1. Research Experience in Molecular Biotechnology & Genomics Summer 2010 Emma E. Balfanz 1,2, Erin E. Sandford 1, Michael G. Kaiser 1, and Susan J. Lamont 1 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 industry 1 APEC may be transmittable to humans and thus capable of causing urinary tract infections, meningitis, and sepsis 2,5 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 cytokines 3 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 1234 Day 1Day 5 Chal. Not Chal. x 4 severemild 1 1 severemild 1 1 1 1 Differential immunological gene expression following E. coli infection in chickens 1 Department of Animal Science, Iowa State University, Ames, IA 2 Department of Biology, St. Olaf College, Northfield, MN Gene expression levels evaluated with quantitative PCR (RT-qPCR) C(t) Value Adjustment: 40 – [C(t) test gene mean + (C(t) 28s median – C(t) 28s mean)] × (slope test gene / slope 28s ) Statistical Analysis with JMP® program Program supported by the National Science Foundation Research Experience for Undergraduates DBI-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 10 8 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. 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: 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 Pathology for 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 Studies Increase 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