentrations, Se supplementation did not consistently prevent FR nor accelerate recovery from FR over the 62 week treatment period compared with no Se supplementation. Selenium supplementation did decrease the percentage of healthy ewes that acquired FR during the first 20 weeks of the study; however, we did not observe a similar effect in wks 28, 40, and 60. In a companion paper, we reported that the greatest treatment success with oxytetracycline was observed in ewes receiving the highest Se-yeast dosage (24.5 mg Se/wk) compared with Se-yeast dosages of 0, 4.9, or 14.5 mg Se/wk 
. In the current analysis, we observed an improvement in FR prevalence in FR affected sheep receiving Na-selenite and Se-yeast supplementation compared with no Se and Na-selenate supplementation at wk 60, but not at earlier time points.
In general, the prevalence of FR changed according to the ewe production cycle and management practices. At baseline, ewes were selected for 50% FR prevalence, and treatment groups were stratified for FR severity and age of ewe. Ewes were kept on pastures and supplemented with grass hay during the breeding and gestation seasons. Footrot prevalence decreased to 30±2% during this period. Ewes were moved from pasture into the barn for lambing around wk 20 and fed alfalfa hay and shelled corn. By wk 28 the FR prevalence had increased to 82±2%. Serum Se concentrations were decreasing by week 27 after increasing from week 0 to week 14. The decrease in blood Se concentrations reflected an increased Se transfer from ewes to lambs in late gestation and early lactation 
. A corresponding decrease in WB-Se concentrations was not observed until week 40, likely because of the longer half-life of RBC.
Housing ewes in the barn at higher stocking density for the 3-month period around lambing (up to week 30) likely contributed to increased FR prevalence and severity. It is known that environmental factors play an important role in determining infection rate and progression of FR severity, with heavier infection rates occurring under warm moist conditions 
. Dichelobacter nodosus
, one of the main organisms associated with FR is an anaerobic and fastidious bacterium that colonizes the interdigital epithelial tissue more readily during the wet seasons of spring, fall, and winter. Higher stocking density is also more conducive to heavy infection rates 
. Crowding and moist bedding conditions could explain the higher FR prevalence in the flock in general during this time period.
Once the ewes were returned to pasture and eating grass forage at wk 30, the FR prevalence decreased to 39±3% at wk 40. In part, this reflects the administration of oxytetracycline antibiotic to the more severely FR-affected ewes at wk 28 (52% of all ewes received oxytetracycline). Again, at wk 40, oxytetracycline treatment of the more severely affected ewes (16% of all ewes) was repeated. Although beneficial in helping decrease FR prevalence in these ewes from 100% to 59±9%, the overall FR prevalence increased between wk 40 and wk 60, to 48±3%.
Selenium is not recommended as the sole treatment for FR in sheep. Administration of topical or systemic antibiotics, foot paring, foot bathing in disinfectants, and vaccination with a commercially available vaccine for footrot (Footvax, MSD) containing multiple serotypes of D. nodosus
, reviewed by Duncan et al. 
, have all been suggested for use in treating sheep with advanced FR lesions. Ewes that do not respond to treatments are often culled; this was not the case in our study, which explains why older ewes represented the majority of cases of FR infection in our study and another 
. Early detection of disease and prompt treatment with parenteral long-term acting oxytetracycline were control measures resulting in sheep being significantly more likely to recover from FR lesions and lameness within 5 days of treatment compared with sheep that were foot trimmed with or without parenteral administration of antibacterials 
. In our study, ewes with FR severity scores of II (with foot scores of 4) and higher received parenteral long-term acting oxytetracycline injections at 28 and 40 wk. In hindsight, a more intensive parenteral antibiotic treatment regimen in the current study may have been beneficial in decreasing pathogen load and, thus, infection challenge. This might have allowed us the opportunity to see more benefits associated with Se supplementation.
It is unclear why ewes receiving no Se supplementation were so resistant to FR infection. One explanation is that dietary Se status was not the most limiting factor for FR infection in this flock. Footrot is a multifactorial disease 
and, therefore, the optimal Se supplementation dosage may vary depending on nutritional and management conditions, as well as the sheep’s immune system. Another explanation is that inflammation, and thus progression of foot lesions with marginal Se deficiency, is dampened. In support, we have shown in a companion paper 
that WB-neutrophil gene expression profiles are shifted in an anti-inflammatory direction (increased GPx4 and SEPS1) with no Se supplementation. Others have shown that moderate Se deficiency in mice down-regulates inflammation-related genes and reduces myeloperoxidase and lysozyme activities in Se-restricted leukocytes 
. Myeloperoxidase is expressed in neutrophils and monocytes and generates ROS that are important for antimicrobial and cytotoxic effects, as well as modulation of the immune response via nuclear factor kappa B (NF-κB) signaling. Down regulation of inflammation under marginal Se deficiency requires further study.
The goal of immunonutrition is to enhance immunity and increase resistance to disease. We are interested in supranutritional levels of Se, to determine if supplementing Se at concentrations above those currently recommended for sheep (supranutritional) can modulate the immune response in a way that reduces the severity and/or improves recovery from a disease process. Using FR as our disease model, we have shown in a companion paper that supranutritional supplementation of these ewes with Se-yeast at 24.5 mg Se/wk improved lamb growth and ewe health compared with maximal FDA-allowed levels of Se-yeast 
. In this study, Se supplementation did not prevent FR, but did improve innate and humoral immune functions negatively affected by FR. Future studies are warranted to evaluate whether Se supplementation enhances innate and adaptive immune responses and provides protection against other bacterial or viral pathogens.