Download Document

Research Experience in Molecular Biotechnology & Genomics
Summer 2010
Differential immunological gene expression following
E. coli infection in chickens
Emma E.
1Department
1,2
Balfanz ,
Erin E.
1
Sandford ,
Michael G.
1
Kaiser ,
and Susan J.
1
Lamont
of Animal Science, Iowa State University, Ames, IA 2Department of Biology, St. Olaf College, Northfield, MN
Introduction
Discussion
Materials and Methods
Avian Pathogenic Escherichia coli (APEC) is a
•
Day-old male broilers, split into four
experimental replicates (120/ group)
gram-negative bacteria that causes Colibacillosis in
Adjusted C(t) values of IL-6 and IL-1β were
significantly higher in Challenged birds than in
Non-challenged, suggesting higher level of gene
chickens.
•
Resulting mortality and reduced
At four weeks of age, Challenged
birds received 108 cfu of APEC via
intra-air sac injection.
Non-challenged birds were mock
injected
productivity responsible for multimillion dollar losses in the poultry industry1
1
2
120
APEC may be transmittable to humans and thus
3
120
4
120
120
•
capable of causing urinary tract infections,
meningitis, and sepsis2,5
Day 1
Gene expression will be assayed in:
Interleukin (IL)-10: anti-inflammatory cytokine that
suppresses expression of pro-inflammatory genes
Day 5
expression in response to APEC
Indicative of pro-inflammatory response of the innate
immune system
Higher expression levels result of upregulation in
Necropsies conducted 1 and
5 days post-injection. Birds’
level of pathology classified
as Mild or Severe
these genes or increased cell migration
Day post-challenge had significant effect on gene
expression levels of IL-6; higher Adjusted C(t) values
Not
Chal.
Chal.
1
1
1
1
mild
1
severe
x4 •
1
mild
severe
IL-6: pro-inflammatory cytokine assisting in growth
Current study involved six
spleen samples from each of
the four experimental
replicates, making 24 total
experimental samples.
and differentiation of T- and B-cells
resulted in Day 1 birds than in Day 5
Suggestive of enhanced pro-inflammatory response
which is reduced by Day 5
Severe birds yielded significantly higher Adjusted C(t)
•
Gene expression levels evaluated with quantitative PCR (RT-qPCR)
IL-1β: another pro-inflammatory cytokine that
Figure 1: Data from Opticon Monitor 2, displaying the RT-qPCR
results of IL-10 expressed in spleen samples of 24 broilers
enhances inflammation by T-cell and macrophage
values for IL-10 and IL-6 than Mild birds
More pronounced bacterial infection may have
activation
produced increased expression as means of combating
effects of APEC
Granzyme A (GzmA): protease located in granules of
cytotoxic T cells which, when released, enters an
Indicates pro- and anti-inflammatory responses
infected cell and initiates apoptosis. May also have
increasing in parallel, illustrating the balance of a host
a role in activating pro-inflammatory cytokines3
immune response to an invading pathogen.
•
Hypotheses:
C(t) Value Adjustment:
Significant interaction of Day Post-Challenge and Level
40 – [C(t)test genemean + (C(t)28smedian – C(t)28smean)] × (slopetest gene/ slope28s)
Exposure to APEC will result in differential gene
•
of Pathology in GzmA. Severe, Day 1 birds had
Statistical Analysis with JMP® program
expression levels of IL-10, IL-6, and IL-1β between
highest Adjusted C(t) values while Severe, Day 5 had
Challenged and Non-challenged birds
lowest expression
Results
Furthermore, APEC will induce a higher level of GzmA
agreement with Sarson et al. (4)
Objectives
Figure 2. Effect of Day on Adjusted C(t)
values of all 24 samples analyzed
28
28
P =0.028
P =0.004
24
20
Chal.
Non-chal.
P =0.013
16
Adj C(t) LS Mean
level in Challenged birds than Non-challenged, in
Figure 1. Effect of Challenge on Adjusted C(t)
values of all 24 samples analyzed
Adj C(t) LS Mean
gene expression in Day 1 birds than Day 5, and higher
24
16
in challenged birds.
Long-term goal: identify genes that may be
manipulated to augment the chicken’s resistance to
References
1. Barnes H. J. and W. B. Gross. 1997. Diseases of Poultry. pp. 131–141.
2. Bauchart P. et al. 2010. Microb Pathog. [Epub ahead of print].
3. Irmler M. et al. 1995. J. Exp. Med. 181:1917–1922.
4. Sarson A. J. et al. 2009. BMC Genomics. 10:260.
5. Tivendale K.A. et al. 2010. Infect Immun. [Epub ahead of print]
GzmA
P =0.010
P =0.027
24
20
mild
severe
16
Future Studies
Use lesion scores to analyze gene expression levels
within individual tissues
Analyze additional genes within same samples
28
P =0.009
24
Samples from the thymus, bursa, bone marrow, and
20
Day 1
Day 5
16
white blood cells are also available for each bird
12
12
IL-10
APEC infection.
IL-6
IL-1β
Gene
Figure 4. Effect of Day on adjusted C(t)
values of 16 Challenged birds
28
stages
Increase sample size to strengthen reliability of results
IL-10
Adj C(t) LS Mean
of analysis post-challenge, and level of pathology
GzmA
IL-6
IL-1β
Gene
IL-10
GzmA
IL-6
IL-1β
Gene
GzmA
Figure 5. Interaction of Day Post-Challenge X Level of Pathology
for 16 Challenged birds (P-value of 0.025)
Adj C(t) LS Mean
male broilers, with respect to APEC challenge, day
IL-6
IL-1β
Gene
Figure 3. Effect of Level of Pathology on
Adjusted C(t) values of 16 Challenged birds
Adj C(t) LS Means
expression of IL-1β, IL-6, IL-10 and GzmA among 24
Day 1
Day 5
12
IL-10
antimicrobial role is more pronounced than at later
P =0.005
20
12
Determine whether there is differential gene
Within Severe classification, GzmA’s early
18
A
AB
16
14
AB
mild
severe
* Endpoints without
B
12
1
Day
5
common superscript
resulted in P ≤ 0.05,
according to Student’s
t-test
Acknowledgements
The authors wish to thank members of the Lamont lab group for
their support and guidance throughout the program. Also
thanks to Dr. Max Rothschild and Justin Rice for their
dedication in making this summer a valuable and memorable
experience.
Author email: [email protected]
Program supported by the National Science Foundation Research Experience for Undergraduates
DBI-0552371