Source: UNIV OF MINNESOTA submitted to NRP
DEVELOPING PREDICTIVE TOOLS TO RESPOND TO THE CHANGING ECOLOGICAL LANDSCAPE OF E. COLI IN POULTRY PRODUCTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
1022835
Grant No.
2020-67015-31678
Cumulative Award Amt.
$500,000.00
Proposal No.
2019-05908
Multistate No.
(N/A)
Project Start Date
Jul 1, 2020
Project End Date
Jun 30, 2024
Grant Year
2020
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
COLLEGE OF VETERINARY MEDICINE
Non Technical Summary
E. coli is a bacteria that causes devastating disease problems for chickens and turkeys being raised for meat consumption. Even though E. coli is present in every chicken or turkey, we do not fully understand why some birds get sick and others do not. Vaccines are often used to control E. coli disease in birds, but these do not always work as expected. The goal of this project is to better understand the ecology of E. coli populations that circulate within a poultry farm, and how vaccination shifts E. coli populations in different anatomical locations within the bird, and in thebarn environment. To do this, we will partner with turkey producers in Minnesota to compare flocks that have been vaccinated for E. coli versus those that have not been vaccinated, studying the populations of E. coli using their genomes. We will also use these flocks to understand how E. coli might be spread from the parents (breeders) to their progeny that get raised for meat consumption. We will use this information to predict when vaccines will work, and when they will not be effective on the farm. This work will greatly advance our understanding of the dynamics of E. coli within the chicken or turkey, and will enable poultry producers to predict the best strategies by which to control E. coli disease on their farms.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31132301100100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3230 - Turkey, live animal;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Escherichia coli is a bacterial pathogen that has been studied intensively in poultry production. Despite decades of research, diseases caused by E. coli continue to be a top concern for poultry producers. Numerous methods of vaccination have been explored and commercialized, yet comprehensive control of this pathogen remains elusive. While substantial work has focused on defining avian E. coli isolated from clinical disease, there are major gaps in understanding of how these clinical isolates relate to the overall ecology of E. coli in poultry production systems. A baseline understanding of the dynamics of the total E. coli populations within poultry production systems, and their response to mitigation strategies, is necessary to better predict the best approaches for E. coli control. The goal of this work is to define E. coli ecology in poultry production, and utilize data to inform control with an emphasis on predictive approaches for understanding when, where, and how to implement mitigation approaches. Using field-based and controlled studies, the ecological landscape of E. coli in poultry production relative to disease will be defined. Shifts in E. coli ecology and disease will be studied in the context of vaccination. From these analyses, predictive models will be developed to quantify expected changes in E. coli populations in response to mitigation, and these models will be validated using real data. Overall, this study takes innovative approaches towards assessing and responding to changes in pathogen ecology that can serve as a template for applied solutions in animal agriculture. This will be accomplished with the following Objectives:Objective 1. Define the ecological landscape of E. coli in poultry production relative to disease. While disease-associated avian E. coli from poultry have been thoroughly studied, there are major knowledge gaps surrounding the overall ecology of E. coli in poultry production systems. We will leverage ongoing longitudinal studies to comprehensively examine E. coli populations in the gastrointestinal and respiratory tracts in commercial turkey production. These populations will be compared to breeder source populations, and disease-associated E. coli at the breeder and commercial flock levels, to determine the relationships between colonizing E. coli in the bird, and those ultimately causing disease. This objective will also determine transmission dynamics from parent to progeny, relationships between E. coli load and disease, and E. coli clonal diversity in the context of APEC carriage and the existing microbiota.Objective 2. Determine shifts in E. coli ecology and disease relative to vaccination. Commercial and autogenous vaccines are commonly used in poultry production to prevent or reduce E. coli-associated disease. However, we know very little about the impact of such vaccination on colonizing E. coli populations, and whether vaccination specifically targets disease prevention or more broadly targets APEC colonization throughout the bird. Here, we will utilize a large field study to examine the implementation of a commercial E. coli vaccine into an integrated turkey production system that is naïve to such vaccination. Populations of E. coli will be examined across ten different farms within a vertically integrated turkey company, with control and vaccinated barns within each farm followed for multiple flock cycles. We will utilize a top-down approach to assess shifts in E. coli populations within several niches (barn environment, gastrointestinal tract, respiratory tract, and clinical isolates from disease). Mortality, performance, and immune response will also be measured. From these analyses, predictive models will be developed to quantify expected changes in E. coli populations in response to vaccine implementation. Follow-up controlled animal experiments will be performed to validate the model and to further predict when vaccines will lose efficacy in the field.
Project Methods
Objective 1. Define the ecological landscape of E. coli in poultry production relative to disease.Overview. While we have preliminary data highlighting the diversity of APEC among clinical isolates from diseased turkeys across the U.S., very little is known about the underlying ecology of E. coli driving resulting disease. The purpose of this Objective is to define the baseline ecological landscape of E. coli in commercial turkey production. Using a previously conducted trial's sample bank, we will assess the following questions:What is the relationship between E. coli colonizing the turkey GI and respiratory tract, and the barn environment, as it relates clinical disease outcome?Does environmental E. coli load correlate with morbidity and mortality?Do breeder flocks contribute to the dissemination of APEC to commercial poults?Are there correlations between APEC/clone carriage and bacterial community?To address these questions, we will utilize a sample bank from a trial recently performed in collaboration with a turkey hatchery and turkey co-operative in Minnesota. This study took place from May 2017 through November 2018. A single breeder flock was placed with 30,000 hens, and these hens were sampled weekly from 0-9 weeks of age, and every other week from 11-55 weeks of age. Sampling involved collection of cloacal swabs from 5 random birds within the flock per timepoint. Poults from this flock were then single-source placed into two different commercial farms (Commercial #1 and Commercial #2; n=21,000 per flock). These flocks were placed in two consecutive cycles with single-source birds from the breeder flock. Birds from the commercial flocks were sampled weekly from 0-6 weeks of age, then every other week from 8-12 weeks of age. Sampling in the commercial flocks involved collection of matched cloacal and tracheal swabs from 5 random birds per flock and timepoint.In Objective 1, a top-down approach will be used to study E. coli ecology from the collected samples. This will include PCR-based screens for phylogenetic type, APEC virulence plasmid possession, and sequence type. Whole genome sequencing will then be performed on a subset of these samples to determine specific linkages between breeder and commercial bird isolates. E. coli load will be examined using a quantitative PCR to determine if correlations exist between load and disease. Microbiome profiling will be performed to determine if there are correlations between the gut microbiota and carriage of APEC or specific sequence types.Objective 2. Determine shifts in E. coli ecology and disease relative to vaccination.Overview. While we have preliminary data suggesting that APEC populations shift in response to mass vaccination, and similar data exists in human bacterial pathogens, no work has been done to confirm or quantify this effect. Moreover, it is unknown if vaccination against E. coli in poultry impacts only clinical disease, or if it shifts the entire ecology of E. coli in the bird and its environment. The purpose of this Objective is to address these questions using a previously conducted trial's sample bank. The following specific questions will be addressed:Do clinical APEC populations shift in response to mass commercial vaccination?Do respiratory colonizing E. coli populations shift > GI populations?Are shifts primarily strain-level shifts or antigenic shifts due to recombination and mutation?Can these shifts be predicted with modeling and validated with controlled experiments?To address these questions, we will utilize a sample bank from a trial currently being performed in collaboration with an integrated turkey company in Minnesota. This study is taking place from March 2019 through March 2020. Fifteen commercial farms are being used, each containing control barns receiving no vaccine and vaccinated barns receiving the anE. coli vaccine as per manufacturer's recommendations. Samples taken at 0, 3, 6, 9, and 12 weeks of age will be used. Matching tracheal swabs and cloacal swabs are being collected from 10 random birds per barn and per timepoint, and grab litter samples are taken at the same time. Importantly, this company has no history of using any form of E. coli vaccination, so the population should be naïve to E. coli vaccine pressures. Flocks are being placed in two consecutive cycles for each barn, with the same treatments for both flock cycles. Similar approaches will be used to assess these E. coli populations.A system dynamics simulation model of the turkey production system from placement of poults to slaughter will then be developed. This model will include APEC dynamics, using the data from our studies and from the literature. The model will allow the option of vaccinating against APEC, wherein the user will be able to select the APEC strains that should be included in the model, and with that information, imperfect efficacy against these strains will allow population shifts to be predicted. The model will ultimately allow long-term predictions of the impact of specific mitigation strategies, including vaccination, and a sensitivity analysis to identify the parameters with most leverage on the system.Finally, observedchanges in response to vaccination in the field will be further tested in controlled fashion. Using a cocktail of diverse APEC strains, we will assess the effects of commercial vaccination on the success of strains within this cocktail in different niches in the turkey.

Progress 07/01/20 to 06/30/24

Outputs
Target Audience:This is a collaborative project with commercial turkey growers in Minnesota. We have met several times with this industry group to discuss the results. This work has also been presented at the Poultry Science Asociation annual meeting, the North Central Avian Diseases meeting in 2022, and the American Association of Avian Pathologists meeting in 2024. We have also worked independently with four different major poultry producers to educate them about the findings of this work and develop collaborative projects using the tools developed from this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided opportunity for poultry professionals to become familiar with genomic data. In fact, a platform is in development that will enable poultry producers to easily use Salmonella and E. coli genomes for proactive surveillance purposes. This new APEC typing scheme developed in this project has been presented to the poultry industry, and is actively being used internally by two major poultry producers and one major allied industry company. The Mid-Central Research and Outreach Center at the University of Minnesota also collaborates with several companies to provide the new APEC typing scheme for surveillance purposes. How have the results been disseminated to communities of interest?Results have been disseminated widely to the poultry industry and scientific community through conferences and individual meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have collected over 12,000 samples (cloacal swabs, tracheal swabs, and litter samples) from 20 flocks sampled for two successive flocks. These flocks were a part of a field trial with matched barns either not vaccinated (Control) or vaccinated with a commercial E. coli vaccine. From these samples, approximately 2,000 samples from a single farm (brood and growout complexes) have been characterized. Five E. coli colonies were isolated from each sample and subjected to Clermont E. coli phylotyping and APEC plasmid genotyping. Results indicate significant shifts in gut and respiratory E. coli populations in response to commercial vaccination, with shifts occurring at the phylotype level away from the phylotype of the vaccine strain. Vaccination did appear to have a significant impact on the overall proportion of APEC isolates recovered from control versus vaccinated groups, specifically those belonging to the B1 and C phylogenetic groups. A second study was also conducted to examine the landscape of clinical isolates causing colibacillosis in commercial turkeys, compared to cecal E. coli isolates from healthy birds. These isolates were characterized with whole genome sequencing. In contrast to the results above, we identifed high proportions of isolates posessing high virulence potential in clinical isolates (85-90%) and in cecal isolates (55- 65%). From this work, a revised APEC typing scheme targeting high-risk virulent APEC clones has been developed, published, and validated in two different studies. This work overall extends our knowledge of the APEC landscape as it relates to clinical disease, and the overall ecology of E. coli in poultry production.

Publications


    Progress 07/01/22 to 06/30/23

    Outputs
    Target Audience:This is a collaborative project with commercial turkey growers in Minnesota. We have met two times with this industry group to discuss the results and next steps. This work has also been presented at the Poultry Science Asociation annual meeting and the North Central Avian Diseases meeting in 2022. We have also worked independently with four different major poultry producers to educate them about the findings of this work and develop collaborative projects using the tools developed from this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided opportunity for poultry professionals to become familiar with genomic data. In fact, a platform is in development that will enable poultry producers to easily use Salmonella and E. coli genomes for proactive surveillance purposes. This new APEC typing scheme developed in this project has been presented to the poultry industry, and is actively being used internally by two major poultry producers and one major allied industry company. The Mid-Central Research and Outreach Center at the University of Minnesota also collaborates with several companies to provide the new APEC typing scheme for surveillance purposes. How have the results been disseminated to communities of interest?Results have been disseminated widely to the poultry industry and scientific community through conferences and individual meetings. What do you plan to do during the next reporting period to accomplish the goals?During the next project period, remaining isolates will be characterized for APEC-associated traits and selected isolates will be sequenced and analyzed.

    Impacts
    What was accomplished under these goals? We have collected over 12,000 samples (cloacal swabs, tracheal swabs, and litter samples) from 20 flocks sampled for two successive flocks. These flocks were a part of a field trial with matched barns either not vaccinated (Control) or vaccinated with a commercial E. coli vaccine. From these samples, approximately 2,000 samples from a single farm (brood and growout complexes) have been characterized. Five E. coli colonies were isolated from each sample and subjected to Clermont E. coli phylotyping and APEC plasmid genotyping. Results indicate significant shifts in gut and respiratory E. coli populations in response to commercial vaccination, with shifts occurring at the phylotype level away from the phylotype of the vaccine strain. Vaccination did not appear to have a significant impact on the overall proportion of APEC isolates recovered from control versus vaccinated groups. A second study was also conducted to examine the landscape of clinical isolates causing colibacillosis in commercial turkeys, compared to cecal E. coli isolates from healthy birds. These isolates were characterized with whole genome sequencing. In contrast to the results above, we identifed high proportions of isolates posessing high virulence potential in clinical isolates (85-90%) and in cecal isolates (55- 65%). From this work, a revised APEC typing scheme targeting high-risk virulent APEC clones has been developed, published, and validated in two different studies. To further understand the microecology of E. coli in the individual bird and flock, two additional experiments were conducted. In one study, a pen trial was used to collect the following samples from ten individual birds: ileum, jejunum, cecum, and trachea. Ten colonies per tissue/bird were isolated, totaling 400 colonies from the ten birds. These isolates are being characterized with PFGE and whole genome sequencing to determine if there are tissue-specific tropisms for specific APEC clones towards particular anatomical sites. A second study was also performed using these same anatomical sites across 25 birds from random flocks across the United States, totaling 1,000 colonies.

    Publications


      Progress 07/01/21 to 06/30/22

      Outputs
      Target Audience:This is a collaborative project with commercial turkey growers in Minnesota. We have met two times with this industry group to discuss the results and next steps. This work has also been presented at the Poultry Science Asociation annual meeting and the North Central Avian Diseases meeting in 2022. We have also worked independently with four different major poultry producers to educate them about the findings of this work and develop collaborative projects using the tools developed from this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided opportunity for poultry professionals to become familiar with genomic data. In fact, a platform is in development that will enable poultry producers to easily use Salmonella and E. coli genomes for proactive surveillance purposes. This new APEC typing scheme developed in this project has been presented to the poultry industry, and is actively being used internally by two major poultry producers and one major allied industry company. The Mid-Central Research and Outreach Center at the University of Minnesota also collaborates with several companies to provide the new APEC typing scheme for surveillance purposes. How have the results been disseminated to communities of interest?Results have been disseminated widely to the poultry industry and scientific community through conferences and individual meetings. What do you plan to do during the next reporting period to accomplish the goals?During the next project period, remaining isolates will be characterized for APEC-associated traits and selected isolates will be sequenced and analyzed.

      Impacts
      What was accomplished under these goals? We have collected over 12,000 samples (cloacal swabs, tracheal swabs, and litter samples) from 20 flocks sampled for two successive flocks. These flocks were a part of a field trial with matched barns either not vaccinated (Control) or vaccinated with a commercial E. coli vaccine. From these samples, approximately 2,000samples from a single farm (brood and growout complexes) have beencharacterized. Five E. coli colonies were isolated from each sample and subjected to Clermont E. coli phylotyping and APEC plasmid genotyping.Results indicate significant shifts in gut and respiratory E. coli populations in response to commercial vaccination, with shifts occurring at the phylotype level away from the phylotype of the vaccine strain. Vaccination did not appear to have a significant impact on the overall proportion of APEC isolates recovered from control versus vaccinated groups. A second study was also conducted to examine the landscape of clinical isolates causing colibacillosis in commercial turkeys, compared to cecal E. coli isolates from healthy birds. These isolates were characterized with whole genome sequencing. In contrast to the results above, we identifed high proportions of isolates posessing high virulence potential in clinical isolates (85-90%) and in cecal isolates (55- 65%). From this work, a revised APEC typing scheme targeting high-risk virulent APEC clones has been developed, published, and validated in two different studies. To further understand the microecology of E. coli in the individual bird and flock, two additional experiments were conducted. In one study, a pen trial was used to collect the following samples from ten individual birds: ileum, jejunum, cecum, and trachea. Ten colonies per tissue/bird were isolated, totaling 400 colonies from the ten birds. These isolates are being characetrized with PFGE and whole genome sequencing to determine if there are tissue-specific tropisms for specific APEC clones towards particular anatomical sites. A second studywas also performed using these same anatomical sites across 25birds from random flocks across the United States, totaling 1,000 colonies.

      Publications

      • Type: Journal Articles Status: Published Year Published: 2022 Citation: Johnson TJ, Miller EA, Flores-Figeuroa C, Munoz-Aguayo J, Cardona C, Fransen K, Lighty M, Gonder E, Nezworski J, Haag A, Behl M, Kromm M, Wileman B, Studniski M, Singer RS. Refining the definition of the avian pathogenic Escherichia coli (APEC) pathotype through inclusion of high-risk clonal groups. Poultry Science 101:102009.
      • Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Delago J, Miller EA, Flores-Figueroa C, Munoz Aguayo J, Cardona C, Smith AH, Johnson TJ. Survey of clinical and commensal Escherichia coli from commercial broilers and turkeys, with emphasis on high-risk clones using APECTyper. Poultry Science, in press.
      • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Ecology of avian Escherichia coli in health and disease using a refined typing scheme. Poultry Science Association Annual Meeting, San Antonio, TX, July 2022.


      Progress 07/01/20 to 06/30/21

      Outputs
      Target Audience:This is a collaborative project with commercial turkey growers in Minnesota. We met two times in 2021 to discuss the ongoing results of this work. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided opportunity for poultry professionals to become familiar with genomic data. In fact, a platform is in development that will enable poultry producers to easily use Salmonella and E. coli genomes for proactive surveillance purposes. How have the results been disseminated to communities of interest?Results have been disseminated to turkey producers in two meetings in 2021. Results will be presented to the scientific community at the Western Poultry Disease Conference in Vancouver, BC in April 2022. Manuscripts are in preparation. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will further characterize the collection of E. coli from the field trial to determine precise shifts in E. coli ecology in response to vaccination across multiple flocks and farms. Whole genome sequencing will be used to provide better resolution on subsets of isolates. A revised APEC typing panel has been developed that will also be used to screen the broader collection of isolates. Controlled animal experiments are planned to determine the nature of E. coli ecology across the different gut locations, within the respiratory tract, and in resulting fecal droppings. This will build a "roadmap" for E. coli ecology in the bird that is greatly needed.

      Impacts
      What was accomplished under these goals? We have collected over 12,000 samples (cloacal swabs, tracheal swabs, and litter samples) from 20 flocks sampled for two successive flocks. These flocks were a part of a field trial with matched barns either not vaccinated (Control) or vaccinated with a commercial E. coli vaccine. From these samples, approximately 500 samples from a single farm (brood and growout complexes) were further characterized. Five E. coli colonies were isolated from each sample and subjected to Clermont E. coli phylotyping and APEC plasmid genotyping. The former technique characterizes isolates for chromosmal lineage of E. coli, whereas the latter scheme identifies isolates with high virulence potential for causing disease in poultry. We have determined thus far that vaccinated flocks exhibit a shift in E. coli ecology, away from the phylogenetic group C, to which the attenuated commercial vaccine strain belongs. Overall, the proportions of isolates possessing high virulence potential in the cloaca was relative low (5-15%), while slightly higher in tracheal swabs (10-25%). A second study was also conducted to examine the landscape of clinical isolates causing colibacillosis in commercial turkeys (n=396), compared to cecal E. coli isolates from healthy birds (n=600). These isolates were characterized with whole genome sequencing. In contrast to the results above, we identifed high proportions of isolates posessing high virulence potential in clinical isolates (85-90%) and in cecal isolates (55-65%). The reason for the discrepancy between APEC carriage in the cloaca versus ceca is currently unclear. However, the phylogenetic backgrounds of clinical isolates were drastically different than those from the ceca. We then compared strains representing dominant clinical sequence types versus thoserepresenting dominant cecal sequence types in an embryo lethality model. The results showed that clinical STs were significantly more lethal than cecal STs, even though both subsets of strains possessed the APEC plasmid and all of the genes currently used for APEC typing. Thus, the current APEC typing scheme may not effectively discriminate between high and low virulence potential in poultry, and should be revised.

      Publications