This document summarizes research on molecular mechanisms behind lameness in meat chickens. The research found alterations to bone homeostasis and bacterial immune responses that contribute to lameness. Specifically, it was found that bacterial infection dysregulates genes involved in mitochondrial function, dynamics, and biogenesis in bone cells, leading to mitochondrial dysfunction, increased cell death, and disruption of cellular processes. Additionally, genes related to the autophagy pathway were downregulated in lame chickens, suggesting bacterial infection impairs autophagy in bone tissue. The research provides insights into how bacteria may cause lameness at the molecular level by interfering with mitochondrial health and autophagy in leg bones.
(March 14, 2024) Webinar: Validation of DEXA for Longitudinal Quantification ...Scintica Instrumentation
More Related Content
Similar to (November 30, 2022) Webinar: Molecular Mechanisms Behind Lameness in Meat Chickens – Alterations to Bone Homeostasis and Bacterial Immune Responses
Improved outcomes after intratumoral administration of immunostimulatory mRNA...ModernaTherapeutics1
Similar to (November 30, 2022) Webinar: Molecular Mechanisms Behind Lameness in Meat Chickens – Alterations to Bone Homeostasis and Bacterial Immune Responses (20)
(November 30, 2022) Webinar: Molecular Mechanisms Behind Lameness in Meat Chickens – Alterations to Bone Homeostasis and Bacterial Immune Responses
1. Molecular Mechanisms Behind Lameness in
Meat Chickens – Alterations to Bone
Homeostasis and Bacterial Immune Responses
Dr. Alison Ramser
Post-Doctoral Researcher at
Center for Excellence in Poultry Science,
University of Arkansas
10. Experimental Design – in vivo
Wideman, R. F., K. R. Hamal, J. M. Stark, J. Blankenship, H. Lester, K. N. Mitchell, G. Lorenzoni, and I. Pevzner. Poult. Sci. 91:870-883, 2012
• Healthy and BCO affected
• Sampling
– Femur and tibia
– Blood
11. Experimental Design – in vitro
• hFOB 1.19 (ATCC)
– S. agnetis 908 (Courtesy of Dr. Rhoads); MOI 50:1
– Cellular pathway manipulation
– Cyto(chemo)kine exposure
• Primary chondrocytes*
12. Sample Preparation and Analysis
Gene expression
• Trizol method
• rtPCR
• Real-time qPCR
• Student t-test or
one-way ANOVA
– p-value < 0.05
– GraphPad
Protein expression
• Protein lysis buffer
• Western Blot analysis
– AlphaView software
quantification
• Student t-test or one-
way ANOVA
– p-value < 0.05
– GraphPad
Immunofluorescence
• Primary chondrocyte
cells
• Primary; fluorescent
secondary antibodies
– Vectashield
with DAPI
• Imaged using Zeiss
Imager M2 and
AxioVision software
version LE2019
MTT cell viability assay
16. Bacterial infection effect on
mitochondria
Varnesh Tiku, Man-Wah Tan, Ivan Dikic. Mitochondrial Functions in Infection and
Immunity. February 11, 2020DOI:https://doi.org/10.1016/j.tcb.2020.01.006
17.
18.
19. Mitochondrial Biogenesis
C
o
n
tro
l
B
C
O
0
1
2
3
D
-lo
o
p
m
R
N
A
e
x
p
r
e
s
s
io
n
*
C
o
n
t
r
o
l
B
C
O
0 .0
0 .5
1 .0
1 .5
S
k
i
m
R
N
A
e
x
p
r
e
s
s
io
n
C
o
n
t
r
o
l
B
C
O
0
1
2
3
4
5
P
G
C
-
1
m
R
N
A
e
x
p
r
e
s
s
io
n
*
C
o
n
t
r
o
l
B
C
O
0
1
2
3
4
5
P
G
C
-
1
m
R
N
A
e
x
p
r
e
s
s
io
n
*
C
o
n
t
r
o
l
B
C
O
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
T
F
A
M
m
R
N
A
e
x
p
r
e
s
s
io
n
C
o
n
t
r
o
l
B
C
O
0 .0
0 .5
1 .0
1 .5
S
S
B
P
1
m
R
N
A
e
x
p
r
e
s
s
io
n
20. Mitochondrial Dynamics
C
o
n
tro
l
B
C
O
0 .0
0 .5
1 .0
1 .5
O
P
A
1
m
R
N
A
e
x
p
r
e
s
s
io
n
*
C
o
n
t
r
o
l
B
C
O
0 .0
0 .5
1 .0
1 .5
2 .0
O
M
A
1
m
R
N
A
e
x
p
r
e
s
s
io
n
C
o
n
t
r
o
l
B
C
O
0 .0
0 .5
1 .0
1 .5
2 .0
M
F
N
1
m
R
N
A
e
x
p
r
e
s
s
io
n
C
o
n
t
r
o
l
B
C
O
0 .0
0 .5
1 .0
1 .5
M
T
F
P
1
m
R
N
A
e
x
p
r
e
s
s
io
n
C
o
n
t
r
o
l
B
C
O
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
D
N
M
1
m
R
N
A
e
x
p
r
e
s
s
io
n
C
o
n
t
r
o
l
B
C
O
0
1
2
3
4
M
F
N
2
m
R
N
A
e
x
p
r
e
s
s
io
n
*
C
o
n
t
r
o
l
B
C
O
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
M
T
F
R
1
m
R
N
A
e
x
p
r
e
s
s
io
n
C
o
n
t
r
o
l
B
C
O
0
1
2
3
4
5
M
F
F
1
m
R
N
A
e
x
p
r
e
s
s
io
n
21. Mitochondrial Dynamics
OPA1 processing in cell death and disease – the long and short of it
Thomas MacVicar, Thomas Langer
Journal of Cell Science 2016 129: 2297-2306; doi: 10.1242/jcs.159186
22. Mitochondrial Function
C
o
n
tro
l
B
C
O
0 .0
0 .5
1 .0
1 .5
A
N
T
m
R
N
A
e
x
p
r
e
s
s
io
n
*
C
o
n
tro
l
B
C
O
0 .0
0 .5
1 .0
1 .5
C
O
X
5
A
m
R
N
A
e
x
p
r
e
s
s
io
n
*
C
o
n
t
r
o
l
B
C
O
0 .0
0 .5
1 .0
1 .5
C
O
X
IV
m
R
N
A
e
x
p
r
e
s
s
io
n
*
C
o
n
t
r
o
l
B
C
O
0
1
2
3
4
5
a
v
-
U
C
P
m
R
N
A
e
x
p
r
e
s
s
io
n
*
C
o
n
tro
l
B
C
O
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
N
R
F
1
m
R
N
A
e
x
p
r
e
s
s
io
n
C
o
n
t
r
o
l
B
C
O
0 .0
0 .5
1 .0
1 .5
F
O
X
O
1
m
R
N
A
e
x
p
r
e
s
s
io
n
C
o
n
t
r
o
l
B
C
O
0 .0
0 .5
1 .0
1 .5
2 .0
F
O
X
O
3
m
R
N
A
e
x
p
r
e
s
s
io
n
C
o
n
t
r
o
l
B
C
O
0 .0
0 .5
1 .0
1 .5
2 .0
F
O
X
O
4
m
R
N
A
e
x
p
r
e
s
s
io
n
C
o
n
t
r
o
l
B
C
O
0
1
2
3
4
5
K
e
a
p
1
m
R
N
A
e
x
p
r
e
s
s
io
n
C
o
n
t
r
o
l
B
C
O
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
P
P
A
R
m
R
N
A
e
x
p
r
e
s
s
io
n
C
o
n
t
r
o
l
B
C
O
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
P
P
A
R
m
R
N
A
e
x
p
r
e
s
s
io
n
23. Conclusion
• Implicates mitochondrial dysfunction
– Potential:
• ↑ cell death (chondronecrosis)
• Alteration of cellular processes
• Contribution to previously found mechanisms
• Further research needed
26. Bacteria, Bone, and Autophagy
Campoy, E, Colombo, M. 2009. BBA – Mol. Cell Res. (9)1465-1477.
Xiao, L, Xiao, Y. 2019. Front. Endocrinol. (10)490.
27. 1. in vivo study
Initiation
Normal BCO
GAPDH
Beclin1
←37 kDa
←60 kDa
Beclin1
N
o
r
m
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
A
T
G
9
A
m
R
N
A
e
x
p
r
e
s
s
io
n
N
o
r
m
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
A
T
G
1
3
m
R
N
A
e
x
p
r
e
s
s
io
n
*
N
o
r
m
a
l
B
C
O
0
5
1 0
1 5
2 0
2 5
B
e
c
lin
1
/G
A
P
D
H
*
28. Nucleation
Normal BCO
GAPDH
ATG5
←37 kDa
←55 kDa
N
o
r
m
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
U
V
R
A
G
m
R
N
A
e
x
p
r
e
s
s
io
n
N
o
r
m
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
A
T
G
1
4
m
R
N
A
e
x
p
r
e
s
s
io
n
*
N
o
r
m
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
A
T
G
5
/G
A
P
D
H
29. Elongation – Protein Expression
ATG16L
ATG3
LC3A/B
GAPDH
ATG7
ATG12
Normal BCO
←37 kDa
←14,16 kDa
←40 kDa
←53 kDa
←66 kDa
←78 kDa
N
o
r
m
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
L
C
3
/G
A
P
D
H
*
30. Elongation – mRNA Expression
ATG7; ATG3; ATG4A; LC3A; LC3B;
N
o
rm
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
A
T
G
1
2
m
R
N
A
e
x
p
r
e
s
s
io
n
*
N
o
rm
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
L
C
3
C
m
R
N
A
e
x
p
r
e
s
s
io
n
*
N
o
r
m
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
A
T
G
1
6
L
m
R
N
A
e
x
p
r
e
s
s
io
n
*
N
o
r
m
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
A
T
G
2
B
m
R
N
A
e
x
p
r
e
s
s
io
n
*
N
o
r
m
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
A
T
G
1
0
m
R
N
A
e
x
p
r
e
s
s
io
n
*
N
o
r
m
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
A
T
G
9
B
m
R
N
A
e
x
p
r
e
s
s
io
n
*
N
o
r
m
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
A
T
G
4
B
m
R
N
A
e
x
p
r
e
s
s
io
n
*
31. Fusion
Normal BCO
GAPDH
Rab7
←37 kDa
←22 kDa
LAMP2
N
o
r
m
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
S
Q
S
T
M
1
(
p
6
2
)
m
R
N
A
e
x
p
r
e
s
s
io
n
*
N
o
rm
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
R
A
B
7
A
m
R
N
A
e
x
p
r
e
s
s
io
n
*
N
o
r
m
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
R
A
B
7
/G
A
P
D
H
32. 2. in vitro study
Effect of S. agentis 908 on hFOB cell viability
- +
0
5 0
1 0 0
1 5 0
S . a g n e tis 9 0 8
%
o
f
c
o
n
t
r
o
l
v
ia
b
ility *
33. BCO isolate and Autophagy – in vitro
ATG16L
ATG3
LC3A/B
GAPDH
ATG7
ATG12
ATG5
Beclin1
Rab7
S. agnetis 908 - - - + + +
←37 kDa
←14 type I
←16 type II
←40 kDa
←53 kDa
←66 kDa
←78 kDa
←55 kDa
←60 kDa
←22 kDa
A
T
G
7
A
T
G
1
6
L
B
e
c
l
i
n
1
A
T
G
5
A
T
G
1
2
A
T
G
3
R
a
b
7
L
C
3
I
I
/
I
r
a
t
i
o
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
p
r
o
te
in
/G
A
P
D
H
-
+
*
S . a g n e tis 9 0 8
35. Effect of Autophagy Inhibition – in vitro
C
o
n
t
r
o
l
3
-
M
A
C
Q
0
5 0
1 0 0
1 5 0
%
o
f
c
o
n
t
r
o
l
v
ia
b
ility
a
b
c
36. Effect of Autophagy Inhibition on
Machinery in vitro
ATG3
LC3A/B
GAPDH
ATG7
ATG12
Beclin1
Control 3-MA(5mM) CQ (10µM)
←37 kDa
←14 type I
←16 type II
←40 kDa
←53 kDa
←78 kDa
←60 kDa
C
o
n
t
r
o
l
3
-
M
A
C
Q
0
1
2
3
L
C
3
II:L
C
3
I
a
b
c
C
o
n
t
r
o
l
3
-
M
A
C
Q
0 .0
0 .5
1 .0
1 .5
B
e
c
lin
1
/G
A
P
D
H
a
b
b
C
o
n
t
r
o
l
3
-
M
A
C
Q
0 .0
0 .5
1 .0
1 .5
A
T
G
7
/G
A
P
D
H
b
b
a
C
o
n
t
r
o
l
3
-
M
A
C
Q
0 .0
0 .5
1 .0
1 .5
A
T
G
1
2
/G
A
P
D
H
a
a b
b
C
o
n
t
r
o
l
3
-
M
A
C
Q
0 .0
0 .5
1 .0
1 .5
A
T
G
3
/G
A
P
D
H
a
a
b
38. Conclusions
• Dysregulation of autophagy in BCO could be caused
by bacterial manipulation and contribute to BCO
pathology by affecting cell survival and homeostasis
46. Effect of recombinant cyto(chemo)kines
on hFOB cell viability
c
o
n
t
r
o
l
I
L
-
1
I
L
-
8
T
N
F
6 0
8 0
1 0 0
1 2 0
V
ia
b
ilit
y
(
%
o
f
c
o
n
t
r
o
l)
*
*
47. Conclusions
• BCO has systemic effects on pro-inflammatory
factors
• These factors form a unique, detectable
signature of BCO and could contribute to BCO
etiology
50. Materials and Methods
Animal Rearing
•Day1 chicks →
Day10
•Ad libitum food
and water
•Weighed
•Humanely
euthanized
Chondrocyte
Isolation
•Tibia proximal
head
•Cross cut and
shavings from
growth plate
•Serum-free media
•Digestion media
Chondrocyte Culture
•2 x 105 cells/cm2
•Complete media
•DMEM; sodium
pyruvate;
ascorbic acid;
FBS; P/S
•Cytation3 imaging
•Protein and RNA
53. Gene Expression Changes in Culture
d
3
d
7
d
1
1
d
1
4
d
1
8
d
2
1
0
1
2
3
4
A
C
A
N
m
R
N
A
e
x
p
r
e
s
s
io
n
a
ab
b
b
b
b
d
3
d
7
d
1
1
d
1
4
d
1
8
d
2
1
0
1
2
3
4
5
C
O
L
IA
1
m
R
N
A
e
x
p
r
e
s
s
io
n
a
ab
ab
ab
b
b
d
3
d
7
d
1
1
d
1
4
d
1
8
d
2
1
0
2 0
4 0
6 0
8 0
1 0 0
C
O
L
IA
2
m
R
N
A
e
x
p
r
e
s
s
io
n
a
a c
b
b
b c
b c
d
3
d
7
d
1
1
d
1
4
d
1
8
d
2
1
0 .0
0 .5
1 .0
1 .5
C
O
L
II
m
R
N
A
e
x
p
r
e
s
s
io
n
a
ab ab
b
b
c
d
3
d
7
d
1
1
d
1
4
d
1
8
d
2
1
0 .0
0 .5
1 .0
1 .5
S
o
x
9
m
R
N
A
e
x
p
r
e
s
s
io
n
a
b c
b c c
ab
a b c
d
3
d
7
d
1
1
d
1
4
d
1
8
d
2
1
0 .0
0 .5
1 .0
1 .5
C
O
L
X
m
R
N
A
e
x
p
r
e
s
s
io
n
a
b
c c c c
54. Protein Expression Changes in Culture
COL I
COL II
GAPDH
Cell Lysate Media
Day 3 7 11 14 18 21 Day 3 7 11 14 18 21
COLXA1
ACAN
Sox9
COL I
COL II
COLXA1
ACAN
150
83
66
250
56
37
Ponceau S
kDa
3 7 1 1 1 4 1 8 2 1
0
1
2
3
4
5
T im e (d a y s )
p
r
o
te
in
e
x
p
r
e
s
s
io
n
/G
A
P
D
H
C O L I
S ox9
C O L II
A C A N
C O L X A 1
$
#
3 7 1 1 1 4 1 8 2 1
0
1
2
3
4
5
T im e (d a y s )
p
r
o
te
in
e
x
p
r
e
s
s
io
n
/P
o
n
c
e
a
u
S
C O L I
C O L II
A C A N
C O L X A 1
+
*
56. BFA treatment on COLII secretion
COL II
Cell Lysate Media
BFA - - + + BFA - - + +
(1 µg/mL) (1 µg/mL)
GAPDH
COL II
150
37
Ponceau S
0 .0
0 .5
1 .0
1 .5
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
p
r
o
te
in
e
x
p
r
e
s
s
io
n
/G
A
P
D
H
p
r
o
te
in
e
x
p
r
e
s
s
io
n
/P
o
n
c
e
a
u
S
*
B F A - + - +
ly s a te m e dia
( 1 g /m L )
kDa
60. What to DO about BCO?
Primary
Breeder
Grower
Feed and
Nutrition
Processor
1. Prevention
2. Detection
• Phenotyping
• Diagnosing
3. Treatment or Mitigation
62. DXA as a Tool in Avian Physiology Research
What we tested
• Live bird imaging
• Ex vivo leg quarter analysis
• Breast myopathies
• In ovo imaging
What we found
• Skeletal diagnostics and
imaging
• BMD and BMC analysis
across treatment groups*
• Embryonic positioning
*data not shown
66. The Takeaway
• Understanding the molecular mechanisms provides the foundation for
non-invasive biomarkers and therapeutic or selection targets for BCO
• Developing a reliable and relevant in vitro model of avian growth plate
diseases is key for mechanistic studies
• The fast screen time and accuracy make DXA imaging a promising tool in
developing more robust phenotypes for bone health, skeletal disorders,
and in ovo imaging
• Streamlining the process and further research is key!
Leg Bones Increase in 4X in Length & 7X in Diameter
cell utilizes the yellow tetrazolium salt which is metabolized by mitochondrial succinic dehydrogenase activity of proliferating cells to yield a purple formazan product by the mitochondria of viable cell .
Dysregulation of autophagy could be involved in the pathology of the bacterial component of BCO
Dysregulation of autophagy machinery is present in BCO affected femur heads which is indicative of decreased autophagy
Challenge with a known BCO isolate also induces dysregulation of the autophagy machinery
Inhibition of autophagy via separate mechanisms results in not only dysregulated autophagy machinery as seen in tissue and bacterially challenged cells, but also decreased cell viability
Courtesy of Dr. Orlowski
While BCO is bacterially derived, the processes involved point to metabolic, immune, and boen health and also giv ethe foundation for developing non-invasive biomarkers and therapeutic or selection targets for BCO