1. 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

2. 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

3. 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

4. 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

5. 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

6. 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.

7. 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)

8. 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

9. 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

10. 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

11. 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?

12. 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?

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

14. Questions?