SELENIUM DEFICIENCY AND IMMUNITY IN OREGON SHEEP WITH FOOT-ROT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0207449
• Project Start Date: Jun 1, 2006
• Project End Date: Sep 30, 2007
• Program Code: [(N/A)]- (N/A)

Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331

Performing Department
COLLEGE OF VETERINARY MEDICINE

Non Technical Summary
Foot-rot remains the most common cause of lameness in sheep flocks because methods for controlling the disease are problematic. If the incidence or severity of foot-rot infection in sheep could be prevented or reduced with selenium supplementation, the advantages would be numerous: less labor intensive management required, less expense involved in treatment, limits the use of antibiotics in food-producing animals, and avoids the difficulties associated with producing a cross protective vaccine.

Animal Health Component

5%

Research Effort Categories

Basic

90%

Applied

5%

Developmental

5%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3610	1090	100%

Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3610 - Sheep, live animal;

Field Of Science
1090 - Immunology;

Keywords

foot-rot

sheep

selenium supplementation

immune response

Goals / Objectives
Our goal is to characterize the innate and adaptive immune responses in sheep that are deficient in the essential element selenium in order to provide information to Oregon sheep producers that will stress the importance of adequate selenium supplementation for protection of sheep against foot-rot. Specific objectives are 1) to compare blood selenium levels in foot-rot affected versus non foot-rot affected sheep from the same herd receiving similar care, 2) to examine the clinical effects of selenium supplementation on the incidence and severity of foot-rot in sheep over a 1-year supplementation period, 3) to compare humoral and cell-mediated immune responses to a novel antigen (keyhole limpet hemocyanin) in sheep supplemented with selenium versus a control group, and 4) to assess neutrophil phagocytosis in sheep supplemented with selenium versus a control group. We hypothesize that sheep with foot-rot have lower blood selenium levels than non-affected sheep, and that selenium supplementation will reduce the incidence and severity of foot-rot, as well as improve humoral, cell-mediated, and innate immunity responses.

Project Methods
The sheep rancher will be queried concerning flock health status and management practices. Strategic questions will ascertain if the flock has been tested for selenium status, if selenium supplements have been previously used, whether a foot-rot vaccination program is in place, and other relevant questions. Three sheep flocks in central and southern parts of the Willamette Valley will be chosen for the project based on a high incidence of foot-rot and low levels of selenium supplementation. Foot-rot affected sheep will be randomly divided into 2 groups of 12 sheep each on each ranch. One group will serve as the control group, while the other group will serve as the experimental (treatment) group on each ranch. Thus, with 3 sheep flocks we will have 36 control animals and 36 treatment animals total. Sheep will be identified by ear tags. Sheep will be bled at time zero and then rebled at 3-month intervals for one year to collect blood for selenium analysis; thus, blood will be collected at 0, 3, 6, 9, and 12 months. Because of the seasonal incidence of foot-rot, a one-year study will provide more accurate data. Whole blood will be collected in EDTA and the selenium levels measured in the OSU Central Analytical Lab, where normal values have been previously established for sheep. On each blood collection day, the sheep's feet will be examined and scored for foot-rot. A scoring system has been previously reported. Briefly, 0=Normal; 1=Interdigital dermatitis, presence of heat, and characteristic foot-rot odor; 2=Initial underrunning of the hoof wall between toes; 3=Underrunning of the sole; 4=Extensive underrunning of the sole and lateral walls of the hoof. It is expected that marginal selenium deficiency will result in increased foot-rot incidence and severity in sheep, reflected by higher scores in the control groups compared to the selenium-supplemented groups. Initially, and at subsequent 2-month intervals, control sheep will receive a placebo injection of 1 mL saline subcutaneously, whereas selenium-supplemented sheep will receive 5 mg of MU-Se subcutaneously. We will also bleed 12 healthy, non foot-rot affected sheep in each flock at time 0 during peak foot-rot season to determine if their selenium levels are higher than those in the foot-rot control and selenium supplemented groups (at time 0). It is expected that those with foot-rot will have lower selenium levels demonstrating that the health status is impaired in these sheep compared to that of selenium-adequate members of the flock. In vivo cell-mediated and humoral immunity will be assessed as follows. Once selenium-supplementation is underway, we will immunize control and treatment sheep twice, 2-weeks apart, with keyhole limpet hemocyanin (KLH), a novel protein. The day following the second injection, a DTH skin test will be performed. Serum will be collected for KLH antibody titer 2 weeks after the second immunization. We will follow procedures we have used previously for the dog and horse. Phagocytosis of opsonized latex-coated beads by peripheral blood neutrophils will be measured by flow cytometry to assess innate immunity, as previously described in the dog and horse.

Progress 06/01/06 to 09/30/07

Outputs
OUTPUTS: The study was conducted primarily in one commercial sheep flock in Southern Oregon chosen for the project based on a high incidence of foot rot and the owner's willingness to participate in a long-term clinical trial using selenium supplementation. Four other commercial sheep flocks were sampled one time each. Four first- and second-year professional Doctor of Veterinary Medicine program students were mentored and participated in the project. Results have been presented twice at the College of Veterinary Medicine, Oregon State University. The first was an oral abstract presentation (also proceedings abstract) for the 3rd Annual Research Day, College of Veterinary Medicine, Oregon State University, presented September 7, 2006; Corvallis, OR. The second was a senior paper (oral presentation and submitted paper) to the College of Veterinary Medicine, Oregon State University; Corvallis, OR. (Presented September 6, 2007; paper pending acceptance). Future dissemination will include an oral abstract presentation (also published abstract in J Vet Int Med) and a peer-reviewed paper for J Vet Int Med. Both of the latter are national audiences; both have been submitted (pending). An additional peer-reviewed paper is in progress for Veterinary Immunology and Immunopathology. PARTICIPANTS: Jean A. Hall, DVM, PhD, PI/PD. No funding from the agency. Monica M. Skinner, PhD, Research Associate. William Vorachek, PhD, Research Associate was hired after Monica resigned/relocated. Both were post-doctoral research associates who were directly responsible for performing laboratory assays. Katie Thonstad, Professional DVM Program; D. Paul Bailey, Professional DVM Program (Both Second year Professional DVM Program students were funded by the Department of Biomedical Sciences to work on the project during the summer of 2006. No funding from the agency.) Rachel Chinn, Professional DVM Program (First year Professional DVM Program student funded by the Department of Biomedical Sciences to work on the project during the summer of 2007. No funding from the agency.) Rachel Sendek, Howard Hughes Medical Institute Summer Research Program 2007. Undergraduate, pre-veterinary student funded by HHMI to work on the project during the summer of 2007. No funding from the agency.) TARGET AUDIENCES: Target audience includes veterinarians, veterinary students, animal scientists, basic science researchers, commercial sheep producers, and commercial cattle producers. Science-based knowledge has been delivered by oral presentation and abstracts. Peer-reviewed publications are in progress. PROJECT MODIFICATIONS: No major changes. No changes in animal use protocols. Minor changes were as follows. Originally we planned to use three sheep flocks. Foot rot-affected sheep would be randomly divided into 2 groups of 12 sheep each on each ranch. One group was to receive saline injection (placebo control group), while the other group was to receive selenium (experimental treatment group) on each ranch. It became apparent that we would also need a healthy control group of sheep without foot rot. Thus, we added another group of sheep. We elected to use one sheep flock and have higher numbers of foot rot-affected sheep in each group (n=19) for the prospective, placebo-controlled clinical trial of 15-month duration. We surveyed selenium status at one time point in foot rot-affected vs healthy sheep in 4 other flocks. The study duration was 15 months rather than 12 months.

Impacts
Dissemination of the results of this project will increase the knowledge of decision makers in veterinary medicine and of commercial sheep producers and cattle producers about the benefits of selenium supplementation. We anticipate that there will be a change in action to increase selenium supplementation in ruminants. In particular, as we gain information about methodologies to deliver increased selenium to herbivores (other collaborators are working on projects to apply selenium to pastures via selenium fertilization to increase selenium content in forages), our data will encourage adoption of these new recommendations.

Publications

• Dissertation - Bailey, D.P. 2007. Foot rot-affected sheep supplemented with selenium exhibit significant improvement in foot scores. Senior paper, College of Veterinary Medicine, Oregon State University. Corvallis, OR. (Presented September 6, 2007; pending)
• Abstract - Hall, J.A., and Van Saun, R. J. 2008. Parenteral selenium supplementation benefits foot rot-affected sheep. J Vet Int Med (abstract) and Annual Meeting American College of Veterinary Internal Medicine Forum, San Antonio, Texas. June 2008. (Pending)
• Journal Article - Hall, J.A., Bailey, D.P., Thonstad, K.N., and Van Saun, R. J. 2008. Effect of parenteral selenium supplementation on the prevalence and recovery from foot rot in sheep. J Vet Int Med (Submitted January 2008; pending)
• Journal Article - Hall, J.A., Bailey, D.P., Thonstad, K.N., Sendek, R.S., Chinn, R., Wang, Y., Forsberg, N.E., Vorachek, W.R., and Van Saun, R. J. 2008. Effect of parenteral selenium supplementation on immunity in sheep with foot rot. Veterinary Immunology and Immunopathology (pending)
• Abstract - Thonstad, K.N., Bailey, D.P., Skinner, M.M., and Hall, J.A. 2006. Selenium status and neutrophil function in sheep with foot-rot. Page 1-2 in Proc. 3rd Annual Research Day, College of Veterinary Medicine, Oregon State University, presented September 7, 2006. Corvallis, OR.

Progress 01/01/06 to 12/31/06

Outputs
We are conducting a 1-year study on injectable selenium (Se) supplementation in sheep and monitoring the morbidity of foot-rot (FR) in the herd. A sheep flock in southern Oregon was chosen for the project based on a high incidence of FR and the owner's willingness to participate in a 1-year trial. FR affected sheep were randomly divided into 2 groups of 19 sheep each. An additional group of 19 sheep without FR were identified. Sheep feet were examined, trimmed, and scored for FR using a scale of 0 (no FR) to 4 (extensive under-running of the sole and lateral walls of the hoof). Scores from each foot were summed to generate a total score for each sheep. At 3-month intervals, sheep feet are being reexamined, trimmed and rescored. Sheep are bled at time 0 and then at 3-month intervals to collect whole blood for Se analysis (0, 3, 6, 9, and 12 months). Nineteen FR affected sheep are being treated with 5 mg injectable sodium selenite at 1-month intervals; the other 19 FR affected sheep are being given a 1-mL injection of saline solution at 1-month intervals. The 19 sheep without FR at the beginning of the study receive no treatment. To assess cell-mediated immunity (CMI) and humoral immunity, after 3 months of Se supplementation all sheep were immunized twice, 2-weeks apart with keyhole limpet hemocyanin (KLH), a novel protein. The day following the second injection, a DTH skin test was performed. Intradermal injections of KLH were administered at the ear tip and in 2 wool-free sites on the abdomen. Measurements of wheal diameter and thickness (ear tip) were taken at 30 min, and at 24, 48, 72, and 96 h. Results of foot scores: After 3 months of treatment, foot scores were compared to baseline scores (4.4 +/- 0.5; mean +/- SEM). FR affected sheep receiving saline had foot scores of 3.1 +/- 0.5 with a net decrease of 1.3 +/- 0.5 from baseline. FR affected sheep receiving Se had foot scores of 1.9 +/- 0.5 with a net decrease of 2.5 +/- 0.5 from baseline (P = 0.11). After 6 months of treatment, foot scores were again compared to baseline scores. FR affected sheep receiving saline had foot scores of 2.7 +/- 0.5 with a net decrease of 1.7 +/- 0.4 from baseline. FR affected sheep receiving Se had foot scores of 1.4 +/- 0.4 with a net decrease of 3.2 +/- 0.5 from baseline (P < 0.05). Results of Se levels: At the beginning of the study, healthy control sheep had higher whole blood Se concentrations (255 +/- 11 ng/mL; mean +/- SEM) compared to sheep with FR (208 +/- 9; P< 0.05). After 3 months of injectable Se supplementation, whole blood Se concentrations increased in sheep with FR (266 +/- 7) and were comparable to healthy control sheep (243 +/- 12), whereas sheep with FR receiving saline placebo had lower whole blood Se concentrations (204 +/- 13; P<0.05). Results of CMI responses to KLH: After 3 months of treatment, healthy control sheep had a superior CMI response based on the DTH wheal test results. There were no differences between FR affected sheep supplemented with Se vs FR affected sheep given saline. Results for 9 and 12 month evaluations are pending.

Impacts
Preliminary conclusions: Sheep with foot-rot have lower blood selenium (Se) concentrations compared to healthy sheep. Parenteral Se supplementation increases blood Se concentrations and is associated with a decrease in the severity of foot-rot based on foot scores. Thus, by reducing foot-rot severity in the herd, selenium supplementation has the potential to enhance economic opportunities for agricultural producers

Publications

• Abstract - Bailey D.P., Thonstad K.N., Skinner M.M., Hall JA. 2006. Effect of injectable selenium supplementation on immune function of adult sheep with foot-rot. Oregon State University, College of Veterinary Medicine, 3rd Annual Research Day; September 7, 2006; Corvallis, OR.
• Abstract - Thonstad KN, Bailey DP, Skinner MM, Hall JA. 2006. Selenium status in sheep with foot-rot. Oregon State University, College of Veterinary Medicine, 3rd Annual Research Day; September 7, 2006; Corvallis, OR.