Evaluation of Immune Protection Elicited by Recombinant Antigen EtsC 1st Year Undergraduate Honors Research Mentor Program Department of Food Science and Human Nutrition David Couri Research Mentors: Dr. Melha Mellata and Dr. Angelica Van Goor

Background Avian Pathogenic Escherichia coli (APEC) Vaccine Antigen Disease causing E.coli in poultry responsible for colibacillosis Coughing, sneezing, reduced appetite, poor growth, dejection (sad) Responsible for economic loss in livestock Vaccine Decreasing disease by providing protection via immune response Stimulate antibody production Antigen Capable of inducing an immune response Specific to the pathogen it inhabits. Recombinant Antigen Antigen that is artificially reproduced Elicit same immune response as pathogen with antigen

Background EtsC Efflux protein Common in APEC and associated with disease Recombinant antigen EtsC Genetically engineered etsC gene from  plasmid  E.coli isolate/purify protein χ 7122 PBS EtsC 1 Day old White Leghorns 1st rAg vacc. 2nd vacc. (Boost) Sera collection Day 1 Day 4 Day 18 Day 29

Overview of Methods How prevalent is etsC among APEC? Polymerase Chain Reaction (PCR) - Determine existence of etsC in different APEC strains by PCR - Amplify a specific region of DNA - Visualize the presence of gene, by running PCR samples of amplified DNA segments in a gel electrophoresis If used as a vaccine, does etsC elicit antibodies? Enzyme-linked immunosorbent assay (ELISA) - Identify presence of specific antibodies in serum - 9 chickens vaccinated with EtsC and 9 chickens had PBS injection If antibodies are elicited, are they protective? Sera Bactericidal Assay - Measure capability of serum (antibodies) to kill bacteria Hypothesis: Administration of rAg EtsC in chickens will elicit and immune response for the production of antibodies that have the capability to kill APEC that express this antigen

Prevalence of etsC using PCR Purpose: To experimentally determine the existence gene etsC in 14 strains of APEC Procedure: Add GoTaq mastermix to wells Run through PCR machine Conditions: Initial Denaturation (95°C for 2 minutes) Denaturation (95°C for 1 minute) Annealing (50°C for 1 minute) Extension (72°C variable) Final Extension(72°C for 5 minutes) Soak 4°C MG 1655 (-control) APEC 02 APEC #9 O1 APEC #13 O1 APEC #8 O18 APEC #41 O18 APEC #23 O55 APEC #32 O55 APEC #37 O55 APEC #71 O55 APEC #2 O78 APEC #3 O78 APEC #4 O78 χ7122 (+control) 35 cycles

Detection of Antibodies using ELISA Purpose: Measure immune response of chickens that were 29 days old and vaccinated with recombinant antigen EtsC. (Blood serum) Compare vaccinated to non-vaccinated samples and each had a technical replicate    EtsC serum     TMB substrate rabbit anti-chicken IgY Samples randomized using random number generator and run in duplicate.

Ability of sera to kill bacteria using Sera Bactericidal Assay Purpose: Experimentally measure the ability of the antibodies to kill various APEC. APEC isolates from chickens that had Colibacillosis Strains represent different serogroups that are commonly isolated in field cases Procedure: Serum from 10 chickens was pooled within each treatment group Bacteria added (102 CFU) 96 well plates used for the assay Dilutions -2 and -3 plated on MacConkey agar MG 1655 (-control) APEC 02 APEC #9 O1 APEC #13 O1 APEC #8 O18 APEC #41 O18 APEC #23 O55 APEC #32 O55 APEC #37 O55 APEC #71 O55 APEC #2 O78 APEC #3 O78 APEC #4 O78 χ7122 (+control)

PCR Results and Analysis Results: Visualized using gel electrophoresis APEC #37, O55 χ7122 (+control) APEC #9, O1 APEC #13, O1 APEC #8, O18 APEC #41, O18 APEC #23, O55 APEC #32, O55 APEC #71, O55 APEC #2, O78 APEC #3, O78 APEC #4, O78 APEC O2 MG1655 (no DNA) - + - + - - - - + - + - - + + 1 kb- Length of etsC strand (1371 bp) 6/14 strains had etsC genes

ELISA Results and Analysis ___ v ___ v ___ nv ___ v ___ nv ___ v Read absorbance at 450nm with plate reader *P<0.05 using T test in Prims software* (0.0027) Results indicate that administration on a vaccine with recombinant antigen EtsC stimulates the production of antibodies

Sera Bactericidal Assay Results and Analysis Results: # of colonies were counted on each plate and CFU’s/ml of each plate calculated In 6 strains vaccinated samples had lower bacterial count In 2 strains, no growth at all   O1 O18 O55 O78

Conclusions PCR ELISA Sera Bactericidal Assay Unexpected results? The etsC genes are present in APEC and among different serogroups ELISA When chickens are vaccinated with rAg EtsC, they will produce antibodies Sera Bactericidal Assay Many of the vaccinated serum samples were able to kill APEC bacteria Unexpected results? Many strains without etsC gene were killed by vaccinates serum samples. Although some strains don’t have etsC, maybe the antibodies from rAg EtsC recognize other antigens with a similar structure to EtsC. Strains with etsC gene don’t reflect hypothesis that serum from vaccinated chickens will reduce APEC with the gene etsC Not all strains expressed?

Future Experimentation Repeat Sera Bactericidal Assay Confidence that there is an immune response in cells Mix bacteria with splenocytes in chickens given vaccination Are cells able to kill bacteria via a means other than antibodies?

Acknowledgments Dr. Mellata Dr. Van Goor Caroline Treadwell ISU Honors for First Year Honors Mentor Program Grant ($450)

Questions?