Source: OHIO STATE UNIVERSITY submitted to NRP
A NOVEL, TRANSLATIONAL, MULTIDISCIPLINARY APPROACH TO CONTROL POULTRY RESPIRATORY DISEASES IN THE UNITED STATES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1005721
Grant No.
2015-68004-23131
Cumulative Award Amt.
$7,230,000.00
Proposal No.
2014-08054
Multistate No.
(N/A)
Project Start Date
Apr 1, 2015
Project End Date
Mar 31, 2023
Grant Year
2019
Program Code
[A5152]- Global Food Security: Minimizing Losses from Pests and Diseases of Livestock
Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691
Performing Department
OARDC Food Animal Health
Non Technical Summary
Respiratory diseases continue to be a major concern to poultry producers because losses induced by respiratory diseases have significant local and national economic impact to the industry. Protection of poultry by effective prevention and control of diseases is critical to maintain wholesome poultry products, which is the number one animal protein consumed in the U.S. Such efforts make a significant contribution towards national food security. Our goal is to develop knowledge-based integrated approaches to detect, control and prevent endemic, emerging and re-emerging poultry respiratory diseases in the U.S. In this project, the efforts of multiple institutions across the country will concentrate on the following four specific objectives: 1) Understand the ecology of poultry respiratory diseases; 2) Investigate the multifactorial etiology involving poultry respiratory diseases; 3) Develop new and improved diagnostic tools, vaccines, and novel preventive measures; and4) Educate stakeholders for prevention and control of respiratory diseases.
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
3113299107040%
3113299110030%
3113299110130%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1101 - Virology; 1070 - Ecology; 1100 - Bacteriology;
Goals / Objectives
Our goal is to develop knowledge-based integrated approaches to detect, control and prevent endemic, emerging and re-emerging poultry respiratory diseases in the US. In this project, the efforts of multiple institutions across the country will concentrate on the following four specific objectives:1) Understand the ecology of poultry respiratory diseases. The surveillance effort will be built upon the foundation laid by the existing multistate research program (NC-1180). Participants will gather disease surveillance information from each state and share the data among participants and institutions. A standardized and easily accessible data reporting and dissemination system will be developed. Metagenomic-based approaches will be incorporated to better understand the multi-etiological nature of respiratory diseases and also to assess the contributions of the commensal respiratory microbiota towards the establishment of disease.2) Investigate the multifactorial etiology involving poultry respiratory diseases. Research on pathogenesis of respiratory diseases involves exploration of the intricate and complex interactions among pathogen, host, and environment. Collaborative efforts will be taken emphasizing contemporary approaches to understand multifactorial interactions of infections impacting respiratory disease of poultry. Participants will collaborate to determine co-infecting viral and bacterial agents involved in respiratory disease, and host status predisposing to disease.3) Develop new and improved diagnostic tools, vaccines, and novel preventive measures. We will focus on validation of diagnostic tests, and development and commercialization of new vaccines and antibiotic-free antimicrobial approaches. Effort will be made for standardization of molecular diagnostic tests among laboratories. Novel vaccines for viral pathogens and small molecule-based alternative control strategies for bacterial pathogens will be developed.4) Educate stakeholders for prevention and control of respiratory diseases. Our efforts will focus on translating research findings into practices that will reach both industry and government stakeholders under real world conditions. We will work closely with producers to adopt and implement effective intervention strategies and evaluate their effect on overall respiratory disease prevalence. Extension activities will be designed to work with individuals and groups, including educational programs developed to help producers better understand new management practices as well as preventative and emergency procedures so that appropriate measures can be taken to reduce disease risk and transmission of disease agents. Relevant government stakeholders will be engaged in establishing standardized test protocols, and in validating and applying newly developed diagnostic tests and vaccines.
Project Methods
Objective 1. Understand the ecology of poultry respiratory diseases. Activity 1.1: Expand the nationwide surveillance and research network and develop a standardized and easily accessible data reporting and dissemination system. This project will connect and coordinate poultry disease data archiving and collection in the US in order to develop a comprehensive data base with standardized reporting system and easy access for poultry professionals. This collaborative data depository will be developed to facilitate our understanding of poultry diseases in the US and spur more effective epidemiological studies and enhance the ability to respond to emerging, re-emerging and endemic diseases outbreaks. Activity 1.2: Targeted surveillance in conjunction with microbiome and metagenomic analysis. We will establish a baseline respiratory microbiome in birds by following multiple commercial flocks over extended time points. We will identify at least six commercial flocks to sample during brood and grow-out periods depending on the species (broiler, layer, and turkey). Objective 2. Investigate the multifactorial etiology involving poultry respiratory diseases. Activity 2.1: Study the interactions of relevant respiratory and immunosuppressive agents based on species (broiler, layer, and turkeys), their clinical outcomes, transmission patterns, and potential changes in vaccination and diagnostic strategies. Based on field observation and literature, different combinations of respiratory pathogens were selected for each species to reproduce the clinical outcome. Some combinations of respiratory pathogens may cause mild or subclinical disease that would be economically inconsequential. Combining these infectious agents with environmental stress or IBDV induced immune suppression may dramatically alter the clinical outcome of the respiratory disease. 1) Co-infection of broiler chickens with LPAIV, IBV and Mycoplasma to reproduce bronchial casts and mortality reported with LPAIV infections in the field; 2) 4) Effect of IBDV on the clinical outcomes and transmission patterns of the respiratory pathogens in broiler and layer chickens Activity 2.2: Study potential interactions between the vaccines used against respiratory disease. The will test combination of vaccines can affect the development and longevity of immunity when multiple live attenuated vaccines and recombinant vaccines are given either simultaneously or sequentially to pullets, and co-infections with LPAIV, IBV variants, NDV, ILTV or mycoplasma at the time of vaccination can compromise protection.Activity 2.3: Evaluate the effects of air quality on the onset (infection), transmission and severity of respiratory disease caused by IBV as a model. We will test that reduced air quality in poultry houses can damage cilia in the respiratory tract and predispose chickens to infection by both primary and secondary pathogens. In addition, the physiological effects of reduced air quality will influence vaccine efficacy and development of immunity, as well as exacerbate the incidence, transmission, and outcome of respiratory disease. Objective 3. Develop new and improved diagnostic tools, vaccines, and novel preventive measures. Activity 3.1: Unification of molecular detection methods for poultry respiratory pathogens.Wet bench analysis comparing primers and probes with de novo synthesized samples will provide a discriminating factor if more than 3 tests are being considered. For the top two tests, diagnostic laboratories in the network will run on clinical samples the test that they normally run and the "new" tests to generate data from field samples.A different pathogen will be evaluated each year. In the fifth year, proficiency panels will be sent to participating diagnostic laboratories to provide assurance that the tests are being performed on a satisfactory basis. Activity 3.2: Development of broadly reactive vaccine against respiratory and immunosuppressive viral pathogens. Generation of a broadly reactive vaccine that can confer protection across serotypes or variants has been a long sought goal for viruses that continuously evolve. In this study, three new approaches will be utilized to develop broad-spectrum vaccines for IB, ND, and IBD. All three approaches target IB which makes it easier for comparative evaluation using similar challenge protocol. Considering the flexibility of each system, once validated with IB and/or ND/IBD, the platform can quickly be utilized to develop vaccines against different respiratory pathogens of interest. 1) Development of novel nanoparticle-based vaccines for IB; 2) Optimization of recombinant NDV expressing IBV S2 to confer protection across IBV serotypes; 3) Use of HVT vector vaccines to protect against important respiratory and immunosuppressive diseases of poultry. Activity 3.3: Novel non-antibiotic compounds for the control of colibacillosis and Mycoplasma infection in poultry. The use of small molecules is a highly practical approach because 1) they can be selected to inhibit a specific enzymatic reaction that does not exert selective pressure on the targeted bacterium, which is therefore less likely to develop resistance to these compounds; 2) use of conventional antibiotics in chickens is complicated by the emergence of resistant bacteria; 3) small size of small molecules allows them to diffuse readily into target cells, making them highly suitable for mass application, a prerequisite in a commercial poultry setting; and, 4) these molecules can be modified to enhance qualities desired for specific applications, such as solubility and stability under adverse environmental conditions. Small molecules will be customized to target a APEC and Mycoplama without exerting toxic side effects on both the host and commensal bacteria. Objective 4. Develop extension and outreach program to facilitate collaboration among researchers and stakeholders and poultry industries to adopt effective prevention and control strategies A major obstacle in the control of infectious diseases in poultry is non-science based management practices and lack of proper understanding of the real issues and practical control strategies among the stakeholders including poultry industries and small backyard flock owners. Science-based information on control and prevention of poultry respiratory diseases needs to be disseminated effectively and in a timely manner to our stakeholders. Our efforts will focus on translating research findings into practices that will reach both industry and government stakeholders under real world conditions. We will work closely with producers to adopt and implement effective intervention strategies, train for management of respiratory diseases and evaluate their effect on overall respiratory disease prevalence.

Progress 04/01/15 to 03/31/23

Outputs
Target Audience:Poultry industry including backyard poultry farmers; Government and state officials involved in control of animal diseases. Scientists in the field of infectious diseases, animal production, Ag-engineering, virology, bacteriology, immunology & vaccinology. Extension and outreach specialists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project support training of more than several undergraduate and graduate students, post-docs, research assistants, research associates and scientists (listed below). In addition to hand-on experiences, students and post-docs presented the results at the local and national meetings and published the results in scientific journals. How have the results been disseminated to communities of interest?We developed the main project website (https://prdcap.com/) to share our projects with professionals, stakeholders and the general public. The results from individual projects were presented at the international and regional meetings. Several manuscripts are being prepared for publication in timely manner. Furthermore, our Extension group actively conduct comprehensive educational training program on the importance of controlling respiratory diseases for Veterinarians, Extension Educators, Gamebird Producers, Small Organic and Pastured Poultry Operations and Backyard, Hobby, and Exhibition Growers and the general public through distribution of brochures, presentation at a poster, presentation at local, regional, and national meetings, workshops and specific training programs. A working online platform for poultry mapping has been developed and could be ready for use in a timely manner; this could be a useful tool in case the need arises. We have identified that poultry production companies that do perceive value in sharing this type of information; and currently, collaborations are on-going to use retrospectively collected routine data to produce "case studies" that will serve as examples for the industry on how mapping platforms/ spatial information/ publicly available data can be used to inform disease risk. Results were presented in multiple professional and poultry producers meetings. The created maps along with the contact information of all trained veterinarians were shared with relevant parties including, but not limited to, the Ohio Poultry Association, the Ohio Breeders Association, the Ohio Veterinary Medical Association, the Iowa Poultry Association, the Iowa Veterinary Medical Association and others to promote the use of services of participant veterinarians by noncommercial poultry owners. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. Ecology and Networking This project supported several new SEED projects to stimulate new and innovative research and expand collaboration. We established project website (https://prdcap.com/) to share our projects with professionals and stakeholders; 3) Toward creating a centralized poultry disease work group and developing a database, we conducted a nationwide survey to assess the perception of poultry professionals toward disease mapping and monitoring programs and to identify groups of poultry professionals with similar perceptions and attitudes toward these projects. For the successful development of poultry disease mapping and monitoring programs, various groups of poultry professions were engaged in different manners to achieve maximum participation in the voluntary program that could be used in case of disease outbreaks. Understanding issues around participation on poultry disease mapping/ disease control projects: Major challenges identified amongst survey respondents included 'increased potential for misinformation; and 'accidental release of premise locations'. Within the national survey, we were able to identify 2 diverse clusters (groups) of poultry professionals that had differing attitudes and opinions regarding poultry disease: The first group was characterized by perceiving very high levels of benefits but also high levels of risks, nervousness about data being accidentally released and getting in the hands of animals' rights group and/or being open to misinterpretation by third party groups. The second group perceived lesser benefits and were characterized by mostly being production and service technicians. Mapping systems and risk analysis for poultry disease control using relevant examples (several important respiratory poultry diseases): We were able to exemplify the uses of mapping systems and risk analysis for disease control. The goal of these pieces was to present and publish this information and use it to demonstrate the uses of this type of project in the long-term. Objective 2. Multifactorial Respiratory Microbiome of poultry. We have defined the microbiome in the respiratory tracts of commercial broilers, layers, and turkeys in relation to health status and performance to facilitate disease prediction and prevention and identification of alternatives to antibiotics. Furthermore, we have constructed BiomeSeq, a bioinformatics pipeline was instrumental for analysis of the viromes of different poultry species. To study the respiratory virome of backyard poultry.A protocol for sample collection by visiting backyard flocks, transport, storage, processing for library prep and bioinformatics analysis has been optimized. Ribodepletion before library preparation was helpful in increasing viral reads. Gallid herpesvirus-1 was detected in all respiratory cases and picornaviruses like chicken picornavirus and plant type picornaviruses were detected in apparently healthy chickens along with retroviruses and bacteriophages. Evaluate the effects of air quality on the onset (infection), transmission and severity of respiratory disease caused by IBV as a model.The results indicate that commercial broilers exposed to moderate levels of ammonia are not more susceptible to IBV challenge if they are appropriately vaccinated, but nonvaccinated-challenged birds experience more severe clinical signs and a higher incidence of airsacculitis. Combinations of live attenuated NDV, IBV, ILTV vaccines in pullets and the effect of these vaccines in pullets co-infected with other respiratory pathogens. A typical pullet vaccination program consisting of serially administered live attenuated vaccines against multiple respiratory pathogens can result in the development of protective immunity against each disease agent. Objective 3. Diagnostic tools and preventive measures Core genome MLST assay for ORT. Developed Core Genome Multilocus Sequence Typing (cgMLST) assay for ORT. This study provides a standardized, portable sequence typing assay that allows ORT sequence typing results to be shared at a national and international scale and improves our understanding of ORT population and epidemiology, which in turn can improve prevention, control and eradication efforts. Improved real time PCR and real time RT-PCR tests for common poultry respiratory diseases. Six TaqMan™-based quantitative real-time RT-PCR assays for Infectious bronchitis ( Universal, Ark, Mass, DE/GA98, GA07, GA08) were developed. Further validation of specificity using clinical and biological specimens was also successful. We also created a panel of quantitative real-time PCR (qPCR) assays for other common Respiratory viral diseases such as NDV, ILTV and AMPV. Further validation of specificity using clinical and biological specimens was also performed. Novel antimicrobials for bacterial pathogens of poultry. We have identified several novel non-antibiotic compounds (small molecules and antimicrobial peptides) that inhibit avian pathogenic E. coli (APEC) and M. gallisepticum for administration in drinking water of chickens. Development of in ovo-compatible NS1-truncated live attenuated influenza vaccines. This study demonstrates that targeted genetic mutations can be applied to further attenuate and enhance the safety of live attenuated influenza vaccines to develop future in ovo vaccines for poultry. Development of Novel Peptide Nanoparticle-based Vaccine for infectious bronchitis virus. We have successfully developed the IBV peptide SANP FluoDot vaccine and tested for the immunogenicity and challenge studies in SPF chickens. This vaccine will be cost-effective and easier to transport without refrigeration. Vectored vaccine technology for poultry diseases. Results demonstrated that a adding a cytokine to the construct enhances protection of the recombinant vaccine. The industry would benefit significantly if vaccination with live Ark-type viruses and possibly other IBV serotypes could be replaced by priming or boosting with tailored rLS. Objective 4. Extension and Outreach -A series of three videos in relation to ammonia measurement and ammonia control were created and published: 1)Video: "Paper Test Strips"; 3 min 18 sec. 2)Video: "Pull Tubes and Diffusion Tubes"; 5 min 29 sec. 3)Video: "Instrument Measurement Comparisons"; 4 min 57 sec. The videos can be found athttps://extension.psu.edu/poultry-houses-and-ammonia -A series of factsheets have been published Technical bulletin: Detecting ammonia in poultry housing using inexpensive instruments.https://extension.psu.edu/ammonia-monitoring-in-barns-using-simple-instruments Technical bulletin: Poultry dust - what you need to know about impact on bird health.https://extension.psu.edu/poultry-dust-what-you-need-to-know-about-impact-on-bird-health Technical bulletin: Inlets for mechanical ventilation systems in animal housing.https://extension.psu.edu/inlets-for-mechanical-ventilation-systems-in-animal-housing Poultry Disease Workshop for Ohio and Iowa Practicing Veterinarians. A curriculum for the one-day workshop has been developed. The learning objectives of this curriculum are to equip non-poultry specialist practicing veterinarians with enough knowledge to enable them to accept noncommercial poultry cases. Six separate workshops were conducted in four different locations across the state of Ohio. Poultry Disease Workshop for Minnesota. Extension Poultry and Veterinary Diagnostic Laboratory (VDL) team at the University of Minnesota conducted in-person and online webinars. Videos of each webinar were made available on the UMN Extension YouTube channel, 1 to 2 weeks after the presentations.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Hofstetter, D., Fabian E., D. Dominguez, and Lorenzoni A.G. Automatically controlled dust generation system using Arduino. Sensors 2022, 22, 4574. https://doi.org/10.3390/s22124574.
  • Type: Journal Articles Status: Submitted Year Published: 2023 Citation: M. R. Campler, T-Y. Cheng, C. Hofacre, C-W. Lee, G. Lossie, M. El-Gazzar, A. G. Arruda. Spatial factors influencing infectious bronchitis virus (IBV) antibody titers at slaughter in broiler chickens. Under Review in Research in Veterinary Science
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: M. R. Campler, A. Hashish, M. El-Gazzar, A. G. Arruda. Spatio-temporal clusters and molecular characterization of O. rhinotracheale and P. multocida in turkeys. 2021 Conference of Research Workers in Animal Diseases (CRWAD). Jan 22-24, 2023. Chicago, IL, USA. (poster).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: T-Y. Cheng, R. Campler, A. G. Arruda. Assessment of IBV exposure risks in broiler chicken using spatial analysis, machine learning, and risk factor analysis. 2022 American Association of Avian pathologists (AAAP) Annual Meeting. July 28-Aug02, 2022. Philadelphia, PA, USA (oral).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: M. R. Campler, T-Y. Cheng, C. L. Hofacre, A. G. Arruda. Epidemiological tools for informing disease risk: the example of infectious bronchitis virus (IBV). 71st Western Poultry Disease Conference. Apr 03-06, 2022. Vancouver, BC, Canada (oral).
  • Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Dominguez, D., Hofstetter, D., Buiatte, V., Parett, M., Dunn, P., Lorenzoni A.G. Effect of hardwood dust and ammonia gas on the respiratory integrity of broiler chickens. Avian Diseases.
  • Type: Other Status: Published Year Published: 2022 Citation: 1) Technical bulletin: Detecting ammonia in poultry housing using inexpensive instruments. https://extension.psu.edu/ammonia-monitoring-in-barns-using-simple-instruments
  • Type: Other Status: Published Year Published: 2023 Citation: 3) Technical bulletin: Inlets for mechanical ventilation systems in animal housing. https://extension.psu.edu/inlets-for-mechanical-ventilation-systems-in-animal-housing
  • Type: Other Status: Published Year Published: 2022 Citation: 2) Technical bulletin: Poultry dust - what you need to know about impact on bird health. https://extension.psu.edu/poultry-dust-what-you-need-to-know-about-impact-on-bird-health
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Hofstetter, D., Fabian E., and Lorenzoni A.G. (2021). Ammonia generation system for poultry health research using Arduino. Sensors (21)6664: 1-20. https://doi.org/10.3390/s21196664.
  • Type: Other Status: Published Year Published: 2021 Citation: A series of three videos in relation to ammonia measurement and ammonia control were created and published: 1) Video: Paper Test Strips; 3 min 18 sec. 2) Video: Pull Tubes and Diffusion Tubes; 5 min 29 sec. 3) Video: Instrument Measurement Comparisons; 4 min 57 sec. https://extension.psu.edu/poultry-houses-and-ammonia
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: 2. Sakhrie, A., J. Ding, A Kalluri, Z Helal, C. V. Kumar and M. I. Khan. Fluodot Nanoparticle  A Promising Novel Delivery System for Veterinary Vaccine. International Journal of Nanoparticle Research, 2020; 3:14. DOI: 10.28933/ ijonr-2020-08-1205
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: 1. Li Jianping., Z. H. Helal, C. P. Karch, N. Mishra,T. Girshick, A. Garmendia, P. Burkhard, M. I. Khan. A self-adjuvanted nanoparticle-based vaccine against infectious bronchitis virus. PLoS ONE 13(9): e0203771
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: 1. Mo, J., M. Angelichio, L. Gow, V. Leathers and M. W. Jackwood. Validation of specific quantitative real-time RT-PCR assay panel for Infectious Bronchitis using synthetic DNA standards and Clinical Specimens. J. Virological Methods. 276: https://doi.org/10.1016/j.jviromet.2019.113773 2020
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: 2. Mo, J., M. Angelichio, L. Gow, V. Leathers and M. W. Jackwood. Quantitative real-time assays for the concurrent diagnosis of infectious laryngotracheitis virus, Newcastle disease virus and avian metapneumovirus in poultry. J. Veterinary Science, 2022, doi.org/10.4142/jvs.21153
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Jackwood, M. W., R. Clark, S. Cheng, B. J. Jordan. Protection following simultaneous vaccination with three or four different attenuated live vaccine types against infectious bronchitis virus. Avian Pathol. 49:335-341. 2020
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Aston, E.J., M.W. Jackwood, R.M. Gogal, Jr., D.J. Hurley, B.D. Fairchild, D.A. Hilt, S. Cheng, L.R. Tensa, M. Garcia, B.J. Jordan. Ambient ammonia does not inhibit the immune response to infectious bronchitis virus vaccination and protection from homologous challenge in broiler chickens. Veterinary Immunology and Immunopathology 217, 2019, doi.org/10.1016/j.vetimm.2019.109932
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Aston, E.J., B.J. Jordan, S. M. Williams, M. Garcia, M.W. Jackwood. Effect of pullet vaccination on development and longevity of immunity. Viruses, 11, 135, doi: 10.3390/v11020135 2019.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: T. J. Beyene, C. W. Lee, G. Lossie, A. G. Arruda. Poultry professionals perception of participation in voluntary disease mapping and monitoring programs in the United States: a cluster analysis. Avian Diseases. 65(1): 67-76. https://doi.org/10.1637/aviandiseases-D-20-00078.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Z. Khalid*, L. He, Q. Yu, C. Breedlove, K. Joiner, H. Toro (2021). Enhanced Protection by Recombinant Newcastle Disease Virus Expressing Infectious Bronchitis Virus Spike-Ectodomain and Chicken Granulocyte-Macrophage Colony-Stimulating. Avian Diseases 65: 364-372
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Ngunjiri JM, Taylor KJM, Ji H, Abundo MC, Ghorbani A, Kc M, Lee CW. Influenza A virus infection in turkeys induces respiratory and enteric bacterial dysbiosis correlating with cytokine gene expression. PeerJ. 2021 Jul 22;9:e11806. doi: 10.7717/peerj.11806. PMID: 34327060; PMCID: PMC8310620
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: KC M, Ngunjiri JM, Ghorbani A, Abundo MEC, Wilbanks KQ, Lee K, Lee, CW. Assessment of TLR3 and MDA5- Mediated Immune Responses Using Knockout Quail Fibroblast Cells. Avian Dis. 2021. 2021 Sep;65(3):419-428. doi: 10.1637/0005-2086-65.3.419.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Kathayat D, Closs G Jr, Helmy YA, Deblais L, Srivastava V, Rajashekara G. In Vitro and In Vivo Evaluation of Lacticaseibacillus rhamnosus GG and Bifidobacterium lactis Bb12 Against Avian Pathogenic Escherichia coli and Identification of Novel Probiotic-Derived Bioactive Peptides. Probiotics Antimicrob Proteins. 2021 Aug 30. doi: 10.1007/s12602-021-09840-1. PMID: 34458959.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Kathayat, D.; Lokesh, D.; Ranjit, S.; Rajashekara, G. Avian Pathogenic Escherichia coli (APEC): An Overview of Virulence and Pathogenesis Factors, Zoonotic Potential, and Control Strategies. Pathogens 2021, 10, 467. https://doi.org/10.3390/pathogens1004046.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Kathayat D, Closs G Jr, Helmy YA, Lokesh D, Ranjit S, Rajashekara G. Peptides affecting outer membrane lipid asymmetry (MlaA-OmpC/F) system reduce avian pathogenic Escherichia coli (APEC) colonization in chickens. Appl Environ Microbiol. 2021 Jun 16:AEM0056721. doi: 10.1128/AEM.00567-21. Online ahead of print.PMID: 34132592
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Abundo MC, Ngunjiri JM, Taylor KJM, Ji H, Ghorbani A, KC M, Weber BP, Johnson TJ, Lee CW. Assessment of two DNA extraction kits for profiling poultry respiratory microbiota from multiple sample types. PLoS One. 16(1):e0241732. 2021.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Sakhrie A, Ding J, Kalluri A, Helal Z, Kumar CV, Khan MI. Fluodot Nanoparticle - A Promising Novel Delivery System for Veterinary Vaccine. International Journal of Nanoparticle Research. 3:14. 2020
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zegpi RA, He L, Yu Q, Joiner KS, van Santen VL, Toro H. Limited Protection Conferred by Recombinant Newcastle Disease Virus Expressing Infectious Bronchitis Spike Protein. Avian Diseases. 64:53-59. 2020
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Kathayat D, Antony L, Deblais L, Helmy YA, Scaria J, Rajashekara G. Small molecule adjuvants potentiate colistin activity and attenuate resistance development against Escherichia coli by affecting pmrAB system. Infection and Drug Resistance 2020:13 22052222. 2020
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Helmy YA, Kathayat D, Ghanem M, Jung K, Closs Jr G, Deblais D, Srivastava V, El-Gazzar M, Rajashekara G. Identification and characterization of novel small molecule inhibitors to control Mycoplasma gallisepticum infection in chickens. Veterinary Microbiology: 108799. 2020
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Krunkosky M, Garc�a M, Beltran G, Williams SM, Hurley DJ, Gogal RM Jr. Ocular Exposure to Infectious Laryngotracheitis Virus Alters Leukocyte Subsets in the Head-associated Lymphoid Tissues and Trachea of 6-week-old White Leghorn Chickens. Avian Pathology.49: 4, 404-417. 2020
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Abundo MC, Ngunjiri JM, Taylor KJM, Ji H, Ghorbani A, KC M, Elaish M, Jang H, Weber BP, Johnson TJ, Lee CW. Evaluation of Sampling Methods for the Study of Avian Respiratory Microbiota. Avian Diseases. 64(3):277-285. 2020
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Taylor KJM, Ngunjiri JM, Abundo MC, Jang H, Elaish M, Ghorbani A, Kc M, Weber BP, Johnson TJ, Lee CW. Respiratory and Gut Microbiota in Commercial Turkey Flocks with Disparate Weight Gain Trajectories Display Differential Compositional Dynamics. Appl Environ Microbiol. 86(12):e00431-20. 2020
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Abundo MC, Ngunjiri JM, Taylor KJM, Ji H, Ghorbani A, KC M, Weber BP, Johnson TJ, Lee CW. Assessment of two DNA extraction kits for profiling poultry respiratory microbiota from multiple sample types. PLoS One. 16(1):e0241732. 2021
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Ngunjiri JM, Taylor KJM, Abundo MC, Jang H, Elaish M, KC M, Ghorbani A, Wijeratne S, Weber BP, Johnson TJ, Lee CW. Farm Stage, Bird Age, and Body Site Dominantly Affect the Quantity, Taxonomic Composition, and Dynamics of Respiratory and Gut Microbiota of Commercial Layer Chickens. Appl Environ Microbiol. 85(9). pii: e03137-18. 2019
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Johnson TJ, Youmans BP, Noll S, Cardona C, Evans NP, Karnezos TP, Ngunjiri JM, Abundo MC, and Lee CW. A consistent and predictable commercial broiler chicken bacterial microbiota in antibiotic-free production displays strong correlations with performance. Applied and Environmental Microbiology 84: e00362-18. 2018.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Mulholland, K.A. and C.L. Keeler, Jr. (2019) BiomeSeq: A tool for the characterization of animal microbiomes from metagenomic data. bioRxiv DOI: https://doi.org/10.1101/800995https://www.biorxiv.org/content/10.1101/800995v1.full
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Beltr�n, G., D. J. Hurley, R. M. Gogal Jr., S. Sharif, L. R Read, S. M. Williams, C. F. Jerry, D. A Maekawa, and M. Garc�a. Immune Responses in the Eye-Associated Lymphoid Tissues of Chickens after Ocular Inoculation with Vaccine and Virulent Strains of the Respiratory Infectious Laryngotracheitis Virus (ILTV). Viruses 11 (77) 635, https://doi.org/10.3390/v11070635. 2019.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Dipak Kathayat, Linto Antony, Loic Deblais, Yosra A. Helmy, Joy Scaria, and Gireesh Rajashekara. Small molecule adjuvants potentiate colistin activity and attenuate resistance development against Escherichia coli by affecting pmrAB system. Manuscript under review, Frontiers in Microbiology.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Vagnozzi AE, Beltr�n G, Zavala G, Read L, Sharif S, Garc�a M. Cytokine gene transcription in the trachea, Harderian gland, and trigeminal ganglia of chickens inoculated with virulent infectious laryngotracheitis virus (ILTV) strain. Avian Pathol. 47(5):497-508. 2018.
  • Type: Other Status: Published Year Published: 2018 Citation: Kathayat D, Helmy YA, Deblais L, Rajashekara G. Novel small molecules affecting cell membrane as potential therapeutics for avian pathogenic Escherichia coli. Sci Rep. 17;8(1):15329. 2018.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Helmy YA, Deblais L, Kassem II, Kathayat D, Rajashekara G. Novel small molecule modulators of quorum sensing in avian pathogenic Escherichia coli (APEC). Virulence. 9(1):1640-1657. 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Li J, Helal ZH, Karch CP, Mishra N, Girshick T, Garmendia A, Burkhard P, Khan MI. A self-adjuvanted nanoparticle based vaccine against infectious bronchitis virus. PLoS One. 14;13(9):e0203771. 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Derksen T.J., Lampron R., Hauck R., M. Pitesky, R. A. Gallardo. Biosecurity Assessment and Seroprevalence of Respiratory Diseases in Backyard Poultry Flocks Located Close and Far from Commercial Premises. Avian Diseases. 62(1):1-5. 2018
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Fatma Eldemery, Kellye S. Joiner, Haroldo Toro, Vicky L. van Santen. Protection against infectious bronchitis virus by spike ectodomain subunit vaccine. Vaccine. 35:5864-5871. 2017
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zegpi R.A., C. Breedlove, V.L. van Santen, C.R. Rasmussen-Ivey, H. Toro. Kidney Cell-Adapted Infectious Bronchitis ArkDPI Vaccine is Stable and Protective. Avian Diseases. 61:221-228. 2017
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Karch, C., P., J. Li, C. Kulangara, S.M. Paulillo, S.K. Raman, S. Emadi, A. Tan, Z.H. Helal, Q. Fan, M. I. Khan, P. Burkhard. Vaccination with self-adjuvanted protein nanoparticles provides protection against lethal influenza challenge. Nanomedicine: Nanotechnology, Biology and Medicine.13(1): 241-251. 2017
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Garc�a, M. Current and future vaccines and vaccination strategies against infectious laryngotracheitis (ILT) respiratory disease of poultry. Veterinary Microbiology
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Reddy SM, Izumiya Y, Lupiani B. 2016. Mareks disease vaccines: Current status, and strategies for improvement and development of vectory vaccines. Veterinary Microbiology.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Hulet R. M., E. Wallner-Pendleton, P. Clauer, G. Martin, P. Dunn, and P. Patterson. Educational Program Development in Response to 2014-2015 Avian Influenza Outbreak. Poultry Sci. 95 (Suppl.1): 326P. 2016
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Ghorbani A, Ngunjiri JM, Edward C Abundo M, Pantin-Jackwood M, Kenney SP, Lee CW. Development of in ovo-compatible NS1-truncated live attenuated influenza vaccines by modulation of hemagglutinin cleavage and polymerase acidic X frameshifting sites. Vaccine. 2023 Mar 10;41(11):1848-1858. doi: 10.1016/j.vaccine.2023.01.018. Epub 2023 Jan 18. PMID: 36669965.


Progress 04/01/21 to 03/31/22

Outputs
Target Audience:Poultry industry including backyard poultry farmers; Goverment and state officials involved in control of animal diseases. Scientists in the field of infectious diseases, animal production, Ag-engineering, virology, bacteriology, immunology & vaccinology. Extension and outreach specialists. Changes/Problems:The PI was changed to Dr. Rajashekara since the original PI, Dr. Chang Won Lee left the OSU in August 2021 What opportunities for training and professional development has the project provided?This project support training of more than several undergraduate and graduate students, post-docs, research assistants, researchassociates and scientists (listed below). In addition to hand-on experiences, students and post-docs presented the results atthe local and national meetings and published the results in scientific journals. How have the results been disseminated to communities of interest?We developed the main project website (PRDCAP.com) to share our projects with professionals, stakeholders and thegeneral public. The resultsfrom individual projects were presented at the international and regional meetings. Several manuscripts are being preparedfor publication in timely manner. Furthermore, our extension group actively conduct comprehensive educational trainingprogram on the importance of controlling respiratory diseases for Veterinarians, Extension Educators, Gamebird Producers,Small Organic and Pastured Poultry Operations and Backyard, Hobby, and Exhibition Growers and the general public through distribution of brochures, presentation at a poster, presentation at local, regional, and national meetings, workshops andspecific training programs. Working online platform for poultry mapping, has been developed and could be ready for use in a timely manner; this could be a useful tool in case the need arises. We have identified that poultry production companies that do perceive value in sharing this type of information; and currently, collaborations are on-going to use retrospectively collected routine data to produce "case studies" that will serve as examples for the industry on how mapping platforms/ spatial information/ publicly available data can be used to inform disease risk. Results were presented in multiple professional and poultry producers meetings. The created maps along with the contact information of all trained veterinarians were shared with relevant parties including, but not limited to, the Ohio Poultry association, The Ohio breeders association, The Ohio Veterinary Medical association, Iowa Poultry Association, Iowa Veterinary Medical Association and others to promote the use of services of participant veterinarians by noncommercial poultry owners. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue working on our examples to demonstrate the use of spatial data and publicly available datasets to inform diseases risk in poultry respiratory diseases. We will do that through the publication of at least two manuscripts and presentations on Conferences of interest. We also plan to use opportunities to disseminate our findings locally, regionally and nationally, and advocate for large-scale uses of mapping platforms and routinely collect data on poultry respiratory diseases to improve disease prevention and control. Finish data analysis to understand the underlying immunological mechanisms of protection by live vaccinesin ovoand after hatch. Develop commercially viable formulations by combining different small molecules with currently used antibiotics (to reduce antibiotic use and resistance), determine their effects against multi-drug resistant APEC infections, and identify and validate their antibacterial targets using thermal proteome profiling and immunoblot assays. Synthesize derivatives of effective small molecule to increase their efficacy. Test the LGG and LGG derived AMPs in large number of chickens under field simulated conditions and validate the target of peptides by determining drug-target binding affinities. Design of nanoparticle based IBV vaccine prototypes-The intracellular delivery and expression of EGFP mRNA being evaluated and tested in the HeLa cells.SPF chickens are being monitored and samples are being taken from the inoculated conjugated BSA-PEHA-S1mRNA vaccine trial for immunogenicity studies. We plan to continue working on our examples to demonstrate the use of spatial data and publicly available datasets to inform diseases risk in poultry respiratory diseases. We will do that through the publication of at least two manuscripts and presentations on Conferences of interest. We also plan to use opportunities to disseminate our findings locally, regionally and nationally, and advocate for large-scale uses of mapping platforms and routinely collected data on poultry respiratory diseases to improve disease prevention and control.

Impacts
What was accomplished under these goals? Objective 1. Ecology and Networking We have continued to work on establishing novel framework for assessing risk factors for exposure for infectious bronchitis virus (IBV), and confirmed outbreaks and spatial distribution over time forO. rhinotrachealis and P.multocida.The main product of this project, a working online poultry mapping platform, has been developed and could be ready for use in a timely matter; this could be a useful tool in case the need arises. Project participation for voluntary data sharing has not dramatically increased despite loosened COVID-19 restrictions. Reluctance of data sharing remains a key issue that needs to be addressed, thus it's vital to be able to present new findings that will sway stakeholders into seeing potential benefits in doing so. There is great value in presenting findings using shared company data, albeit currently anonymously, as the perceived challenges associated with data sharing may be mitigated by the potential benefits to the industry derived herein. Objective 2. Multifactorial etiology We have defined the microbiome in the respiratory tracts of commercial broilers, layers, and turkeys in relation to health status and performance tofacilitate disease prediction and prevention and identification of alternatives to antibiotics. Furthermore, we have constructedBiomeSeq, a bioinformatics pipeline that will be instrumental for analysis of the viromes of different poultry species. A protocol for sample collection by visiting backyard flocks, transport, storage, processing for library prep and bioinformatics analysis has been optimized. We have compared different kits for library preparation with and without ribodepletion. Ribodepletion before library preparation was helpful in increasing viral reads. We investigated the relationships between LPAI virus replication, cytokine gene expression, and respiratory and gut microbiota disruption in specific-pathogen-free turkeys. Overall, our data suggest a potential relationship where bacterial community diversity and enrichment or depletion of several bacterial genera in the gut and respiratory tract are dependent on the level of LPAI virus replication. Our studies shows that a typical pullet vaccination program consisting of serially administered live attenuated NDV, IBV and ILTV vaccines against multiple respiratory pathogens can result in the development of protective immunity against each disease agent. Objective 3. Diagnostic tools and preventive measures Developed a standardized, portable sequence typing assay that allows ORT sequence typing cgMLST) results to be shared at a national and international scale. This assay could reflect the accurate relatedness between isolates circulating in the field during a specific timeframe. Six TaqMan™-based quantitative real-time RT-PCR assays for IBV (Universal, Ark, Mass, DE/GA98, GA07, GA08) were developed and examined the sensitivity and specificity for each assay. Assays were developed targeting the hypervariable region in the S1 gene subunit. All developed assays performed equivalently when using synthetic DNA templates as standard material. Further validation of specificity using clinical and biological specimens was also successful. We created a panel of quantitative qPCR assays forNewcastle disease (ND), infectious laryngotracheitis (ILT) and avian metapneumovirus (AMPV)suitable for rapid and type specific diagnostic purposes.All developed assays performed equivalentlywithlinearity over a 5 log10dynamic range with a reproducible (R2≥0.99)limit of detection of ≤10 target copies per reaction, with high calculated amplification efficiencies ranging between86.8%-98.2%.Further validation of specificity using clinical and biological specimens was alsoperformed. We developed recombinant vaccine technology to eliminate type-specific live vaccination. We have focused on IBV Arkansas (Ark)-type spike (S) trimeric ectodomain (Se), i.e., S1 subunit + S2 subunit without transmembrane domain and cytoplasmic tail, as both a subunit vaccine and expressed from a recombinant virus vaccine. Evaluate the effects of air quality on the onset (infection), transmission and severity of respiratory disease caused by IBV as a model. Two bird studies have been completed. The results indicate that commercial broilers exposed to moderate levels of ammonia are not more susceptible to IBV challenge if they are appropriately vaccinated, but nonvaccinated-challenged birds experience more severe clinical signs and a higher incidence of airsacculitis. We determined if multiple IBV vaccines given simultaneously can equally immunize birds.The results are important for the control of IBV because they indicate that simultaneous vaccination with up to 4 different IBV vaccine types can provide adequate protection against challenge for each type. In addition, multiple IBV vaccine types given simultaneously can provide partial protection against heterologous viruses. We have identified several novel non-antibiotic compounds (small molecules and antimicrobial peptides) that inhibit avian pathogenicE. coli(APEC) andM. gallisepticum.Administration of these compounds in the drinking water of chickens in a field simulated conditions for 7 days, reduced the mortality, lesions, and APEC load in internal organs, which was more than the reduction caused by sulfadimethoxine treatment, that is currently used in the industry. Additionally, similar body weight gain (BWG) and feed conversion ratio (FCR) were observed when treated with these products compared to non-infected, non-treated control chickens. Further, the potential targets for these small molecule or peptides have been identified as outer membrane proteins of APEC, targeting outer membrane will less likely enable bacteria to develop resistance unlike the current antibiotics currently used to treat APEC infections Objective 4. Extension and Outreach A series of three videos in relation to ammonia measurement and ammonia control were created and published: Video: "Paper Test Strips"; 3 min 18 sec. Video: "Pull Tubes and Diffusion Tubes"; 5 min 29 sec. Video: "Instrument Measurement Comparisons"; 4 min 57 sec. The videos can be found athttps://extension.psu.edu/poultry-houses-and-ammonia A series of factsheets have been published Technical bulletin: Detecting ammonia in poultry housing using inexpensive instruments.https://extension.psu.edu/ammonia-monitoring-in-barns-using-simple-instruments Technical bulletin: Poultry dust - what you need to know about impact on bird health.https://extension.psu.edu/poultry-dust-what-you-need-to-know-about-impact-on-bird-health Technical bulletin: Inlets for mechanical ventilation systems in animal housing.https://extension.psu.edu/inlets-for-mechanical-ventilation-systems-in-animal-housing Conducted 12 different workshops in 2 different states which has created a list of practicing veterinarians who are trained, willing and able to provide veterinary services to the noncommercial poultry population. This list of veterinarians are geographically positioned to allow easier access to veterinary services for this severely underserved noncommercial poultry population. While we were able to measure the impact of this Extension and educational effort on participating veterinarians, the bigger impact is predicted to be on the noncommercial poultry. More importantly, this project provides an example to follow which can be repeated in other states and across the nation to provide a solution to this problem. The Extension Poultry and Veterinary Diagnostic Laboratory (VDL) team at the University of Minnesota conducted in-person and online webinars. Videos of each webinar were made available on the UMN Extension YouTube channel, 1 to 2 weeks after the presentations. There were more than 70 views for these presentations on YouTube. All in-person workshops were also well attended.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: - Amir Ghorbani, John M. Ngunjiri, Michael C. Abundo, Chang-Won Lee. Development of an in ovo compatible live attenuated influenza vaccine by targeting multiple viral proteins. 102nd Conference of Research Workers in Animal Diseases, December 3-7, 2021, Chicago, IL, USA. (Poster Presentation)
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: 2. Mo, J., M. Angelichio, L. Gow, V. Leathers and M. W. Jackwood. Quantitative real-time assays for the concurrent diagnosis of infectious laryngotracheitis virus, Newcastle disease virus and avian metapneumovirus in poultry. J. Veterinary Science, 2022, doi.org/10.4142/jvs.21153.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: M. R. Campler, T-Y. Cheng, A. G. Arruda. Investigation of geospatial risk factors on infectious bronchitis virus (IBV) antibody titer levels in Midwestern poultry farms. 2021 Conference of Research Workers in Animal Diseases (CRWAD). Dec 04-08, 2021. Virtual (Oral virtual presentation by Dr. Campler).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: M. R. Campler, T-Y. Cheng, A. G. Arruda. Epidemiological tools for informing disease risk: the example of infectious bronchitis. Presented orally by Dr. Arruda, at the 71st Annual Western Poultry Disease Conference. April 3-6, 2022.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Hofstetter, D., Fabian E., Lorenzoni A.G. (2021). Ammonia generation system for poultry health research using Arduino. Sensors (21)6664: 1-20. https://doi.org/10.3390/s21196664.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Dominguez, D., Hofstetter, D.,; Buiatte, V., ; Paret, M., Dunn, P. Lorenzoni, A.G. (2022). Effect of atmospheric ammonia and hardwood-dust on the performance and respiratory integrity of broiler chickens. International Poultry Scientific Forum.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Hofstetter, D. W., Gino, L., & Fabian, E. E. (2021). Dust generator for maintaining a set indoor airborne dust concentration for poultry health research. ASABE Annual International Virtual Meeting, July 13-15, 2021. (doi:10.13031/aim.202100671).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Hofstetter, D. W., Dominguez, D., Fabian, E. E., & Gino, L. (2021). Update on ammonia generator for maintaining a set indoor gas concentration for poultry health research. 2021 ASABE Annual International Virtual Meeting, July 13-15, 2021. (doi:10.13031/aim.202100667).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: D. Lokesh1, D. Kathayat, G. Rajashekara. Elucidation of mechanism of action of GI-7, a novel small molecule inhibitor of avian pathogenic E. coli (APEC). CRWAD, Dec 7 -13, Chicago, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Amor Hashish, Mohamed El-Gazzar, Tim Johnson, Mostafa Ghanem, Yuko Sato. Development of Core Genome Multilocus Sequence Typing Scheme (cgMLST) for Ornithobacterium rhinotracheale. 72nd North Central Avian Disease Virtual Conference
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Amor Hashish, Mohamed El-Gazzar and Yuko Sato Poultry Medicine Workshops. 72nd North Central Avian Disease Virtual Conference
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Ngunjiri JM, Taylor KJM, Ji H, Abundo MC, Ghorbani A, Kc M, Lee CW. Influenza A virus infection in turkeys induces respiratory and enteric bacterial dysbiosis correlating with cytokine gene expression. PeerJ. 2021 Jul 22;9:e11806. doi: 10.7717/peerj.11806. PMID: 34327060; PMCID: PMC8310620. Copy Ngunjiri JM, Taylor KJM, Ji H, Abundo MC, Ghorbani A, Kc M, Lee CW. Influenza A virus infection in turkeys induces respiratory and enteric bacterial dysbiosis correlating with cytokine gene expression. PeerJ. 2021 Jul 22;9:e11806. doi: 10.7717/peerj.11806. PMID: 34327060; PMCID: PMC8310620.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 3. Kathayat D, Closs G Jr, Helmy YA, Lokesh D, Ranjit S, Rajashekara G. Peptides affecting outer membrane lipid asymmetry (MlaA-OmpC/F) system reduce avian pathogenic Escherichia coli (APEC) colonization in chickens. Appl Environ Microbiol. 2021 Jun 16:AEM0056721. doi: 10.1128/AEM.00567-21. Online ahead of print.PMID: 34132592.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 4. Kathayat, D.; Lokesh, D.; Ranjit, S.; Rajashekara, G. Avian Pathogenic Escherichia coli (APEC): An Overview of Virulence and Pathogenesis Factors, Zoonotic Potential, and Control Strategies. Pathogens 2021, 10, 467. https://doi.org/10.3390/pathogens1004046.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 5. Kathayat D, Closs G Jr, Helmy YA, Deblais L, Srivastava V, Rajashekara G. In Vitro and In Vivo Evaluation of Lacticaseibacillus rhamnosus GG and Bifidobacterium lactis Bb12 Against Avian Pathogenic Escherichia coli and Identification of Novel Probiotic-Derived Bioactive Peptides. Probiotics Antimicrob Proteins. 2021 Aug 30. doi: 10.1007/s12602-021-09840-1. PMID: 34458959.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 6. KC M, Ngunjiri JM, Ghorbani A, Abundo MEC, Wilbanks KQ, Lee K, Lee, CW. Assessment of TLR3 and MDA5-Mediated Immune Responses Using Knockout Quail Fibroblast Cells. Avian Dis. 2021. 2021 Sep;65(3):419-428. doi: 10.1637/0005-2086-65.3.419.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 7. Ngunjiri JM, Taylor KJM, Ji H, Abundo MC, Ghorbani A, KC M, Lee CW. Influenza A virus infection in turkeys induces respiratory and enteric bacterial dysbiosis correlating with cytokine gene expression. PeerJ. 2021 Jul 22;9:e11806.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 8. Abundo MC, Ngunjiri JM, Taylor KJM, Ji H, Ghorbani A, KC M, Weber BP, Johnson TJ, Lee CW. Assessment of two DNA extraction kits for profiling poultry respiratory microbiota from multiple sample types. PLoS One. 16(1):e0241732. 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Sakhrie, Aseno., Ankaro Kalluri, Chela V. Kumar, and M. I. Khan Study of expression of EGFP using cationic BSA-polyamine Nano complex. TechConnect World Innovation Conference, National Harbor, MD, June 13-15, 2022.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2022 Citation: 2. Anita Kumari, Vikash K. Singh, David Suarez, Michaela C. Olson, Sagar M. Goyal, Sally L. Noll, H. Wayne Martin, Robert E. Porter, and Sunil Kumar Mor. Understanding the Respiratory virome of backyard poultry in Minnesota. Accepted for poster presentation at the AAAP meeting, July 30-Aug. 2, 2022.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Z. Khalid*, L. He, Q. Yu, C. Breedlove, K. Joiner, H. Toro (2021). Enhanced Protection by Recombinant Newcastle Disease Virus Expressing Infectious Bronchitis Virus Spike-Ectodomain and Chicken Granulocyte-Macrophage Colony-Stimulating. Avian Diseases 65: 364-372


Progress 04/01/20 to 03/31/21

Outputs
Target Audience:Poultry industry including backyard poultry farmers; Goverment and state officials involved in control of animal diseases. Scientists in the field of infectious diseases, animal production, Ag-engineering, virology, bacteriology, immunology & vaccinology. Extension and outreach specialists. Changes/Problems:The COVID-19 pandemic significantly affected the progress of the all the studies within this project. Significant unexpected delay in research due to occupancy and other restrictions created difficulty in maintaining personnel committed to the project and thus the budget. We have obtained no-cost extension approval in advance to complete the project and will focus on completing the key experiment and data analysis that are essential for publication of the work. What opportunities for training and professional development has the project provided?This project support training of more than 20 undergraduate and graduate students, post-docs, research assistants, research associates and scientists (listed below). In addition to hand-on experiences, students and post-docs presented the results at the local and national meetings and published the results in scientific journals. How have the results been disseminated to communities of interest?We developed the main project website (PRDCAP.com) to share our projects with professionals, stakeholders and the general public. To further expand the network, the annual meeting was held on in conjunction with the NC-1180. The results from individual projects were presented at the international and regional meetings. Several manuscripts are being prepared for publication in timely manner. Furthermore, our extension group actively conduct comprehensive educational training program on the importance of controlling respiratory diseases for Veterinarians, Extension Educators, Gamebird Producers, Small Organic and Pastured Poultry Operations and Backyard, Hobby, and Exhibition Growers and the general public through distribution of brochures, presentation at a poster, presentation at local, regional, and national meetings, workshops and specific training programs. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Ecology & Network: Continuing efforts will be made for the expansion and maintenance of the nationwide poultry respiratory disease network; We will continue working with poultry industry to collect and update the poultry disease data and provide relevant information in real-time as a model to expand nationwide; Coordinated effort among Ohio, Minnesota and Delaware groups on microbiome project will continue. In addition to testing surveillance samples, we are conducting and collaborating with the investigators in this project to sample the respiratory microbiome using models of respiratory infection (refer to Objective 2) and in commercial flocks experiencing respiratory infections. Efforts will continue to evaluate the utility of BiomeSeq using data derived from other animal species and from biological niches other than the respiratory tract. Objective 2. Multifactorial etiology: All the proposed studies in Objective 2 involve extensive animal experiments, which have generated a large amount of descriptive data. Collaborative efforts will continuously be made among PIs to collectively interpret the data in relation to multiple factors including respiratory pathogens, immunosuppressive agents, vaccine statuses, and environmental stress for each individual species to mimic the natural condition and better understand the pathogenesis. Newly funded SEED projects will be completed. Objective 3. Diagnostic tools & preventive measures: Diagnostic research will be geared toward improving the sensitivity of NGS testing using a cost- effective approaches. Once the most effective approaches have been identified, we will simplify the testing procedure and reduce costs, likely through using barcodes to run more samples per run. We will develop the core genome MLST for ORT which was identified as emerging disease and supported by SEED funding. Vaccine and non-antibiotic small molecule development will focus on validation of safety and efficacy in experimental settings and also in field conditions. Efforts will also be made for patent application and exploring licensing options. Objective 4. Extension & outreach: We will finalize and reorganize many extension materials including videos and websites. We will heavily promote the extension material to poultry producers, small flock owners and public while on presentations at local, regional and national extension meetings/workshops. We will continue the yearly 3 full day poultry disease workshops. We will accumulate and analyze the data from last 5 years and publish a peer review manuscript that details the program. We will maintain a map and a list of trained veterinarians which will be promoted and solicited to the relevant groups in each state. The low cost vehicle disinfection system has shown potential and will further improve the disinfection efficacy and outreach will be conducted on vehicle biosecurity, and how to implement a low-cost vehicle disinfection system.

Impacts
What was accomplished under these goals? Objective 1. Ecology and Networking 1) We are continuously expanding the nationwide poultry disease research network and new participants from Mississippi and additional member from Georgia joined in our NC-1180 multistate project; 2) Since 2016, we have supported new SEED projects to stimulate new and innovative research and expand collaboration. We also maintain project website (PRDCAP.com) to share our projects with professionals and stakeholders; 3) Toward creating a centralized poultry disease work group and developing a database, we conducted a nationwide survey to assess the perception of poultry professionals toward disease mapping and monitoring programs and to identify groups of poultry professionals with similar perceptions and attitudes toward these projects. The cluster analysis yielded two distinct groups of respondents; Cluster 1 subjects could be characterized as optimistic, perceiving major benefits of sharing farm-level poultry disease information. However, they had major concerns, mostly related to potential accidental data release and providing competitive advantages to rival companies. Cluster 2 subjects were characterized as perceiving lesser degree of benefits from sharing farm-level poultry disease information. This second cluster mostly included production and service technicians. The roles and perceptions of risk and benefits of the participants contributed significantly to cluster assignment, while the represented commodity and geographic location in the US did not. For the successful development of poultry disease mapping and monitoring programs, various groups of poultry professions will be approached in different manners to achieve maximum participation in the voluntary program. Objective 2. Multifactorial etiology 1) The complexity of avian gastrointestinal and respiratory tracts presents a major challenge regarding the choice of sample types along with sample acquisition and processing methods for rigorous profiling of poultry gut and respiratory microbiome via high throughput sequencing. To this end, we have optimized sample acquisition and processing methods for different sample types including nasal and tracheal washes and swabs, lower respiratory tract lavage, and cecal and ileal contents and homogenates. Using these tools, we conducted a comprehensive study of commercial layers to understand the baseline microbiota associated with optimal health and egg production. Interestingly, Lactobacillus, a genus with species associated with enhanced health and production performance in chickens, was among several genera commonly found in the gut-respiratory core. We subsequently found similar core microbiota in a research flock maintained under specific-pathogen-free (SPF) conditions which justifies the use of SPF birds for future controlled studies. Optimal performance of both commercial and research flocks corresponded with very low levels of γ-proteobacteria (<5% abundance). A surge of performance-inhibiting bacteria such as γ-proteobacteria can arise from disruption of microbiota by pathogens lurking in the barn environment as we have demonstrated using avian influenza virus (AIV), infectious bursal disease virus, and infectious bronchitis virus (IBV) in young turkeys and layer chickens. These baseline observations pave the way for future work studying ways to positively manipulate the microbiome to enhance performance and reduce disease in flocks; 2) In regards to virome work, we have found that new ribodepletion kit from Illuminais good for depletion of host and bacterial part. Clontech library preparation kit is efficientto deal withlow concentration of ribodepleted RNA;3) The utility of BiomeSeq, a comprehensive bioinformatic tool to identify and quantify microbiomes was examined for avian species other than chicken (which was done in previous year) and for locations other than the respiratory tract (sinus, cloaca). We have confirmed that BiomeSeq can be used to determine virome composition from ducks, quail and turkey and demonstrated its expanded utility; 4) Respiratory diseases involve multiple pathogens, and they interact with each other. Our study highlighted the role of avian Mycoplasma in exacerbating the clinical outcome of poultry co-infected with respiratory viruses (AIV and IBV) and also inducing side effect from normal vaccination (eg. infectious laryngotracheitis virus (ILTV)). Our co-infection studies in different environmental conditions also provided important and much needed information on the interaction of respiratory pathogens in poultry, which will help improve diagnostics and vaccination strategies needed to control respiratory syndromes in poultry. Objective 3. Diagnostic tools and preventive measures 1) The project includes several lines of research related to diagnostics for poultry respiratory pathogens. ORT is a re-emerging disease problem in commercial turkey production and SEED funding was provided to develop Core Genome Multilocus Sequence Typing (cgMLST) assay. Whole genome sequences of 66 diverse isolates from chickens and turkeys isolated from 1995 - 2020 were determined. By analyzing the gnome of these isolates along with 58 isolates from Minnesota from 2009 - 2018 and 3 complete genomes available on NCBI database, we successfully identified a preliminary draft core genome for ORT which is critical for development of the assay in the coming year; 2) Vaccination has been a widely used tool in the poultry industry to prevent or control diseases caused by infectious disease agents. In addition to four different vaccine platforms that have been developed and being tested for broadly reactive protective efficacy, basic research on cell mediated immunity which is supported by SEED funding is being conducted. This study showed a clear correlation between early memory CTLs (Cytotoxic T lymphocytes)and inhibition of challenged ILTV replication in the trachea in CEO (egg-origin vaccine) vaccinated chickens for the first time. It was also demonstrated that, in partially protected chickens, a suboptimal CTL response in the trachea is compensated with a delayed natural killer (NK) cell response. Therefore, mechanisms to accelerate and promote NK responses may enhance protection of the recombinant (HVT-LT) and tissue culture origin (TCO) vaccines. 3) To overcome the issue of antibiotic resistance and reconcile the demand of antibiotic withdrawal with maintaining animal health and food security, we have identified several novel non-antibiotic compounds that inhibit avian pathogenic E. coli (APEC) and Mycoplasma gallisepticum which show low toxicity. These small molecules are highly suitable for commercial application because of their small size, specificity, and stability. Further, the identification of new antibacterial targets such as LptD and MlaA which are likely not allow bacteria to develop resistance can have huge implications in future drug development against APEC and related pathogens. Objective 4. Extension and Outreach Our extension group have been conducting a comprehensive and effective educational training program on the importance of controlling respiratory diseases (including recent outbreak of infectious coryza) for veterinarians, extension educators, gamebird producers, small organic and pastured poultry operations and backyard, hobby, and exhibition growers, state and federal government stakeholders, and the general public. For example, Nebraska group developed and launched the Big Red Biosecurity Program. The program website is designed for lay people and is presented in an easy-to-follow questions and answers format. The program also offers assistance in training through on-line educational videos and slide sets that are available free of charge, 24/7. Through this program there is an opportunity to earn a biosecurity training certificate. The training modules have also been translated in Spanish.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Taylor KJM, Ngunjiri JM, Abundo MC, Jang H, Elaish M, Ghorbani A, Kc M, Weber BP, Johnson TJ, Lee CW. Respiratory and Gut Microbiota in Commercial Turkey Flocks with Disparate Weight Gain Trajectories Display Differential Compositional Dynamics. Appl Environ Microbiol. 86(12):e00431-20. 2020.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Abundo MC, Ngunjiri JM, Taylor KJM, Ji H, Ghorbani A, KC M, Elaish M, Jang H, Weber BP, Johnson TJ, Lee CW. Evaluation of Sampling Methods for the Study of Avian Respiratory Microbiota. Avian Diseases. 64(3):277-285. 2020.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Krunkosky M, Garc�a M, Beltran G, Williams SM, Hurley DJ, Gogal RM Jr. Ocular Exposure to Infectious Laryngotracheitis Virus Alters Leukocyte Subsets in the Head-associated Lymphoid Tissues and Trachea of 6-week-old White Leghorn Chickens. Avian Pathology.49: 4, 404-417. 2020.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Helmy YA, Kathayat D, Ghanem M, Jung K, Closs Jr G, Deblais D, Srivastava V, El-Gazzar M, Rajashekara G. Identification and characterization of novel small molecule inhibitors to control Mycoplasma gallisepticum infection in chickens. Veterinary Microbiology: 108799. 2020.
  • Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Beyene TJ, Lee CW, Lossie G, El-Gazzar MM, Arruda AG. Poultry professionals perception of participation in voluntary disease mapping and monitoring programs in the United States: a cluster analysis. Avian Diseases. In press.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Abundo MC, Ngunjiri JM, Taylor KJM, Ji H, Ghorbani A, KC M, Weber BP, Johnson TJ, Lee CW. Assessment of two DNA extraction kits for profiling poultry respiratory microbiota from multiple sample types. PLoS One. 16(1):e0241732. 2021.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Kathayat D, Antony L, Deblais L, Helmy YA, Scaria J, Rajashekara G. Small molecule adjuvants potentiate colistin activity and attenuate resistance development against Escherichia coli by affecting pmrAB system. Infection and Drug Resistance 2020:13 22052222. 2020.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zegpi RA, He L, Yu Q, Joiner KS, van Santen VL, Toro H. Limited Protection Conferred by Recombinant Newcastle Disease Virus Expressing Infectious Bronchitis Spike Protein. Avian Diseases. 64:53-59. 2020.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Jackwood MW, Clark R, Cheng S, Jordan BJ. Protection following simultaneous vaccination with three or four different attenuated live vaccine types against infectious bronchitis virus. Avian Pathology. 49:335-341. 2020.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Sakhrie A, Ding J, Kalluri A, Helal Z, Kumar CV, Khan MI. Fluodot Nanoparticle - A Promising Novel Delivery System for Veterinary Vaccine. International Journal of Nanoparticle Research. 3:14. 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Maekawa D, Riblet SM, Whang P, Alvarado I, Garc�a M. Safety and Protection Efficacy of a Cell Line Adapted Infectious Laryngotracheitis Virus (ILTV) BDORFC Strain Following In ovo and/or Spray Vaccination with and without rHVT-LT Vaccine. American Veterinary Medical Association. Annual Meeting. August 4-5, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Alvarado I, Maekawa D, and Garc�a M. Replication and Efficacy of HVT Single (HVT-LT) and Double (HVT-ND-LT) Constructs. American Veterinary Medical Association. Annual Meeting. August 4-5, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Helmy YA, Kathayat D, Deblais L, Closs G Jr, Srivastava V,Rajashekara G. Novel Anti-virulence Compound to Control Avian Pathogenic Escherichia coli (APEC) Infections in Poultry. The CFAES Annual Research Conference, College of Food, Agriculture and Environmental Sciences. The Ohio State University. April 22, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Kathayat D, Helmy YA, Deblais L, Srivastava V, Closs G Jr, Rajashekara G. Small molecule targeting outer membrane lipopolysaccharide transporter complex (LptD/E) reduces avian pathogenic Escherichia coli (APEC) infection in poultry. The CFAES Annual Research Conference, College of Food, Agriculture and Environmental Sciences. The Ohio State University. April 22, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Kathayat D, Closs G Jr, Helmy YA, Deblais L, Srivastava V, Rajashekara G. Probiotic reduces avian pathogenic Escherichia coli (APEC) infection in poultry by releasing small bioactive peptides. MOSES conference, La Crosse, Wisconsin, February 27-29. 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Khalid Z, Yu Q, Kitchens C, Joiner K, Toro H. Enhanced Protection by rNDV Expressing IBV Spike Ectodomain and GM-CSF. Auburn University, College of Veterinary Medicine, Phi Zeta Annual Meeting. November, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Dan Hofstetter, Eli Baker, Eileen Fabian, Gino Lorenzoni. Ammonia Generator for Maintaining a Set Indoor Gas Concentration for Poultry Health Research. American Society of Agricultural and Biological Engineers. Annual meeting. July 13-15. 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Beyene TJ, Lossie G, Lee CW, Arruda AG. Perceived risks and benefits for participation in poultry disease monitoring programs in the United States: a cluster analysis. Ohio State University. CVM Research Day. April, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Arruda AG, Lossie G, Lee CW. Assessing perceived risks and benefits for participation in poultry disease monitoring projects. The American Association of Avian Pathologists (AAAP) Annual meeting. August 4 -5. 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Beyene TJ, Lee CW, Lossie G, El-Gazzar MM, Arruda AG. Perceived risks and benefits for participation in poultry disease monitoring programs in the US: a cluster analysis" Conference of Research Workers in Animal Diseases. Annual Meeting. December 4-8. 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Kathayat D, Closs G Jr, Rajashekara G. Peptides affecting membrane phospholipid transport as novel therapeutics against avian pathogenic Escherichia coli (APEC). Conference of Research Workers in Animal Diseases. December 5-8, 2020.


Progress 04/01/19 to 03/31/20

Outputs
Target Audience:Poultry industry including backyard poultry farmers; Goverment and state officials involved in control of animal diseases. Scientists in the field of infectious diseases, animal production, Ag-engineering, virology, bacteriology, immunology & vaccinology. Extension and outreach specialists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This projects support training of many undergraduate and graduate students, post-docs, research assistants, research associates and scientists (listed below). In addition to hand-on experiences, students and post-docs presented the results at the local and national meetings and published the results in scientific journals. Auburn University: - Ramon Zegpi, PhD student - Developing vaccine and conducting experiment. 0.2 FTE. - Zubair Khalid, MS student - Testing different vaccine regimes in chickens. 0.7 FTE. University of Connecticut: - Aseno Sakhrie, PhD Student: Involved in developing, production and immunogenicity studies of the nanoparticles. 0.5 FTE - Maia Annunziato, Undergraduate Student: Helping in virus propagation and serological testing. - Qialian Su, Research Associate: Involved in virus propagation, purification and immunogenicity studies, 0.1 FTE. University of Delaware: - Kelly Mulholland, PhD student: developing the bioinformatic pipeline for determining the avian respiratory nicrobiome. 1 FTE. - Monique Robinson, MS student. Determining the composition of the respiratory microbiome of a flock of commercial broiler birds sampled weekly from placement through processing. 1 FTE. - Daniel Hougentogler, senior research associate. Involved in project development, training, system evaluation, and system demonstration. 0.1 FTE - 10 undergraduate students (Makayla Musgrove, Adrianna Szostek, Lillie Binder, Emily Hudson, Kasey Hobart, Reese Hardy, Brianna Ames, Will Tammaro, Rose Benn, and Emma Redman). The undergraduate students participated in implementing the laboratory and field disinfection trials. Adrianna Szostek, Brianna Ames, and Emma Redman each had individual projects and provided leadership opportunities with their projects. Adrianna Szostek parlayed the experience into admission at veterinary school. University of Minnesota: - Elizabeth Miller, PhD student: Responsible for bioinformatic analyses of microbiome data. 0.2 FTE. - Jeong Hoon, Undergraduate student: Worked16 hours in a week. Assisted in samples collection and processing for molecular work. - Brittanie Winfield, Undergraduate: Worked in hatch-brood analyses and participated in manuscript preparation. Ohio State University: - Geoffrey Lossie (Poultry Specialist): Communication specialist worked with poultry industry and developting poultry network platform. 0.5 FTE. - Tariku Beyene (Post-Doctoral Fellow): Responsible for organizing, analyzing and publishing the results from the survey, updating data to the mapping platform, working with CURA in further platform developmental aspects. - Michael Abundo, Research Assistant: Serving as project manager for project coordination. 1 FTE - John Ngunjiri, Research Scientist - Surveillance and microbiome study coordination. 0.1 FTE - Mahesh KC, PhD student - sample collection, processing, and sequence analysis. 0.5 FTE - Amir Ghorbani, PhD student - sample collection, processing, and sequence analysis. 0.5 FTE - Dipak Kathayat, PhD student: Responsible for conducting studies related to APEC growth inhibitors and mini-grant and assisting in other studies of this project. 1 FTE. - Yosra A. Helmy, Research Associate: Responsible for conducting research on APEC quorum sensing inhibitors and Mycoplasma in addition to assisting in other aspects of this project. 0.5 FTE. Penn State University: - Eli Baker, Undergraduate student: Worked on a project validating the ammonia sensors we are currently using in our machines. - Vinicius Buiatte, PhD student: Collecting and conditioning dust samples for a larger experiment. 0.3 FTE. Southeast Poultry Research Laboratory, USDA: - Several post-doctoral researchers participating in this project includes Josh Parris (40%), Henry Karithii (20%), Iryna Gorachuk (10%), Kiril Dimitrov (10%) who have all worked on the NGS development, bioinformatics, and analysis of samples. - Two temporary technicians (Katie Camp and Edna Espinoza 10% each) were involved in the AmPV RRT-PCR testing. University ofNebraska - J. Arumugam, PhD student: Studied the effect that beta-glucans have on the early development of the bird's immune system by assessing the acquired and innate immune responses. 0.1 FTE Iowa State University - Amor Hashish, Postdoctoral research associate: Generating and analyzing next generation full genome sequencing data and developing and validating core genome MLST assays. Also involved in organizing three workshops provided in 2019 and to helped collection and analysis of the data for the whole project. 1 FTE. How have the results been disseminated to communities of interest?We developed the main project website (PRDCAP.com) to share our projects with professionals, stakeholders and the general public. To further expand the network, the annual meeting washeld on in conjunction with the NC-1180 and the U.S. Animal Health Association Meeting, and received full attention from our stakeholders. The results from individual projects were presented at the international and regional meetings. Several manuscripts are being prepared for publication in timely manner. Furthermore, our extension group actively conduct comprehensive educational training program on the importance of controlling respiratory diseases for Veterinarians, Extension Educators, Gamebird Producers, Small Organic and Pastured Poultry Operations and Backyard, Hobby, and Exhibition Growers and the general public through distribution of brochures, presentation at a poster, presentation at local, regional, and national meetings, workshops and specific training programs. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Ecology & Network: Continuing efforts will be made for the expansion and maintenance of the nationwide poultry respiratory disease network; We will continue working with Ohio poultry industry to collect and update the poultry disease data and provide relevant information in real-time as a model to expand nationwide; Coordinated effort among Ohio, Minnesota and Delaware groups on microbiome project will continue. In addition to testing surveillance samples, we are conducting and collaborating with the investigators in this project to sample the respiratory microbiome using models of respiratory infection (refer to Objective 2) and in commercial flocks experiencing respiratory infections. We will establish a link between virus-induced dysbiosis, suppression of innate immune responses, and enhancement of virus replication in superinfected chickens. Efforts will continue to evaluate the utility of BiomeSeq using data derived from other animal species and from biological niches other than the respiratory tract. Objective 2. Multifactorial etiology: All the proposed studies in Objective 2 involve extensive animal experiments, which have generated a large amount of descriptive data. Collaborative efforts will continuously be made among PIs to collectively interpret the data in relation to multiple factors including respiratory pathogens, immunosuppressive agents, vaccine statuses, and environmental stress for each individual species to mimic the natural condition and better understand the pathogenesis. Newly funded SEED projectswill be completed. Objective 3. Diagnostic tools & preventive measures: Diaganostic research plans will be geared toward improving the sensitivity of NGS testing using a cost- effective approaches. Once the most effective approaches have been identified, we will simplify the testing procedure and reduce costs, likely through using barcodes to run more samples per run. Ultimately once a research platform is defined, efforts to look at field samples from poultry farms and use the metadata from the flocks to make associations with disease pathogens will be conducted; Vaccine and non-antibiotic small molecule development will focus on validation of safety and efficacy in experimental settings and also in field conditions. Efforts will also be made for patent application and exploring licensing options. Objective 4. Extension & outreach: This is the final year of the project and we will finalize and reorganize many extension materials including videos and websites. We will heavily promote the extension material to poultry producers, small flock owners and public while on presentations at local, regional and national extension meetings/workshops. We will continue the yearly 3 full day poulty disease workshops in 2020. We will accumulate and analyze the data from last 4 years and publish a peer review manuscript that details the program. We will maintain a map and a list of trained veterinarians which will be promoted and solicited to the relevant groups in each state. The low cost vehicle disinfection system has shown potential and will further improve the disinfection efficacy and outreach will be conducted on vehicle biosecurity, and how to implement a low-cost vehicle disinfection system.

Impacts
What was accomplished under these goals? Objective 1. Ecology and Networking 1) We continuously expanded the nationwide poultry disease research network with the most recent participant from New York in our existing NC-1180 multistate project. Since 2016, we have supported new SEED projects to stimulate new and innovative research and expand collaboration. We funded four new proposals in 2019. We also maintain project website (PRDCAP.com) to share our projects with professionals and stakeholders; 2) Toward creating a centralized poultry disease work group and developing a database in Ohio, we have successfully established a mapping platform in collaboration with The Ohio State University's Center for Urban and Regional Analysis (CURA). This platform is able to accommodate different levels of data sharing and disease visualization tools. We will continue working with Ohio poultry industry to collect and update the data in real-time as a model to expand nationwide; 3) We have established and evaluated multiple respiratory tract sampling methods and found that live-bird swabs can detect the dominant microbes captured by invasive techniques and also respiratory microbiota sampling is highly reproducible especially in the trachea and lower respiratory tract; 4) BiomeSeq, a comprehensive bioinformatic tool to identify and quantify microbiomes (eukaryotic viruses, bacteria, bacteriophage, fungi) using Next Generation Sequencing data (DNASeq and RNASeq) was completed and evaluated. The tool is available on the Docker Hub for use by the research community; 5) We completed analyzing respiratory microbiota from healthy commercial turkeys flocks along the different phases of production. In agreement with the commercial chicken flock surveillance, the body site and age/farm stage had very dominant effects on the quantity, taxonomic composition, and dynamics of core bacteria. Interestingly, microbiota in the ileum and upper respiratory tract (URT) were compositionally more related to each other than to cecum; 6) An analysis of eukaryotic respiratory viral microbiome of a healthy broiler flock from hatch through processing was completed. In general, the broiler tracheal microbiome ecology was found to change in composition and to increase in diversity during growth of the flock. The baseline data generated in this study are necessary for the development of effective microbiome-based interventions to enhance production performance and to prevent and control disease in commercial poultry. Objective 2. Multifactorial etiology Respiratory diseases involve multiple pathogens, and they interact with each other. For example, our study highlighted the role of avian Mycoplasma in exacerbating the clinical outcome of poultry co-infected with respiratory viruses (LPAIV and IBV) and also inducing side effect from normal vaccination (eg. ILTV). Thus, researchers cannot study one pathogen but must look at how the host reacts; that can vary depending on the health condition of the host. The environment, including the air quality on the farm might affect the disease. Our co-infection studies in different environmental conditions provided important and much needed information on the interaction of respiratory pathogens in poultry, which will help improve diagnostics and vaccination strategies needed to control respiratory syndromes in poultry. Specifically, our studies provide practical information on what to expect in regard to clinical outcomes of co-infections with respiratory pathogens in different environmental and host conditions. Our ongoing studies will address the role of co-infections on susceptibility and transmission of respiratory pathogens in poultry. Objective 3. Diagnostic tools and preventive measures 1) The project includes several lines of research related to diagnostics for poultry respiratory pathogens. As part of a related research project, avian metapneumovirus A (AmPV A) was detected in Mexico. New real-time RT-PCR diagnostic tests were bench validated for avian metapneumovirus A, B and C that has better sensitivity and are easier to use than traditional RT-PCR based tests. These tests can be rapidly implemented in the U.S. NAHLN system if an outbreak of these viruses occurs. Separately, next generation sequencing has shown it can detect a variety of pathogens directly from clinical samples in a sequence independent approach. Although our study shows that low sensitivity remains an issue, many different approaches can be used to increase the sensitivity with the expectation that NGS will become a frontline diagnostic method in less than 5 years; 2) Vaccination has been a widely used tool in the poultry industry to prevent or control diseases caused by infectious disease agents. However, in spite of extensive vaccination programs, respiratory pathogens continue to evolve and cause enormous economic losses. Four different vaccine platforms have been successfully developed and being tested for broadly reactive protective efficacy that can confer protection across serotypes or variants. Patent application has been filed for two of the platforms. In addition, commercial company is testing one of the developed vaccinse for commercialization. In addition, newly developed vaccine for emerging disease, ornithobacterium rhinotracheale, is being tested in field condition in collaboration with the industry; 3) To overcome the issue of antibiotic resistance and reconcile the demand of antibiotic withdrawal with maintaining animal health and food security, we have identified several novel non-antibiotic compounds that inhibit avian pathogenic E. coli and Mycoplasma gallisepticum which show low toxicity. These small molecules are highly suitable for commercial application because of their small size, specificity, and stability. We have identified several novel small molecule antimicrobials that are effective in chickens against colibacillosis and mycoplasmosis. Further, these studies have resulted in a patent application and efforts are underway to explore potential licensing options in collaboration with university technology licensing and commercialization office. Objective 4. Extension and Outreach A major obstacle in the control of infectious diseases in poultry is non-science based management practices and lack of proper understanding of the real issues and practical control strategies among the stakeholders including poultry industries and small backyard flock owners. Science-based information on control and prevention of poultry respiratory diseases needs to be disseminated effectively and in a timely manner to our stakeholders. Our extension group have been conducting a comprehensive and effective educational training program on the importance of controlling respiratory diseases (including recent outbreak of infectious coryza and continuing outbreak of Newcastle disease) for veterinarians, extension educators, gamebird producers, small organic and pastured poultry operations and backyard, hobby, and exhibition growers, state and federal government stakeholders, and the general public. We also developed and held a Poultry Respiratory Health Seminar which directly introduced the science-based information obtained mainly from this project. Furthermore, we developed Poultry Disease Workshop specifically for veterinarians and conducting annual workshops in 2 different states (Ohio and Iowa) has created a list of practicing veterinarians who are trained, willing and able to provide veterinary services to the noncommercial poultry population. These trained veterinarians are geographically positioned to allow easier access to veterinary services for this severely underserved noncommercial poultry population. A low-cost vehicle disinfection system was developed and approximately eight rounds of testing have showed the practical value of the system. Many of the extension efforts are highly successful and have merit to be expanded to regional and national level.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Taylor KJM, Ngunjiri JM, Abundo MC, Jang H, Elaish M, KC M, Ghorbani A, Weber BP, Johnson TJ, Lee CW. RESPIRATORY AND GUT MICROBIOTA IN COMMERCIAL TURKEY FLOCKS WITH DISPARATE WEIGHT GAIN TRAJECTORIES DISPLAY DIFFERENTIAL COMPOSITIONAL DYNAMICS. Submitted.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Mulholland, K.A. and C.L. Keeler, Jr. (2019) BiomeSeq: A tool for the characterization of animal microbiomes from metagenomic data. bioRxiv DOI: https://doi.org/10.1101/800995https://www.biorxiv.org/content/10.1101/800995v1.full.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Beltr�n, G., D. J. Hurley, R. M. Gogal Jr., S. Sharif, L. R Read, S. M. Williams, C. F. Jerry, D. A Maekawa, and M. Garc�a. Immune Responses in the Eye-Associated Lymphoid Tissues of Chickens after Ocular Inoculation with Vaccine and Virulent Strains of the Respiratory Infectious Laryngotracheitis Virus (ILTV). Viruses 11 (77) 635, https://doi.org/10.3390/v11070635. 2019.
  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Dipak Kathayat, Linto Antony, Loic Deblais, Yosra A. Helmy, Joy Scaria, and Gireesh Rajashekara. Small molecule adjuvants potentiate colistin activity and attenuate resistance development against Escherichia coli by affecting pmrAB system. Manuscript under review, Frontiers in Microbiology.
  • Type: Journal Articles Status: Accepted Year Published: 2020 Citation: R. A. Zegpi, L. He, Q. Yu, K. S. Joiner, V. L. van Santen, and H. Toro (2020). Limited Protection Conferred by Recombinant Newcastle Disease Virus Expressing Infectious Bronchitis Spike Protein. Avian Diseases 64: (in press).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Chang-Won Lee, John M. Ngunjiri, Michael C. Abundo, Kara Taylor, Mohamed Elaish, Hyesun Jang, Hana Ji, Mahesh KC, Amir Ghorbani. Toward Understanding the Underappreciated Poultry. Respiratory Microbiome. American Society for Microbiology. ASM Microbe 2019. June 20-24, San Francisco, CA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: John Ngunjiri, Kara Taylor, Daral Jackwood, Michael Abundo, Hana Ji, Linda Michel, Mahesh KC, Hyesun Jang, Mohamed Elaish, Chang-Won Lee. Microbiota signatures of viral infections in turkeys and chickens. Poultry Science Association 108th Annual Meeting, July 15-18, 2019, Montr�al, Canada.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Chang-Won Lee, John M. Ngunjiri, Michael C. Abundo, Kara Taylor, Mohamed Elaish, Hyesun Jang, Hana Ji, Mahesh KC, Amir Ghorbani. Toward Understanding the Respiratory Microbiota of Poultry. The American Association of Avian Pathologists (AAAP) Annual meeting, August 2-5, 2019, Washington, D.C.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Khan, M. I. Novel nanoparticle based vaccine against respiratory viruses. 36th Euro Global summit and Expo on Vaccines & Vaccination, London, UK. June 3-4, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Woldt, Wayne E., Ph.D., P.E., Reynolds, Donald L., Ph.D., D.V.M., and Oetjen, M. Unmanned Aircraft Systems for Enhanced Biosecurity Through Aerial Surveillance and Response. Invited conference proceedings and poster presentation. Annual International Meeting of the Association of Unmanned Vehicle Systems International: XPONENTIAL 2019, Chicago, IL  April 28, 2019-May 2, 2019.
  • Type: Other Status: Published Year Published: 2019 Citation: Reynolds, Don and Amy Schmidt. Enhanced Biosecurity Through In-House Composting of Routine Mortalities. Oral presentation. Nebraska Poultry Industries Annual Meeting. April 17  18, 2019. Columbus, NE.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Reynolds, Don and Amy Schmidt. Enhanced Biosecurity Through In-House Composting of Routine Mortalities. 70th North Central Avian Disease Conference, Minneapolis, MN. March 11-12, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Reynolds, Don, Reddy, Jay and Rajkumar Arumugam. Enhancing Innate Immunity for Better Respiratory Disease Protection. Western Poultry Disease Conference - Association of Poultry Science Specialists (ANECA) International meeting. Puerto Vallarta, Mexico. April 2-6, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Reynolds, D., Reddy, J. Arumugam, R. The effects of beta glucan on thrombocytes and innate immunity. International Nutritional Science and Food Chemistry Conference (OLCNSFC-2019). Venice, Italy. November 11-12, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Michael C. Abundo, John M. Ngunjiri, Kara Taylor, Mahesh KC, Amir Ghorbani, Hana Ji, Bonnie P. Youmans, Timothy J. Johnson and Chang-Won Lee. Invasive and non-invasive methods of sampling chicken respiratory microbiota. 100th Conference of Research Workers in Animal Diseases, November 2-5, 2019, Chicago, IL, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: A. G. Arruda. Application of epidemiological tools in investigating disease from voluntary diseases databases. American Association of Avian Pathologists Annual Meeting. Aug 2019, Washington, DC, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kara Taylor, John M. Ngunjiri, Michael C. Abundo, Hana Ji, Mahesh KC, Amir Ghorbani, Bonnie P. Youmans, Timothy J. Johnson and Chang-Won Lee. Progress toward integrating the dynamics of gut and respiratory microbiota into poultry health. 100th Conference of Research Workers in Animal Diseases, November 2-5, 2019, Chicago, IL, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Johnson, TJ. Models for Development of Antibiotic Alternatives in Food Animal Production. Invited presentation at the Microbiome Movement Animal Health and Nutrition Meeting, St Louis, MO, October 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: A. G. Arruda. Update on OH poultry disease mapping platform. 2019 Ohio Poultry Association Annual Board Meeting. Dec 2019, Columbus, OH, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ramon A. Zegpi, C. Breedlove, Steve Gulley, Q. Yu, Vicky van Santen, Haroldo Toro (2019). Protection Conferred by IBV S-ectodomain Expressed from Recombinant NDV LaSota. Annual Convention AVMA/ American Association of Avian Pathologists, Aug 2-6, Washington DC.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: E.R. Benson, R.L. Alphin, D.P. Hougentogler, A.K. Szostek, and J.R. Improving biosecurity through the use of a low cost, open source vehicle cleaning and disinfection system. Poultry Science Association annual meeting. Montreal, Quebec. July 15, 2019  July 19, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: D.P. Hougentogler, E.R. Benson, J.R. Moyle, and R.L. Alphin. 2019. Use of a low cost vehicle wash station. National Meeting on Poultry Health, Processing, and Live Production. Ocean City, MD. October 9, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Hougentogler, D.P., A.K. Szostek, E.R. Benson, R.L. Alphin. 2019. Evaluation of a Low Cost Vehicle Disinfection System. Northeast US Animal Health Association. April 8, 2019. Annapolis, MD.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: E.R. Benson, J.R. Moyle, D.P Hougentogler, and R.L. Alphin. 2019. The Importance of Proper Containment for Biosecurity. Mid Atlantic Nutrition Conference. April 10  11, 2019. Hunt Valley, MD.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Gino Lorenzoni. Presentation at the Pennsylvania Poultry Sales and Service Conference. Infectious Coryza, background and global history. Sept 11-12, 2019. Conference Center Hotel, State College, PA.
  • Type: Other Status: Other Year Published: 2019 Citation: Gino Lorenzoni. Presentation at the Lancaster Poultry Health and Management Seminar. Infectious coryza, infectious bronchitis and mycoplasma. Background. Nov 13, 2019. Farm and Home Center. Lancaster, PA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Pantin-Jackwood, M., Smith, D., Leyson, C., Jackwood, M., Fergunson-Noel. Infections that may affect Avian Influenza pathogenesis - Infectious Bronchitis and Mycoplasma synoviae. Guatemala National Poultry Congress, Guatemala, August 29 to 30, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Moyle, J.R., E.R. Benson, J.R Rhodes, and N.L. Tablante. 2019. Impact of extension educational programs on commercial broiler flock biosecurity. Poultry Science Association annual meeting. July 15  19, 2019. Montreal, Quebec.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Maekawa D., P. Whang, D. Hurley and M. Garc�a. Assessment of T lymphocyte and monocyte dynamics and tissular changes in the trachea of vaccinated challenged chickens against ILTV. Southern Conference on Avian Disease (SCAD). Atlanta, GA, USA, January 28, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Mullholland, K.A., M. Robinson, S.J. Keeler, T. Johnson and C.L. Keeler, Jr., (August 2-5, 2019) Whats in the tracheal microbiome of a commercial broiler poultry flock? Annual Meeting of the American Association of Avian Pathologists, Washington, DC.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Reddy S., Khan, O, Al-Mohmood, M. and Lupiani, B. Use of HVT vector vaccines to express protective antigens from important viral pathognes. American Association of Avian Pathologists (AAAP), AVMA Annual Convention, Washigton, DC. August 2-5, 2019.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Ngunjiri JM, Taylor KJM, Abundo MC, Jang H, Elaish M, KC M, Ghorbani A, Wijeratne S, Weber BP, Johnson TJ, Lee CW. Farm Stage, Bird Age, and Body Site Dominantly Affect the Quantity, Taxonomic Composition, and Dynamics of Respiratory and Gut Microbiota of Commercial Layer Chickens. Appl Environ Microbiol. 85(9). pii: e03137-18. 2019.


Progress 04/01/18 to 03/31/19

Outputs
Target Audience:Poultry industry including backyard poultry farmers; Goverment and state officials involved in control of animal diseases. Scientists in the field of infectious diseases, animal production, Ag-engineering, virology, bacteriology, immunology & vaccinology. Extension and outreach specialists. Changes/Problems:Because of complexity and extensive coordination is required for the project, some of the activity may not be completed in time. In such situation, we will request for no-cost extension to USDA. What opportunities for training and professional development has the project provided?This projects support training of many undergraduate and graduate students, post-docs, research assistants and research associates (listed below). In addition to hand-on experiences, students and post-docs presented the results at the local and national meetings and published the results in scientific journals. Auburn University: Ramon Zegpi, PhD student - Developing vaccine and conducting animal experiment. 0.1 FTE. Fatma Eldemery, PhD student - Production and assessment of recombinant S ectodomain proteins. 0.1 FTE. University of California, Davis: Robin Isobel Lampron, Undergraduate student. Assisted surveys and sampling at small flocks, performed ELISAs. Part-time student. Theodore Derksen, MS student. Performed trainings at the 4-H seminar, conducted surveys and data analysis. 0.5 FTE. Rüdiger Hauck, Post-doctoral scholar. Directed the grad student and help the data analysis and experimental design. 0.1 FTE. University of Connecticut: Jianping Li, PhD student - implementing immunogenicity studies of nanoparticle vaccine. 1 FTE Zeinab Helal, Postdoctoral Fellow - implementing immunogenicity studies of nanoparticle vaccine. 0.5 FTE Shobin Chen, Undergraduate student - Assisting immunogenicity studies of nanoparticle vaccine. 0.1 FTE. Christropher Karch, postdoctoral fellow - development and optimizing of nanoparticles. 0.1 FTE University of Delaware: Monique Robinson, MS student - Conducted microbiome sample collection, nucleic acid purification and data analysis. 1 FTE. Kelly Mulholland, Ph.D. student - developed the bioinformatic pipeline for determining the avian respiratory virome. 1 FTE Laura Donohue, undergraduate student in Animal and Food Sciences - Assisted investigating the microbiome of the respiratory tract of broiler chickens. 0.1 FTE Darian Abreu, Lucas Serge, Dianna Kitt, Xiaolun Guo: All undergraduate engineering students- developing the low cost vehicle undercarriage wash station. Part-time students Alissa Moritz, Adrianna Szostek, Melanie Lopez (designated as a project intern), Laura Burton, and Brittney Anderson: Animal Science and/or Preveterinary Medicine and Animal Bioscience graduate and undergraduate students - Extensively involved in the symposium, disinfection research, and training programs. Part-time. University of Georgia: Emily Aston, PhD student - Conducted animal experiments, and coordinated necropsy and sample collection and testing. Dr. Aston also analyzed the data generated from this experiment. 0.41 FTE. Jongseo Mo, Ph.D. Student - Conducting studies on multiple disease agent identification in a clinical sample to be used to identify polyvalent vaccine. 0.2 FTE. Madelyn Krunkosky, MS student - trained and conducted basic immunology and molecular biology related to ILTV infection in chickens. 0.33 FTE Gabriela Beltrán PhD student - Development and optimization of protocols for processing CALT, HG and trachea samples for reverse transcriptase PCR and flow cytometry analysis. 0.5 FTE. Marianne Dos Santos, Research Professional - animal experiment coordination. 0.2 FTE Victoria Drouet, Masters of Avian Medicine student. Sample collection and data analysis. 0.2 FTE Rachel Jude, MS student. Sample collection and data analysis. 0.4 FTE Mohammadreza Ehsan; Sample collection and data analysis 0.2 EFT University of Minnesota: Bonnie Youmans, PhD student - Coordinating and implementing microbiome analysis. 0.3 FTE. Ohio State University: Michael Abundo, Research Assistant - served as project manager for project coordination. 1 FTE Hana Ji, Research Assistant - assistingin vitro and in vivo experiment. 0.5 FTE John Ngunjiri, Research Associate - Surveillance coordination. 0.1 FTE Mahesh KC, MS student - sample collection, processing, and sequence analysis. 0.5 FTE Dipak Kathayat, PhD student - Learning and implementing small molecule study as antibiotic alternatives. 1 FTE Yosra Mohamed, Postdoctoral Research Associate - Learning and implementing small molecule study. 0.3 FTE Gary Closs Jr., Research associate. Trained in number of assays related to drug screening as well as in animal studies. 0.1 FTE. Penn State University: Lisa Kitto, Technician - extension coordination. 0.03FTE Southeast Poultry Research Laboratory, USDA: Nikolai Lee, Undergraduate Student - trained for sample preparation and processing, and helps with analyzing samples. 0.5 FTE. Andrea Ayala, Research Associate - conducting and coordinating BSL-2 experiment. 1 FTE Seth Cook, Sumer Fink, James Dornbush, Fred Torpy and Benjamin Williams: undergraduate students - work or have worked 15 to 20 hours per week during the reporting period assisting field work Tim Olivier, Research associate. Laboratory analyses. 0.2 FTE Texas A&M University: Owais Khan, postdoctoral fellow - expression of foreign genes against additional pathogens. 0.5 FTE. Aijun Sun & Guoqing Zhuang (Post-doctoral Fellows), Kanika Bajwa, Liao Yifei, and Mohammad Al-Mahmood, Ph.D. student (all Ph.D. students) also involved in vitro and in vivo experiments as part-time. How have the results been disseminated to communities of interest?We developed the main project website (PRDCAP.com) to share our projects with professionals, stakeholders and the general public. To further expand the network, the annual meeting is held on in conjunction with the NC-1180 and the U.S. Animal Health Association Meeting, and received full attention from our stakeholders. The results from individual projects were presented at the international and regional meetings. Several manuscripts are being prepared for publication in timely manner. Furthermore, our extension group actively conduct comprehensive educational training program on the importance of controlling respiratory diseases for Veterinarians, Extension Educators, Gamebird Producers, Small Organic and Pastured Poultry Operations and Backyard, Hobby, and Exhibition Growers and the general public through distribution of brochures, presentation at a poster, presentation at local, regional, and national meetings, workshops and specific training programs. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Ecology & Network: 1) Continuing efforts will be made for the expansion of the nationwide poultry respiratory disease network by expanding both the participants (researchers and stakeholders) and states involved. We willbuild and maintain a digital map of poultry production addlayer of data on this map; 2) Coordinated effort among Ohio, Minnesota and Delaware groups on microbiome project will continue.In addition to testing surveillance samples, we are collaborating with the investigators in this project to sample the respiratory microbiome using models of respiratory infection (refer to Objective 2) and in commercial flocks experiencing respiratory infections. We expect to determine the changes that microbial communities undergo over the course of respiratory pathogen infection. In addition, we expect to identify specific microbial populations that might be favored, altered, or reduced during the infection. Objective 2. Multifactorial etiology: 1) We will conduct follow up studies using commercial chickens and turkeys to compare with the results obtained with the SPF birds. Two studies are planned, one examining coinfection of broiler chickens with LPAIV, IBV and MS, and one examining coinfection of commercial turkeys with LPAIV, NDV and MS. All the data including commercial vs SPF birds data will be collectively analyzed to draw final conclusion of the study; 2) We will continue to assess the effect of IBDV on co-infection of respiratory pathogens.In addition, we will determine the effect of IBDV infection on microbiome and AIV transmission. In addition, information gathered from the 16S microbiome analysis will be used in devising effective control strategies for multifactorial respiratory diseases; 3) We will refineco-infection studies with MS, NDV and IBV vaccines in broilers. We will evaluate the effect of time and sequence of MS infection on NDV and IBV vaccine replication. The information from this study will be used to evaluate poultry industry's management ofMS infected flocks; 4)We will determine the distribution, activation state and function of lymphocytes in trachea and in peripheral blood of chickens after virulent Infectious laryngotracheitis virus (ILTV) challenge that were previously vaccinated with live attenuated or recombinant viral vector ILTV vaccines. Unravelling the differential immune response elements elicited by live attenuated and by recombinant vaccines, relative to virulent challenge, will provide the fundamental understanding of the immunological cascade needed to establish enhanced protection from ILT vaccines. All the proposed studies in Objective 2 involve extensive animal experiments, which have generated a large amount of descriptive data. Collaborative efforts will continuously be made among PIs to collectively interpret the data in relation to multiple factors including respiratory pathogens, immunosuppressive agents, vaccine statuses, and environmental stress for each individual species to mimic the natural condition and better understand the pathogenesis. Objective 3. Diagnostic tools & preventive measures: 1) The current Newcastle disease test was developed without an internal positive control. A recently described internal control based on the enhanced green florescent protein is being evaluated for inclusion in the NDV matrix and fusion tests. This multiplex RT-PCR addition uses de novo synthesized RNA internal control and a short target to make the test easy to implement and at a reasonable cost. The need for an internal control is critical as the recent outbreak in California relies on reliable rRT-PCR diagnostics for the detection and virulence determination of outbreak viruses. Efforts will also continue to optimize the NGS sequencing technology using the Minion sequencer;2) We will optimize the nanoparticle vaccine and vaccination regimen and the protective efficacywill be tested using an established challenge model and compared with other vaccine platforms; 3) Parenteral delivery of trimeric recombinant S1+S2 ectodomain proteins showed that extension of the S1 domain with S2 improves protection against IBV. We will evaluateprotection conferred by S1+S2 ectodomain expressed from recombinant NDV against IBV challenge;4) Regarding the development of HVT vectored vaccine: we will complete evaluation of stability of recombinant viruses by cell culture andIn vivo experiments will be conducted to examine antibody response in chicken.Based on these results, we are negotiating agreements with Elanco, a global animal health company, to potentially exploit commercialization of candidate vaccines; 5) We will evaluate safety and efficacy of live Temperature sensitive (Ts) mutant ORT strain in experimental turkeys. We will also validate the efficacy of the vaccine in field condition; 6) We will evaluate the long-lasting immunity of chimeric NDV-vectored AI vaccination strategy in layer chickens and turkeys against HPAI viruses. Heterologous prime-booster strategy will be utilized by immunization of 1-day-old chicks with chimeric NDV followed by boosting with NDV strain LaSota expressing the HA or HA/NA proteins; 7) We will determinethe efficacy of small molecule growth inhibitor lead (GI-7), probiotics [Lactobacillus rhamnosus GG (LGG), Bifidobacterium lactis Bb12 (Bb12)] and/or peptides (P-1, P-2 and P-3) combinations to control APEC infection in chickens. We will also validate, under field simulated conditions, the efficacy of most effective combination of GI-7, probiotic(s) and peptides to control APEC infection in chickens. Objective 4. Extension & outreach: 1) The UDE & UMD team willcohost poultry respiratory disease training sessions with Delaware Ag Week sessions oriented for regional broiler growers anda one day Poultry Respiratory Disease program. In addition, the team will improve and validate the low-cost vehicle cleaning and disinfection system by adding a low cost solar tank heater to the system. Outreach will be conducted on vehicle biosecurity, and how to implement a low-cost vehicle disinfection system; 2) The primary focus of Connecticut team will continue to be on presentations at local, regional and national extension meetings/workshops and to provide producers, educators and service people with new educational materials and methods of detecting and preventing respiratory disease in poultry;3) Penn State team willdevelop on-line course for small flocks that will include husbandry, biosecurity, disease recognition and prevention.A symposium at yearly AAAP meetings is scheduled on decreasing respiratory diseases in small flocks. The presentations by the team will be recorded and placed on department website for distribution worldwide on management and respiratory disease in small flocks;4) Iowa team will continue the yearly 3 full day workshops, each targeting 15 - 20 small animals and mixed veterinary practitioners. Data collection will be done through initial in-workshop-survey in addition to 4 follow-up surveys, once each quarter, to measure the impact of these workshops on participating veterinarians. We will maintain a map and a list of trained veterinarianswhich will be promoted and solicited to the relevant groups in each state; 5) Georgia group will finalize and distribute an informational video series on Proper hatchery, Vaccination, and Management;6) Nebraska team will continue to develop the Big Red Biosecurity Program website and online educational videos which will be available through the website. We will also further our work with drone technology incorporating thermal imaging and plan on utilizing the technology with several different types of poultry operations. We will also continue to evaluate the feasibility of using in-house composting as a more biosecure method of disposing of routine mortalities. Coordinated effort will be made among participants for effective generation and validation of extension and education materials and approaches.

Impacts
What was accomplished under these goals? Objective 1. Ecology and Neworking:1) We continued to expand the nationwide poultry disease research network. In addition to existing NC-1180 participants and institutions from the majority of poultry producing states, we now have additional participants and collaborators from Mississippi, Texas, California, Nebraska, and Iowa, and Maryland. In addition, a Request for Proposals (RFP) is made each year to stimulate new and innovative research in critical areas which are not included in the current project. We funded four new proposals in 2018. We also maintain project website (PRDCAP.com) to share our projects with professionals and stakeholders.We have also prepared and disseminated newsletters (can be found in website) to highlight the progress of our project to relevant stakeholders and research community;2) We are making progress in creating a centralized disease work group and developing a database that captures the size and the distribution of poultry species and poultry diseases in Ohio. We have addressed the confidentiality issue and companies are taking part in the study and have shared their data for disease mapping using ArcGIS-portal.We will continue working with Ohio poultry industry to collect and update the data in real-time as a model to expand nationwide; 3) We determined baseline respiratory microbiota to understand the microbiome's role in host susceptibility and response to respiratory pathogens and vaccines. We first compared the bacterial communities that were present in the upper and lower respiratory tract of chickens. In addition, we evaluated field-utilized respiratory swab collection methodswhich showed microbiome from tracheal swab and wash are distinct from each other while the tracheal and choanal swabs share a high degree of similarity. We also have sampled healthy flocks of commercial chickens and turkeys along the different phases of production. In general, the body site and age/farm stage had very dominant effects on the quantity, taxonomic composition, and dynamics of core bacteria. Interestingly, microbiota in the ileum and upper respiratory tract (URT) were compositionally more related to each other than to cecum. The overlapping bacteria also contained various levels of several genera with well-recognized avian pathogens. The baseline data generated in this study are necessary for the development of effective microbiome-based interventions to enhance production performance and to prevent and control disease in commercial poultry. Objective 2.Multifactorial etiology: Wehave identified and tested key combinations of respiratory pathogens, immunosuppressive agents, vaccines, and environmental factors: 1)An increase in Mycoplasma Synoviae (MS) shedding was found in birds co-infected with avian influenza virus (AIV) and infectious bronchitis virus (IBV). Tracheal damage more pronounced than LPAIV+IBV co-infection, indicating that MS are necessary to reproduce the lesion in the field; 2)Infectious laryngotracheitis virus (ILTV) and IBV vaccination in MS-infected broilers lead to significant increases in airsacculitis and mortality compared to non-infected broilers; and MS infection also affect vaccine efficacy;3) Co-infection of infectious bursal disease virus (IBDV) enhanceAIV replication and clinical signs while significantly lowering antibody titers. Microbiome study showed that several core microbiota were disruptedwhich was dependent on the body site, the virus, and the occurrence of coinfection;4)ILTV inhibited apoptosis of monocyte-derived macrophages which suggest thegland can be a source of infected mononuclear phagocytes which facilitate virus spread while evading the host immune response; 5)Pullets serially administered live attenuated vaccines against IBV, NDV, and ILTV were protected against homologous challenge with IBV, NDV, or ILTV for at least 36 weeks. Additionally, immunity to individual vaccine viruses was not compromised despite serial administration of multiple live attenuated vaccines; 6) Commercial broilers exposed to moderate levels of NH3 are equally protected against IBV challenge if appropriately vaccinated, and the absence of robust immune activation in vaccinated, challenged birds suggests that the challenge virus was efficiently neutralized before establishing infection. In contrast, NH3 exposure was associated with a higher incidence of airsacculitis in non-vaccinated, challenged birds, despite the apparent lack of differences in the immune response between birds in the NH3-exposed and NH3 control groups. Objective 3.Diagnostic tools andpreventive measures: 1) The Sequence-Independent-Single-Primer-Amplification protocol allowed us to detect and fully characterize genomes of AIV, NDV, and IBV with genome coverage ranging from 94.9% to 100% for all three viruses. Using this approach up to 96 samples can be processed at the same time, which brings the cost per sample down to less than $100 for a complete genome. These newly developed protocols can greatly reduce time and costs necessary to identify the pathogens; 2) IBV-Flagellin self-assembling protein nanoparticle (SAPN) vaccine was developed, induced high antibody responses in chickens, and showed a significant reduction of tracheal virus shedding and tracheal lesion against challenge; 3) We developed new Herpes virus vector to incorporate NDV, IBV and IBDV genes toprotect chickens against three diseases simultaneously with a single vaccine dose in ovo or at hatch which entered into a licensing agreement with Elanco Animal Health for potential commercialization; 4) We generated chimeric NDV expressing the AIV HA protein for prime and LaSota expressing the HA protein for boost. In chicken and turkey studies, we demonstrated a potential use of heterologous prime and boost vaccination to protect poultry against H7 HPAI viruses;5) We identified novel antimicrobials (small molecules) that are effective in chickens against colibacillosis and mycoplasmosis. The study resulted in a patent application and undergoing licensing options for commercialization. Objective 4. Extension and Outreach:1) Univ. of MD and DE group developed a one-day Poultry Respiratory Health Seminar and utilized Delaware Agriculture Week to link back the science developed through this project directly to target audiences.In addition, a low-cost vehicle disinfection system was developed whichiseasily assembled by a grower and uses parts either available on the farm or commonly available;2) Univ. of CT and Penn State group reached both small flock owners and commercial poultry industry.Multiple small flock owner workshops were held throughout Southern New England and Pennsylvania.Posters were distributed and displayed at county fairs, organic and pastured poultry meetings, and regional extension meetings to educate on the danger of respiratory disease in small flocks;3) Iowa Stategroup held three workshops to train 33 actively practicing veterinarians. The distribution of represented clinics across Iowa promotes better access of veterinary services for noncommercial poultry owners;4) Univ. of GA group are developing poultry health video series. The first two videos in the series have been recorded, andare ready to be distributed once the USDA logo has been added to the videos. The next three videos in the series are currently being recorded all at once;5) Univ. of NE team developed and launched the Big Red Biosecurity Program for Poultry website which is designed for lay people with minimal understanding of biosecurity principles. In addition, we have developedmethods to enhance biosecurity in poultry operations utilizing small unmanned aircraft system technology. We also successfully evaluated the feasibility of using in-house composting as a more biosecure method of disposing of routine mortalities.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Derksen T.J., Lampron R., Hauck R., M. Pitesky, R. A. Gallardo. Biosecurity Assessment and Seroprevalence of Respiratory Diseases in Backyard Poultry Flocks Located Close and Far from Commercial Premises. Avian Diseases. 62(1):1-5. 2018.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Li J, Helal ZH, Karch CP, Mishra N, Girshick T, Garmendia A, Burkhard P, Khan MI. A self-adjuvanted nanoparticle based vaccine against infectious bronchitis virus. PLoS One. 14;13(9):e0203771. 2018.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Helmy YA, Deblais L, Kassem II, Kathayat D, Rajashekara G. Novel small molecule modulators of quorum sensing in avian pathogenic Escherichia coli (APEC). Virulence. 9(1):1640-1657. 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Kathayat D, Helmy YA, Deblais L, Rajashekara G. Novel small molecules affecting cell membrane as potential therapeutics for avian pathogenic Escherichia coli. Sci Rep. 17;8(1):15329. 2018.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Vagnozzi AE, Beltr�n G, Zavala G, Read L, Sharif S, Garc�a M. Cytokine gene transcription in the trachea, Harderian gland, and trigeminal ganglia of chickens inoculated with virulent infectious laryngotracheitis virus (ILTV) strain. Avian Pathol. 47(5):497-508. 2018.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Johnson TJ, Youmans BP, Noll S, Cardona C, Evans NP, Karnezos TP, Ngunjiri JM, Abundo MC, and Lee CW. A consistent and predictable commercial broiler chicken bacterial microbiota in antibiotic-free production displays strong correlations with performance. Applied and Environmental Microbiology 84: e00362-18. 2018.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Ngunjiri JM, Taylor KJM, Abundo MC, Jang H, Elaish M, Kc M, Ghorbani A, Wijeratne S, Weber BP, Johnson TJ, Lee CW. Farm stage, bird age and body site dominantly affect the quantity, taxonomic composition, and dynamics of respiratory and gut microbiota of commercial layer chickens. Appl Environ Microbiol. 2019. [Epub ahead of print]
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Aston EJ, Jordan BJ, Williams SM, Garc�a M, Jackwood MW. Effect of Pullet Vaccination on Development and Longevity of Immunity. Viruses. 11(2). 2019.
  • Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Aston, E.J.; Jackwood, M.W.; Gogal, R.M. Jr.; Hurley, D.J.; Fairchild, B.D.; Hilt, D.A.; Cheng, S.; Tensa, L.R.; Garcia, M.; Jordan, B.J. Ambient ammonia does not inhibit the immune response to infectious bronchitis virus vaccination and protection from homologous challenge in broiler chickens. Vet. Immunol. and Immunopath. 2019. submitted and under review.
  • Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Ayala, A.J., Hernandez, S.M., Olivier, T.L., Welch, C.N., Dimitrov, K.M., Goraichuk, I.V, Afonso, C.L., and P.J. Miller. Experimental Infection and Transmission of Lentogenic Newcastle Disease Virus in Passerines. 2019. Submitted and under review.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Reynolds, Donald L. Developing and Evaluating a Biosecurity Plan. Nebraska Poultry Industries Annual Meeting. Columbus, NE. February 21-22, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Reynolds, Donald L. Wayne Woldt and Marcia Oetjen. The Big Red Biosecurity Program for Poultry Develops Drone Technology for Bio-surveillance. American Association of Avian Pathologists / American Veterinary Medical Association annual convention meeting. Denver, CO. July 13  17, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Woldt, Wayne E., Ph.D.,P.E., Reynolds, Donald L. DVM, Ph.D., Oetjen, M., and Bhatti, S. Unmanned Aircraft Systems for Enhanced Biosecurity Through Aerial Surveillance. Annual International Meeting of the American Society of Agricultural and Biological Engineers, Detroit, MI  July 29-Aug 3, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Aston, E., Jackwood, M., Gogal, R., Garcia, M., Fairchild, B., Hilt, D., & Jordan, B. Effect of Ammonia on the Immune Response to Infectious Bronchitis Virus Vaccination and Protection from Homologous Challenge in Broiler Chickens. International Avian Respiratory Disease Conference, Athens GA. May 29-June 1, 2018.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Pantin-Jackwood, Mary, Diane Smith, Christina Leyson, Sungsu Youk, Mark Jackwood, Maricarmen Garcia, Naola Fergunson-Noel. Co-infection of Chickens with Low Pathogenicity Avian Influenza Virus, Infectious Bronchitis Virus and Mycoplasma Synoviae. Proceedings of the 2018 International Avian Respiratory Disease Conference, Athens, GA, May 29-June 1, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Pantin-Jackwood, Mary, Diane Smith, Christina Leyson, Sungsu Youk, Mark Jackwood, Maricarmen Garcia, Naola Fergunson-Noel. Co-infection studies with respiratory viruses and/or Mycoplasma synoviae in chickens and turkeys. American Association of Avian Pathologists (AAAP) Annual meeting in Denver, Colorado, July 13-17, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Pantin-Jackwood, Mary, Diane Smith, Christina Leyson, Sungsu Youk, Mark Jackwood, Maricarmen Garcia, Naola Fergunson-Noel, Erica Spackman E., Chris Stephens. Co-infection studies with AIV, IBV, IBDV and Mycoplasma synoviae in chickens and turkeys. Animal Health FLU SEMINAR organized by Boehringer Ingelheim, Playa del Carmen, Mexico, November 7 and 8, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Valerie C Marcano, Susan M Williams, Maricarmen Garci?a, Marianne Dos Santos, and Naola Ferguson-Noel. Tracheal Lesion Evaluation of broilers co-infected with Mycoplasma synoviae and Infectious Laryngotracheitis. American Veterinary Medical Association (AVMA) Annual Convention, Denver, CO. July 13th -17th, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Khan, M. I. Novel Nanoparticles based vaccines to protect against respiratory viral infections in poultry. Shandong Academy of Agriculture. Seminar series. Jinan City, Shandong, China. August 23, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Johnson T. Antibiotic-free alternatives to improve health and performance in commercial turkeys. The Conference for Research Workers in Animal Diseases, Chicago, IL, December 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Weber B and Johnson T. Effects of turkey poult hatch-brood incubation on the gastrointestinal microbiota before and after placement on commercial farms. The Poultry Science Association Annual Meeting, San Antonio, TX, July 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Johnson, T. Microbiome-Based Approaches for Probiotic Development in Poultry Production. Microbiome Movement Animal Health Conference, St. Louis, MO, May 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Johnson, T. The Poultry Microbiome: Strategies to Modulate, Mitigate, and Mediate. Animal Microbiome Congress USA, Kansas City, MO, March 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Yosra A. Helmy, Dipak Kathayat, Mostafa Ghanem, Loic Deblais, Gary Closs, Vishal Srivastava, Mohamed El-Gazzar, and Gireesh Rajashekara. Novel small molecules with antimicrobial activities against Mycoplasma gallisepticum infections in poultry. The 99th annual conference of research workers in animal Diseases (CRWAD). Chicago. IL. December 1 - 4. 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Kathayat D, Helmy YA, Deblais L, Rajashekara G. Small molecule adjuvants potentiate the antimicrobial activity of colistin against avian pathogenic Escherichia coli. 99th Annual Conference of Research Workers in Animal Diseases (CRWAD). Chicago. IL. December 1 - 4. 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Johnson,T. Microbiome Manipulation: Prebiotics and Probiotics. New York Academy of Sciences Controlling Antimicrobial Resistance in Food Animal Production meeting, New York, NY, September 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Beltr�n, G., S. M. Riblet, D. J. Hurley, M. Garc�a. Phenotypic changes of T and B cells in the conjunctiva associated-lymphoid tissue and Harderian gland during infection or vaccination with Infectious laryngotracheitis virus (ILTV). XV Avian Immunology Research Group (AIRG) Meeting. Oxford, UK, September 3 - 5, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Beltr�n, G., S. M. Riblet, D. J. Hurley, M. Garc�a. Changes in CD4+/CD8+ T cells in the conjunctiva associated-lymphoid tissue (CALT) and Harderian gland (HG) during infection or vaccination with Infectious laryngotracheitis virus (ILTV). Annual meeting of the American Association of Avian Pathologists (AAAP). Denver, CO., July 13 to 14, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Beltr�n, G., S. M. Williams, G. Zavala, J. S. Guy and M. Garc�a. The replication patterns of infectious laryngotracheitis virus (ILTV) pathogenic strain and chicken embryo origin (CEO) vaccine are ruled by the route of inoculation. 2nd International Avian Respiratory Disease Conference. Athens, GA., May 29 to June 1, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: John M. Ngunjiri, Michael C. Abundo, Kara J. Taylor, Mahesh KC, Hyesun Jang, Mohamed Elaish, Amir Ghorbani, Hana Ji, Bonnie P. Youmans, Timothy J. Johnson, Chang-Won Lee. Respiratory microbiota of chicken layers: diversity, dynamics, and correlations with vaccines & infections. The 99th Annual Conference of Research Workers in Animal Diseases. Chicago, Illinois, USA. December 1-4, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Kara J. Taylor, John M. Ngunjiri, Michael C. Abundo, Mahesh KC, Hyesun Jang, Mohamed Elaish, Amir Ghorbani, Hana Ji, Chang-Won Lee. Disruption of turkey core gut and respiratory microbiome by reovirus and influenza virus. The 99th Annual Conference of Research Workers in Animal Diseases. December 1-4, 2018. Chicago, Illinois, USA. December 1-4, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Chang-Won Lee. Poultry Respiratory Disease Coordinated Agricultural Project (PRD-CAP). The 99th Annual Conference of Research Workers in Animal Diseases. Chicago, Illinois. December 1-4, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: John M. Ngunjiri, Mahesh KC, Michael Abundo, Mohamed Elaish, Amir Ghorbani, Hyesun Jang, Chang-Won Lee. Influenza virus infection and respiratory microbiome in chickens infected with infectious bursal disease virus.10th International Symposium on Avian Influenza Avian Influenza in Poultry and Wild Birds. Brighton, UK. April 15 - 18, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Michael C. Abundo, John M. Ngunjiri, Hyesun Jang, Mohamed Elaish, Mahesh KC, Amir Ghorbani, Bonnie P. Youmans, Timothy J. Johnson and Chang-Won Lee (2018). Microbiome in the upper and lower respiratory tract and standardization of sample collection methods in clinically healthy chicken layers. The American Association of Avian Pathologists (AAAP) Annual meeting. Denver, Colorado. July 13-17, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: John M. Ngunjiri, Mohamed Elaish, Michael Abundo, Amir Ghorbani, Hyesun Jang, Mahesh K C, Michael Abundo, Chang-Won Lee. Influenza virus infection and respiratory microbiome of turkeys.10th International Symposium on Avian Influenza Avian Influenza in Poultry and Wild Birds. Brighton, UK. April 15 - 18, 2018.
  • Type: Websites Status: Published Year Published: 2019 Citation: A Novel, Translational, Multidisciplinary Approach to Control Poultry Respiratory Diseases in the United States. PRD-CAP project main website. PRDCAP.com
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Reynolds, Donald L. and Amy Schmidt. Enhanced Biosecurity Through In-House Composting of Routine Mortalities. 70th North Central Avian Disease Conference, Minneapolis, MN. March 11-12, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Michaela C. Olson, Robert E. Porter, Sagar M. Goyal, Sally L. Noll, H. Wayne Martin, Sunil K. Mor. Investigating the respiratory virome of backyard poultry and need for owner education about respiratory pathogens. 70th North Central Avian Disease Conference, Minneapolis, MN, March 12, 2019.


Progress 04/01/17 to 03/31/18

Outputs
Target Audience:Poultry industry including backyard poultry farmers; Goverment and state officials involved in control of animal diseases. Scientists in the field of infectious diseases, animal production, Ag-engineering, virology, bacteriology, immunology & vaccinology. Extension and outreach specialists. Changes/Problems:The poultry industry in the majority of states has 3 major segments (broilers, layers, and turkeys) with very little communication between them. The concept of having a centralized database and data sharing is unfamiliar in the poultry industry and there are concerns about confidentiality and data security. Working closely with the poultry association will enable us to move forward with the project. With tremendous effort in 2017 exercising multiple meetings and communications, we have obtained several formal participation intents from major producers in Ohio. We will continue working with Ohio poultry industry and OhioPoultry Associationto collect and update the data in real-time. What opportunities for training and professional development has the project provided?This projects support training of many undergraduate and graduate students, post-docs, research assistants and research associates (listed below). In addition to hand-on experiences, students and post-docs presented the results at the local and national meetings and also publishedthe resultsin scientific journals. Auburn University: Ramon Zegpi, PhD student - Developing vaccine and conducting animal experiment. 0.1 FTE. Fatma Eldemery, PhD student - Production and assessment of recombinant S ectodomain proteins. 0.1 FTE. University of California, Davis: Robin Isobel Lampron, Undergraduate student. Assisted surveys and sampling at small flocks, performed ELISAs. Part-time student. Theodore Derksen, MS student. Performed trainings at the 4-H seminar, conducted surveys and data analysis. 0.5 FTE. Rüdiger Hauck, Post-doctoral scholar. Directed the grad student and help the data analysis and experimental design. 0.1 FTE. University of Connecticut: Jianping Li, PhD student - implementing immunogenicity studies of nanoparticle vaccine. 1 FTE Zeinab Helal, Postdoctoral Fellow - implementing immunogenicity studies of nanoparticle vaccine. 0.5 FTE Shobin Chen, Undergraduate student - Assisting immunogenicity studies of nanoparticle vaccine. 0.1 FTE. Christropher Karch, postdoctoral fellow - development and optimizing of nanoparticles. 0.1 FTE University of Delaware: Monique Robinson, MS student - Conducted microbiome sample collection, nucleic acid purification and data analysis. 1 FTE. Kelly Mulholland, Ph.D. student - developed the bioinformatic pipeline for determining the avian respiratory virome. 1 FTE Laura Donohue, undergraduate student in Animal and Food Sciences - Assisted investigating the microbiome of the respiratory tract of broiler chickens. 0.1 FTE Darian Abreu, Lucas Serge, Dianna Kitt, Xiaolun Guo: All undergraduate engineering students - developing the low cost vehicle undercarriage wash station. Part-time students Alissa Moritz, Adrianna Szostek, Melanie Lopez (designated as a project intern), Laura Burton, and Brittney Anderson: Animal Science and/or Preveterinary Medicine and Animal Bioscience graduate and undergraduate students - Extensively involved in the symposium, disinfection research, and training programs. Part-time. University of Georgia: Emily Aston, PhD student - Conducted animal experiments, and coordinated necropsy and sample collection and testing. Dr. Aston also analyzed the data generated from this experiment. 0.41 FTE. Madelyn Krunkosky, MS student - trained and conducted basic immunology and molecular biology related to ILTV infection in chickens. 0.33 FTE Gabriela Beltrán PhD student - Development and optimization of protocols for processing CALT, HG and trachea samples for reverse transcriptase PCR and flow cytometry analysis. 0.33 FTE. Marianne Dos Santos, Research Professional - animal experiment coordination. 0.2 FTE Victoria Drouet, Masters of Avian Medicine student. Sample collection and data analysis. 0.2 FTE Rachel Jude, MS student. Sample collection and data analysis. 0.4 FTE University of Minnesota: Bonnie Youmans, PhD student - Coordinating and implementing microbiome analysis. 0.3 FTE. Ohio State University: Michael Abundo, Research Assistant - served as project manager for project coordination. 1 FTE Hyesun Jang, PhD student - implementing infectious animal experiment for microbiome study. 0.5 FTE John Ngunjiri, Research Associate - Surveillance coordination. 0.1 FTE Mahesh KC, MS student - sample collection, processing, and sequence analysis. 0.5 FTE Dipak Kathayat, PhD student - Learning and implementing small molecule study as antibiotic alternatives. 1 FTE Yosra Mohamed, Postdoctoral Research Associate - Learning and implementing small molecule study. 0.3 FTE Penn State University: Lisa Kitto, Technician - extension coordination. 0.03FTE Southeast Poultry Research Laboratory, USDA: Nikolai Lee, Undergraduate Student - trained for sample preparation and processing, and helps with analyzing samples. 0.5 FTE. Andrea Ayala, Research Associate - conducting and coordinating BSL-2 experiment. 1 FTE Seth Cook, Sumer Fink, James Dornbush, Fred Torpy and Benjamin Williams: undergraduate students - work or have worked 15 to 20 hours per week during the reporting period assisting field work Tim Olivier, Research associate. Laboratory analyses. 0.2 FTE Texas A&M University: Owais Khan, postdoctoral fellow - expression of foreign genes against additional pathogens. 0.5 FTE. Aijun Sun & Guoqing Zhuang (Post-doctoral Fellows), Kanika Bajwa, Liao Yifei, and Mohammad Al-Mahmood, Ph.D. student (all Ph.D. students) also involved in vitro and in vivo experiments as part-time. How have the results been disseminated to communities of interest?We developed the main project website (PRDCAP.com) to share our projects with professionals, stakeholders and the general public. To further expand the network, the annual meeting is held on in conjunction with the NC-1180 and the U.S. Animal Health Association Meeting, and received full attention from our stakeholders. The results from individual projects were presented at the international and regional meetings. Several manuscripts has been published and are being prepared for publication in timely manner. Furthermore, our extension group actively conduct comprehensive educational training program on the importance of controlling respiratory diseases for Veterinarians, Extension Educators, Gamebird Producers, Small Organic and Pastured Poultry Operations and Backyard, Hobby, and Exhibition Growers and the general public through distribution of brochures, presentation at a poster, presentation at local, regional, and national meetings, workshops and specific training programs. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Understand the ecology of poultry respiratory diseases 1) Continuing efforts will be made for the expansion of the nationwide poultry respiratory disease network by expanding both the participants (researchers and stakeholders) and states involved. We will continue working with poultry industry to collect and update the data of poultry farms in Ohio. We will then build and maintain a digital map of poultry production in Ohio. We will add another layer of data on this map including the poultry disease data. 2) Coordinated effort among Ohio, Minnesota and Delaware groups on microbiome project will continue. In addition to bacterial and viral community, we will profile baseline fungal communities in the respiratory system (trachea) and intestine (ileum). In addition to testing surveillance samples, we are collaborating with the investigators in this project to sample the respiratory microbiome using models of respiratory infection (refer to Objective 2) and in commercial flocks experiencing respiratory infections. We expect to determine the changes that microbial communities undergo over the course of respiratory pathogen infection. In addition, we expect to identify specific microbial populations that might be favored, altered, or reduced during the infection. Objective 2. Investigate the multifactorial etiology involving poultry respiratory diseases 1) Co-infection of SPF turkeys will be conducted with a LPAIV isolate, NDV, and MS. This study will complement the previous co-infection study in turkeys by determining the role of NDV vaccine strains in co-infections in turkeys. In addition, we will study the effect of co-infections on the susceptibility and transmissibility of respiratory pathogens in chickens. 2) We will continue to assess the effect of IBDV on co-infection of respiratory pathogens.In addition, we will determine the effect of IBDV infection on microbiome and highly pathogenic AIV transmission. 3) We plan to continue the co-infection studies with MS and NDV vaccines in broilers. We will evaluate the effect of time and sequence of MS infection on NDV vaccine replication. We will conduct a co-infection study with MS and IBV vaccine, and evaluate clinical signs as well as shedding of the vaccine. 4)We willdetermine if multiple vaccinations given simultaneously can equally immunize birds. We will vaccinate SPF leghorn chicks with 3 live attenuated vaccines (IBV, NDV and ILTV) or 4 live attenuated vaccines (IBV-Mass, IBV-GA08, NDV and ILTV) to determine if the birds can sort out and respond immunologically to all of the different vaccines. Challenge will also be conducted to assess protection. 5) We will evaluate the leukocyte phenotypic shifts in the respiratory lympohoid tissues after ocular infection with ILTVor vaccination. We will also assess innate immune gene expression vaccinated via the ocular and oral route. If time and resources permit, we will perform a additional experiment where the immune response after co-vaccination with ILTV and IBV will be assessed. 6) We will evaluate the effects of reduced air quality (high ammonia levels) on the onset (infection), transmission and severity of respiratory disease caused by IBV alone, IBV+ILTV given simultaneously and IBV+MS given simultaneously. In addition, a group of birds vaccinated for IBV will be included to access the role that IBV plays in the severity of disease in the presence or absence of high ammonia levels. Objective 3. Develop new and improved diagnostic tools, vaccines, and novel preventive measures 1) We will compare specificity and sensitivity of commonly used diagnostic tests with newly developed techniques and protocols. Efforts to complete the bench validation of AIV and NDV will be completed, and the test will be transferred to the National Veterinary Services Laboratories to do additional evaluations on reference and clinical samples. The currently available IBV molecular tests will be compared and evaluated. The goal of the proposal is to identify the best tests for each respiratory pathogen that will be shared with all interested diagnostic laboratories. 2) Further testing/repeat of various IBV nanoparticle constructs for immunogenicity and heterologous protection will be conducted against challenge with pathogenic strains. The protective efficacy of the nanoparticle vaccines will be tested using an established challenge model. 3) Although our initial results using the S2 gene of IBV were very promising, new constructs using the S2 sequence of other IBV have failed to show the same effectiveness. Because our results using trimeric recombinant S1+S2 ectodomain proteins show that extension of the S1 domain with S2 improve protection against IBV, we will use two different platforms for vaccine delivery to maximize protection efficacy. 4) We will evaluate new strategies to improve the single and simultaneous high efficient expression of IBV S1 and IBDV VP2 genes. To achieve this goal, we will test different promoters and gene orientations in the cloning sites already identified. Replication of recombinant vaccines in chickens, and their ability to induce immune response will be examined. 5) We will conduct pilot studies in chickens under field conditions by infecting the chickens orally and treating the most promising small molecules forprevention of colibacillosisorally in water. We will also determine the effect of the lead growth inhibitors for Mycoplasma in chickens. Objective 4. Extension and outreach program 1) The UDE & UMD team will use two pronged training approach in which (a) cohosting poultry respiratory disease training sessions with Delaware Ag Week sessions oriented for regional broiler growers and (b) a one day Poultry Respiratory Disease program. 2) The primary focus ofUConn will be presentations at local, regional and national extension meetings/workshops and to provide producers, educators and service people with new educational materials and methods of detecting and preventing respiratory disease in poultry. This approach is especially useful for the small scale producers. 3) In addition to Ohio, the Poultry Medicine Workshops for veterinarians will be held in the state of Iowa. This will strengthen the results and conclusions of this study and Extension effort bycollecting and utilizing datafrom two different states. 4) Penn State University will continue to work on greater distribution on a regional and national basis will be attempted by regional meetings in New England and Pennsylvania.We are also planning an on-line course for small flocks that will include husbandry, biosecurity, disease recognition and prevention. 5) Georgia group will shoot an informationalvideo series which the contents have been already developed in detail. 6) UC Davis group will continue with the backyard flock seminars, network analysis and surveillance. 7)UNE grouphas plan on training and educational modules developed and ready for use by the end of the calendar year. We are planning to offer this training via workshops / conferences and through online means. We will continue our work exploring the most resource effective way to utilize drone technology for more effective biosecurity programs and program auditing. We expect that some of the proposed research and extension effort will be highly successful and have merit to be expanded to regional and national level. In addition, we expect to identify need for additional extension effort as new findings accumulate from the proposed researches. The host institution maintains funds to support additional extension effort annually. Coordinated effort will be made among participants for effective generation and validation of extension and education materials and approaches.

Impacts
What was accomplished under these goals? Objective 1. Understand the ecology of poultry respiratory diseases 1) We continued to expand the nationwide poultry disease research network. In addition to existing NC-1180 participants and institutions from the majority of poultry producing states, we now have additional participants and collaborators from Mississippi, Texas, California, Nebraska, and Iowa. The 3rd annualmeeting was held in conjunction with the NC-1180 and the U.S. Animal Health Association meetings on October 18-19, 2017 and received full attention from our stakeholders. In addition, a Request for Proposals (RFP) is made each year to stimulate new and innovative research in critical areas which are not included in the current project. We funded two new proposals in 2017. The proposals for 2018 SEED funding are currently under review. We also maintain project website (PRDCAP.com) to share our projects with professionals and stakeholders. All progress reports presented at the annual meeting have been uploaded to our website. All these efforts will support the expansion of the national poultry disease research network. 2) We are making progress in creating a centralized disease work group and developing a database that captures the size and the distribution of poultry species and poultry diseases in Ohio. We have addressed the data confidentiality issue and have obtained several formal participation intents from major producers in Ohio. We will continue working with Ohio poultry industry to collect and update the data in real-time as a model platform to expand nationwide. 3) Respiratory microbiome and metagenomic profiling is being conducted in all 3 species or types: broilers, layers, and turkeys. We conducted extensive surveillance in all 3 species in Delaware, Minnesota, and Ohio. The following outlines key findings: a) Anatomical sites examined are distinct; b) There is clear and predictable microbiome succession by age;3) The tracheal microbiome is dominated by Lactobacillus species; and 4) Specific bacterial taxa strongly correlate with enhanced bird performance. We also developed methods which can identify both DNAand RNA virusesin respiratory samples. Objective 2. Investigate the multifactorial etiology involving poultry respiratory diseases Weidentified and tested key combinations of respiratory pathogens, immunosuppressive agents, vaccines, and environmental factors and thefollowing outlines key findings: 1)An increase in Mycoplasma Synoviae (MS) shedding was found in birds co-infected with low pathogenic avian influenza virus (LPAIV) and infectious bronchitis virus (IBV) compared to birds only infected with MS. The damage to the trachea was more pronounced than seen in our previous LPAIV+IBV co-infection studies, indicating that MS are likely necessary to reproduce the lesion in the field. 2) Co-infection of infectious bursal disease virus (IBDV) enhancedLPAIV replication and clinical signs. In addition, significantly lower antibody titers were observed in birds from the co-infection group. 3) Infectious laryngotracheitis (ILT) vaccination in MS-infected broilers lead to significant increases in airsacculitis and mortality.MS infectionalso induced more severe clinical signs and higher mortality rates after ILTV challenge. 4) We have collected extensive data which are being evaluated to determine the effect of vaccination schedule on development of immunity and how co-infections with different pathogens can compromise protection. 5) Innate immune gene expression showed that ILTV alters signaling pathways which favor viral replication in the upper respiratory tract. At the same time, selected cells within the lymphoid tissues and trachea are capableof rapidly establishing antiviral state. 6) High ammonia exposure was associated with decreased body weights and increased airsacculitis in non-vaccinated challenged birds. Objective 3. Develop new and improved diagnostic tools, vaccines, and novel preventive measures 1) Sequence-Independent-Single-Primer-Amplification protocol was developed whichcan characterize the genomes of AIV, NDV, and IBV simultaneously in single clinical sample.This newly developed protocol can greatly reduce time and costs necessary to identify the pathogens. 2) We designed novel IBV vaccines using self-assembled peptide nanoparticle (SAPN) platform andadded flagellin for immunostimulation. The self-adjuvanted SAPN significantly reduced IBV shedding and pathology. 3) We showed that S2 domain of IBV contributes substantially to binding affinity which may confer protection against challenge virus. We produced recombinant LaSota virus expressing a codon-optimized S ectodomain for protection study. 4)We demonstratedthe stability of kidney-cell-adapted IBV that precludes emergence of vaccine virus subpopulationsand also reduce the potential forthe emergence of subpopulations from challenge virus. 5) We developed new Herpes virus vector to incorporate NDV, IBV and IBDV genes toprotect chickens against three correspondingdiseases simultaneously with a single vaccine dose in ovo or at hatch. 6) We identified novel non-antibiotic compounds (smallmolecules)for the control of colibacillosis and Mycoplasma infection. These compounds demonstratedgood safety in vitro andefficacy in wax moth model. Objective 4. Extension and outreach program 1) University of MD (UMD)and UDE extension group: A one-day Poultry Respiratory Health Seminar was developed and added onto a five-day Emergency Poultry Disease Response Course to link back the science developed through this project directly to target audiences and to translate research findings into practices.The team also leveraged external funding and opportunities to provide regional (Delaware and Pennsylvania) and agency (USDA National Veterinary Stockpile) specific training on foam depopulation and provided two multiple day training programs for international veterinarians on emergency poultry disease response. 2) UConn and Penn State extension group reached both small flock owners and commercial poultry industry.Multiple small flock owner workshops were held throughout Southern New England and Pennsylvania.Posters were distributed and displayed at county fairs, organic and pastured poultry meetings, and regional extension meetings to educate on the danger of respiratory disease in small flocks. Four brochures were prepared and 5000+ were distributed to the youth and adults from the poultry meetings. 3) The Ohio State extension group provided specific education and training, in the form of a full day workshop, for practicing veterinarians in Ohio who will provide a much needed basic veterinary services to the noncommercial poultry. During 2016 and 2017, through the 6 workshops, we added 98 practicing veterinarians that are newly trained for poultry medicine at an entry level. 5) UC Davisgroup has developed an extensive map of more than 600 small flocks using a Geo survey database. From the mapped flocks, the group has sampled from flocks located close and far from commercial poultry premises. The study showed that wearing dedicated shoes decreased pathogen-specific antibody. History of wild bird contact had a clear effect on an increased seroprevalence to several pathogens. 6) UNE team recently joined the project in 2017 and are developing the Big Red Biosecurity Program for Poultry in the state.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Derksen T.J., Lampron R., Hauck R., M. Pitesky, R. A. Gallardo. Biosecurity Assessment and Seroprevalence of Respiratory Diseases in Backyard Poultry Flocks Located Close and Far from Commercial Premises. Avian Diseases. 2017. In Press.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Fatma Eldemery, Kellye S. Joiner, Haroldo Toro, Vicky L. van Santen. Protection against infectious bronchitis virus by spike ectodomain subunit vaccine. Vaccine. 35:5864-5871. 2017.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Fatma Eldemery, Yufeng Li, Qingzhong Yu, Vicky L. van Santen, and Haroldo Toro. Infectious Bronchitis Virus S2 of 4/91 Expressed from Recombinant Virus Does Not Protect Against Ark-type Challenge. Avian Diseases. 61: 397-401. 2017.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zegpi R.A., C. Breedlove, V.L. van Santen, C.R. Rasmussen-Ivey, H. Toro. Kidney Cell-Adapted Infectious Bronchitis ArkDPI Vaccine is Stable and Protective. Avian Diseases. 61:221-228. 2017.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Karch, C., P., J. Li, C. Kulangara, S.M. Paulillo, S.K. Raman, S. Emadi, A. Tan, Z.H. Helal, Q. Fan, M. I. Khan, P. Burkhard. Vaccination with self-adjuvanted protein nanoparticles provides protection against lethal influenza challenge. Nanomedicine: Nanotechnology, Biology and Medicine.13(1): 241-251. 2017.
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Li, J., Karch, C., Tan, A., Helal, Z., Fan, Q., Garmendia, A., Girshick, T., Burkhard, P. and Khan, M. I. Flagellin Built-in Novel Nanoparticle-based vaccine induces specific neutralizing antibodies against infectious bronchitis virus. Under Review.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Krunkosky, M., M. Garc�a, L.G. Beltran, E. Karpuzoglu-Belgin, J Levin, R.J. Williams, R.M. Gogal Jr. Seeding of the Mucosal Leukocytes in the HALT and Trachea of White Leghorn Chickens. Journal of Immunoassays and Immunohistochemisty. In Press.
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Helmy YA, Rajashekara G et al. Identifying novel quorum sensing inhibitors to control avian colibacillosis. Under Review.
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Kathayat D, Helmy YA, Deblais L, and Rajashekara G. Novel small molecule compounds with antimicrobial activities against avian pathogenic Escherichia coli. Under Review
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Timothy Johnson, Bonnie Youmans, Sally Noll, Carol Cardona, Nicholas Evans, Peter Karnezos, John Ngunjiri, Michael Abundo, and Chang Lee. A consistent and predictable commercial broiler chicken bacterial microbiome in antibiotic-free production displays strong correlations with performance. Under Review.
  • Type: Other Status: Published Year Published: 2017 Citation: Lee CW. Helping chickens breathe easier. Retaking the Field. SoAR Foundation. Volume 2, March 2017.
  • Type: Other Status: Published Year Published: 2017 Citation: Lee CW. Global health is for the birds: Studying avian diseases to protect human health. Scientia. 113:132-135. 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: H. Toro, R.A. Zegpi, V.L. van Santen. Increased Population Homogeneity in ArkDPI Vaccine Precludes Emergence of Subpopulations after Challenge. XXth World Veterinary Poultry, 4th-8th Sept., Edinburgh, UK. 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Derksen T., R. Lampron, R. Hauck, R. A. Gallardo. 2017. California backyard poultry as a reservoir for respiratory diseases. 66th Western Poultry Disease Conference, Sacramento, CA. March 20, 2017.
  • Type: Other Status: Other Year Published: 2017 Citation: Gallardo RA. Small flock poultry biosecurity and management for 4H volunteers. San Luis Obispo, CA. October, 2017.
  • Type: Other Status: Other Year Published: 2017 Citation: Khan, Mazhar I. Development of novel nanoparticle-base vaccines for avian respiratory diseases. Beijing Academy of Agriculture, Beijing, China, August 28, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Karch, Christopher, Jianping Li , Caroline Kulangara , Sara M. Paulillo , Senthil K. Raman, Sharareh Emadi , Anmin Tan , Zeinab H. Helal, Qing Fan, Mazhar I. Khan and Peter Burkhard. Vaccination with self-adjuvanted protein nanoparticles provides protection against lethal influenza challenge. Influenza Vaccines for the World. Lausanne, Switzerland. April 19-21, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zeinab H. Helal, Jianping Li, Christopher Karch, Peter Burkhard and Mazhar I. Khan. Immunogenicity and Protective Efficacy of Peptide Nanoparticle Based Vaccine against Infectious Bronchitis Virus Infections. ASM Microbiology, New Orleans, LA. June 1-5, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Jianping Li, Zeinab H. Helal, Christopher Karch, Peter Burkhard and Mazhar I. Khan. The Development of a Vaccine Prototype for Infectious Bronchitis Virus Using Peptide Nanoparticles. Annual Meeting of the American Association of Avian Pathologists. Indianapolis, Indiana. July 21-25, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: R.L. Alphin, A.H. Moritz, D.P. Hougentogler, E.R. Benson. 2017. Evaluation of foam applied disinfectants and chemicals for inactivating infectious bronchitis virus with and without the presence of high organic load. Poultry Science Association annual meeting. Orlando, Florida. July 17-20, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Robinson, M., S. J. Keeler, C. M. Boettger, T. J. Carr, Jr., D. A. Bautista and C. L. Keeler, Jr. The respiratory microbiome of chickens: Developing protocols for virome analysis. Annual Meeting of the American Association of Avian Pathologists. Indianapolis, IN. July 21-25, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Victoria Drouet Pratt, Naola Ferguson-Noel, Maricarmen Garc�a, C. Stephen Roney, Marianne Dos Santos, Ruth Wooten, Tyler Gamble, and D.G. Sandu. Evaluation of Infectious Laryngotracheitis CEO Vaccine in Mycoplasma synoviae Positive Broilers. Annual Meeting of the American Association of Avian Pathologists. Indianapolis, IN. July 21-25, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Beltr�n, G., S. M. Riblet, L. Read, S. Sharif. Local innate responses in conjunctiva, Harderian gland and trachea after ocular or oral inoculation with a virulent strain of infectious laryngotracheitis virus (ILTV). American Veterinary Medical Association. Indianapolis, Indiana. July 21 -25. 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Johnson, T.J. Modulating the turkey host and its microbiome: host-microbe interactions following antibiotic and probiotic administration. Poultry Science Association annual meeting. Orlando, Florida. July 17-20, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Johnson, T.J. Understanding/defining/modulating the broiler microbiome. Midwest Poultry Federation. Saint Paul, MN. March 14-16, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Johnson, T.J. What is the microbiome and why should I care about it? Midwest Poultry Federation Purina-sponsored symposium. Saint Paul, MN. March 14-16, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Youmans, B., Karnezos, T.P., Evans, N.P., and Johnson, T.J. Inter-relationships between the ileal, cecal, tracheal, and litter bacterial microbiomes in antibiotic-free commercial broilers, and correlations with performance. Gut Health Symposium. Saint Louis, MO. November 13-15, 2017.
  • Type: Other Status: Other Year Published: 2017 Citation: Pendleton, Eva. Diagnosis, Treatment, and Prevention of Respiratory Disease in Small Flocks. Pennsylvania Veterinary Practitioners Meeting. Harrisburg, PA, November, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Hulet R. M. Vegetative Buffers for Air Quality. Poultry Science Association annual meeting. Orlando, Florida. July 17-20, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Martin, G. Operational Biosecurity  Continual Improvement. National Webinar and Extension Workshop. International Poultry Expo. Atlanta, GA. January 31, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Martin, G. Practical Biosecurity. Midwest Poultry Convention, Midwest Poultry Federation. Saint Paul, MN. March 14-16, 2017.
  • Type: Other Status: Other Year Published: 2017 Citation: Martin, G. Practical Biosecurity, Regional Manure Handlers Meeting. Lancaster, PA. March, 2017.
  • Type: Other Status: Other Year Published: 2017 Citation: Pendleton, E. Biosecurity for Game Bird Production. North American Game Bird Association. Biloxi, MS. January, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Dipak Kathayat, Yosra A. Helmy, Loic Deblais, Huang-Chi Huang and Gireesh Rajashekara. Identification of novel small molecule compounds with antimicrobial activities specific to Avian Pathogenic E. Coli. OARDC Annual Research Conference. Columbus, OH. April 20, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: antimicrobial compounds for the control of Avian Pathogenic E. Coli (APEC). ASM Microbiology. New Orleans, LA. June 1-5,2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Yosra A. Helmy, Issmat I Kassem, Loic Deblais, and Gireesh Rajashekara. Control of Avian Pathogenic E. coli (APEC) using Quorum Sensing Inhibitor Small Molecules. The American Association of Avian pathologist (AAAP). Indianapolis, IN. July 21-25, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Kathayat D., Helmy Y.A., Deblais L., Logue C.M., Nolan L.K., and Rajashekara G. Novel small molecule compounds with antimicrobial activities against avian pathogenic Escherichia coli. The 98th annual Conference of Research Workers in Animal Diseases (CRWAD). Chicago. IL. Dec 3-5. 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: John M. Ngunjiri, Amir Ghorbani, Hyesun Jang, Mohamed Elaish, Sumyya Waliullah, Mahesh KC, Michael C. Abundo, Chang-Won Lee. Specific pathogen free turkey model for emerging reovirus and assessment of microbiome in reovirus-infected Turkeys. American Society for Virology 36th Annual Meeting. Madison, WI. June 24-28, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: J.M. Ngunjiri, M.C. Abundo, H. Jang, M. Elaish, M. KC, A. Ghorbani, B.P. Youmans, T.J. Johnson, C.-W. Lee. Understanding the respiratory microbiome of commercial turkeys and chicken layers. 98th Annual Conference of Research Workers in Animal Diseases meeting. Chicago, IL. December 3-5, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Michael C. Abundo, John M. Ngunjiri, Hyesun Jang, Mohamed Elaish, Mahesh KC, Amir Ghorbani, Bonnie P. Youmans, Timothy J. Johnson, Chang-Won Lee. Successional changes in respiratory microbiome in clinically healthy chicken layers. 98th Annual Conference of Research Workers in Animal Diseases meeting. Chicago, IL. December 3-5, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Aston, E. Effect of pullet vaccination on development and longevity of immunity. Science of Veterinary Medicine Symposium. University of Georgia, Athens, GA. October, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Aston, E., B. Jordan, M. Garcia and M. Jackwood. Effect of pullet vaccination on development and longevity of immunity. American Association of Avian Pathologists. Indianapolis, IN. July 21-25, 2017.
  • Type: Other Status: Other Year Published: 2017 Citation: Aston, E. Air quality and poultry respiratory disease. Poultry Respiratory Health Seminar. University of Delaware. Newark, DE. June, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Aston, E., B. Jordan, M. Garcia and M. Jackwood. Effect of pullet vaccination on development and longevity of immunity, IPPE, Atlanta, GA, January 31, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Lee CW. USDA-NIFA funded Poultry Respiratory Disease Coordinated Agricultural Project (PRD-CAP). United States Animal Health Association annual meeting. San Diego, California. October 12-18, 2017.
  • Type: Websites Status: Published Year Published: 2017 Citation: Lee CW. A Novel, Translational, Multidisciplinary Approach to Control Poultry Respiratory Diseases in the United States. PRD-CAP project main website. PRDCAP.com.


Progress 04/01/16 to 03/31/17

Outputs
Target Audience: Poultry industry including backyard poultry farmers; Goverment and state officials involved in control of animal diseases. Scientists in the field of infectious diseases, animal production, Ag-engineering, virology, bacteriology, immunology & vaccinology. Extension and outreach specialists. Changes/Problems: The poultry industry in the majority of states has 3 major segments (broilers, layers, and turkeys) with very little communication between them. The concept of having a centralized data base and data sharing is unfamiliar in the poultry industry and there are concerns about confidentiality and data security. Working closely with the poultry association will enable us to move forward with the project. Another approach is to start with smaller subgroups within each sector first and combine them later. We will continue our efforts towards testing transmission of respiratory microorganisms in backyard chickens under different biosecurity treatments. Previous experience in the last six months has taught us the necessity of compartmentalizing certain tasks to prevent cross-contamination (chicken handling vs. wild bird handling) as well as the need for more resources to train skilled student ornithologists and engineers. For experiment proposed in Objective 2, raising birds to maturity is not without its challenges. Aggressive male behavior required us to separate the sexes. Although we lost more birds than anticipated, we still had enough birds for statistically relevant data at each necropsy. What opportunities for training and professional development has the project provided? This projects support training of many undergraduate and graduate students, post-docs, research assistants and research associates (listed below). In addition to hand-on experiences, students and post-docs presented the results at the local and national meetings. Auburn University: Ramon Zegpi, PhD student - Developing vaccine and conducting animal experiment. 0.1 FTE. University of California, Davis: Robin Isobel Lampron, Undergraduate student. Conducted surveys and sampling at small flocks, performed ELISAs. 0.2 FTE. Theodore Derksen, MS student. Performed trainings at the 4-H seminar, conducted surveys and sampling at small flocks. 1 FTE. Rüdiger Hauck, Post-doctoral scholar. Performed talks in quality assurance meetings, took part of the laboratory experiments and the 4-H seminar. 0.1 FTE. University of Connecticut: Jianping Li, PhD student - Implementing immunogenicity studies of nanoparticle vaccine. 1 FTE Zeinab Helal, Postdoctoral Fellow - Implementing immunogenicity studies of nanoparticle vaccine. 0.5 FTE Shobin Chen, Undergraduate student - Assisting immunogenicity studies of nanoparticle vaccine. 0.1 FTE. Christropher Karch, postdoctoral fellow - Development and optimizing of nanoparticles. 0.1 FTE University of Delaware: Monique Robinson, MS student - Learning and implementing sample collection, nucleic acid purification and sequence analysis. 1 FTE. Albert D'Agostino, undergraduate student - Investigating the microbiome of the respiratory tract of broiler chickens. 0.1 FTE Darian Abreu, Lucas Serge, Dianna Kitt, Xiaolun Guo: All undergraduate engineering students - developing the low cost vehicle undercarriage wash station. Part-time. University of Georgia: Emily Aston, PhD student - Conducted animal experiments, and coordinated necropsy and sample collection and testing. Dr. Aston also analyzed the data generated from this experiment. 0.4 FTE. Madelyn Krunkosky, MS student - Development and optimization of protocols for processing CALT, HG and trachea samples for reverse transcriptase PCR and flow cytometry analysis 0.33 FTE Gabriela Beltrán PhD student - Development and optimization of protocols for processing CALT, HG and trachea samples for reverse transcriptase PCR and flow cytometry analysis 0.33 FTE. University of Minnesota: Bonnie Youmans, PhD student - Learning and implementing microbiome analysis. 0.3 FTE. Ohio State University: Michael Abundo, Research Assistant - Project coordination. 1 FTE Hyesun Jang, PhD student - Implementing infectious animal experiment for microbiome study. 0.5 FTE John Ngunjiri, Research Associate - Surveillance coordination. 0.1 FTE Mahesh KC, MS student - Sample collection, processing, and sequence analysis. 0.5 FTE Dipak Kathayat, PhD student - Learning and implementing small molecule study as antibiotic alternatives. 1 FTE Yosra Mohamed, Postdoctoral Research Associate - Learning and implementing small molecule study. 0.3 FTE Penn State University: Lisa Kitto, Technician - Extension coordination. 0.03FTE Southeast Poultry Research Laboratory, USDA: Nikolai Lee, Undergraduate Student - Trained for sample preparation and processing, and helps with analyzing samples. 0.5 FTE. Texas A&M University: Owais Khan, postdoctoral fellow - Expression of foreign genes against additional pathogens. 0.5 FTE. How have the results been disseminated to communities of interest? We developed the main project website (PRDCAP.com) to share our projects with professionals, stakeholders and the general public. To further expand the network, the annual meeting is held on in conjunction with the NC-1180 and the U.S. Animal Health Association Meeting, and received full attention from our stakeholders. The results from individual projects were presented at the international and regional meetings. Several manuscripts are being prepared for publication in timely manner. Furthermore, our extension group actively conduct comprehensive educational training program on the importance of controlling respiratory diseases for Veterinarians, Extension Educators, Gamebird Producers, Small Organic and Pastured Poultry Operations and Backyard, Hobby, and Exhibition Growers and the general public through distribution of brochures, presentation at a poster, presentation at local, regional, and national meetings, workshops and specific training programs. What do you plan to do during the next reporting period to accomplish the goals? Objective 1. Understand the ecology of poultry respiratory diseases. Continuing efforts will be made for the expansion the nationwide poultry respiratory disease network by expanding both the participants (researchers and stakeholders) and states involved. We will continue working with Ohio poultry industry and with Ohio Poultry Association to collect and update the data of poultry farms in Ohio. We will then build and maintain a digital map of poultry production in Ohio. We will add another layer of data on this map including the poultry disease data. Formation of the centralized body is planned to be on year 3. We will continue to perform sampling on commercial layers, broilers, turkeys in Ohio, Delaware, Minnesota and Iowa. Although sampling time points will differ by the species because of the length of age of the bird, we will ultimately generate the same type of data for commercial birds to determine and compare the baseline respiratory microbiota of broilers vs layers vs turkeys. In addition to testing surveillance samples, it is also planned to collaborate with the investigators in this project to sample the respiratory microbiome using models of respiratory infection (refer to Objective 2) and in commercial flocks experiencing respiratory infections. We expect to determine the changes that microbial communities undergo over the course of respiratory pathogen infection. In addition, we expect to identify specific microbial populations that might be favored, altered, or reduced during the infection. We will continue our efforts towards testing transmission of respiratory microorganisms in backyard chickens under different biosecurity treatments. Objective 2. Investigate the multifactorial etiology involving poultry respiratory diseases. We plan to conduct coinfection studies: 1) Co-infection of SPF leghorn chickens with H5N2 LPAIV, IBV (virulent strain) and MS; 2) Co-infection of SPF broiler chickens with H5N2 LPAIV, IBV (virulent strain) and MS; and 3) Co-infection of SPF turkeys with a LPAIV isolate, NDV (vaccine strain), and Mycoplasma gallisepticum (MG). In addition, co-infection study with immunosuppressive agent (IBDV) will be conducted. We will continue the co-infection studies with MS and ILTV vaccine in broilers. We will evaluate the effect of time and sequence of MS infection on ILT CEO vaccine efficacy. We will conduct a co-infection study with MS and IBV vaccine, and evaluate clinical signs as well as shedding of the vaccine. We will determine if protection is compromised when multiple live attenuated vaccines are given to pullets at the same time they are co-infected with either LPAIV, IBV, NDV, ILTV or mycoplasma. Two independent studies to evaluate the immune responses in CALT, HG and trachea after CEO vaccination and after IBV vaccination will be performed. Cytokine gene expression and immune cells phenotypes changes in CALT, HG and trachea will be assessed by RT-PCR and flow cytometry. We will evaluate the effect of environment by rearing groups of broiler chickens in an environment similar to what is found in a commercial poultry house and in one group reduce the air quality by increasing the relative humidity to above 70%, which will in turn raise ammonia levels to a target of > 40ppm. Birds will be evaluated after vaccination and challenge. Objective 3. Develop new and improved diagnostic tools, vaccines, and novel preventive measures. We will compare specificity and sensitivity of commonly used diagnostic tests for AIV and IBV. After initial bench validation of AIV test, the test will be transferred to the National Veterinary Services Laboratories to do additional evaluations on reference and clinical samples. The initial bioinformatics analysis of IBV has been conducted and different RT-PCR test will be compared and validated to identify the best test that will be shared with all interested diagnostic laboratories. We will further optimize IBV long-long form nanoparticle vaccine. Protective efficacy against challenge with a pathogenic IBV strain will be determined. Based on our results using trimeric recombinant S1+S2 ectodomain proteins which showed improved protection against IBV, we will insert the entire S ectodomain into our NDV vector and evaluate protection against challenge. We will generate HVT vectors that are able to express two genes from the same construct. We will express NDV-F and IBD-VP2. This construct is expected to protect against MD, ND and IBD. We will also express IBDV-S1 and IBD-VP2, which is expected to protect against MD, IB and IBD. Regarding the development of non-antibiotic preventive measure, we will test the selected APEC QS inhibitors for their effect in chickens. We also plan to identify molecular targets of those inhibitors that significantly impact APEC infection of chickens. For mycoplasma study, we will conduct in silico analysis of the preliminary hits to select subset of compounds that are novel and likely to possess drug like properties. Objective 4. Extension and outreach program. The University of Delaware and Maryland team is using a two pronged training approach in which (a) cohosting poultry respiratory disease training sessions with Delaware Ag Week sessions oriented for regional broiler growers; and (b) a one day PRD program for summer 2017. The primary focus of Connecticut will be presentations at local, regional and national extension meetings/workshops and to provide producers, educators and service people with new educational materials and methods of detecting and preventing respiratory disease in poultry. This approach is especially useful for the small scale producers. The Ohio State University aims to hold 3 full day workshop targeting 15 - 20 small animals and mixed veterinary practitioners. The workshops will be held in each of three large urban centers in Ohio including Columbus, Cleveland and Cincinnati to make it easier for participating veterinarians to attend. By the end of the project we aim to train roughly 200 veterinarians practicing in the state of Ohio to provide basic veterinary services to noncommercial poultry. Penn State University will continue to work on greater distribution on a regional and national basis will be attempted by regional meetings in New England and Pennsylvania. The presentations for these meetings will also be presented via a webinar for national distribution. The presentations will also be packaged as a hard copy and digital copies off our website. University of Georgia group will develop an informational video series detailing how biosecurity, house management, and farm operations effect respiratory disease in commercial poultry. University of California group will continue to provide backyard flock seminars. The group will identify and analyze networks as well as doing surveillance in a more systematic and statewide approach targeted to 4-H volunteers and small flock owners. Coordinated effort will be made among participants for effective generation and validation of extension and education materials and approaches.

Impacts
What was accomplished under these goals? Objective 1. Understand the ecology of poultry respiratory diseases We continued to expand the nationwide poultry disease network. In addition to existing NC-1180 participants and institutions from the majority of poultry producing states, we now have additional participants and collaborators from Mississippi, Texas, and California. To further expand the network, the annual meeting is being held in conjunction with the NC-1180 and the U.S. Animal Health Association Meeting. In addition, a Request for New Proposals is made each year to stimulate new and innovative research in critical areas which are not included in the current proposal. We funded three new preproposals in 2016. We expect to fund two new proposals in 2017. We maintain project website (PRDCAP.com) to share our projects with professionals and stakeholders. As an initial effort to create a centralized disease work group and develop a database that captures the size and the distribution of poultry species and poultry diseases, the poultry industry in Ohio has been introduced to the concept of a centralized disease epidemiology committee. The formation of such a central disease network in Ohio is on-going and will be presented as a model that could be emulated and extended to a regional or the national level. As a new collaborative initiative, respiratory microbiome and metagenomic profiling is being conducted in all 3 species or types: broilers, layers, and turkeys. Based on method we established in Year 1, we conducted extensive surveillance in all 3 species in Delaware, Minnesota, and Ohio. Overall, our studies have started to yield crucial baseline data that will enhance our understanding on the relationship between respiratory microbiome and susceptibility or resistance disease. A newly funded SEED grant awardee studies the transmission of respiratory microorganisms from backyard chickens to peridomestic songbirds which would be reduced by enhancing backyard chicken husbandry practices. Objective 2. Investigate the multifactorial etiology involving respiratory diseases Research on pathogenesis of respiratory diseases involves exploration of the intricate and complex interactions among pathogen, host, and environment. We established key combinations of respiratory pathogens, immunosuppressive agents, vaccine status, and environmental factors for each species to mimic the natural condition and explore the pathogenesis. We conducted a study in which we co-infected SPF Leghorn chickens with low pathogenic avian influenza viruses and infectious bronchitis virus (IBV). This study showed that other pathogens are necessary to reproduce the bronchial casts reported and morbidity reported in the field. We have completed a study with Mycoplasma Synoviae (MS) and Infectious laryngotracheitis virus (ILTV) on evaluation of ILTV CEO vaccine in MS-positive broilers. Our study highlighted the benefit of controlling MS infection as well as the risk of ILT CEO vaccine in MS-infected broiler flocks. We monitored the development and longevity of protective immunity elicited by the combinations of live attenuated NDV, IBV, ILTV vaccines in pullets and the effects of these vaccines in pullets co-infected with other respiratory pathogens. We showed that ILTV modulates the conjunctival associated lymphoid tissues, Harderian gland, and trachea immune response to promote viral replication and tissue damage. This knowledge is critical in order to appropriately develop new vaccines and adjuvants. Some of the planned studies have been delayed to coordinate with other ongoing animal studies to maximize the collaboration and resources. All the proposed study in Objective 2 involves extensive animal experiments which will produce large amount of descriptive data. Objective 3. Develop new and improved diagnostic tools, vaccines, and novel preventive measures Based on a comprehensive bioinformatics, two real-time RT-PCR was developed for AIV. Once bench validation is completed, the test will be transferred to the National Veterinary Services Laboratories to do additional evaluations on reference and clinical samples. We designed novel IBV vaccines using self-assembled peptide nanoparticle platform. In vivo study in chickens vaccinated with IBV long-long form of construct showed significant antibody response. We will further optimize the IBV long-long nanoparticle and determined protective efficacy against challenge. We developed an improved Ark-type attenuated vaccine by adaptation to chicken kidney cells (CEK). CEK-adaptation eliminated emergence of subpopulations in vaccinated chickens, and protected against challenge. We developed two independent HVT (herpesvirus of turkey) constructs that either expressed F (fusion) gene of NDV or S1 gene of IBV. The F and S1 genes were under the control of CVM IE promoter and bovine growth hormone polyA signal sequences. The HVT genomic site that allowed stable expression of foreign genes was identified by expression of infectious bursal disease VP2 gene expression. The use of small molecules is a highly practical approach as a novel non-antibiotic compounds for the control of colibacillosis. We have selected 10 compounds that significantly inhibited the quorum sensing AI-2 activity, identified by screening a library of ~4,200 compounds, which showed minimal cytotoxicity. In addition, we developed chicken infection model to test these compounds in chickens. To identify novel small molecule inhibitors of Mycoplasma gallisepticum (MG), we screened a library of ~4,200 compounds and identified 584 small molecules that completely inhibited MG growth. Further characterization of these potential leads would facilitate development of novel therapeutics to augment MG control in poultry. Objective 4. Extension and outreach program University of Delaware and Maryland extension group provided training to the National Association of County Agriculture Agents on biosecurity. The team leveraged project activity along with separately funded sessions, providing synergistic biosecurity and response training for USDA National Veterinary Stockpile 3-D contractors, USDA Veterinary Services personnel, and for international veterinarians. University of Connecticut group held 13 small flock owner workshops throughout Southern New England reaching out to more than 320 people about best poultry management practices, including health and disease management. The Ohio State University extension group provided specifically developed education and training, in the form of a full day workshop, for practicing veterinarians in Ohio, who are witnessing an increasing number of poultry patients. Trained veterinarians will provide a much needed basic veterinary services to the increasingly popular noncommercial poultry. Penn State University group developed a Farm Plan Program for flocks dealing with catastrophic disease events. In addition, educational brochures and posters were created to inform Veterinarians, Extension Educators, Gamebird Producers, Small Organic and Pastured Poultry Operations and Backyard, Hobby, and Exhibition Growers and the general public on the importance of controlling respiratory diseases. Over 100,000 contacts were made through distribution of brochures, presentation at a poster, presentation at local, regional, and national meetings. University of Georgia group was newly awarded with SEED grant in 2016 on project developing an informational video series detailing how biosecurity, house management, and farm operations effect respiratory disease in commercial poultry. The videos will be 2-5 minutes in length and distributed through multiple channels. University of California group (new 2016 SEED grant awardee) has developed an extensive map of more than 600 small flocks using a Geo survey database. The group also worked on understanding the small poultry networks using Social Network Analysis (SNA) detecting that 4-H volunteers are the best targets for extension work in California.

Publications

  • Type: Websites Status: Other Year Published: 2016 Citation: Lee CW. A Novel, Translational, Multidisciplinary Approach to Control Poultry Respiratory Diseases in the United States. PRD-CAP project main website. PRDCAP.com.
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Garc�a, M. Current and future vaccines and vaccination strategies against infectious laryngotracheitis (ILT) respiratory disease of poultry. Veterinary Microbiology. In Press.
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Reddy SM, Izumiya Y, Lupiani B. 2016. Mareks disease vaccines: Current status, and strategies for improvement and development of vectory vaccines. Veterinary Microbiology. In Press.
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: R.A. Zegpi, C. Breedlove, V.L. van Santen, C.R. Rasmussen-Ivey, H. Toro (2017) Kidney Cell-Adapted Infectious Bronchitis ArkDPI Vaccine is Stable and Protective. Avian Diseases. In Press.
  • Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Beltr�n, G., S. M. Williams, G. Zavala and M. Garc�a. Replication patterns of infectious laryngotracheitis virus (ILTV) virulent strain and chicken embryo origin (CEO) vaccine. Avian Pathology. Submitted.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Hulet R. M., E. Wallner-Pendleton, P. Clauer, G. Martin, P. Dunn, and P. Patterson. Educational Program Development in Response to 2014-2015 Avian Influenza Outbreak. Poultry Sci. 95 (Suppl.1): 326P. 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Lee CW. A novel, translational, multidisciplinary approach to control poultry respiratory diseases in the United States. Proceedings of 65th Western Poultry Disease Conference. Vancouver, BC, Canada. April 24-27, pp.147-148. 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Aston, E. J., B. J. Jordan, M. Garc�a, M. W. Jackwood. Effect of pullet vaccination on development and longevity of immunity. International Poultry Scientific Forum, Southern Conference on Avian Diseases. Atlanta GA. January 30, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Krunkosky, M., G. Beltr�n, R. M. Gogal Jr., D. J. Hurley and M. Garc�a. Tracking leukocyte phenotype changes in CALT, Harderian gland, trachea and spleen of chickens ocularly-infected with Infectious laryngotracheitis virus (ILTV). XIVth Avian Immunology Research Group (AIRG) Meeting. Herrsching, Germany. September, 7 -10, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Beltr�n, G. and M. Garc�a. Cytokines expression in CALT Harderian gland and trachea after ocular or oral inoculation with infectious laryngotracheitis virus (ILTV). Convention of the American Veterinary Medical Association, San Antonio, Texas. August 5 -9, 2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Li, J., Tan, A., Helal, Z. Karch, C., and Burkhard, P. Khan, M. I. Novel Nanoparticles-based vaccine for infectious bronchitis virus. AVMA, San Antonio, Texas. August 5-9, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Zeinab H. Helal, Jianping Li, Christopher Karch, Peter Burkhard and Mazhar I. Khan. Peptide nanoparticle-based vaccine for infectious bronchitis virus. International Vaccine Conference, Boston, US, October2-5, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Yosra A. Helmy, Issmat I Kassem, Anand Kumar, Loic Deblais, and Gireesh Rajashekara (2015). High Throughput Screening to Identify Quorum Sensing Inhibitors to Enhance the Control of Avian Pathogenic E. coli. Conference of Research Workers in Animal Diseases (CRWAD). 6-8 December, Chicago, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Vicky L. van Santen, Fatma E. Eldemery, Saiada Farjana, Kellye S. Joiner, Haroldo Toro. Role of spike S2 ectodomain in attachment and selection of ArkDPI IBV vaccine subpopulations in chickens. Annual Meeting AAAP, 08/2016, San Antonio, TX.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Yosra A. Helmy, Issmat I Kassem,Anand Kumar, Loic Deblais, and Gireesh Rajashekara (2016). Quorum Sensing Inhibitors to Enhance the Control of Avian Pathogenic E. coli. The 65th Western Poultry disease conference, 24-27th April, Vancouver, Canada.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Yosra A. Helmy, Issmat I Kassem,Anand Kumar, Loic Deblais, and Gireesh Rajashekara (2016). Novel Quorum Sensing Inhibitors for the Control of Avian Pathogenic E. coli. OARDC Annual Research Conference. 21st April, Ohio State University, Columbus, Ohio State, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Dipak Kathayat, Yosra A. Helmy, Loic Deblais, Issmat I Kassem and Gireesh Rajashekara (2016). Discovery of Novel Narrow Spectrum Small Molecule Growth Inhibitors for Avian Pathogenic E. coli. Conference of Research Workers in Animal Diseases (CRWAD). 4-6 December, Chicago, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Haroldo Toro, Vicky van Santen, Cassandra Breedlove. Characterization of Kidney Cell-Adapted IBV ArkDPI Vaccine during Back-Passages in Embryonated Eggs. Annual Meeting AAAP, 08/2016, San Antonio, TX.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Moyle, J. and G. Martin, 2016. Assessing How Poultry Growers Respond to Current Biosecurity Demands. National Poultry Extension Workshop. New Orleans, LA, July 11-14.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Martz, Michael, 2016. Biosecurity Plan for Dealing with Pathogenic Avian Influenza, 2016. Pennsylvania Game Bird Meeting, State College, PA, February 22 - 24.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Rodrigo A. Gallardo. Seroprevalence of respiratory diseases in small flocks close and far from commercial poultry premises. California poultry quality assurance meeting. Ontario, CA. September 28, 2016.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Rodrigo A. Gallardo. Seroprevalence of respiratory diseases in small flocks close and far from commercial poultry premises. California poultry quality assurance meeting. Modesto, CA. September 30, 2016.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Rodrigo A. Gallardo. Seroprevalence of respiratory diseases in small flocks close and far from commercial poultry premises. Shell egg food safety quality assurance meeting . Sacramento, CA October 26, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Rodrigo A. Gallardo. Seroprevalence of respiratory diseases in small flocks close and far from commercial poultry premises. California poultry federation annual meeting, Monterey, CA. September 16, 2016.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Mohamed El-Gazzar. Bird Flu: What is it? And how it happened? All Ohio 4-H educators in service meeting. Columbus, OH. 2/2/16.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Mohamed El-Gazzar. Basic Approach for Poultry Disease Diagnosis. All Ohio 4-H educators in service meeting. Columbus, OH. 2/2/16.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Mohamed El-Gazzar. Avian Influenza and Infectious Disease Biosecurity. Noncommercial poultry owners meeting, Medina County, OH. 2/6/16.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Mohamed El-Gazzar. Avian Influenza A Foreign Disease Eradication and Impact. Dark County stakeholders with Dark County Extension Office. Greenville, OH. 2/19/16
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Mohamed El-Gazzar. Avian Influenza Updates and Brochure with (ODA/OPA/USDA). PULLORUM TESTING SCHOOL. Wooster, OH. 5/3/16.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Mohamed El-Gazzar. Avian Influenza Updates and Brochure with (ODA/OPA/USDA). PULLORUM TESTING SCHOOL. Bucyrus, OH. 5/4/16.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Lee CW. Multidisciplinary, Multistate Collaborative Project to Control Poultry Respiratory Diseases in the United States. 67th North Central Avian Disease Conference. St. Paul, MN. March 14-15, 2016.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Mohamed El-Gazzar. Avian Influenza Updates and Brochure with (ODA/OPA/USDA). PULLORUM TESTING SCHOOL. Columbus, OH. 5/5/16.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Mohamed El-Gazzar. Bird Flu: What is it? And how it happened? All Ohio Extension Ag Educators meeting, Columbus, OH. 6/16/16.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Mohamed El-Gazzar. Network of Disease Control Committee. Poultry Producers with OPA. Columbus. OH. 11/11/16
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Lee CW. Poultry Respiratory Diseases Coordinated Agricultural Project (PRD-CAP). 2016 AAAP/AVMA Meeting, San Antonio, TX. August 6-9, 2016.
  • Type: Other Status: Other Year Published: 2016 Citation: Mohamed El-Gazzar. Avian Influenza (AI) testing sample collection: Sauders Eggs with (ODA/OPA/USDA). Bellville, OH. 6/9/16.
  • Type: Other Status: Other Year Published: 2016 Citation: Mohamed El-Gazzar. Avian Influenza (AI) testing sample collection: Kalmbach Feeds. Reynoldsburg, OH. 7/6/16


Progress 04/01/15 to 03/31/16

Outputs
Target Audience:Poultry industry including backyard poultry farmers; Goverment and state officials involved in control of animal diseases; Scientists in the field of infectious diseases,animal production,virology, bacteriology,immunology & vaccinology. Changes/Problems:Objective 1. Understand the ecology of poultry respiratory diseases. The poultry industry in the majority of states has 3 major segments (broilers, layers, and turkeys) with very little communication between them. The concept of having a centralized data base and data sharing is unfamiliar in the poultry industry and there are concerns about confidentiality and data security. Working closely with the poultry association will enable us to move forward with the project. Another approach is to start with smaller subgroups within each sector first and combine them later. Since we are trying to establish a baseline respiratory microbiome in poultry for the first time, the biggest challenge was to determine an effective sampling strategy for respiratory tissues. We attempted direct extraction from swabs and tissue homogenates and also compared the effectiveness of a wide range of saline volumes for tracheal wash. The most effective approach was to wash several times using moderate volumes (5-50 mL) of saline followed by pelleting of the bacterial cells and DNA extraction. Moving forward, we will utilize this approach for sampling. With the threat of avian influenza, it has been difficult to get the cooperation of broiler integrators. However, although there was some delay in initiating the project, persistent efforts overcame this obstacle. Meanwhile fresh biological material for method development was obtained from the University Poultry Diagnostic Laboratory and also from research flocks. Objective 2. Investigate the multifactorial etiology involving poultry respiratory diseases. One of the challenges that we have encountered is in replicating the field clinical picture without resorting to overly severe and artificial methods of inoculation. In this study, the mortality and clinical signs were greater than anticipated. We are evaluating alternative methods of inoculation and evaluation, including a less-virulent strain of MS. Objective 3. Develop new and improved diagnostic tools, vaccines, and novel preventive measures. None Objective 4. Extension and outreach program Respiratory diseases and other fast moving diseases are a significant challenge. In January 2016 in Indiana, a case of low pathogenic avian influenza mutated to highly pathogenic avian influenza. Control measures included quarantine, biosecurity, depopulation, disposal, and cleaning and disinfection. Low temperature operation limited depopulation options in the field and this is an example of the type of challenges that continue to face emergency animal management options. The HPAI outbreaks in 2015 overshadowed focus on other respiratory disease problems and became the major focus of outreach programs. The solution was to help producers understand that the same biosecurity measures implemented for HPAI can be used to help prevent other respiratory diseases of concern. The HPAI in 2015 demanded redirection of the Extension education efforts. More effort was need in the professional poultry industry to aid in understanding the disease and its control and eradication efforts. However, noncommercial population was also reached through mass media and Extension educational efforts and printed material. What opportunities for training and professional development has the project provided?This projects support training of 6graduate students and three post-docs. In addition, 2 undergraduate students were involved as part of the research program. In addition to hand-on experiences, students and post-docs presented the results at the local and national meetings. How have the results been disseminated to communities of interest?We developed the main project website (PRDCAP.com) to share our projects with professionals, stakeholders and the general public. To further expand the network, the annual meeting was held on October 26-28, 2015 in conjunction with the NC-1180 and the U.S. Animal Health Association Meeting, and received full attention from our stakeholders. The results from individual projects were presented at the international and regional meetings. Several manuscripts are being prepared for publication in timely manner. Furthermore, our extension group actively conduct comprehensive educational training program on the importance of controlling respiratory diseases for Veterinarians, Extension Educators, Gamebird Producers, Small Organic and Pastured Poultry Operations and Backyard, Hobby, and Exhibition Growers and the general public through distribution of brochures, presentation at a poster, presentation at local, regional, and national meetings, workshops and specific training programs. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Understand the ecology of poultry respiratory diseases. Continued effort will be made to expand the nationwide poultry respiratory disease network by expanding both the participants (researchers and stakeholders) and states involved. A Master database that includes both the digital map of poultry production and disease data in Ohio will be developed and used as a model (together with other successful model) to be subsequently applied to other states. An established ftp connection will be used to distribute raw sequence data to the individual groups. The pipelines for data analysis will be available via cloud computing to the microbiome study groups. In addition to testing surveillance samples, it is also planned to collaborate with the investigators in this project to sample the respiratory microbiome using models of respiratory infection (refer to Objective 2) and in commercial flocks experiencing respiratory infections. Following the experimental design of each investigator, tracheal sampling will be included in the collection protocol for control and treatment groups. We expect to determine the changes that microbial communities undergo over the course of respiratory pathogen infection. In addition, we expect to identify specific microbial populations that might be favored, altered, or reduced during the infection. Objective 2. Investigate the multifactorial etiology involving poultry respiratory diseases. We will conduct a follow up study in which chickens will be co-infected with AIV, IBV and Mycoplasme synoviae (MS). A co-infection study will also be done in turkeys using different respiratory pathogens including AIV, NDV, and Mycoplasma meleagridis (MG). We will continue the co-infection studies with MS and ILTV vaccine in broilers. We will evaluate the effect of time and sequence of application on clinical signs. We will conduct a co-infection study with MS and IBV vaccine, and evaluate clinical signs as well as shedding of the vaccine. We will also evaluate the effect of MS infection on ILT CEO vaccine efficacy. We will use 1 week old SPF and commercial broilers to inoculate with IBDV (T1) then 1 week later repeat the co-infection of these birds with H5N2 LPAIV, IBV (vaccine strain) and MS.Combinations of respiratory pathogens that cause little or no clinical disease in broiler and layer chickens will be targeted.These combinations will be repeated in chickens that have been given a previous or concurrent inoculation of IBDV. We will vaccinate layer and broiler pullets with live attenuated IBV, NDV, and ILTV vaccines as well as ILTV recombinant vaccine using a typical pullet vaccination program and monitor the replication of the vaccines in the upper respiratory tract. We will also examine the development of mucosal immunity (IgA) in tracheal washes and tears by collecting a temporal set of samples post-vaccination. We will hold the birds for up to 36 weeks of age (18 weeks following the last vaccination) challenging subsets of the vaccinated birds periodically to assess protection. We will continue the immunophenotyping experiments in trachea, HG, and CALT of chickens inoculated with either the CEO vaccine or virulent ILTV strain administered via the intratracheal and ocular routes.A second objective for year 2 is to develop trachea cell culture system to evaluate "in situ" functional innate immune responses. We will evaluate the effect of environment by rearing groups of broiler chickens in an environment similar to what is found in a commercial poultry house and in one group reduce the air quality by increasing the relative humidity to above 70%, which will in turn raise ammonia levels to a target of > 40ppm. Birds will be evaluated after vaccination and challenge. Objective 3. Develop new and improved diagnostic tools, vaccines, and novel preventive measures. We will compare specificity and sensitivity of commonly used diagnostic tests for IBV, ILTV, aMPV, and Mycoplasma.Wet bench analysis comparing primers and probes with de novo synthesized samples will provide a discriminating factor if more than 3 tests are being considered. For the top two tests, diagnostic laboratories in the network will run on clinical samples the test that they normally run and the "new" tests to generate data from field samples.The goal of the proposal is to identify the best tests that will be shared with all interested diagnostic laboratories. Immunogenicity studies in chickens will be performed using optimized IBV nanoparticle vaccine candidates (IBV-SAPNS). Vaccine candidates will be selected for challenge study against pathogenic IBV. Therecombinant NDV-IBV-S2 vaccine will be characterized by the embryo mean death time (MDT), egg infectious dose 50% (EID50), and intracerebral pathogenicity index (ICPI) tests. It will also be tested in protection/challenge trials in animals to assess its protective capabilities. We will evaluate the stability of the HVT recombinant viruses generated. Specifically we are planning on studying which of the three genomic sites used for insertion of the gene of interest will allow stable expression of foreign genes. Stable HVT recombinants will be further characterized by multi-step growth kinetics in CEF. Regarding the development of non-antibiotic preventive measure, we will evaluate the effect of QS inhibiting molecules on the adhesion, invasion and intracellular survival of APEC O78 in Caco-2 intestinal epithelial cells and assess the effect of QS inhibiting compounds on other APEC serotypes (O1 and O2). We will also screen small molecule library for novel inhibitors to enhance the control of Mycoplasma synoviae and M. gallisepticum in poultry. Objective 4. Extension and outreach program We will complete the development of a web based biosecurity training / certification program. The recent outbreak of HPAI in Indiana highlights the need for continued biosecurity vigilance. This program will be rolled out and tested for the Delmarva region in Q2 of Year 2. Based on feedback and results, the web based biosecurity program will be adjusted and released nationally by Q4 of Year 2. For the Year 3 training program, a detailed syllabus will be developed and implementation dates and locations determined by Q3.Prospective guest instructors, ideally including several from this project, will be identified and recruited.Ideally, 25% of the in house instructed topic modules will be developed and completed by Q3 of Year 2, with 33% of the in house instructed topic modules completed by Q4. Qualtrics web based pre-course and post-course assessment will be developed by Q4. We plan to hold 3 full day workshops targeting 15 - 20 small animal and mixed veterinary practitioners in Ohio. The workshops will be held in each of three large urban centers in Ohio including Columbus, Cleveland and Cincinnati to make it easier for participating veterinarians to attend. We will focus on the development of a full day National Small Flock Workshop that will allow stakeholders to learn about poultry health and diseases.The workshop will be held in multiple locations to allow for convenient access for our national audience.Information presented at the meetings will be compiled into a manual that will be available for attendees as well as for others electronically. We expect that some of the proposed research and extension effort will be highly successful and have merit to be expanded to regional and national level. In addition, we expect to identify need for additional extension effort as new findings accumulate from the proposed researches. The host institution maintains funds to support additional extension effort annually. Coordinated effort will be made among participants for effective generation and validation of extension and education materials and approaches.

Impacts
What was accomplished under these goals? Objective 1. Understand the ecology of poultry respiratory diseases. We continue to expand the nationwide poultry disease network. In addition to existing NC-1180 participants and institutions from the majority of poultry producing states, we currently have additional participants and collaborators from Mississippi, Texas, and California. To further expand the network, the annual meeting was held on October 26-28, 2015 in conjunction with the NC-1180 and the U.S. Animal Health Association Meeting, and received full attention from our stakeholders. In addition, a Request for New Proposals (RFP) was made in November 2015 to stimulate new and innovative research in critical areas which are not included in the current proposal. We expect to fund two new proposals by April, 2016. We also developed the main project website (PRDCAP.com) to share our projects with professionals, stakeholders and the general public. As an initial effort to create a centralized disease work group and develop a database that captures the size and the distribution of poultry species and poultry diseases, the poultry industry in Ohio has been introduced to the concept of a centralized disease epidemiology committee. The formation of such a central disease network in Ohio will be presented as a model that could be emulated and extended to a regional or the national level. Development of a master database and the structure of a centralized disease committee in Ohio are on-going. We are also developing an ftp (file transfer protocol) site which will be available within a month that will share all of the sequencing data related to metagenomics and microbiome studies. The site will include the raw sequence data as well as mothur analysis of the data. We ran a pilot experiment examining the feasibility of examining sinus and trachea washes for respiratory microbiome study. We determined that trachea and sinus washes are feasible for DNA extraction and 16S rRNA amplification. We also determined that there are different bacterial populations in the trachea versus the sinus washes. We also tested different DNA/RNA extraction kits and protocols, and optimized based on the tissue or wash samples. Extensive longitudinal sampling of both respiratory and intestinal tissues was started from commercial broiler, layer and turkey flocks. Sequencing and data analysis is on-going. We will translate this information into improved diagnostics that can be used by the producer to determine their flock health according to microbiome content, and the most effective mitigation strategies to prevent disease. Objective 2. Investigate the multifactorial etiology involving poultry respiratory diseases. Research on pathogenesis of respiratory diseases involves exploration of the intricate and complex interactions among pathogen, host, and environment. We established key combinations of respiratory pathogens, immunosuppressive agents, vaccine statuses, and environmental factors for each species to mimic the natural condition and explore the pathogenesis. In co-infection study of AIV and IBV, co-infected birds shed more virus and severe clinical signs compared to single AIV-infected birds but no bronchial casts or mortality were observed, indicating other pathogens might be necessary to reproduce what is seen in the field. We evaluated the impact of a commercial ILT CEO vaccine on clinical signs in MS-infected commercial broilers. The preliminary results highlight the benefit of controlling MS infection as well as the risk of ILT CEO vaccine in MS-infected broiler flocks. We evaluated key innate immune pathways activated by ILTV, IBV, NDV and MG vaccinations. We showed that infection with a virulent ILTV strain activates innate immune pathways and cells that favor viral replication, persistence and disease. This information is critical as we expect that it will contrast with pathways activated by the CEO vaccine. Some of the planned studies have been delayed to coordinate with other ongoing animal studies to maximize the collaboration and resources. All the proposed study in Objective 2 involves extensive animal experiments which will produce large amount of descriptive data. Collaborative effort will continuously be made among PIs to select and focus on combinations of respiratory pathogens, immunosuppressive agents, vaccine statuses, and environmental factors for each species to mimic the natural condition and explore the pathogenesis. Objective 3. Develop new and improved diagnostic tools, vaccines, and novel preventive measures. Because of the highly pathogenic avian influenza (HPAI) outbreak in the US, APHIS requested us to examine the H5 subtype influenza test to solve the issues related to detection. Using the SNP method for identifying primers from highly divergent viruses, several alternative tests were developed that can better identify H5 viruses. Once validated, it will be used by USDA for national surveillance and emergency detection of HPAI virus. The early detection of HPAI virus is critical for the control and eradication of HPAI. Three different vaccine platforms are being successfully developed: 1) Nanoparticle based IBV vaccine prototypes were designed and successfully synthesized. Immunogenicity studies in chickens using selected vaccine candidate is being performed; 2) IBV S2 gene was synthesized with codons optimized for chicken and cloned into the Newcastle disease virus (NDV) LaSota (LS) vaccine vector. The infectious clone was used for rescue of the recombinant vaccine virus; 3) We cloned VP2 gene of IBD and F gene of NDV into HVT genome and demonstrated successful expression of VP2. IN addition, using HVT genomic BAC clone, we generated a recombinant HVT vector containing IBV S gene. Three recombinant viruses were generated to test in animals. We identified several novel non-antibiotic compounds that inhibit avian pathogenic E. coli (APEC) and show low cytotoxicity. Further characterization of these potential leads would facilitate development of novel therapeutics to augment APEC control in poultry. Objective 4. Extension and outreach program. University of Delaware and Maryland group have provided regional and agencies (USDA National Veterinary Stockpile (3 sessions), USDA Veterinary Services (1 session), Delaware Department of Agriculture (2 sessions) and Virginia) specific training on foam depopulation, and are providing a week long training program for international veterinarians on emergency poultry disease response. The team is evaluating the development of a biosecurity certification program for growers. To date, the project team has worked with approximately 1425 participants. University of Connecticut group have contacted over 1000 small and medium flock owners in Connecticut and New England to inform and educate about the HPAI situation. Due to the group's effort, both commercial and small flock owners implemented increased biosecurity and disease surveillance programs. For example, the largest commercial layer farm in CT tightened their existing biosecurity and constructed a truck/vehicle wash area outside the entrance to their facility. A large organic producer in MA instituted truck/vehicle washing and the use of more PPE for their personnel and each person was only allowed into one coop on the property to prevent any cross contamination. OSU and PSU group conducted a comprehensive and effective educational training program on the importance of controlling respiratory diseases for Veterinarians, Extension Educators, Gamebird Producers, Small Organic and Pastured Poultry Operations and Backyard, Hobby, and Exhibition Growers and the general public. Over 100,000 contacts were made through distribution of brochures, presentation at a poster, presentation at local, regional, and national meetings.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Pendleton E. Avian Influenza Outbreak Update. 2015 Veterinary Practitioners Meeting. State College, PA. May 1st, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Pendleton E. HPAI and Sullivan Poultry. Centre Hall, PA. April 23. 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Pendleton E. Biosecurity and recognition of disease. PDA Certified Poultry Technician Course. State College, PA. May 12, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Pendleton, E. Avian Influenza Outbreak and Lessons Learned. PADLS Conference. September 2, PA. 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Pendleton, E. Necropsy and Tissue Submission. 2016 Pennsylvania Game Breeders and Hunting Preserves Conference. State College, PA. February 22. 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Dunn P. How to Deal with the FDA Feed Additive Ruling. 2016 Pennsylvania Game Breeders and Hunting Preserves Conference. State College, PA. February 22. 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Hulet RM. Incubation and Disease Prevention and Game Bird Production. NE SARE Project Professional Development in Applied Poultry Science. Freeport, ME. October 21- 23. 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Clauer P. Risk of Avian Influenza. Mainstream Penn States Sustainability News. University Park, PA. December 18 -19, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Pendleton E. Avian Influenza and What You Should Do to Protect Your Backyard Poultry Flock. Ag Progress. Russell E. Larsen Agricultural Research Center. PA. August 18, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Clauer P. Best Management Practice for the Care of Backyard Poultry. Ag Progress. Russell E. Larsen Agricultural Research Center. PA. August 18 - 19, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Martin G. Small Scale Poultry Processing Methods for a Quality Food Product. Ag Progress. Russell E. Larsen Agricultural Research Center. PA. August 19,2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Beltr�n-Garza LG, Zavala G, Williams SM, Garc�a M. Replication of infectious laryngotracheitis virus (ILTV) chicken embryo origin (CEO) vaccine after different mucosal inoculation routes. American Veterinary Medical Association Meeting. Boston, MA. July 10 to 14, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Beltr�n-Garza LG, Garc�a M. Protection elicited by infectious laryngotracheitis virus (ILTV) chicken embryo origin (CEO) vaccine after different after different mucosal inoculation routes. 58th Annual Meeting of The Southern Conference of Avian Diseases. Atlanta, GA. January 25 and 26, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Krunkosky M, Levin J, Beltr�n-Garza LG, Karpuzoglu-Belgin E, Garc�a M, Williams RJ, Hurley DJ, Gogal Jr. RM. Immunophenotyping Avian Leukocytes in the Mucosal Tissues of the Head During Aging in the White Leghorn. 58th Annual Meeting of The Southern Conference of Avian Diseases. Atlanta, GA. January 25 and 26, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Costa-Hurtado M, Smith D, Jackwood M, Spackman E, Suarez D, Pantin-Jackwood M. Experimental co-infection of SPF chickens with low pathogenicity avian influenza virus subtypes H9N2, H5N2 and H7N9 and infectious bronchitis virus. American Association of Avian Pathologist (AAAP) Annual Meeting, Boston, July 11-14, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Helmy YA, Kassem II, Kumar A, Deblais L, Rajashekara G. High-Throughput Screening to Identify Quorum Sensing Inhibitors to Enhance the Control of Avian Pathogenic E. coli. Conference of Research Workers in Animal Diseases (CRWAD). p 115, December 6-8, Chicago, IL. 2015
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Toro H, Ghetas AM, Thaxton GE, van Santen VL. Adaptation of Embryo Attenuated Arkansas IBV Vaccine to Kidney Cells Reduces Population Heterogeneity. 2015 Annual Meeting AAAP. July 12-14, Boston, MA. 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Martin G. Biosecurity, Cleaning and Disinfection. 2016 Pennsylvania Game Breeders and Hunting Preserves Conference. State College, PA. February 22. 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Martin G. HPAI Review. County Commissioners Association Meeting. Grantville, PA. October 27; November 5, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Martin G. Biosecurity and Bird Disposal. Governors Cabinet tabletop exercise with PEMA emergency management area coordinators. Harrisburg, PA. November 9. 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Khan MI, Li J, Karch C, Burkhard P, Tan A, Helal Z., Fan Q. Immunogenicity Studies on Nanoparticle Based Vaccines for Bird Flu AVMA, Boston, MA. July 23-26, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Pendleton E. HPAI and Necropsy Demonstration. Poultry Medicine Lecture Series for 3 Rivers Veterinary Symposium. PA. November 8, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: AM Ghetas, GE Thaxton, C Breedlove, VL van Santen, H Toro. Adaptation of Embryo Attenuated Infectious Bronchitis Virus Arkansas to Kidney Cells. 58th Annual Meeting of The Southern Conference of Avian Diseases. Atlanta, GA. January 25 and 26, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Li J, Karch C, Tan A, Helal Z, Fan Q, Garmendia A, Girshick T, Burkhard P, Khan MI. Novel nanoparticle-based induced specific neutralizing antibodies against bird flu. GARAD 2015. Strand Campus, Kings College, London, UK, June 29- July1, 2015.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Li J, Helal Z, Karch C, Burkhard P, Khan MI. Peptide Nanoparticle-based Vaccine for Infectious Bronchitis Virus. AVMA, San Antonio, TX July 23-26, 2016. To be presented.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: El-Gazzar M. Poultry Disease Prevention and Control 4-H Poultry Clinic, OARDC March 28, Wooster, OH. 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: El-Gazzar M. HPAI and Biosecurity Presentation in the annual OPA meeting April 16, Columbus, OH. 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Li J, Karch C, Tan A, Helal Z, Fan Q, Garmendia A, Girshick T, Burkhard P, Khan MI. Novel Nanoparticle-based vaccine induces specific neutralizing antibodies against avian influenza. Nanoscale Science & Engineering for Agriculture & Food Systems Gordon Research Conference, Bentley University, Waltham, MA. June7-12, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: El-Gazzar M. Bird Flu: What is it? ? And how it happen? All Ohio 4-H educators in service. November 9, Columbus, OH. 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: El-Gazzar M. Avian Influenza (AI) testing sample collection: Swabbing the upper respiratory system of poultry (ODA/OPA/USDA). February 1. Reynoldsburg, OH. 2016
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Lee CW. A novel, translational, multidisciplinary approach to control poultry respiratory diseases in the United States. WPDC. 65th Western Poultry Disease Conference, Vancouver, Canada, April 25?27, 2016
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Lee CW. Multidisciplinary, Multistate Collaborative Project to Control Poultry Respiratory Diseases in the United States. North Central Avian Disease Conference. March 14-15. St. Paul. MN. 2016.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Lee CW. Poultry Respiratory Disease Coordinated Agricultural Project (PRD-CAP). American Veterinary Medical Association Convention, San Antonio, TX August 5-9, 2016.