Research Project: Intervention Strategies to Mitigate Avian Escherichia coli Infections and Antimicrobial Resistance in the Poultry Environment

Location: Poultry Research

2024 Annual Report

Objectives
1. Use proteomics, genomics, and systems biology approaches to identify molecular determinants of pathogenesis, strain variation, and tissue tropism of different E. coli strains. 2. Identify immunological targets that will confer cross-protection against prevalent E. coli strains in poultry production and develop vaccine platforms that are effective in very young birds, provide cross- protection, and can be easily administered. 2.a. Identify genetic determinants for antigenicity and pathogenicity of E. coli through comparative genomics and analyses. 2.b. Identification of immunological targets will provide a cross protection against different strains of Avian Pathogenic Escherichia coli (APEC). 2.c. Assess in ovo vaccination technology for delivery of live attenuated APEC vaccines. 3. Develop systems-level capabilities to evaluate the effects of commercial-scale, poultry management practices on animal health and production; microbial ecology, development of antimicrobial resistance and bacterial pathogen transmission to develop mitigation strategies. 3.a.1. Evaluate performance of three bio-aerosol samplers for collecting airborne E. coli attached to dust particles from poultry production environments. 3.a.2. Quantify concentration and size distribution of airborne E. coli in representative US broiler and layer houses. 3.a.3. Evaluate electrostatic particle ionization (EPI) and ultraviolet (UV) radiation to reduce airborne E. coli. 3.b.1. Evaluate effects of litter amendment application rate on E. coli populations in broiler litter. 3.b.2. Assess E. coli populations and antibiotic growth promoter (AGP) residue concentrations in biochar-treated and untreated litter (live study). 3.b.3. Evaluate effects of litter management [top-dressed (TD) vs non-top-dressed] and bedding type (pine vs switchgrass) on litter E. coli populations over successive flocks.

Approach
Proteomic, genomic, and systems biology approaches will be applied to identify molecular determinants of pathogenesis, strain variation, and tissue tropism of different E. coli strains. The E. coli strains analyzed will be isolated from varying diseased poultry flocks and strain genomic & proteomic characteristics and isolate epidemiological factors will be applied model development for greater understanding of pathogenic E. coli and associated disease. To further protect against pathogenic E. coli, immunological targets will be identified that will confer cross-protection against prevalent E. coli strains in poultry production. The genomic and plasmid sequences of various E. coli strains will be aligned, and antigenic factors will be determined. Immunological targets will be identified and assessed via challenge models that provide cross-protection against pathogenic E. coli. In addition, vaccination platforms that are effective in very young birds, provide cross-protection, and can be easily administered will be developed. In ovo technologies will be assessed for delivery of protective vaccines and associated protocols developed. To increase the understanding of environmental E. coli and evaluate risks to poultry and associated antimicrobial resistance, studies will evaluate airborne E. coli associated with dust particles and E. coli linked to poultry litter. Further, mitigation means will be assessed for their impact on environmental E. coli populations.

Progress Report
Per Objective 1, 261 E. coli genomes were completely sequenced. Ten of the genomes (E1-E10) were sequenced using both paired-end Illumina MiSeq platform and MinION Oxford Nanopore Technologies, revealing a high level of consistency between the platforms. Sequences were compared to 1,463 publically available sequences with results indicating a large extent of genomic, serological, pathogenicity, and antimicrobial resistance diversity among E. coli isolates in southern United States poultry. In line with Sub-Objective 2A, 184 clinical samples were compared with 3,381 sequences from public data revealing geographic, lineage, and pathogenicity specific genetic signatures. Additionally, proteomic analysis of 9 APEC and 9 commensal E. coli isolates identified 4 outer membrane proteins which were specific to APEC strains which may serve as viable, polyvalent vaccine candidates. Per Sub-Objective 2C, 101 E. coli isolates were characterized including presence of virulence associated genes, antibiotic resistance, and multilocus sequence typing (MLST). A high throughput MLST protocol was developed to assist with characterization and has been published in Poultry Science. An additional 29 APEC isolates were evaluated in the chicken embryo lethality model. Studies were conducted to develop intra-tracheal infection models in young layer pullets and broiler chicks for determination of pathogenicity and host immune response to APEC isolates. A PhD dissertation was published including the embryo lethality and intra-tracheal infection models. A total of 13 vaccine candidates were used to identify target antigens with pagP identified as a potential novel vaccine candidate. An electron-beam killed bacterin-based vaccine has been produced and is ready for assessment as a means of protection against APEC challenge among broiler and layer chickens. For Sub-Objective 3B2, data from a live bird study investigating the effect of biochar on litter E. coli was analyzed and found no differences in E. coli populations between treatments. Additionally, litter and chicken cecal content samples were sequenced revealing a significant effect of treatment on litter and cecal microbiome.

Accomplishments
1. High throughput multilocus sequence typing (MLST) on avian E. coli isolates. A high throughput multilocus sequence typing (MLST) protocol was successfully developed by ARS researchers at the Mississippi State University location, using Oxford Nanopore Technology was used to determine the sequence type of 308 E. coli isolates. This protocol provides an economic and rapid MLST method for large-scale E. coli research that will substantially increase the speed at which Avian Pathogenic E. coli strains can be identified.

2. Avian Pathogenic E. coli Electron Beam Killed Vaccine. A highly virulent Avian Pathogenic E. coli (APEC) strain was irradiated ARS researchers at the Mississippi State University location, utilizing Electron Beams to produce a vaccine which will be applied to control of the poultry pathogen.

3. Association of Avian Pathogenic E. coli Sequence Type and Antibiotic Resistance with Pathogenicity in Animals. Avian Pathogenic E. coli (APEC) refers to any E. coli strain isolated from internal lesions of chickens. However, it is generally unknown if these isolates caused disease in the birds or over grew in the face of other stressors. This research ARS researchers at the Mississippi State University location, provides much needed information on the ability of APEC isolates from infected birds to produce disease in naïve birds along with the association between predicted severity based on multilocus sequence typing (MLST) and disease severity in birds.

4. Research Facility upgrades. Poultry Disease related facilities were renovated and improved ARS researchers at the Mississippi State University location. Improvements included enhanced environment and updating biological isolation units, incubator/hatching facilities, chicken rearing facilities, and the feed mill.

Review Publications
Jia, L., Arick Ii, M.A., Hsu, C., Peterson, D.G., Evans, J.D., Robinson, K., Adhikari, P., Zhang, L. 2024. Complete genome sequences of two avian pathogenic Escherichia coli strains isolated from broilers exhibiting colibacillosis in Mississippi. Microbiology Resource Announcements. 13:e01020-23. https://doi.org/10.1128/mra.01020-23.
Nguyen, X.D., Zhao, Y., Evans, J.D., Lin, J., Schneider, L., Voy, B., Hawkins, S.A., Purswell, J.L. 2021. Evaluation of bioaerosol samplers for collecting airborne E. coli carried by dust particles from poultry litter. ASABE Annual International Meeting. https://doi.org/10.13031/aim.202100065.
Nguyen, X.D., Zhao, Y., Lin, J., Purswell, J.L., Tabler, T., Voy, B., Hawkins, S., Evans, J.D. 2023. Modeling long distance airborne transmission of highly pathogenic avian influenza carried by dust particles. Scientific Reports. 13:16255. https://doi.org/10.1038/s41598-023-42897-2.
Ovi, F., Zhang, L., Nabors, H., Jia, L., Adhikari, P. 2023. A compilation of virulence-associated genes that are frequently reported in avian pathogenic Escherichia coli (APEC) compared to other E. coli. Journal of Applied Microbiology. 134(3). Article lxad014. https://doi.org/10.1093/jambio/lxad014.
Joseph, J., Jennings, M., Barbieri, N., Zhang, L., Adhikari, P., Ramachandran, R. 2023. Characterization of avian pathogenic Escherichia coli isolated from broiler breeders with colibacillosis in Mississippi. Poultry. 2(1):24-39. https://doi.org/10.3390/poultry2010004.
Fatemi, S.A., Lindsey, L.L., Evans, J.D., Collins Elliott, K.E., Leigh, S.A., Robinson, K., Mousstaaid, A., Gerard, P.D., Peebles, E.D. 2023. Effects of the in ovo injection of an Escherichia coli vaccine on the hatchability and quality characteristics of commercial layer hatchlings. Poultry Science. 102(11):103057. https://doi.org/10.1016/j.psj.2023.103057.
Nguyen, X.D., Zhao, Y., Evans, J.D., Lin, J., Schneider, L., Voy, B., Hawkins, S.A., Purswell, J.L. 2022. Evaluation of bioaerosol samplers for airborne Escherichia coli carried by poultry litter particles. Journal of the ASABE. 65(4):825-833. https://doi.org/10.13031/ja.15057.
Feng, A., Akter, S., Leigh, S.A., Wang, H., Pharr, G.T., Evans, J.D., Branton, S.L., Landinez, M.P., Pace, L., Wan, X. 2023. Genomic diversity, pathogenicity and antimicrobial resistance of Escherichia coli isolated from poultry in the southern United States. BMC Microbiology. 23:15. https://doi.org/10.1186/s12866-022-02721-9.
Fatemi, S.A., Mousstaaid, A., Williams, C.J., Deines, J., Poudel, S., Poudel, I., Collins Elliott, K.E., Walters, E.R., Forcier, N., Peebles, E.D. 2023. In ovo administration of the Marek's disease vaccine in conjunction with 25-hydroxyvitamin D3 and its subsequent effects on the performance and immunity-related characteristics of Ross 708 broiler hatchlings. Poultry Science. 103(1). Article 103199. https://doi.org/10.1016/j.psj.2023.103199.