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

2.
Efficient
Affordable
Popular
Sustainability
Welfare
Robustness

3.
Day 1 Day 42
Dynamic Molecular Reorganization
Fast Growth and the Leg Bones of the
Modern Broiler
Courtesy of Dr. Wideman

4.
The Modern Broiler and BCO
Wideman, R. 2016. Poult Sci (95)2:325-344

5.
The Avian Growth Plate and BCO
Wideman, R. 2016. Poult Sci (95)2:325-344

6.
Wideman, R. 2016. Poult Sci (95)2:325-344
The Avian Growth Plate and BCO

7.
Lameness, BCO, and the Broiler
BCO
Welfare
Food
Safety
Economic
https://www.allaboutfeed.net/animal-feed/feed-additives/research-probiotics-reduces-
lameness-in-broilers/

8.
How do Bacteria Cause BCO?

9.
dsRNA
NLRP3
Inflammasome
IL-1β
↓ Cell
Viability
Dicer1
Mitochondria
Dysfunction
Autophagy
Dysregulation

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

13.
Double-stranded RNA in BCO

14.
dsRNA and DICER1 Dysregulation in BCO
Greene E, et al. Am J Pathol. 2019. doi: 10.1016/j.ajpath.2019.06.013

15.
dsRNA
NLRP3
Inflammasome
IL-1β
↓ Cell
Viability
Dicer1
Mitochondria
Dysfunction
Autophagy
Dysregulation

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.
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

18.
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

19.
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

20.
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

21.
Conclusion
• Implicates mitochondrial dysfunction
– Potential:
• ↑ cell death (chondronecrosis)
• Alteration of cellular processes
• Contribution to previously found mechanisms
• Further research needed

22.
dsRNA
NLRP3
Inflammasome
IL-1β
↓ Cell
Viability
Dicer1
Mitochondria
Dysfunction
Autophagy
Dysregulation

23.
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.

24.
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
*

25.
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

26.
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
*

27.
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
*

28.
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

29.
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 *

30.
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

31.
Mechanisms for Autophagy Inhibition
InvivoGen http://www.invivogen.com/autophagy-inhibitors

32.
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

33.
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

34.
Summary
Killian, S. 2012. AIDS Research and Therapy. 9(1):16

35.
Conclusions
• Dysregulation of autophagy in BCO could be caused
by bacterial manipulation and contribute to BCO
pathology by affecting cell survival and homeostasis

36.
dsRNA
NLRP3
Inflammasome
IL-1β
↓ Cell
Viability
Dicer1
Mitochondria
Dysfunction
Autophagy
Dysregulation

37.
Effect of plasma from normal and BCO
broilers on hFOB cell viability
N
o
r
m
a
l
B
C
O
0 .0
0 .5
1 .0
1 .5
%
v
ia
b
ility
o
f
c
o
n
t
r
o
l
*

38.
Cytokines
Bone Blood
T
N
F

I
L
-
4
I
L
-
1
8
I
L
-
3
I
L
-
6
I
L
-
1
0
I
L
-
1
2
B
I
L
-
1
7
0
1
2
3
4
5
m
R
N
A
e
x
p
r
e
s
s
io
n
N o rm a l
B C O
* *
T
N
F

I
L
-
4
I
L
-
1
8
I
L
-
1

I
L
-
6
I
L
-
1
0
I
L
-
1
2
B
I
L
-
1
7
0
1
2
3
4
5
m
R
N
A
e
x
p
r
e
s
s
io
n
N o rm a l
B C O
*

39.
Chemokines
Bone Blood
C
C
L
-
4
C
C
L
-
2
0
C
C
L
L
-
4
C
X
C
L
-
1
4
C
C
L
-
5
C
R
P
I
L
-
8
L
1
0
2
4
6
m
R
N
A
e
x
p
r
e
s
s
io
n
N o rm a l
B C O
*
*
C
C
L
-4
C
C
L
-2
0
C
C
L
L
-4
C
X
C
L
-1
4
C
C
L
-5
C
R
P
IL
-8
L
1
IL
-8
L
2
0
1
2
3
4
m
R
N
A
e
x
p
r
e
s
s
io
n
N o rm a l
B C O
*

40.
Inflammasomes
Bone Blood
N
L
R
C
3
N
L
R
C
5
N
L
R
X
1
N
L
R
P
3
0
1
2
3
4
5
m
R
N
A
e
x
p
r
e
s
s
io
n
N o rm a l
B C O
*
N
L
R
C
3
N
L
R
C
5
N
L
R
X
1
0 .0
0 .5
1 .0
1 .5
2 .0
m
R
N
A
e
x
p
r
e
s
s
io
n
N o rm a l
B C O

41.
FGF23 Pathway
Bone Blood
F
G
F
2
3
F
G
F
R
1
K
l
o
t
h
o
0
1
2
3
4
m
R
N
A
e
x
p
r
e
s
s
io
n
N o rm a l
B C O
F
G
F
2
3
F
G
F
R
1
K
l
o
t
h
o
0 .0
0 .5
1 .0
1 .5
2 .0
m
R
N
A
e
x
p
r
e
s
s
io
n
N o rm a l
B C O
*

42.
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)
*
*

43.
Conclusions
• BCO has systemic effects on pro-inflammatory
factors
• These factors form a unique, detectable
signature of BCO and could contribute to BCO
etiology

44.
dsRNA
NLRP3
Inflammasome
IL-1β
↓ Cell
Viability
Dicer1
Mitochondria
Dysfunction
Autophagy
Dysregulation

45.
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

46.
https://musculoskeletalkey.com/cartilage-and-chondrocytes/
Jul 3, 2016 | Posted by admin in RHEUMATOLOGY

47.
Primary Chondrocytes within Culture
Day 3 7 11
14 18 21

48.
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

49.
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
+
*

50.
Protein Expression Changes in Culture
DAPI COL II
DAPI COL II
DAPI COL I
DAPI COL I
Day 7
Day 18

51.
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

52.
Conclusion
Chondroprogenitor Proliferating
chondrocyte
Hypertrophic
chondrocyte
Dedifferentiated
chondrocyte
Early-culture
d3 – d7
Mid-culture
d11 – d14
Late-culture
d18 – d21
Cell
Secreted
COLII
Sox9
COLII
COLXA1
ACAN
Cell
Secreted
COLII
COLI
COLXA1
COLII
COLI Cell
Secreted
COLI
COLXA1
ACAN
COLI

53.
dsRNA
NLRP3
Inflammasome
IL-1β
↓ Cell
Viability
Dicer1
Mitochondria
Dysfunction
Autophagy
Dysregulation

54.
Audience Poll

55.
What to DO about BCO?
Primary
Breeder
Grower
Feed and
Nutrition
Processor
1. Prevention
2. Detection
• Phenotyping
• Diagnosing
3. Treatment or Mitigation

56.
Improved Mechanisms of
Measurement
• DXA vs. other means

57.
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

58.
Live bird imaging

59.
Live bird imaging

60.
In ovo and Newly Hatched Imaging

61.
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!

62.
Q&A Session
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