The Effects of Selenium

Supplementation on Bacterial Killing in Sheep with Foot-Rot

Rachel SendekDr. Jean Hall

HHMI Summer 2007

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.

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.

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 + H2O2 → GS–SG + 2H2O

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.

Methods

Heparinized blood collected from sheep in Riddle, OR

Placed on ice for transport

Neutrophils isolated using Percoll gradient technique; mRNA purified

RT-qPCRMeasure abundance of neutrophil

marker mRNAs

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!

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.

Percent bacterial killing after neutrophil innoculation with MOI=0.01 L. lactis and

incubation for 1 h.

Control (n=8)FR-Saline

(n=9)FR-Se (n=7)

0.70

0.75

0.80

0.85

0.90

0.95

Percent bacterial killing

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.

MPO-halide-H2O2 mechanism of killing

Neutrophilic granules contain myeloperoxidase (MPO)

Chloride ion (Cl-) is ubiquitous

Cl- + H2O2 + H+ MPO> HOCl + H2O

Hypochlorous acid (HOCl) is a potent oxidant and antimicrobial agent

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

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

L-selectin IL-8R MPO

Fold Change

Saline

Selenium

Effect of Selenium Supplementation on Levels of mRNA in Foot-Rot Sheep

N = 8 to 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.

Questions for future investigations

Determine dosage of selenium optimal for bacterial killing by neutrophils.

Organic vs. inorganic selenium supplements?

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