Download The Effect of Selenium Supplementation in Sheep with Foot

The Effects of
Selenium
Supplementation on
Bacterial Killing in
Sheep with Foot-Rot
Rachel Sendek
Dr. 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 oxygendependent 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
Neutrophils isolated using Percoll
gradient technique; mRNA purified
Placed on ice for transport
RT-qPCR
Measure abundance of neutrophil
marker mRNAs
Methods
Culture Lactococcus lactis to use as an
ex vivo model to assess bacterial killing.
Neutrophils kill bacteria by
phagocytosis and NETs. Incubate
for enumeration of colonies that
represent surviving bacteria.
Calculate percent killing!
Mix bacteria with isolated,
activated neutrophils to
reproduce the immune
response.
Bacterial Killing Results
Percent bacterial killing after neutrophil
innoculation with MOI=0.01 L. lactis and
incubation for 1 h.
Percent bacterial killing
0.95
0.90
Control (n=8)
FR-Saline
(n=9)
FR-Se
(n=7)
0.85
0.80
0.75
0.70
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.
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
Effect of Selenium Supplementation on
Levels of mRNA in Foot-Rot Sheep
N = 8 to 13
40.00
35.00
Fold Change
30.00
25.00
Saline
20.00
Selenium
15.00
10.00
5.00
0.00
L-selectin
IL-8R
MPO
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