Research Experience in Molecular Biotechnology & GenomicsSummer 2010

Emma E. Balfanz1,2, Erin E. Sandford1, Michael G. Kaiser1, and Susan J. Lamont1

Avian Pathogenic Escherichia coli (APEC) is a

gram-negative bacteria that causes Colibacillosis in

chickens.

Resulting mortality and reduced

productivity responsible for multi-

million dollar losses in the poultry industry1

APEC may be transmittable to humans and thus

capable of causing urinary tract infections, meningitis,

and sepsis2,5

Gene expression will be assayed in:

Interleukin (IL)-10: anti-inflammatory cytokine

that suppresses expression of pro-inflammatory

genes

IL-6: pro-inflammatory cytokine assisting in

growth and differentiation of T- and B-cells

IL-1β: another pro-inflammatory cytokine that

enhances inflammation by T-cell and

macrophage activation

Granzyme A (GzmA): protease located in

granules of cytotoxic T cells which, when released,

enters an infected cell and initiates apoptosis. May

also have a role in activating pro-inflammatory

cytokines3

Hypotheses:

Exposure to APEC will result in differential gene

expression levels of IL-10, IL-6, and IL-1β

between Challenged and Non-challenged birds

Furthermore, APEC will induce a higher level of

GzmA gene expression in Day 1 birds than Day 5, and

higher level in Challenged birds than Non-challenged,

in agreement with Sarson et al. (4)

Introduction Materials and Methods

120 120 120 120

1 2 3 4

Day 1 Day 5

Chal.

Not Chal.

x 4

severemild 1

1 severemild

1

1

1

1

Differential immunological gene expression following E. coli infection in chickens

1Department of Animal Science, Iowa State University, Ames, IA 2Department of Biology, St. Olaf College, Northfield, MN

• Gene expression levels evaluated with quantitative PCR (RT-qPCR)

• C(t) Value Adjustment:

40 – [C(t)test genemean + (C(t)28smedian – C(t)28smean)] × (slopetest gene/ slope28s)

• Statistical Analysis with JMP® program

Program supported by the National Science Foundation Research Experience for UndergraduatesDBI-0552371

Figure 1: Data from Opticon Monitor 2, displaying the RT-qPCR results of IL-10 expressed in spleen samples of 24 broilers

• Day-old male broilers, split into four experimental replicates (120/ group)

• At four weeks of age, Challenged birds received 108 cfu of APEC via intra-air sac injection.Non-challenged birds were mock injected

• Necropsies conducted 1 and 5 days post-injection. Birds’ level of pathology classified as Mild or Severe

• Current study involved six spleen samples from each of the four experimental replicates, making 24 total experimental samples.

AcknowledgementsThe 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: balfanze@stolaf.edu

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 expression in

response to APEC

Indicative of pro-inflammatory response of the

innate immune system

Higher expression levels result of upregulation in

these genes or increased cell migration

Day post-challenge had significant effect on gene

expression levels of IL-6; higher Adjusted C(t) values

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)

values for IL-10 and IL-6 than Mild birds

More pronounced bacterial infection may have

produced increased expression as means of combating

effects of APEC

Indicates pro- and anti-inflammatory responses

increasing in parallel, illustrating the balance of a host

immune response to an invading pathogen.

Significant interaction of Day Post-Challenge and Level

of Pathology in GzmA. Severe, Day 1 birds had

highest Adjusted C(t) values while Severe, Day 5 had lowest

expression

Within Severe classification, GzmA’s early

antimicrobial role is more pronounced than at later

stages

Discussion

Results

Figure 5. Interaction of Day Post-Challenge X Level of Pathologyfor 16 Challenged birds (P-value of 0.025)

Figure 2. Effect of Day on Adjusted C(t) values of all 24 samples analyzed

Figure 1. Effect of Challenge on Adjusted C(t) values of all 24 samples analyzed

Figure 4. Effect of Day on adjusted C(t) values of 16 Challenged birds

Figure 3. Effect of Level of Pathology on Adjusted C(t) values of 16 Challenged birds

P =0.004

P =0.013

* Endpoints without common superscript resulted in P ≤ 0.05, according to Student’s t-test

Determine whether there is differential gene

expression of IL-1β, IL-6, IL-10 and GzmA among 24 male

broilers, with respect to APEC challenge, day of analysis

post-challenge, and level of pathology in challenged

birds.

Long-term goal: identify genes that may be

manipulated to augment the chicken’s resistance to

APEC infection.

Objectives

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]

References

Future StudiesIncrease sample size to strengthen reliability of results

Use lesion scores to analyze gene expression levels

within individual tissues

Analyze additional genes within same samples

Samples from the thymus, bursa, bone marrow, and

white blood cells are also available for each bird