Progress 01/01/23 to 12/31/23
Outputs
Target Audience:This study targets researchers in poultry disease, vaccine development, and biosensor development, as well as veterinarians, diagnosticians, and poultry industry personnel. Additional target audiences are post-doctoral fellows, graduate students, DVM trainees, and undergraduate trainees. Changes/Problems:There was a delay in animal experiments due to animal housing availability. However, we are convinced that we can complete the proposed experiments in time before the project end date. What opportunities for training and professional development has the project provided?The project has trained three graduate students and one Post-doctoral Associate, who continue to work on the project. Four undergraduate students were also trained under the project. Undergraduate student training included learning the best laboratory practices, general microbiology and molecular biology techniques, chicken experiments, and biosensor technology. Training has also included writing and presentation skills. How have the results been disseminated to communities of interest?Results have been disseminated through presentations and manuscripts. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period: (i) complete the passive immunization experiment mentioned under Objective 2, (ii) assess the immunogenicity and efficacy of the APEC-vectored vaccine against Salmonella Enteritidis challenge, (iii) present research results at the AAAP meeting in June 2024, (iv) submit 3-4 manuscripts, and (v) complete the project and submit the final report.
Impacts
What was accomplished under these goals?
During this review period: Objective 1. Assess the immunogenicity and efficacy of InvG protein of Salmonella Enteritidis against a challenge of S. Enteritidis in chickens.Chickens were vaccinated three times intramuscularly with recombinant InvG at 2, 4, and 6 weeks of age and then challenged with Salmonella Enteritidis orally one-week post-vaccination. The vaccinated chickens elicited a robust serum antibody response (IgG/IgY) against InvG as determined by ELISA.However, there was no significant difference in the mucosal antibody response (IgA) in the intestinal mucosa of the vaccinated birds compared to the unvaccinated controls when the vaccine was administered intramuscularly. Further, there was a reduction of Salmonella counts in the ceca and spleen of vaccinated chickens compared to the unvaccinated control group. There was no significant difference in the bacterial counts in the livers of vaccinated and unvaccinated chickens. Objective 2. Determine if antibodies against InvG can transfer passively from vaccinated hens to progeny chickens, thus providing protection against homologous and heterologousSalmonella. This experiment is currently underway. We vaccinated hens in production by administering recombinant InvG intramuscularly three times two weeks apart, starting from 21 weeks of age. Vaccinated hens produced robust serum IgG response as determined by ELISA. We will continue to monitor serum IgG response and IgY (IgG) response in the egg yolk for four months. Eggs will be collected for hatching, and hatched chickens as well as vaccinated hens, will be challenged with Salmonella Enteritidis, Salmonella, Typhimurium, Salmonella Kenturcky. Objective 3. Construct an E. coli-vectored vaccine expressing InvG to minimize colonization of chicken intestines and ovaries by nontyphoidal Salmonella. The initial construct did not express the InvG protein in vivo as expected. We changed the vector construct and are currently verifying its ability to express InvG. Chicken experiments are planned for July 2024. Objective 4. Establish a proof of concept, high throughput, rapid test for targeting InvG on the surface of Salmonella found in shell eggs. Both Clemson University and Iowa State University have been working on detecting infectious pathogens using electrochemical impedance methods based on laser-inducedgraphene and stimuli-responsive brush platforms in complex sample matrices.In addition to Salmonella, they targeted Listeria monocytogenes, Escherichia coli, norovirus, and SARS-CoV-2. Although recombinant InvG could be used as capture material to detect Salmonella, the efficiency was not as efficient as the use of DNA aptermers as the capture material. While improving the efficiency of InvG protein-based assay, we are currently working on the use of monoclonal antibodies as capture material as an alternative approach. We immunized Balb/c mice with small synthetic peptides of InvG purchased from GenScript and are in the process of hybridoma generation and screening.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2023
Citation:
Hjort, R. G., Pola, C. C., Soares, R. R. A., Oliveira, D. A., Stromberg, L., Claussen, J. C., Gomes, C. L. 2023. Advances in biosensors for detection of foodborne microorganisms, toxins and chemical contaminants. In: Encyclopedia of Food Safety, 2nd Edition. Elsevier. https://doi.org/10.1016/B978-0-12-822521-9.00187-8.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
McLamore, E.S. (2023) Pathogen detection in agricultural systems using portable biosensors. Introduction to Biosystems Engineering course, Clemson University, Guest Lecture, October 10.
Oliveira, D. A., Pola, C. C., Soares, R. R. A., Hjort, R., Claussen, J. C., McLamore, E. S., Gomes, C. L. 2023. Rapid and label-free bacterial pathogen detection based on graphene and stimuli-responsive nanobrushes for in-field water quality monitoring. American Chemical Society, ACS National Meeting and Expo Fall 2023, San Francisco, CA, August.
Neelawala, R., Edison, L. K., Gottapu, C., Kariyawasam, S. 2023. An avian pathogenic E. coli vector system for delivering heterologous vaccine antigens in poultry. Annual Meeting of the American Association of Avian Pathologists (AAAP). Jacksonville, FL, June 11-14.
Neelawala R, Edison LK, Gottapu C, Kariyawasam S. 2023. An avian pathogenic E. coli vector system for delivering heterologous vaccine antigens in poultry. University of Florida College of Veterinary Medicine Research and Phi Zeta Celebration. Gainesville, FL, April 13-14.
Hjort, R., Soares, R. R.A., Miliao, G. L., Pola, C. C., Almeida, C. J., Claussen, J. C., Gomes, C. L. 2023. Future of biological sensing for food, agricultural, and environmental applications: opportunities and challenges. Institute of Biological Engineering annual meeting. Ames, IA, April.
Pola, C. C., Rangnekar, S. V., Szydlowska, B. M., Sheets, R. L., Hersam, M. C., Claussen, J. C., Gomes, C. L. 2023. Electrochemical detection of viruses using printed graphene-based immunosensors. Institute of Biological Engineering annual meeting. Ames, IA, April.
Oliveira, D. A., Soares, R. S., Pola, C. C., Hjort, R. G., Claussen, J. C., Gomes, C. L. 2023. Electrochemical sensors based on laser induced graphene for food safety monitoring Pittcon Conference & Expo 2023, Philadelphia, PA, March.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Neelawala, R., Gorrapu, C., Roberts, J.F., Edison, L., Butcher, G. D. 2024. Preliminary evaluation of InvG as a novel Salmonella vaccine candidate: Immunogenicity and efficacy in layer chickens. Annual meeting of the American Association of Avian Pathologists (AAAP). St. Louis, MO, July 9-11.
Neelawala, R., Gorrapu, C., Roberts, J.F., Edison, L., Butcher, G. D. 2024. Assessment of a novel poultry Salmonella vaccine candidate: efficacy, immunogenicity, and future perspectives. University of Florida College of Veterinary Medicine Research and Phi Zeta Celebration. Gainesville, FL, April 18.
Casso-Hartmann, L., Moreira, G., Tzeng, T-R., Vanegas D., McLamore, E. S. 2024 Electrochemical Detection of Salmonella enterica using a laser inscribed graphene biosensor. ACS Spring Meeting, New Orleans LA. Electrochemical Sensors for Healthcare and the Environment. 1st place prize, Graduate student category. March 19.
Casso-Hartmann, L., Moreira, G., Tzeng, T-R., Vanegas D., McLamore, E. S. 2024 Electrochemical Detection of Salmonella enterica using a laser inscribed graphene biosensor. Global Alliance for Rapid Diagnostics (GARD) Annual Symposium, March 15.
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience:Audiences have included researchers and educators in avian medicine, veterinary medicine, molecular biology and vaccinology, biological engineering, veterinarians, veterinary students, graduate and undergraduate students. Changes/Problems:One of our chicken experiments was faced with a contamination issue. We believe the contamination occurred in the egg incubation room because in ovo Salmonella experiments were done in the same room but in different incubators. The chicken housing facility, including the isolators and the incubators, was decontaminated. New experiments are currently in progress. What opportunities for training and professional development has the project provided?This project has trained one PhD student, one Master's student, and one under-represented (female, African American) research scientist who are continuing to work on the project. A new Post-doctoral Fellow recruited last year has also been working on the project. A couple of undergraduate and DVM trainees were also trained. Training has consisted of learning best laboratory practices, general microbiology and molecular biology techniques, and conducting chicken experiments. Training has also included writing and presentation skills. How have the results been disseminated to communities of interest?Results have been disseminated through presentations and manuscripts. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to: (i) complete vaccine efficacy studies stated under Objective 1, (ii) start the passive immunization experiment mentioned under Objective 2, (iii) assess the immunogenicity and efficacy of the APEC-vectored vaccine against S.Enteritidis challenge, and (iv) present research results at the AAAP meeting in June 2023.
Impacts
What was accomplished under these goals?
Objective 1. Assess the immunogenicity and efficacy of InvG protein of Salmonella Enteritidis against a challenge of S. Enteritidis in chickens. We completed immunogenicity studies by vaccinating the chickens with recombinant InvG twice two weeks apart via intramuscular route. Chickens showed high serum antibody titers as determined by an ELISA developed under this project during Year 1. We challenged the vaccinated birds with S.Enteritidis to determine the vaccine efficacy, but the negative control group (unvaccinated, unchallenged) was contaminated invalidating the entire experiment. We are in the process of repeating the experiment. Objective 3. Construct an E. coli-vectored vaccine expressing InvG to minimize colonization of chicken intestines and ovaries by nontyphoidal Salmonella. We have completed constructing the avian pathogenic E. coli (APEC)-vectored vaccine expressing InvG. First, we created an APEC double mutant strain by deleting aroA and asd genes.This mutant construct was tested for safety in 1-day old-chickens by administering the vaccine strain via the intramuscular route. Then, we transformed the double mutant strain with the Asd-expressing plasmid pYA4515 carrying the invG gene to create the vaccine strain. We demonstrated the InvG is expressed on APEC cell surface in vitro by subcellular fractionation.More experiments are currently underway to characterize the expressed protein and to confirm plasmid stability in the APEC vector strain. Objective 4. Establish a proof of concept, high throughput, rapid test for targeting InvG on the surface of Salmonella found in shell eggs. We were able to preserveInvG in its native form by adding X1 critical micelle concentration of SDS into the protein preparation. Adding SDS at this concentration did not affect the downstream biosensor workflow. During this reporting period,we have made major progress in the development of our flow through system as summarized below in the following four tasks: 4.1 Screening of binding kinetics using biolayer interferometry. We have developed a framework for using high throughput protein-ligand interaction kinetics in the design of EIS biosensors using different concentrations of InvG in the presence of S. Enteritidis. These interaction studies help to optimize surface coverage optimization (maximize binding kinetics and thus minimize steric hindrance). Ongoing work is repeating these studies with different matrix challenges (e.g., exogenous LPS, non-target cells). 4.2. Establishment of graphene electrode platform for high throughput analysis. In this task, a quality control (QC) study was conducted for improving the translation of our work. Along these lines, we have recently developed numerous open source protocols to improve repeatability by other research groups. We developed a simple technique based on repetitive sweeps of CV to condition LIG working electrodes (this technique is common to electrochemistry or sensor labs and is widely available). Analysis of CV data for each sweep (peak redox and charge/discharge dynamics) were used to characterize electrode performance. In addition, an LIG biochip (working, counter and reference electrode in one sensor platform) was also designed and tested. A protocol is under development for sharing this technique using the open source Protocol I/O. 4.3. Laser patterning of cavities for high sample throughput. During our preliminary analysis, we have tested various cavity densities to determine the affect(s) of carbon electrode perforation on electrochemical signal transduction. These preliminary data indicate our ability to control the perforated cavity location as well as diameter. We used a CO2 laser to pattern various microchannel arrays in LIG electrodes, starting from single channel electrodes to orthogonal arrays. We have characterized the electrochemical behavior of micro-cavities. By measuring the change in current during sample filtration, we are currently creating a library of digital fingerprints for various samples. Based on the area under the curve analysis of time series, the test will be used to determine one of two control systems as a binary alarm for false positives/negatives: i) if the sample viscosity is within the range of 10% egg yolk (sample control), or ii) if the capture system was over saturated with bacteria-sized particles (confirmation of contamination). 4.4. Loading of biorecognition element. Our proof of concept work to date has focused on adsorption of an aptamer that is highly selective for InvG (control study). In the next report we will show the biofunctionalization of LIG electrodes with InvG and compare the DNA aptamer coating. Before loading aptamer, the decapped solution was heated to 90° C for 5 minutes (to unfold DNA), and then cooled to room temperature for 10 minutes (to refold). For loading experiment, an aliquot of 10 µl decapped, refolded aptamer solution was drop cast on the surface of nPt-LIG working area, incubated in the BSL2 hood for 10 minutes, gently rinsed with 500 µl bicarbonate buffer, and then connected to the potentiostat for EIS analysis. This process was repeated for five subsequent loading steps.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Yifan Tang, Diana Vanegas and Eric McLamore. Quality Control for LIG Electrodes. Annual IBE conference. Athens, GA. April 7, 2022.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Roshen N. Neelawala, Lekshmi Edison, Chaitanya Gottapu, and Subhashinie Kariyawasam. An Avian Pathogenic E. coli Vector System for Delivering Heterologous Vaccine Antigens in Poultry. UF-CVM Phi Zeta Research Celebration. Gainesville, Florida. April 2023.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Roshen N. Neelawala, Lekshmi Edison, Chaitanya Gottapu, and Subhashinie Kariyawasam. An Avian Pathogenic E. coli Vector System for Delivering Heterologous Vaccine Antigens in Poultry. Annual Meeting of American Assocation of Avian Pathologists. Jacksonville, Florida. June 2023.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2023
Citation:
One manuscript under review describing the electrode system (Tang et al., submitted to RSC Analyst)
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