1. The Effects of Selenium Supplementation on Bacterial Killing in Sheep with Foot-Rot Rachel Sendek Dr. Jean Hall HHMI Summer 2007

2. What is Foot-Rot?  Co-infection with the anaerobic bacteria Dichelobacter nodosus and Fusobacterium necrophorum.  Lameness, sensitivity with hoof trimming, bleeding, and pungent odor. Leads to poor weight gains and loss of productivity.  Decreased immune function may play a role in the development of the disease.

3. Why Selenium?  Pacific Northwest has unusually low soil and forage selenium levels.  “White-Muscle Disease”  Selenium deficiency causes a ↓ in glutathione peroxidase ( GSH-Px) activity and impairs neutrophil and macrophage activity.  Immunodeficiency ↑ susceptibility to bacterial infections such as foot-rot.

4. Hypothesis Selenium supplementation in sheep with foot-rot will improve neutrophil killing of bacteria. o Production of O2-  o These reactive oxygen species can act by either of two oxygen- dependent mechanisms leading to the destruction of microorganisms. o Glutathione peroxidase (GSH-Px) is involved in the reduction of oxidative species such as hydrogen peroxide. 2GSH + H 2 O 2 → GS–SG + 2H 2 O

5. Study Design  15 Month Ongoing Study With 3 Groups of Sheep:  Sheep with no evidence of foot-rot (n=19)  Sheep with foot-rot receiving supplemental selenium (n=19)  5 mg (1 ml MU-Se) once monthly SQ injection  Sheep with foot-rot receiving saline as placebo (n=19)  1 ml saline once monthly SQ injection  Neutrophil function was evaluated by assessing bacterial phagocytosis ex vivo at 15 months. mRNA was also examined for abundance of neutrophil markers at 15 months.

6. Methods Heparinized blood collected from sheep in Riddle, OR Placed on ice for transport Neutrophils isolated using Percoll gradient technique; mRNA purified RT-qPCR Measure abundance of neutrophil marker mRNAs

7. Methods Culture Lactococcus lactis to use as an ex vivo model to assess bacterial killing. Mix bacteria with isolated, activated neutrophils to reproduce the immune response. Neutrophils kill bacteria by phagocytosis and NETs. Incubate for enumeration of colonies that represent surviving bacteria. Calculate percent killing!

8. Bacterial Killing Results Control sheep demonstrated the highest % bacterial killing (P<0.05). There was not a significant difference in % bacterial killing between foot-rot sheep supplemented with saline or selenium.

9. Neutrophil Marker mRNAs L-selectin-- cell surface glycoprotein involved in binding and rolling of leukocytes along endothelial walls facilitating migration to site of inflammation. IL-8 Receptor-- receptor that controls the interaction of neutrophils with the epithelial cell barrier. MPO (myeloperoxidase)-- contained in neutrophilic granules and used to create potent hypochlorous acid involved in phagocytosis. It has been shown in a previous microarray study in dairy cattle that mRNA for these genes are increased by dietary supplements designed to stimulate the immune response (Omnigen). Samples were analyzed with RT-qPCR to determine relative mRNA levels, which allows assessment of gene activity resulting from treatment.

10. MPO-halide-H 2 O 2 mechanism of killing  Neutrophilic granules contain myeloperoxidase (MPO)  Chloride ion (Cl - ) is ubiquitous  Cl - + H 2 O 2 + H + MPO > HOCl + H 2 O  Hypochlorous acid (HOCl) is a potent oxidant and antimicrobial agent

11. Phagocytic leukocytes are directed to sites of infection through interactions between adhesion molecules induced by cytokines

12. Effect of Selenium Supplementation on Levels of mRNA in Foot-Rot Sheep N = 8 to 13

13. Discussion Although no difference in % bacterial killing was detected between foot-rot affected sheep supplemented with saline and those supplemented with selenium, control sheep without foot-rot were shown to have higher bacterial killing ability in ex vivo phagocytosis assays than sheep affected with foot-rot. Thus, other factors besides selenium status may be involved in protecting these sheep from infectious agents (genetics?). However, RT-qPCR data suggests that the neutrophil markers MPO and perhaps IL-8R are up-regulated in sheep supplemented with selenium vs. saline (when compared with controls), indicating selenium supplementation increases mRNA markers of neutrophil function in sheep.

14. Questions for future investigations  Determine dosage of selenium optimal for bacterial killing by neutrophils.  Organic vs. inorganic selenium supplements?

15. Acknowledgements  Dr. Jean Hall  Bill Vorachek and the Kerkvliet Lab  Sonka family and sheep flock  Rockey Lab  Yongqiang Wang  Elena Gorman  Dr. Kevin Ahern  Howard Hughes Medical Institute