A MOLECULAR SEROLOGY APPROACH FOR SURVEILLANCE OF AVIAN PATHOGENIC E. COLI IN GEORGIA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028363
• Grant No.: 2022-67015-36878
• Cumulative Award Amt.: $297,000.00
• Proposal No.: 2021-06944
• Project Start Date: Mar 1, 2022
• Project End Date: Feb 28, 2025
• Grant Year: 2022
• Program Code: [A1221]- Animal Health and Production and Animal Products: Animal Health and Disease

Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016

Performing Department
(N/A)

Non Technical Summary
Colibacillosis is, arguably, the most common and costly disease for the poultry industry worldwide, resulting in mortality at the farm and carcass condemnation at slaughter. The causative agent is Avian Pathogenic E. coli (APEC). For many years, most studies reported the most common APEC serogroups associted with disease included the serogroups O1, O2 and O78. These three serogroups were identified as representing over 90% of all APEC isolates, with commercial vaccines available specifically targeting them. In a preliminary study, using recently collected isolates (2018-2020), we found that only 25 % of isolates were from serogroup O2 or O78, and none of the isolates were of the O1 serotype, indicating a shift in the APEC causing disease in modern poultry pridcution. The goal of this study is to monitor the APEC O serogroups that are causing disease and create a rapid tool for serogroup monitoring that allows us to better predict vulnerable flocks and identify potential strategies for collibacilosis control at the farm level. Specifically, we will: 1) Build and characterize a collection of avian Escherichia coli: APEC, AFEC (avian fecal E. coli) and litter E. coli isolates; 2) Perform molecular analysis of these isolates in relation to virulence. Epidemiological analysis of our data will allow us to identify new emerging strains and defining traits of the environment and the host that potentially lead to E. coli-associated disease in poultry. Having a better knowledge of the emerging serogroups associated with production and disease, we will be able to identify new potential targets for collibacilosis control and development of non-antimicrobial related strategies.

Animal Health Component

10%

Research Effort Categories

Basic

80%

Applied

10%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3299	1040	90%
311	4010	1080	10%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other; 4010 - Bacteria;

Field Of Science
1040 - Molecular biology; 1080 - Genetics;

Keywords

apec

colibacillosis

molecular serology

Goals / Objectives
The long-term goal to which this project contributes is to further our understanding of Avian Pathogenic Escherichia coli (APEC) ecology and epidemiology of cellulitis and collibacilosis and use that knowledge to design effective strategies for colibacillosis control, thereby significantly improving bird health, and welfare and industry performance. In addition, we wish to ascertain what factors during production favor selection of APEC. A potential secondary impact of our work is a reduction in the zoonotic risk of APEC for humans. Our main goal will be to identify the new emerging APEC O types that are causing colibacillosis. Our primary hypotheses are: (i) Vaccines being used to prevent collibacilosis are shifting the pathogenic E. coli population. (ii) The E. coli serogroup present in the environment, commensal and litter are also shifting and can be linked to a more pathogenic APEC. This research proposes to address issues in assessing the relationship between poultry production, and animal health with a view to identifying new emerging E. coli O types applying an epidemiological-based approach to assess the influence of production practice with the specific vaccine serogroup use on health status and as a source for population selection pressure. Specifically, we will pursue the following two objectives: 1) Build and characterize a current collection of avian pathogenicEscherichia coli: APEC, AFEC (avian fecal E. coli) and litter E. coli isolates; perform molecular serogroup analysis; perform the APEC minimal predictors test; and perform phenotypic characterization for the most common new serogroups identified; 2) Perform molecular analysis: We will perform whole genome sequencing, assembly and annotation of the new characterized serogroups; with the goal of contributing new genomes to the APEC genomic toolbox. Knowledge gained from this Seed Grant will allow us how to better identify targets for health status and control of disease during production - significantly impacting the safety of poultry products - and potentially identifying targets for reducing the overall burden of animal disease. Our goal with this seed grant is to identify and characterize the new emerging APEC O serogroup that are causing colibacillosis, create a rapid tool for serogroup monitoring, and leverage the preliminary data for a full USDA standard grant

Project Methods
Objective 1: Build and characterize a collection of avian Escherichia coli:We will perform an epidemiological study to identify the emerging O serogroups that are causing collibacilosis,cellulitis and systemic disease in poultry production. We will identify the O serogroups most frequently found in the litterand those of non-pathogenic E. coli in order to assess what are the most common current E. coli types present inproduction broilers and potentially identify newly emerging clones that could be targets for further investigation, in orderto improve surveillance and disease control.Specific Aim 1A: Build a collection of APEC, AFEC (avian fecal E. coli) and litter E. coli and perform molecularserogroup analysis.To identify the new current O serogroups present, a total of 1500 isolates will be used. The APEC will beisolated at GPLN (Georgia Poultry Laboratory Network) from diagnostic cases and 1 day old chick surveillance. Theywill be collected between April 2021 and December of 2022. We will create a new multiplex PCR to cover the most frequent serogroups found in the new collection. We will perform the multiplex PCR using primers targeting these mostfrequent serogroups. With this data we will be able to identify the serogroups from the farm isolates.Specific Aim 1B: Perform a minimal predictors test analysis to evaluate the APEC and APEC-like presence inthe collection. Here 1500 isolates from diseased birds and 832 E. coli isolated from the production farms (litter andAFEC), will be screened using a multiplex PCR panel that targets 9 genes associated with APEC chromosomal andplasmid virulence genes, that indicate the minimal predictor genes of APEC. We will correlate O serogroups to thepresence of the 9 minimal predictor genes to identify the serogroups that correlate to the most virulent isolates. Thisresult will give us updated information as to what are the new serogroups emergent that correspond to the O1, O2 and O78 ofprevious years.Specific Aim 1C: Perform phenotypic characterization and function elucidation for the most common newserogroups identified.Though much progress has been made in recent years in understanding APEC's virulence, one important traitunderlying its environmental persistence, antimicrobial resistance and acquisition of virulence genes from exogenoussources remains poorly understood. Here 15-20 new isolates from the top 5 new APEC associated serogroups will beinvestigated for better characterization for the ability to form biofilm and the ability to adherence and invasion ofeukaryotic cells.Specific Aim 1C1: Biofilm formation. To assess the ability of different O serogroups to form biofilm, new isolates will be screened for biofilm formation in M63 minimal media uisng the crystal violet assay. The biofilms will be classified as strong(OD600>blank x4), moderate (blankx4≥OD600>blank x2), weak (OD600≤blan x2) or negligible OD600

Progress 03/01/24 to 02/28/25

Outputs
Target Audience:This project is aimed at researchers in poultry disease, faculty, veterinarians, producers, other graduate students, poultry ancillary industries e.g. vaccine companies or feed companies or animal health companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has continued to train a PhD student; two undergraduates were also trained in the past year. How have the results been disseminated to communities of interest?The results of this resarch have been presented at a number of venues including The American Association of Avian Pathologists (AAAP) the american society for microbiology (ASM) The Conference of Research Workers in Animal Disease (CRWAD) International Poultry Science Forum(IPSF) - part of IPPE - southern conference of avian diseases SYSS - Young Scientist meeting in Singapore North Central Avian DiseasesConference (NCAD) National Institute for Antimicrobial Resistance Research and Education (NIAMRRE) annual meeting What do you plan to do during the next reporting period to accomplish the goals?Genome sequence analysis is to be completed and finalizing the last of the research papers - we anticipate at least three more from this work.

Impacts
What was accomplished under these goals? The project has completed a number of publicaitons in the past year which were all accepted in late December with some published in early January. Surveillance found novel and multiple different types of APEC were responsible for disease in Georgia poultry and the project was able to identify the most common strains implicated in disease. A multuplex PCR targeting these strains was developed - named Klao9 seroPCR that targeted the most common APEC. The multuplex was able to target the top 9 serogroups and had a detection rate over 36% when tested with a collection of blind typed isolates.The studies have also extensively characterized APEC from various bird types and lesions and characterized them for phylogeneitc group, harboring pathogenicity associated genes and antimicrobial resistance profiles (see papers for detail). The study also noted a slight shift in serogroup prevalence in the second year of the study reflecting the emergence of new serogroups and competition that may be occurring in poultry hosts. Of interest, phylogenetic group G seemed to become more prevalent and further charcaterization of this phylogroup is warranted to understand its place in APEC and emergence. Additional work focused on serogroup O25-ST131 a serogroup that has been associated with human disease. These studies point out that the strain type also causes disease in birds and the strain types are similartostrains associated with human uropathogenic E. coli. Objective 1 has been completed and published including a second year analysis Objective 2 molecular characterization has been completed and genome sequence analysis is ongoing

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Runcharoon, K., Garcia, B., Peterson, B. N., Young, M. M., Favro, M. E., Barbieri, N. L., & Logue, C. M. (2024). Longitudinal study of avian pathogenic Escherichia coli (APEC) serogroups associated with disease in Georgia poultry using molecular serology and virulence gene analysis. Avian Pathology, 113. https://doi.org/10.1080/03079457.2024.2403414
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Klao Runcharoon, Margaret E. Favro, Catherine M. Logue, Longitudinal analysis of avian pathogenic Escherichia coli (APEC) serogroups and pathotypes from avian colibacillosis in Georgia: A continued investigation  year 2 analysis, Poultry Science, Volume 104, Issue 2, 2025, 104722, ISSN 0032-5791, https://doi.org/10.1016/j.psj.2024.104722.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Klao Runcharoon, Margaret E Favro, Catherine M Logue, The pathogenicity traits of avian pathogenic Escherichia coli O25-ST131 associated with avian colibacillosis in Georgia poultry and their genotypic and phenotypic overlap with other extraintestinal pathogenic E. coli, Journal of Applied Microbiology, Volume 136, Issue 1, January 2025, lxaf015, https://doi.org/10.1093/jambio/lxaf015

Progress 03/01/24 to 02/29/24

Outputs
Target Audience:Target audience for this work includes poultry researchers and microbiologists interested in infectious disease of poultry Other groups of interest include poultry and allied industries such as producers, growers, vaccine compaies, and poultry health companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training of graduate student, undergraduates (biology and microbiology majors), DVM students. How have the results been disseminated to communities of interest?Disseimantion of the resarch at professional meetings including American Society for Microbiology, American Associaiton of Avian Pathologists, Southern Conference for Avian Disease (SCAD) at IPPE, What do you plan to do during the next reporting period to accomplish the goals?Complete whole genome sequencing and analysis, preparation and submission of additonal research papers to be completed. Completeanimal challenge models that are planned. Additonal presentations at professional meetings.

Impacts
What was accomplished under these goals? Work of the grant has included characterization of APEC, identification of the common serogroups of APEC causing disease in Georgia using molecular serology and development of a multiplex PCR targeting common serogroups found. Additonal work has performed whole geniome sequening on selected novel APEC straons found and will assess their pathogeneisis in chicken models including embryos, chicks and mature birds.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: 6. Runcharoon, K., Garcia, B., Peterson, B.N., Young, M.M., Favro, M.E., Barbieri, N.L., Waltman, D., Flores, B., Dinh, E., and Logue, C.M. (2024) Longitudinal Study of Avian Pathogenic Escherichia coli (APEC) Serogroups Associated with Disease in Georgia Poultry Using Molecular Serology and Virulence Gene Analysis. Avian Pathology 1-13 https://doi.org/10.1080/03079457.2024.2403414

Progress 03/01/23 to 02/29/24

Outputs
Target Audience:The target audience for this research includes academic researchers in poultry disease, veterinarians, poultry clinicians, diagnosticians, faculty and staff with interests in poultry disease and health,poultry producers including growers, hatchery and breeder farms, commercial poultry companies, allied poultry industries such as vaccine and feed companies and federal and legislative agencies such as the USDA and FDA and CDC. This work has relevance to both chicken and turkey industries. Additional audiences include postdoctoral researchers, DVM trainee students, graduate students and undergraduate students. Changes/Problems:No changes or problems have occurred during this project rather we have generated many new avenues for future research. What opportunities for training and professional development has the project provided?The project continues to train 1 PhD student who is still working on the project 2 undergraduates have worked on the project for research experience (voluntary) 1 DVM student participated in the research as a summer scholar Training consisted of learning molecular tools, culture work (bacteriology), DNA preparation and analysis for serogroups, viruelnce genes and additional pathogen screening such as biofilms, embryo lethality, and chick assays. Training in oral and poster presentation skills as well as report writing have also been emphasised. How have the results been disseminated to communities of interest?The results of the research have been disseminated through oral and poster presentaiton at national and internationalmeetings. these have includedthe International poultry science forum (IPSF) meeting in Atlanta, the American Society for Microbiology (ASM) internationalmeeting, teh american association for avian pathologists (AAAP) meeting, the SYSS symposium and meeting SIngapore, (Jan 2024), and the north central avian diseases (PEAK) meeting. The summer scholar veterinariain presented the research at the Veterinary Scholars Symposium (formerly Meriel symposium) held in Costa Rica. The students have also presented their work internally at UGA CVM as part of the Giguere Research Symposium and the Veterinary Summer Scholars Symposium. What do you plan to do during the next reporting period to accomplish the goals?The next phase of the work is to validate the emerhiong serogroups (additonal strains and evaluate in vitro and in vivo assays). Complete validation of the APEC O group rapid assay using blinded strains. Complete whole genome sequening and analysis of novel APEC strains and get them complete for publicaiton Submit the Draft of the serogroup analysis - paper is complete and awaiting some final approvals. Draft additonal papers in areas of the APEC ST131 strains, and novel APEC in vitro and in vivo stuidies. Recruit a veterinarian summer scholar for summer 2024 research

Impacts
What was accomplished under these goals? Our work to date has continued surveillance of APEC recovered from Georgia poultry and characterizing strains of interest. We have expanded the databases for an additional year of isolates where the collection now contains more than 1200 isolates of which more than 569 are from adult birds. The collection includes birds from a range of production systems and types including breeders, pullets and cockerels, broilers, game birds and layers. Of interest the majority of isolates are recovered from liver (24.9%) followed by bone marrow (15.1%). The majority of serogroups are O78 (28%) followed by O2 (16%), O25 (15%), O8 (8%) and O1 (8%), of interest, about 46% of APEC could not be assigned to a serogroup. The study has noted more frequent associations between serogroups and bird type from which the strain was isolated. Based on our prevalence data and odds analysis we were able to design a rapid screening multiplex PCR that targets the most common APEC serogroups we see in Georgia poultry. This multiplex protocol targets for O groups O2, O8, O1, O25, O78, O86, O18 and O15 with greater approximately 60% accuracy. Although the assay was designed to target APEC of Georgia we have also applied this test to isolates out of the state and it still provides a rapid means to narrow screening. Additonal work ongoing has identified novel APEC serogroups that would appear to be emergent in Georgia poultry these have included about 10 new novel serogroups including O25, O15, O91, O152, O161, O86, O88, O115 O62/O68, and O45. These strains have been assayed both phenotypically and genotypically and using in vivo assays for embryo lethality and chick survival. Using the serum resistance (chicken serum) assay survival of some strains was evident after 4 h incubation for most strains except O25. Most strains examined had lethality scores in embryo lethality of 80-100% with an LD50 of 2-3 days. Serogroups O88 and O62/O62 showed the least virulence in embryos. We also observed no correlation between embryo lethality and virulence genes harbored by the test strains. Similarly, when challenged in day old chicks, and assessed for lethality using a lethality score (which represents the mortality and over the duration of the study), strains O15, O91 and O88 showed significantly lower lethality score than the APEC O18 WT control strain. All other strains had scores that averaged around 5-6. Ongoing work will assess if these observations represent the serogroups and will be re-evaluated with additional strains from the O group. During the course of these studies we also identified a significant number of APEC O25 ST 131 strains - and ExPEC stain known as a high risk clone with potential for causing human disease and has been linked with urinary tract infection. These isolates were assessed for traits including phylogenetic groups, virulence gene carriage, biofilm formation, serum resistance and ability to grow in human urine. Most of the strains tested identify as phylogenetic group B2 and harbor some traits consistent with uropathogenic E. coli, more than 78.4% were serum resistant and 67% from strong biofilms, with more than 60% able to grow in urine. In addition, most of the O25 isolates screened show high resistance to ciprofloxacin, (>50%), chloramphenicol, ((60%), and trimethoprim sulfa drug resistance (25%) while lower prevalence of resistance to streptomycin, gentamicin, tetracycline, ceftriaxone, cefoxitin, and meropenem (range 5-15%) were also observed. Genotypically, these resistances were confirmed by detection of genes linked to the phenotype.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Runcharoon, K., Logue, C.M. (2023) Characterizing antimicrobial resistance (AMR) genes in Avian Pathogenic E. coli (APEC) O25/ST131 from Georgia colibacillosis cases. Poster presentation at NIAMRRE Annual meeting Gainesville FL 05/23
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Runcharoon, K., Garcia, B., Logue, C.M., (2023) Characterizing the O Serogroups of Avian Pathogenic E. coli (APEC) Associated with Colibacillosis in Georgia Poultry and their Relationship with Production Types. NCAD/ PEAK  Invited TED Talk (Grad Students) from abstract submission 03/23.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Izhar UI Haque, M., Runcharoon, K., Garcia, B., Young, M.M., Logue, C.M. (2023) Molecular Serology and Virulence Characteristics of Avian Pathogenic Escherichia coli (APEC) Recovered from Georgia Poultry. AAAP Annual Meeting 06/23
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., Barbieri, N.L., Logue, C.M. (2023) Assessing the Relationship between O Serogroups and Virulence Gene Carriage in Georgia Poultry Colibacillosis Cases. AAAP Annual Meeting 06/23
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., Barbieri, N.L., Logue, C.M. (2023) Characterizing O serogroups and virulence genes of Avian Pathogenic E. coli (APEC) associated with colibacillosis in Georgia Poultry. ASM Microbe 06/23
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Izhar UI Haque, M., Runcharoon, K., Garcia, B., Young, M.M., Logue, C.M. (2023) Molecular Serology and Virulence Characteristics of Avian Pathogenic Escherichia coli (APEC) Recovered from Georgia Poultry. IPPE/SCAD Annual Meeting presented 01/23 (poster)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., and Logue, C.M. (2023) Characterizing O-Serogroups of Avian Pathogenic E. coli (APEC) Associated with Colibacillosis in Georgia Poultry Production and Their Relationship with Production Types. IPPE/SCAD Annual Meeting presented 01/23 (oral)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Runcharoon, K., Ienes Lima, J., Tsai, Y-Y., Logue, C.M. (2023) Characterizing Pathogenicity Traits of Emerging Serogroups of Avian Pathogenic E. coli (APEC) using in vitro and in vivo Assays. Submitted to SCAD 11/23 #Winner of the C.S. Edison Student Award for Research Excellence (Oral Presentation) SCAD 2024 (Jan 2024)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Runcharoon, K., Logue, C.M. (2023) Characterization of Avian Pathogenic E. coli (APEC) O25 ST 131 High Risk clones recovered from avian colibacillosis cases and their potential as a zoonotic pathogen. SYSS meeting, Singapore 2024
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Runcharoon, K., Logue, C.M. (2023) Pathogenicity traits of emerging serogroups of avian pathogenic E. coli (APEC) from colibacillosis cases in Georgia. Giguere Science of Veterinary Medicine Symposium 10/23
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Perryman, T., Runcharoon, K., Logue, C.M. (2023) Surveillance and Characterization of Avian Pathogenic E., coli using molecular serology Giguere Science of Veterinary Medicine Symposium 10/23
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Perryman, T., Runcharoon, K., and Logue C.M. (2023) Characterization of Avian Pathogenic E. coli (APEC) using Molecular Serogrouping and Pathogenicity Assays. CVM Summer Scholars Symposium 08/23  Oral Presentation.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Perryman, T., Runcharoon, K., and Logue C.M. (2023) Characterization of Avian Pathogenic E. coli (APEC) using Molecular Serogrouping and Pathogenicity Assays. 2023 Veterinary Scholars Symposium, Costa Rica 08/23  Poster Presentation.

Progress 03/01/22 to 02/29/24

Outputs
Target Audience:The target audience for this final report includes, researchers in poultry health and disease, poultry industry and poultry industry affiliates, stakeholders such as growers, producers and processors. Other agencies of interest include USDA, FDA, CDC and NIH. In additon poultry professional organizations such as microbiology societies (ASM, Applied Microbiology, Microbiology Society etc), and poultry societies such as AAAP, Poultry Science, and Animal Health societies as examples. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Additionaloutcomes of this work were multiple presentations at professional meetings for the graduate student and major professor, training of professional (vet students, including an international veterinary student), undergraduates (Microbiology and Biology majors), and a professional masters of avian medicine student.One undergraduate undertook UGAs CURO program and presented her research at the event. Training activities included lab bench work from basic bacterial culture analysis, molecular analysis (PCR), challenge trials in embryos, chicks and birds. Students working with the research group also participated in presentation skills (oral and poster) and report writing (undergraduates for credit). The graduate student also participated in the three minute thesis competition. How have the results been disseminated to communities of interest?Results of this research have been presented in multiple arenas including professional meetings, producer meetings, and student affiliated meetings. Meeting incude American Society for Microbiology (ASM), Conference of Research Workers in Animal Disease (CRWAD), American Association of Avian Pathologists (AAAP), Western Poultry DiseaseMeeting, Southern Conference of AvianDisease SCAD - part ofIPSF at IPPE, NIAMRRE annual conference, and international meetings such as SYSS Singapore. Presentations at these types of venues have included oral presentations of the work - typical 15-30 minute talks and poster prsentaitons of the research. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This project has completed all of the goals outlined and some additional work was added to round out the project. We have identified the major serogroups causing disease in Georgia and have developed multiplex PCR assays to rapidly screen isolates for potential common serogroups likely to be contributing to disease in submitted cases. The assay has been validated using blind sets of samples and has a detection rate of about 36% which is low, and reflective of the changing nature of APEC that cause disesae. (See publicaitons on longitudinal studies year 1 and 2). Included in this work was an analysis of the common serogroups causing disease in different types of poultry in Georgia an obsertvation that found some bird types were more restricted in strains causing disease than others but profiles in broiler breeder birds were reflected in the progeny. In additon, there was a seasonal component to some serogroups detected certain parts of the year compared to others suggesting shifting serogroups may be influenced by temperature. We have identified multiple novel APEC serogroups not previously identified as causative agents of disease in poultry and these novel strain types were subjected to whole genome sequencing andanalysis - papers on this studies are pending submission. We have also modeled disease associated with these strain types in embryos, chicks and chickens in an effort to assess the virulence and pathogenesis of these strains. Additonal studies identified novel E. coli O25 ST131 strains that were causing disease in layers silmilar to human UPEC strains and this report is detailed in the paper describing the pathogenicity traits of APEC O25 ST131 and their overlap wiht humab ExPEC type strains.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Runcharoon*, K., Favro, M.E., and Logue, C.M. (2025) The Pathogenicity traits of avian pathogenic E. coli (APEC) O25-ST131 associated with avian colibacillosis in Georgia poultry and their genotypic and phenotypic overlap with other ExPEC. Journal of Applied Microbiology https://doi.org/10.1093/jambio/lxaf015
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Runcharoon, K., Favro, M.E., and Logue, C.M. (2025) Longitudinal Analysis of Avian Pathogenic Escherichia coli (APEC) Serogroups and Pathotypes from Avian Colibacillosis Cases in Georgia: A Continued Investigation  Year 2 Analysis. Journal of Poultry Science. Vol 104:2:104722 https://doi.org/10.1016/j.psj.2024.104722
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Runcharoon, K., Garcia, B., Peterson, B.N., Young, M.M., Favro, M.E., Barbieri, N.L., Waltman, D., Flores, B., Dinh, E., and Logue, C.M. (2024) Longitudinal Study of Avian Pathogenic Escherichia coli (APEC) Serogroups Associated with Disease in Georgia Poultry Using Molecular Serology and Virulence Gene Analysis. Avian Pathology 1-13 https://doi.org/10.1080/03079457.2024.2403414
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Runcharoon, K., Ienes Lima, J., Tsai, Y-Y., and Logue, C.M. (2025) Using animal challenge models to elucidate novel APEC serogroup pathogenicity. Submitted to AAAP 01/25
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Runcharoon, K, Ienes Lima, J., Favro, M.E. and Logue, C.M. (2025) Pathogenicity traits and genomic features of five High risk clone O25-ST131 Avian Pathogenic E. coli (APEC) isolates from colibacillosis cases in Georgia USA. Submitted to ASM Microbe 01/25
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Runcharoon*, K., Ienes-Lima*, J., Tsai*, Y-Y, and Logue, C.M. (2024) Using animal challenge models to elucidate the pathogenicity of novel APEC serogroups. Abstract submitted IPSF 11/24 accepted 11/24 presented 01/25
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Runcharoon#*, K.L., Ienes-Lima*, J., Tsai*, Y-Y., and Logue, C.M. (2024) Using animal challenge models to identify pathogenicity traits of novel avian pathogenic E. coli (APEC) serogroups isolated from Georgia poultry populations. CRWAD abstract submitted 09/24 accepted 10/24 #Winner of the 2025 American College of Veterinary Microbiologists Student Award  1st Place Oral Presentation
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Runcharoon*#, K., Ienes-Lima*, J., Tsai*, Y-Y., and Logue, C.M. (2024) Assessment of virulence in novel Avian Pathogenic Escherichia coli (APEC) serogroups using in vitro and in vivo assays. AAAP 07/24 #Winner of the AS Rosenwald Student poster award 2024 from AAAP
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Runcharoon*, K., and Logue, C.M. (2024) Genotype and phenotype analysis of Avian Pathogenic E. coli (APEC) O25 ST 131 and its association with other ExPEC. ASM microbe 06/24
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Runcharoon*, K., Ienes-Lima*, J., Tsai*, Y-Y., Logue, C.M. (2023) Characterizing Pathogenicity Traits of Emerging Serogroups of Avian Pathogenic E. coli (APEC) using in vitro and in vivo Assays. SCAD 01/24 #Winner of the C.S. Edison Student Award for Research Excellence (Oral Presentation) SCAD 2024 (Jan 2024)
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Runcharoon, K., Logue, C.M. (2023) Characterizing antimicrobial resistance (AMR) genes in Avian Pathogenic E. coli (APEC) O25/ST131 from Georgia colibacillosis cases. Poster presentation at NIAMRRE Annual meeting Gainesville FL 05/23
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Runcharoon, K., Garcia, B., Logue, C.M., (2023) Characterizing the O Serogroups of Avian Pathogenic E. coli (APEC) Associated with Colibacillosis in Georgia Poultry and their Relationship with Production Types. NCAD/ PEAK  Invited TED Talk (Grad Students) from abstract submission 03/23.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., Barbieri, N.L., Logue, C.M. (2023) Assessing the Relationship between O Serogroups and Virulence Gene Carriage in Georgia Poultry Colibacillosis Cases. AAAP Annual Meeting 06/23
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., Barbieri, N.L., Logue, C.M. (2023) Characterizing O serogroups and virulence genes of Avian Pathogenic E. coli (APEC) associated with colibacillosis in Georgia Poultry. ASM Microbe 06/23
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., and Logue, C.M. (2023) Characterizing O-Serogroups of Avian Pathogenic E. coli (APEC) Associated with Colibacillosis in Georgia Poultry Production and Their Relationship with Production Types. IPPE/SCAD Annual Meeting presented 01/23 (oral)
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Logue, C.M., Barbieri, N.L., Peterson, B.N., Young, M.M. (2022) O Serogroups of APEC from Georgia Poultry  An Overview of Molecular Serology Approaches to Characterize APEC. ASM Microbe Meeting 06/22
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Runcharoon, K., Logue, C.M. (2023) Characterization of Avian Pathogenic E. coli (APEC) O25 ST 131 High Risk clones recovered from avian colibacillosis cases and their potential as a zoonotic pathogen. SYSS meeting, Singapore 2024

Progress 03/01/22 to 02/28/23

Outputs
Target Audience:This study reports target researchers in poultry disease, veterinarians, clinicans, diagnosticians, faculty and staff with an interest in poultry disease and health. Additonal target audiences are undergraduate researchers, DVM in training students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has trained one PhD student who is continuing to work on the project 3 undergraduate students who have worked on the project (voluntary) 1 DVM trainee summer scholar (international) who worked on the project as part of a summerexperience. Training has consited of learning molecular tools for diagniostics, culture work (bacteriology), DNA preparation and results and analysis for serogroups and pathogen screening. Training has alos included writing and presentaiton skills (poster and oral). How have the results been disseminated to communities of interest?Results have been disseminated through oral and poster presentations at national and international meetings. These have included IPSF (International Poultry Scinece Forum) meeting in Atlanta, the ASM annual meeting (American Society for Microbiology); the AAAP meeting (American Association of Avian Pathologists). Presentation at the CURO scholar program at UGA - The center for undergraduate research opportunities Summer scholar program in UGA CVM and the Meriel Summer scholars symposium in Minnesota (DVM trainee participated) Audiences have included researchers, poultry veterinarians and poultry reserchers, commerical companies including vaccine companies, other veterinary students, other undergraduates interested in research and future veterinarians that are curently in training. What do you plan to do during the next reporting period to accomplish the goals?The next stage of progress is to address validation of the multiplex PCR developed that targets new O serogroups emgerging in Georgia and determine accuracy of the new test using a model validation approach; We will identify novelserogroups of APEC causing disease and prepare these for whole genome sequencing, analysis and publicaiton, Conduct molecular and phenotyope analysis of novel strains including biofilm, invasion and adhesion of tissue culture lines. Prepare additonal abstracts and reports for presentation at upcoming meetings including AAAP; ASM and North Central Avian Diseases Conference. Preparation of the first paper on detection of O serogroups and analysis is omngoing and planned for summer/ fall 2023 Recruiting of additonal undergraduate researchers (2) in microbiology for training in research and methodology Recruitment of a summer scholar (veterinarian) for summer project 2023

Impacts
What was accomplished under these goals? Avian Pathogenic Escherichia coli (APEC) the causative agent of colibacillosis causes systemic or localized infections in poultry including airsacculitis, septicemia, pericarditis, perihepatitis, salpingitis, and cellulitis. Serogrouping based on the detection of somatic O-antigensis a useful tool to classify APEC and relate to disease pathogenicity. There are approximately 180 E. coli serogroups and the most common types associated with colibacillosis includeO1, O2, and O78. APEC harbor several virulence-associated genes (VAGs) including those linked with invasins, adhesins, iron acquisition systems, and toxins which influence bacterial pathogenicity and disease severity. Currently, there is limited information on APEC O-groups and VAGs in Georgia poultry. This study assessed the prevalence of O-types and VAGs in Georgia APEC. A total of 603 isolates collected between March 2021 - 2022 from submitted diagnostic cases were analyzed and screened for O-serogroups and VAGs using multiplex polymerase chain reaction (PCR). Overall, 309 isolates were identified as serogroups O78 (41%), O2 (19%), O25 (16%), O8 (9%), O1 (9%), O86, O18, and O15 (5%). Chi-square and odd ratios analysis were employed to identify the relationship between APEC O-serogroups, bird types, and VAGs. The odds of O25 were 1.16 higher in broilers than any other bird type, while in the broiler breeder, the odds of O1 were 9.31. Broiler breeder pullet and cockerel had an odds ratio for O78 of 1.81 and pet/hobby birds were more likely to harbor O8 (OR 2.94). The prevalence of VAGs among all isolates ompT and hlyF (84.7%), iss (82.5%), iroN (82.2%), and aerJ (73.4%) cvaC (42.7%) and ireA (42.4%) etsB (24.6%) and papC (24.2%). The number of genes detected per isolate ranged from 0 (41 isolates, 13.27%) to 9 (3 isolates, 0.97%). The most common virulence gene patterns included cva-iroN-ompT-hlyF-iss-aerJ-ireA-papC (33 isolates, 10.68%), iroN-ompT-hlyF-etsB-iss-aerJ (29 isolates, 9.39%) and iroN-ompT-hlyF-iss-aerJ-ireA (28 isolates, 9.06%). The relationship between O-serogroups and VAGs for O2 was likely to have an odds ratio for cvaC, ireA and papC of 2.45, 1.87, and 2.48 higher than other O-serogroups. O8 tends to harbor aerJ and papC(OR 20 and 27). O1 had odd ratios of 1.94 for cvaC and 13.81 for papC. O25 tends to harbor etsB (OR 7.4), while O78 is likely to have ireA (OR 4.18). Lastly, O15 had an odds ratio for cvaC and etsB(22.24 and 3.3). The data has helped us identify the common O types occurring in Georgia Poultry and we have designed a multiplex toto rapidly screen isolates for the common serogroups - this tool can be validated with a second set of isolates also collected and are currently not identified. This will lead to more accurate typing tools for implemenataion in the diagnostic setting. Further phenotypic characterization and molecular research (currently ongoing) will allow us to develop diagnostic tools for detecting and lessening APEC associated with colibacillosis in Georgia. The broad impact of this work to date is development of a new assay for E. coli serogroup identification which has potential for implementaiton in routine diagnostics alongside our current path panel analysis which can lead to rapid identificaiton of pathogenic E. coli causing disease in Georgia poultry. For the producer and veterinarians rapid testing and turnaround can lead to better understanding and identificaiton of sources of disease for poultry flocks and production of healthier flocks as well as enhancing the health and welfare of poultry destined for human consumption.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Izhar UI Haque, M., Runcharoon, K., Garcia, B., Young, M.M., Logue, C.M. (2022) Molecular Serology and Virulence Characteristics of Avian Pathogenic Escherichia coli (APEC) recovered from Georgia Poultry. IPPE/SCAD Annual Meeting abstract submitted 11/2022 presented 01/23 (poster)
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., and Logue, C.M. (2022) Characterizing O-Serogroups of Avian Pathogenic E. coli (APEC) Associated with Colibacillosis in Georgia Poultry Production and Their Relationship with Production Types. IPPE/SCAD Annual Meeting abstract submitted 11/2022 presented 01/23 (oral)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Logue, C.M., Barbieri, N.L., Peterson, B.N., Young, M.M. (2022) O Serogroups of APEC from Georgia Poultry  An Overview of Molecular Serology Approaches to Characterize APEC. ASM Microbe Meeting 06/22
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Runcharoon, K., Young, M.M., Garcia, B., Logue, C.M. (2022) New Emerging O-types of Avian Pathogenic E. coli (APEC) in the Georgia Poultry Population Giguere Science of Veterinary Medicine Symposium Fall 2022
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Weitkamp, L.K., Garcia, B., Young, M.M., Logue, C.M. (2022) Analyses of Virulence Genes in Avian Pathogenic Escherichia coli. Presented at the National Veterinary Scholars Symposium, Minneapolis MN 07/2022
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Garcia, B., Young, M.Y., and Logue, C.M. (2022) Molecular Serology Approach for Surveillance of Avian Pathogenic E. coli (APEC). Presented at the CURO Research Symposium, UGA 04/2022
• Type: Conference Papers and Presentations Status: Under Review Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., Barbieri, N.L., and Logue, C.M. (2023) Assessing the relationship between O serogroups and virulence gene carriage in Georgia Poultry with Colibacillosis. AAAP annual meeting
• Type: Conference Papers and Presentations Status: Under Review Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., Barbier, N.L., Logue, C.M. (2023) Characterizing O serogroups and virulence genes of Avian Pathogenic E. coli (APEC) associated with colibacillosis in Georgia Poultry. Submitted to ASM Microbe 01/2023

Progress 03/01/24 to 02/28/25

Outputs
Target Audience:This project is aimed at researchers in poultry disease, faculty, veterinarians, producers, other graduate students, poultry ancillary industries e.g. vaccine companies or feed companies or animal health companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has continued to train a PhD student; two undergraduates were also trained in the past year. How have the results been disseminated to communities of interest?The results of this resarch have been presented at a number of venues including The American Association of Avian Pathologists (AAAP) the american society for microbiology (ASM) The Conference of Research Workers in Animal Disease (CRWAD) International Poultry Science Forum(IPSF) - part of IPPE - southern conference of avian diseases SYSS - Young Scientist meeting in Singapore North Central Avian DiseasesConference (NCAD) National Institute for Antimicrobial Resistance Research and Education (NIAMRRE) annual meeting What do you plan to do during the next reporting period to accomplish the goals?Genome sequence analysis is to be completed and finalizing the last of the research papers - we anticipate at least three more from this work.

Impacts
What was accomplished under these goals? The project has completed a number of publicaitons in the past year which were all accepted in late December with some published in early January. Surveillance found novel and multiple different types of APEC were responsible for disease in Georgia poultry and the project was able to identify the most common strains implicated in disease. A multuplex PCR targeting these strains was developed - named Klao9 seroPCR that targeted the most common APEC. The multuplex was able to target the top 9 serogroups and had a detection rate over 36% when tested with a collection of blind typed isolates.The studies have also extensively characterized APEC from various bird types and lesions and characterized them for phylogeneitc group, harboring pathogenicity associated genes and antimicrobial resistance profiles (see papers for detail). The study also noted a slight shift in serogroup prevalence in the second year of the study reflecting the emergence of new serogroups and competition that may be occurring in poultry hosts. Of interest, phylogenetic group G seemed to become more prevalent and further charcaterization of this phylogroup is warranted to understand its place in APEC and emergence. Additional work focused on serogroup O25-ST131 a serogroup that has been associated with human disease. These studies point out that the strain type also causes disease in birds and the strain types are similartostrains associated with human uropathogenic E. coli. Objective 1 has been completed and published including a second year analysis Objective 2 molecular characterization has been completed and genome sequence analysis is ongoing

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Runcharoon, K., Garcia, B., Peterson, B. N., Young, M. M., Favro, M. E., Barbieri, N. L., & Logue, C. M. (2024). Longitudinal study of avian pathogenic Escherichia coli (APEC) serogroups associated with disease in Georgia poultry using molecular serology and virulence gene analysis. Avian Pathology, 113. https://doi.org/10.1080/03079457.2024.2403414
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Klao Runcharoon, Margaret E. Favro, Catherine M. Logue, Longitudinal analysis of avian pathogenic Escherichia coli (APEC) serogroups and pathotypes from avian colibacillosis in Georgia: A continued investigation  year 2 analysis, Poultry Science, Volume 104, Issue 2, 2025, 104722, ISSN 0032-5791, https://doi.org/10.1016/j.psj.2024.104722.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Klao Runcharoon, Margaret E Favro, Catherine M Logue, The pathogenicity traits of avian pathogenic Escherichia coli O25-ST131 associated with avian colibacillosis in Georgia poultry and their genotypic and phenotypic overlap with other extraintestinal pathogenic E. coli, Journal of Applied Microbiology, Volume 136, Issue 1, January 2025, lxaf015, https://doi.org/10.1093/jambio/lxaf015

Progress 03/01/24 to 02/29/24

Outputs
Target Audience:Target audience for this work includes poultry researchers and microbiologists interested in infectious disease of poultry Other groups of interest include poultry and allied industries such as producers, growers, vaccine compaies, and poultry health companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training of graduate student, undergraduates (biology and microbiology majors), DVM students. How have the results been disseminated to communities of interest?Disseimantion of the resarch at professional meetings including American Society for Microbiology, American Associaiton of Avian Pathologists, Southern Conference for Avian Disease (SCAD) at IPPE, What do you plan to do during the next reporting period to accomplish the goals?Complete whole genome sequencing and analysis, preparation and submission of additonal research papers to be completed. Completeanimal challenge models that are planned. Additonal presentations at professional meetings.

Impacts
What was accomplished under these goals? Work of the grant has included characterization of APEC, identification of the common serogroups of APEC causing disease in Georgia using molecular serology and development of a multiplex PCR targeting common serogroups found. Additonal work has performed whole geniome sequening on selected novel APEC straons found and will assess their pathogeneisis in chicken models including embryos, chicks and mature birds.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: 6. Runcharoon, K., Garcia, B., Peterson, B.N., Young, M.M., Favro, M.E., Barbieri, N.L., Waltman, D., Flores, B., Dinh, E., and Logue, C.M. (2024) Longitudinal Study of Avian Pathogenic Escherichia coli (APEC) Serogroups Associated with Disease in Georgia Poultry Using Molecular Serology and Virulence Gene Analysis. Avian Pathology 1-13 https://doi.org/10.1080/03079457.2024.2403414

Progress 03/01/23 to 02/29/24

Outputs
Target Audience:The target audience for this research includes academic researchers in poultry disease, veterinarians, poultry clinicians, diagnosticians, faculty and staff with interests in poultry disease and health,poultry producers including growers, hatchery and breeder farms, commercial poultry companies, allied poultry industries such as vaccine and feed companies and federal and legislative agencies such as the USDA and FDA and CDC. This work has relevance to both chicken and turkey industries. Additional audiences include postdoctoral researchers, DVM trainee students, graduate students and undergraduate students. Changes/Problems:No changes or problems have occurred during this project rather we have generated many new avenues for future research. What opportunities for training and professional development has the project provided?The project continues to train 1 PhD student who is still working on the project 2 undergraduates have worked on the project for research experience (voluntary) 1 DVM student participated in the research as a summer scholar Training consisted of learning molecular tools, culture work (bacteriology), DNA preparation and analysis for serogroups, viruelnce genes and additional pathogen screening such as biofilms, embryo lethality, and chick assays. Training in oral and poster presentation skills as well as report writing have also been emphasised. How have the results been disseminated to communities of interest?The results of the research have been disseminated through oral and poster presentaiton at national and internationalmeetings. these have includedthe International poultry science forum (IPSF) meeting in Atlanta, the American Society for Microbiology (ASM) internationalmeeting, teh american association for avian pathologists (AAAP) meeting, the SYSS symposium and meeting SIngapore, (Jan 2024), and the north central avian diseases (PEAK) meeting. The summer scholar veterinariain presented the research at the Veterinary Scholars Symposium (formerly Meriel symposium) held in Costa Rica. The students have also presented their work internally at UGA CVM as part of the Giguere Research Symposium and the Veterinary Summer Scholars Symposium. What do you plan to do during the next reporting period to accomplish the goals?The next phase of the work is to validate the emerhiong serogroups (additonal strains and evaluate in vitro and in vivo assays). Complete validation of the APEC O group rapid assay using blinded strains. Complete whole genome sequening and analysis of novel APEC strains and get them complete for publicaiton Submit the Draft of the serogroup analysis - paper is complete and awaiting some final approvals. Draft additonal papers in areas of the APEC ST131 strains, and novel APEC in vitro and in vivo stuidies. Recruit a veterinarian summer scholar for summer 2024 research

Impacts
What was accomplished under these goals? Our work to date has continued surveillance of APEC recovered from Georgia poultry and characterizing strains of interest. We have expanded the databases for an additional year of isolates where the collection now contains more than 1200 isolates of which more than 569 are from adult birds. The collection includes birds from a range of production systems and types including breeders, pullets and cockerels, broilers, game birds and layers. Of interest the majority of isolates are recovered from liver (24.9%) followed by bone marrow (15.1%). The majority of serogroups are O78 (28%) followed by O2 (16%), O25 (15%), O8 (8%) and O1 (8%), of interest, about 46% of APEC could not be assigned to a serogroup. The study has noted more frequent associations between serogroups and bird type from which the strain was isolated. Based on our prevalence data and odds analysis we were able to design a rapid screening multiplex PCR that targets the most common APEC serogroups we see in Georgia poultry. This multiplex protocol targets for O groups O2, O8, O1, O25, O78, O86, O18 and O15 with greater approximately 60% accuracy. Although the assay was designed to target APEC of Georgia we have also applied this test to isolates out of the state and it still provides a rapid means to narrow screening. Additonal work ongoing has identified novel APEC serogroups that would appear to be emergent in Georgia poultry these have included about 10 new novel serogroups including O25, O15, O91, O152, O161, O86, O88, O115 O62/O68, and O45. These strains have been assayed both phenotypically and genotypically and using in vivo assays for embryo lethality and chick survival. Using the serum resistance (chicken serum) assay survival of some strains was evident after 4 h incubation for most strains except O25. Most strains examined had lethality scores in embryo lethality of 80-100% with an LD50 of 2-3 days. Serogroups O88 and O62/O62 showed the least virulence in embryos. We also observed no correlation between embryo lethality and virulence genes harbored by the test strains. Similarly, when challenged in day old chicks, and assessed for lethality using a lethality score (which represents the mortality and over the duration of the study), strains O15, O91 and O88 showed significantly lower lethality score than the APEC O18 WT control strain. All other strains had scores that averaged around 5-6. Ongoing work will assess if these observations represent the serogroups and will be re-evaluated with additional strains from the O group. During the course of these studies we also identified a significant number of APEC O25 ST 131 strains - and ExPEC stain known as a high risk clone with potential for causing human disease and has been linked with urinary tract infection. These isolates were assessed for traits including phylogenetic groups, virulence gene carriage, biofilm formation, serum resistance and ability to grow in human urine. Most of the strains tested identify as phylogenetic group B2 and harbor some traits consistent with uropathogenic E. coli, more than 78.4% were serum resistant and 67% from strong biofilms, with more than 60% able to grow in urine. In addition, most of the O25 isolates screened show high resistance to ciprofloxacin, (>50%), chloramphenicol, ((60%), and trimethoprim sulfa drug resistance (25%) while lower prevalence of resistance to streptomycin, gentamicin, tetracycline, ceftriaxone, cefoxitin, and meropenem (range 5-15%) were also observed. Genotypically, these resistances were confirmed by detection of genes linked to the phenotype.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Runcharoon, K., Logue, C.M. (2023) Characterizing antimicrobial resistance (AMR) genes in Avian Pathogenic E. coli (APEC) O25/ST131 from Georgia colibacillosis cases. Poster presentation at NIAMRRE Annual meeting Gainesville FL 05/23
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Runcharoon, K., Garcia, B., Logue, C.M., (2023) Characterizing the O Serogroups of Avian Pathogenic E. coli (APEC) Associated with Colibacillosis in Georgia Poultry and their Relationship with Production Types. NCAD/ PEAK  Invited TED Talk (Grad Students) from abstract submission 03/23.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Izhar UI Haque, M., Runcharoon, K., Garcia, B., Young, M.M., Logue, C.M. (2023) Molecular Serology and Virulence Characteristics of Avian Pathogenic Escherichia coli (APEC) Recovered from Georgia Poultry. AAAP Annual Meeting 06/23
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., Barbieri, N.L., Logue, C.M. (2023) Assessing the Relationship between O Serogroups and Virulence Gene Carriage in Georgia Poultry Colibacillosis Cases. AAAP Annual Meeting 06/23
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., Barbieri, N.L., Logue, C.M. (2023) Characterizing O serogroups and virulence genes of Avian Pathogenic E. coli (APEC) associated with colibacillosis in Georgia Poultry. ASM Microbe 06/23
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Izhar UI Haque, M., Runcharoon, K., Garcia, B., Young, M.M., Logue, C.M. (2023) Molecular Serology and Virulence Characteristics of Avian Pathogenic Escherichia coli (APEC) Recovered from Georgia Poultry. IPPE/SCAD Annual Meeting presented 01/23 (poster)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., and Logue, C.M. (2023) Characterizing O-Serogroups of Avian Pathogenic E. coli (APEC) Associated with Colibacillosis in Georgia Poultry Production and Their Relationship with Production Types. IPPE/SCAD Annual Meeting presented 01/23 (oral)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Runcharoon, K., Ienes Lima, J., Tsai, Y-Y., Logue, C.M. (2023) Characterizing Pathogenicity Traits of Emerging Serogroups of Avian Pathogenic E. coli (APEC) using in vitro and in vivo Assays. Submitted to SCAD 11/23 #Winner of the C.S. Edison Student Award for Research Excellence (Oral Presentation) SCAD 2024 (Jan 2024)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Runcharoon, K., Logue, C.M. (2023) Characterization of Avian Pathogenic E. coli (APEC) O25 ST 131 High Risk clones recovered from avian colibacillosis cases and their potential as a zoonotic pathogen. SYSS meeting, Singapore 2024
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Runcharoon, K., Logue, C.M. (2023) Pathogenicity traits of emerging serogroups of avian pathogenic E. coli (APEC) from colibacillosis cases in Georgia. Giguere Science of Veterinary Medicine Symposium 10/23
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Perryman, T., Runcharoon, K., Logue, C.M. (2023) Surveillance and Characterization of Avian Pathogenic E., coli using molecular serology Giguere Science of Veterinary Medicine Symposium 10/23
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Perryman, T., Runcharoon, K., and Logue C.M. (2023) Characterization of Avian Pathogenic E. coli (APEC) using Molecular Serogrouping and Pathogenicity Assays. CVM Summer Scholars Symposium 08/23  Oral Presentation.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Perryman, T., Runcharoon, K., and Logue C.M. (2023) Characterization of Avian Pathogenic E. coli (APEC) using Molecular Serogrouping and Pathogenicity Assays. 2023 Veterinary Scholars Symposium, Costa Rica 08/23  Poster Presentation.

Progress 03/01/22 to 02/29/24

Outputs
Target Audience:The target audience for this final report includes, researchers in poultry health and disease, poultry industry and poultry industry affiliates, stakeholders such as growers, producers and processors. Other agencies of interest include USDA, FDA, CDC and NIH. In additon poultry professional organizations such as microbiology societies (ASM, Applied Microbiology, Microbiology Society etc), and poultry societies such as AAAP, Poultry Science, and Animal Health societies as examples. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Additionaloutcomes of this work were multiple presentations at professional meetings for the graduate student and major professor, training of professional (vet students, including an international veterinary student), undergraduates (Microbiology and Biology majors), and a professional masters of avian medicine student.One undergraduate undertook UGAs CURO program and presented her research at the event. Training activities included lab bench work from basic bacterial culture analysis, molecular analysis (PCR), challenge trials in embryos, chicks and birds. Students working with the research group also participated in presentation skills (oral and poster) and report writing (undergraduates for credit). The graduate student also participated in the three minute thesis competition. How have the results been disseminated to communities of interest?Results of this research have been presented in multiple arenas including professional meetings, producer meetings, and student affiliated meetings. Meeting incude American Society for Microbiology (ASM), Conference of Research Workers in Animal Disease (CRWAD), American Association of Avian Pathologists (AAAP), Western Poultry DiseaseMeeting, Southern Conference of AvianDisease SCAD - part ofIPSF at IPPE, NIAMRRE annual conference, and international meetings such as SYSS Singapore. Presentations at these types of venues have included oral presentations of the work - typical 15-30 minute talks and poster prsentaitons of the research. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This project has completed all of the goals outlined and some additional work was added to round out the project. We have identified the major serogroups causing disease in Georgia and have developed multiplex PCR assays to rapidly screen isolates for potential common serogroups likely to be contributing to disease in submitted cases. The assay has been validated using blind sets of samples and has a detection rate of about 36% which is low, and reflective of the changing nature of APEC that cause disesae. (See publicaitons on longitudinal studies year 1 and 2). Included in this work was an analysis of the common serogroups causing disease in different types of poultry in Georgia an obsertvation that found some bird types were more restricted in strains causing disease than others but profiles in broiler breeder birds were reflected in the progeny. In additon, there was a seasonal component to some serogroups detected certain parts of the year compared to others suggesting shifting serogroups may be influenced by temperature. We have identified multiple novel APEC serogroups not previously identified as causative agents of disease in poultry and these novel strain types were subjected to whole genome sequencing andanalysis - papers on this studies are pending submission. We have also modeled disease associated with these strain types in embryos, chicks and chickens in an effort to assess the virulence and pathogenesis of these strains. Additonal studies identified novel E. coli O25 ST131 strains that were causing disease in layers silmilar to human UPEC strains and this report is detailed in the paper describing the pathogenicity traits of APEC O25 ST131 and their overlap wiht humab ExPEC type strains.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Runcharoon*, K., Favro, M.E., and Logue, C.M. (2025) The Pathogenicity traits of avian pathogenic E. coli (APEC) O25-ST131 associated with avian colibacillosis in Georgia poultry and their genotypic and phenotypic overlap with other ExPEC. Journal of Applied Microbiology https://doi.org/10.1093/jambio/lxaf015
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Runcharoon, K., Favro, M.E., and Logue, C.M. (2025) Longitudinal Analysis of Avian Pathogenic Escherichia coli (APEC) Serogroups and Pathotypes from Avian Colibacillosis Cases in Georgia: A Continued Investigation  Year 2 Analysis. Journal of Poultry Science. Vol 104:2:104722 https://doi.org/10.1016/j.psj.2024.104722
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Runcharoon, K., Garcia, B., Peterson, B.N., Young, M.M., Favro, M.E., Barbieri, N.L., Waltman, D., Flores, B., Dinh, E., and Logue, C.M. (2024) Longitudinal Study of Avian Pathogenic Escherichia coli (APEC) Serogroups Associated with Disease in Georgia Poultry Using Molecular Serology and Virulence Gene Analysis. Avian Pathology 1-13 https://doi.org/10.1080/03079457.2024.2403414
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Runcharoon, K., Ienes Lima, J., Tsai, Y-Y., and Logue, C.M. (2025) Using animal challenge models to elucidate novel APEC serogroup pathogenicity. Submitted to AAAP 01/25
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Runcharoon, K, Ienes Lima, J., Favro, M.E. and Logue, C.M. (2025) Pathogenicity traits and genomic features of five High risk clone O25-ST131 Avian Pathogenic E. coli (APEC) isolates from colibacillosis cases in Georgia USA. Submitted to ASM Microbe 01/25
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Runcharoon*, K., Ienes-Lima*, J., Tsai*, Y-Y, and Logue, C.M. (2024) Using animal challenge models to elucidate the pathogenicity of novel APEC serogroups. Abstract submitted IPSF 11/24 accepted 11/24 presented 01/25
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Runcharoon#*, K.L., Ienes-Lima*, J., Tsai*, Y-Y., and Logue, C.M. (2024) Using animal challenge models to identify pathogenicity traits of novel avian pathogenic E. coli (APEC) serogroups isolated from Georgia poultry populations. CRWAD abstract submitted 09/24 accepted 10/24 #Winner of the 2025 American College of Veterinary Microbiologists Student Award  1st Place Oral Presentation
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Runcharoon*#, K., Ienes-Lima*, J., Tsai*, Y-Y., and Logue, C.M. (2024) Assessment of virulence in novel Avian Pathogenic Escherichia coli (APEC) serogroups using in vitro and in vivo assays. AAAP 07/24 #Winner of the AS Rosenwald Student poster award 2024 from AAAP
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Runcharoon*, K., and Logue, C.M. (2024) Genotype and phenotype analysis of Avian Pathogenic E. coli (APEC) O25 ST 131 and its association with other ExPEC. ASM microbe 06/24
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Runcharoon*, K., Ienes-Lima*, J., Tsai*, Y-Y., Logue, C.M. (2023) Characterizing Pathogenicity Traits of Emerging Serogroups of Avian Pathogenic E. coli (APEC) using in vitro and in vivo Assays. SCAD 01/24 #Winner of the C.S. Edison Student Award for Research Excellence (Oral Presentation) SCAD 2024 (Jan 2024)
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Runcharoon, K., Logue, C.M. (2023) Characterizing antimicrobial resistance (AMR) genes in Avian Pathogenic E. coli (APEC) O25/ST131 from Georgia colibacillosis cases. Poster presentation at NIAMRRE Annual meeting Gainesville FL 05/23
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Runcharoon, K., Garcia, B., Logue, C.M., (2023) Characterizing the O Serogroups of Avian Pathogenic E. coli (APEC) Associated with Colibacillosis in Georgia Poultry and their Relationship with Production Types. NCAD/ PEAK  Invited TED Talk (Grad Students) from abstract submission 03/23.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., Barbieri, N.L., Logue, C.M. (2023) Assessing the Relationship between O Serogroups and Virulence Gene Carriage in Georgia Poultry Colibacillosis Cases. AAAP Annual Meeting 06/23
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., Barbieri, N.L., Logue, C.M. (2023) Characterizing O serogroups and virulence genes of Avian Pathogenic E. coli (APEC) associated with colibacillosis in Georgia Poultry. ASM Microbe 06/23
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., and Logue, C.M. (2023) Characterizing O-Serogroups of Avian Pathogenic E. coli (APEC) Associated with Colibacillosis in Georgia Poultry Production and Their Relationship with Production Types. IPPE/SCAD Annual Meeting presented 01/23 (oral)
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Logue, C.M., Barbieri, N.L., Peterson, B.N., Young, M.M. (2022) O Serogroups of APEC from Georgia Poultry  An Overview of Molecular Serology Approaches to Characterize APEC. ASM Microbe Meeting 06/22
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Runcharoon, K., Logue, C.M. (2023) Characterization of Avian Pathogenic E. coli (APEC) O25 ST 131 High Risk clones recovered from avian colibacillosis cases and their potential as a zoonotic pathogen. SYSS meeting, Singapore 2024

Progress 03/01/22 to 02/28/23

Outputs
Target Audience:This study reports target researchers in poultry disease, veterinarians, clinicans, diagnosticians, faculty and staff with an interest in poultry disease and health. Additonal target audiences are undergraduate researchers, DVM in training students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has trained one PhD student who is continuing to work on the project 3 undergraduate students who have worked on the project (voluntary) 1 DVM trainee summer scholar (international) who worked on the project as part of a summerexperience. Training has consited of learning molecular tools for diagniostics, culture work (bacteriology), DNA preparation and results and analysis for serogroups and pathogen screening. Training has alos included writing and presentaiton skills (poster and oral). How have the results been disseminated to communities of interest?Results have been disseminated through oral and poster presentations at national and international meetings. These have included IPSF (International Poultry Scinece Forum) meeting in Atlanta, the ASM annual meeting (American Society for Microbiology); the AAAP meeting (American Association of Avian Pathologists). Presentation at the CURO scholar program at UGA - The center for undergraduate research opportunities Summer scholar program in UGA CVM and the Meriel Summer scholars symposium in Minnesota (DVM trainee participated) Audiences have included researchers, poultry veterinarians and poultry reserchers, commerical companies including vaccine companies, other veterinary students, other undergraduates interested in research and future veterinarians that are curently in training. What do you plan to do during the next reporting period to accomplish the goals?The next stage of progress is to address validation of the multiplex PCR developed that targets new O serogroups emgerging in Georgia and determine accuracy of the new test using a model validation approach; We will identify novelserogroups of APEC causing disease and prepare these for whole genome sequencing, analysis and publicaiton, Conduct molecular and phenotyope analysis of novel strains including biofilm, invasion and adhesion of tissue culture lines. Prepare additonal abstracts and reports for presentation at upcoming meetings including AAAP; ASM and North Central Avian Diseases Conference. Preparation of the first paper on detection of O serogroups and analysis is omngoing and planned for summer/ fall 2023 Recruiting of additonal undergraduate researchers (2) in microbiology for training in research and methodology Recruitment of a summer scholar (veterinarian) for summer project 2023

Impacts
What was accomplished under these goals? Avian Pathogenic Escherichia coli (APEC) the causative agent of colibacillosis causes systemic or localized infections in poultry including airsacculitis, septicemia, pericarditis, perihepatitis, salpingitis, and cellulitis. Serogrouping based on the detection of somatic O-antigensis a useful tool to classify APEC and relate to disease pathogenicity. There are approximately 180 E. coli serogroups and the most common types associated with colibacillosis includeO1, O2, and O78. APEC harbor several virulence-associated genes (VAGs) including those linked with invasins, adhesins, iron acquisition systems, and toxins which influence bacterial pathogenicity and disease severity. Currently, there is limited information on APEC O-groups and VAGs in Georgia poultry. This study assessed the prevalence of O-types and VAGs in Georgia APEC. A total of 603 isolates collected between March 2021 - 2022 from submitted diagnostic cases were analyzed and screened for O-serogroups and VAGs using multiplex polymerase chain reaction (PCR). Overall, 309 isolates were identified as serogroups O78 (41%), O2 (19%), O25 (16%), O8 (9%), O1 (9%), O86, O18, and O15 (5%). Chi-square and odd ratios analysis were employed to identify the relationship between APEC O-serogroups, bird types, and VAGs. The odds of O25 were 1.16 higher in broilers than any other bird type, while in the broiler breeder, the odds of O1 were 9.31. Broiler breeder pullet and cockerel had an odds ratio for O78 of 1.81 and pet/hobby birds were more likely to harbor O8 (OR 2.94). The prevalence of VAGs among all isolates ompT and hlyF (84.7%), iss (82.5%), iroN (82.2%), and aerJ (73.4%) cvaC (42.7%) and ireA (42.4%) etsB (24.6%) and papC (24.2%). The number of genes detected per isolate ranged from 0 (41 isolates, 13.27%) to 9 (3 isolates, 0.97%). The most common virulence gene patterns included cva-iroN-ompT-hlyF-iss-aerJ-ireA-papC (33 isolates, 10.68%), iroN-ompT-hlyF-etsB-iss-aerJ (29 isolates, 9.39%) and iroN-ompT-hlyF-iss-aerJ-ireA (28 isolates, 9.06%). The relationship between O-serogroups and VAGs for O2 was likely to have an odds ratio for cvaC, ireA and papC of 2.45, 1.87, and 2.48 higher than other O-serogroups. O8 tends to harbor aerJ and papC(OR 20 and 27). O1 had odd ratios of 1.94 for cvaC and 13.81 for papC. O25 tends to harbor etsB (OR 7.4), while O78 is likely to have ireA (OR 4.18). Lastly, O15 had an odds ratio for cvaC and etsB(22.24 and 3.3). The data has helped us identify the common O types occurring in Georgia Poultry and we have designed a multiplex toto rapidly screen isolates for the common serogroups - this tool can be validated with a second set of isolates also collected and are currently not identified. This will lead to more accurate typing tools for implemenataion in the diagnostic setting. Further phenotypic characterization and molecular research (currently ongoing) will allow us to develop diagnostic tools for detecting and lessening APEC associated with colibacillosis in Georgia. The broad impact of this work to date is development of a new assay for E. coli serogroup identification which has potential for implementaiton in routine diagnostics alongside our current path panel analysis which can lead to rapid identificaiton of pathogenic E. coli causing disease in Georgia poultry. For the producer and veterinarians rapid testing and turnaround can lead to better understanding and identificaiton of sources of disease for poultry flocks and production of healthier flocks as well as enhancing the health and welfare of poultry destined for human consumption.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Izhar UI Haque, M., Runcharoon, K., Garcia, B., Young, M.M., Logue, C.M. (2022) Molecular Serology and Virulence Characteristics of Avian Pathogenic Escherichia coli (APEC) recovered from Georgia Poultry. IPPE/SCAD Annual Meeting abstract submitted 11/2022 presented 01/23 (poster)
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., and Logue, C.M. (2022) Characterizing O-Serogroups of Avian Pathogenic E. coli (APEC) Associated with Colibacillosis in Georgia Poultry Production and Their Relationship with Production Types. IPPE/SCAD Annual Meeting abstract submitted 11/2022 presented 01/23 (oral)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Logue, C.M., Barbieri, N.L., Peterson, B.N., Young, M.M. (2022) O Serogroups of APEC from Georgia Poultry  An Overview of Molecular Serology Approaches to Characterize APEC. ASM Microbe Meeting 06/22
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Runcharoon, K., Young, M.M., Garcia, B., Logue, C.M. (2022) New Emerging O-types of Avian Pathogenic E. coli (APEC) in the Georgia Poultry Population Giguere Science of Veterinary Medicine Symposium Fall 2022
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Weitkamp, L.K., Garcia, B., Young, M.M., Logue, C.M. (2022) Analyses of Virulence Genes in Avian Pathogenic Escherichia coli. Presented at the National Veterinary Scholars Symposium, Minneapolis MN 07/2022
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Garcia, B., Young, M.Y., and Logue, C.M. (2022) Molecular Serology Approach for Surveillance of Avian Pathogenic E. coli (APEC). Presented at the CURO Research Symposium, UGA 04/2022
• Type: Conference Papers and Presentations Status: Under Review Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., Barbieri, N.L., and Logue, C.M. (2023) Assessing the relationship between O serogroups and virulence gene carriage in Georgia Poultry with Colibacillosis. AAAP annual meeting
• Type: Conference Papers and Presentations Status: Under Review Year Published: 2023 Citation: Runcharoon, K., Young, M.M., Garcia, B., Barbier, N.L., Logue, C.M. (2023) Characterizing O serogroups and virulence genes of Avian Pathogenic E. coli (APEC) associated with colibacillosis in Georgia Poultry. Submitted to ASM Microbe 01/2023