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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