Progress 01/01/24 to 12/31/24
Outputs
Target Audience:Academic and industry scientists working on animal health, especially with respect to genetics, disease transmission, and evolution. K-12 students and poultry producers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?University of Edinburgh employed 2 full-time post-docs and 1 part-time statistician and bio-informatician, who developed expertise in statistical path- and meta-analyses (objectives 2 & 3), bio-informatics and phylodynamics (Objective 2), and statistical and dynamic modelling coupled with Bayesian inference (Objective 3). ILRI in 2024 engaged two Masters- level students as graduate fellows (generating their master's degree theses from the research done) under Objective (s). One student from the Department of Agricultural Economics and Agribusiness at the University of Ghana, and one student from the Master in Agriculture Program at Oklahoma State University (United States). A total of 54 persons (36 males and 18 females) participated in four (1 to 3-day) training events hosted by ILRI on: (1) The group model building approach (2) data collection using focus group discussion techniques (3) data collection using the key informant interview approach and (4) the i-MADE-it tool, a user-friendly web-based interface generated from the quantitative SD model for use by farmers and poultry sector policy makers for farm management/disease management decision-making. Participants were students from the University of Ghana (department of Agricultural Economics and Agribusiness) and regional livestock officers from the government Ministries of Food and Agriculture and Rural Development. Using a training-of-trainer (ToT) approach, ILRI provided technical backstopping to the regional livestock officers who attended the training workshop on the i-MADE-it tool, to further share knowledge on use of the tool within their respective regions. An additional 484 district level livestock and extension officers were trained at events held in 15 of the 17 regions in Ghana, consisting of 401 male (83%) and 83 female (17%) trainees. How have the results been disseminated to communities of interest?Scientific papers Aboah, J., Enahoro, D., Mensah, C., Agyemang, N. A., Kondo, E., & Ayertey, D. (2025). Patterns of systemic problems in Ghana's poultry value chain: Journal of Agriculture and Food Research, 101738. [Funded by BBSRC partner]https://doi.org/https://doi.org/10.1016/j.jafr.2025.101738 Karcher, E.L., D. Bogdanic, and S.M. Cloft. 2024. Engaging high school students' interest, motivation, and knowledge through poultry science workshop. Journal of Extension. Currently in review. Scientific conferences and workshops: Oral Presentations Impact of vaccination and selective breeding on the transmission, survival and virus load of Marek's disease virus: a natural transmission study. Margo Chase-Topping, Jamie Prentice, Christopher Pooley, Jody Mays, Cari Hearn, E. Carol McWilliam Leitch, Barbara Shih, Glenn Marion, Samantha Lycett, John Dunn, Hans Cheng, Andrea Doeschl-Wilson. Avian Research Symposium. Online. May, 2024. Disentangling the role of direct and indirect effects of vaccination against Marek's disease: a path analysis modelling approach. Margo Chase-Topping, Jamie Prentice, Christopher Pooley, Jody Mays, Cari Hearn, E. Carol McWilliam Leitch, Barbara Shih, Glenn Marion, Samantha Lycett, John Dunn, Hans Cheng, Andrea Doeschl-Wilson. Quantitative Biology research seminar, University of Edinburgh, March 2024. Healthy animals for sustainable agriculture and One Health. Andrea Doeschl-Wilson. Invited KSLA Wallenberg lecture, Stockholm, Sweden, February 2024. "BICI: General purpose software for inference and simulation using compartmental models", Biomathematics and Statistic Scotland AGM, Chris Pooley and Glenn Marion, 25-26 November 2024 Influence of vaccination and host genetics on Marek's disease transmission. Andrea Doeschl-Wilson, Margo Chase-Topping, Christopher Pooley, Jody Mays, Cari Hearn, E. Carol McWilliam Leitch, Barbara Shih, Glenn Marion, Samantha Lycett, John Dunn, Hans Cheng. USDA EPVD research seminar, January 2025. Why to breed for reduced host transmission, and how? Andrea Doeschl-Wilson. Invited talk at the Gordon Conference of Quantitative Genetics and Genomics, Lucca, Italy, February 2025 US-UK Collab: Influence of vaccines, host genetics, and mutation rates on the evolution of infectious diseases. John Dunn, Jody Mays, Cari Hearn, Hans, Cheng, Margo Chase-Topping, Carol Leitch, Samantha Lycett, Andrea Doeschl-Wilson. Conference of Research Workers in Animal Diseases, Chicago, IL, January 2025. Comparing the effect of vaccination versus hot resistance on natural transmission of Marek's disease virus. Jody Mays, Hans Cheng, Cari Hearn, Margo Chase-Topping, Samanth Lycett, Andrea Doeschl-Wilson, Jakob Trimpert, John Dunn. International Symposium on Marek's Disease and Avian Herpesviruses. St. Louis, MO, July 2024. Poster Presentations Examination of Gene Evolution and Phylodynamics in Marek's Disease Virus. E. Carol McWilliam Leitch, Hans H. Cheng, Andrea Doeschl-Wilson, John R. Dunn, Nikolaus Osterrieder, Jakob Trimpert and Samantha Lycett. Microbiology Society, Edinburgh, UK. March 2024. One Health, University of Edinburgh, UK. November 2024. Evolutionary dynamics and recombination in the avian coronavirus, infectious bronchitis virus. E. Carol McWilliam Leitch, Hans H. Cheng, Andrea Doeschl-Wilson, John R. Dunn, Nikolaus Osterrieder, Jakob Trimpert and Samantha Lycett. Microbiology Society, Edinburgh, UK. March 2024. Ex-ante Impact and Trade-off Analyses of Marek's Disease Mitigation in Small-Scale Intensive Layer Production in Ghana. Joshua Aboah and Dolapo Enahoro. Tropentag 2024: Annual Conference on research in tropical and subtropical agriculture. Vienna, Austria. 11-13 September 2024. Effects of disease control strategies on the efficiency of chicken production in the Bono region of Ghana. Desmond Ayertey, Freda Asem, Charles Okyere, and Dolapo Enahoro. Tropentag 2024: Annual Conference on research in tropical and subtropical agriculture. Vienna, Austria. 11-13 September 2024. Drivers of Change for Marek's disease control in small-scale layer production system in Ghana: a system dynamic modelling approach. Joshua Aboah and Dolapo Enahoro. 17th International Symposium on Veterinary Epidemiology and Economics. ICC Sydney, Australia. 11-15 November 2024. Stakeholder engagement events: ILRI had two main engagements with poultry sector stakeholders: a stakeholder scoping meeting, and a (first) group model building workshop. The project approaches and updates were presented at both events, including a session presented (virtually) by the USDA partner. ILRI hosted a (two-day) workshop in October 2024 with poultry value chain stakeholders for across Ghana's administrative regions. On day One, the findings of the project research were presented to inform and elict discussion/validation of stakeholders. On day Two, regional livestock and extension officers were trained on a decision-making tool under Objective (3). What do you plan to do during the next reporting period to accomplish the goals?In objective 1, virus isolation will continue for passage 10 samples to allow genotype and phenotype comparison of passage 1 and 10 virus isolates. In objectives 2 and 3, we plan to conduct an extensive sensitivity analyses and rigorous model selection procedures to validate the preliminary model results. Furthermore, we will combine data from all four MD transmission experiments in Objective 1 in a meta-analysis to assess vaccine and host genetic effects on MDV transmission and evolutionary dynamics. Specifically, we aim to determine the role of the feather virus load in chicken as biomarker for individual and flock-level protection from MD. Lastly, we will apply the modelling techniques developed for MDV to study vaccine effects on IBV transmission and evolutionary dynamics. We anticipate at least 4-5 manuscripts for publication in scientific journals.
Impacts
What was accomplished under these goals?
Objective 1:Determine the influence of imperfect vaccines, host genetics, and viral mutation rate on transmission and evolution to higher virulence(95% completion). Four high-resolution empirical datasets for detailed analyses of the effects of HVT vaccination and host genetics on Marek's disease virus (MDV) transmission and virulence evolution have been generated from four large-scale MDV transmission experiments, carried out by USDA-ADOL. These datasets are unique in their scope to 1) assess both the direct and indirect effects of vaccination or host genetic resistance to Marek's disease (MD) on the virus transmission, and to 2) monitor differences between vaccinated and non-vaccinated birds or birds that are genetically resistant / susceptible to MDV, with regards to changes in the virus sequences and associated changes in virulence over 10 successive generations (passages) of virus transmission between naturally infected shedder birds and naïve contact birds. Experimental protocols have been standardized and refined over successive experiments, and documented in detail. Generation of a similar dataset for infectious bronchitis virus (IBV) was completed during this reported period. Objective 2:Validate viral genome polymorphisms associated with increased virulence and the ability of the virus to escape immune surveillance(75% completion). Freie Universität Berlin has continued to perform sequence-based enrichment of MDV samples to determine sequence diversity and fitness-based selection of MDV variants in samples obtained under Objective 1. Both Illumina short read and Oxford Nanopore long read sequencing data confirm low levels of genetic variability in all sample types. However, our results suggest an increase in frequency of certain mutation in HVT vaccinated individuals. Currently, our final analysis focuses on determining the exact nature and frequency of mutations selected by HVT vaccination. Freie Universität Berlin has further developed sequencing protocols to sequence samples derived from tracheal swabs of birds infected with IBV under Objective 1. We are currently sequencing several hundred of those samples from passage 1 to 10 of chicken IBV infection in presence and absence of vaccination. The data generated here will be analyzed for genetic changes in response to vaccination and any indication of mutations associated with resistance development. In collaboration with the Szpara Group at Penn State University, we analysed deep-sequenced reads of 60 pathotyped MDV field strains. Using MCMC time-correlated phylogenetic analysis, we obtained substitution rates for the full genome, 46 diverse genes, and conserved genes. Effective population modelling and vaccine history integration identified genes potentially linked to vaccine escape across three vaccination periods. Genotype-phenotype associations revealed three previously unlinked pathogenicity genes. We also explored MDV microRNAs (miRNAs) and found evolution in 3 LAT-cluster miRNAs. Preliminary findings suggest LAT miRNA evolution may contribute to increasing neuropathy via the neurovirulence factor pp14 and host mRNA factors PABP1 and paip2. Objective 3:Build data-informed evolutionary-epidemiological simulation models to develop strategies to control the ecology, evolution and socio-economic burden of MD(80% completion). Protocols for data curation, filtering and processing have been developed to generate the standardized master datasets for all experiments conducted in this project to be used for modelling. The same procedure is currently applied to generate master data files for the IBV transmission experiment. A Bayesian Inference methodology was developed to convert qPCR CT values into reliable virus load measures, accounting for detection thresholds and measurement errors. Mixed models assessed the effects of vaccination and host genetics on MD transmission. While vaccination (full and reduced dose) and genetic resistance did not prevent MDV infection, they significantly reduced MD pathology and death in vaccinated and resistant birds. Both vaccination and resistance lowered MDV shedding, though the reduction was statistically significant only for full vaccination. Full-dose HVT vaccination also provided significant indirect protection to non-vaccinated contact birds by reducing MD disease, death, and virus shedding. Statistical path analysis revealed these indirect effects were mediated by reduced FVL in infected birds. To study vaccination and host genetics' effects on MDV infection and evolution, individual-based epidemiological models were developed alongside Bayesian inference methods to account for host resistance and infectivity variations. The models classify chickens as exposed ('E') and progressing through infectious states. Data for low-dose vaccinated birds show vaccination significantly reduces disease progression, with fewer deaths in vaccinated birds. Mortality rates rise with disease severity, and few die in the 'E' state compared to many in the severe 'IN' state. A notable finding was the impact of environmental pathogen burden, where higher initial MDV exposure accelerated disease progression. Overall, vaccination offers direct protection to vaccinated birds and indirect protection to the flock by reducing MDV shedding. Comparing estimated rates for individuals' disease progression across 10 passages so far provides no evidence for virus evolution towards increased transmissibility or virulence. The analyses are currently expanded to the other master datasets of this project. System dynamics (SD) models were developed for small-scale poultry production in Ghana to assess how epidemiological and socio-economic factors affect farm and industry performance. A qualitative model identified key intervention areas: research funding, biosecurity adoption, and reduced reliance on imported chicken. A quantitative SD model, combining disease epidemiology, financial analysis, and management decisions, evaluated disease mitigation strategies. Revaccination of locally hatched chicks reduced Marek's disease losses by over 76% in both systematic and cohort production schemes, while losses increased by 25.58% when using imported day-old chicks. Econometric analysis showed higher farm efficiency on farms that culled birds early during extreme weather or disease outbreaks and among older farmers. A gender-disaggregated dataset is being analyzed to explore how gender and demographics influence biosecurity adoption and access to credit for women farmers. Objective 4:Disseminate information on MDV and IBV, and the impact of vaccination to poultry producers and the public through training, workshops, online videos, seminars, and various engagement activities(95% completion). In the UK, an online simulation game, Epi-Evo-Chicken-Sim, hosted on Scratch (https://scratch.mit.edu/projects/333623433). Targeted at high school pupils, veterinary students, and the public, the game introduces methods like vaccination and selective breeding to control infectious disease outbreaks in farmed chickens. Players act as chicken farmers aiming to prevent disease spread cost-effectively. Key objectives include understanding genetic resistance, vaccination, disease spread unpredictability, and pathogen evolution. The game will be featured in future teaching and public engagement activities. Additionally, in 2024, the Elementary PAVE online program, developed last reporting period in the US, was adapted for distribution in the UK. Teachers volunteered to pilot the modules in their classrooms, and a questionnaire was created and distributed to teachers throughout Scotland to gather feedback on the student experience In the US, the primary focus this reporting period was data analysis and manuscript preparation. During this time, data from the High School PAVE program was analyzed, and a manuscript was submitted to theJournal of Extension.
Publications
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Progress 01/01/23 to 12/31/23
Outputs
Target Audience:Academic and industry scientists working on animal health, especially with respect to genetics, disease transmission, and evolution. K-12 students and poultry producers. Changes/Problems:A costed project extension has been provided by the UK Research & Innovation Council (UKRI) to the UK partners to contribute until 31.3.2025 to the project. What opportunities for training and professional development has the project provided?At USDA-ADOL, we hired two post-undergraduates in March 2021 for training and assistance with Objective 1. Freie Universität Berlin hired a PhD student in December 2021 to work on the sequencing of chicken derived MDV and Infectious Bronchitis Virus (IBV) samples for Objective 2. Purdue University hired a graduate student (PhD) for Objective 4. University of Edinburgh employed 2 full-time post-docs and 1 part-time statistician and bio-informatician, who developed expertise in experimental design and statistical path- and meta-analyses (objectives 2), bio-informatics and phylodynamics (Objective 2), and statistical and dynamic modelling and inference (Objective 3). A proto-type of the epidemiological models and inference software for estimating effects of vaccination or host genetics on disease transmission developed within this project was integrated into the 5 day international summer school "Understanding infectious diseases by fusing epidemiology, genetics and modelling" organized by the Swedish University of Agricultural Sciences jointly with the University of Edinburgh and held in Sweden (25.9.-29.9.2023), which was attended by 18 early career scientists from various countries. Two categories of training were initiated by ILRI in the reporting period: (1) short-term capacity development such as training events to prepare enumerators to use specific data collection tools, and (2) longer-term training that extends for six months or more and involves skills training on various aspects (such as scientific writing, field study design, data collection and analytical methods) of the socioeconomic modeling component. ILRI is processing the appointments of two graduate student interns/fellows for long-term training (nine months) under this project that will be directly associated with the students' dissertation research at their respective universities. How have the results been disseminated to communities of interest?In addition to community outreach and focused groups mentioned above, we have also disseminated results through scientific conferences and workshops. Scientific conferences and workshops: Oral Presentations US-UK Collab: Influence of vaccine, host genetics, and mutation rates on the evolution of infectious diseases. Jody Mays, Hans Cheng, Cari Hearn, Margo Chase, Sam Lycett, Barbara Shih, Andrea Doeschl-Wilson, John Dunn. International Herpesvirus Workshop, Missoula, MT. July 2023. Disentangling the role of direct and indirect effects of vaccination against Marek's Disease: a path analysis modelling approach. Margo Chase-Topping, Jamie Prentice, Jody Mays, Christopher Pooley, Cari Hearn, E. Carol McWilliam Leitch, Barbara Shih, Glenn Marion, Samantha Lycett, John Dunn, Hans Cheng, Andrea Doeschl-Wilson.. Epidemics9, Bologna, Italy. November-December 2023. Poster Presentations Combined influence of imperfect vaccines, host genetics, and non-genetic drivers on virus transmission and virulence evolution. Jamie Prentice, E. Carol McWilliam Leitch, Margo Chase-Topping, Christopher Pooley, Glenn Marion, Barbara Shih, Jody Mays, Jacob Trimpert, Klaus Osterrieder, Dolapo Enahoro, Cari Hearn, Elizabeth Karcher, John Dunn, Hans Cheng, Sam Lycett, Andrea Doeschl-Wilson. Evolution, Pathogens and Public Health, Glasgow, UK. October 2023. Epidemics9, Bologna, Italy. November-December 2023. Investigation of Gene Evolution and Phylodynamics in Marek's Disease Virus. E. Carol McWilliam Leitch, Hans H. Cheng, Andrea Doeschl-Wilson, John R. Dunn, Nikolaus Osterrieder, Jakob Trimpert and Samantha Lycett. One Health, University of Edinburgh, UK. November 2023. Epidemics9, Bologna, Italy. November-December 2023. Evolutionary dynamics of the highly recombinant avian coronavirus, infectious bronchitis virus. E. Carol McWilliam Leitch, Hans H. Cheng, Andrea Doeschl-Wilson, John R. Dunn, Nikolaus Osterrieder, Jakob Trimpert and Samantha Lycett. Epidemics9, Bologna, Italy. November-December 2023. What do you plan to do during the next reporting period to accomplish the goals?In objective 1, a comprehensive database with quality assessment, for all experiments combined will be built to enable comparative analysis between the baseline (Experiment 1: full-vaccination) and reduced-dose (Experiment 3) and genetic selection for resistance (Experiment 4). This data will be analysed using both statistical models and a Bayesian network path-analysis approach. Furthermore, the data and results will be used to inform and infer the parameters in the epidemiological, evolutionary and socio-economic models. In Objective 2 and 3, we will complete sequencing of viruses generated from Objective 1, analyze polymorphisms, and complete model development and fitting to the experimental data. We will update the modelling frameworks to incorporate any new experimental approaches, and use the additional data collected to extend model fitting. The bioinformatic pipeline we developed to process MDV Illumina short read sequences to investigate polymorphisms in both consensus sequences and within mutant spectra will be applied to MDV reads generated in Objective 2. The new inference methods developed and assessed for Objective 3 will be utilized alongside recognized methods to investigate epidemiological and phylodynamic processes. A manuscript detailing the experimental designs and statistical methodologies developed in this project, using the results from Experiment 1 as illustration, will be submitted for publication. Regarding the socio-economic modelling in Objective 3, the data collected from the farmer group discussions and using the in-depth interview questionnaires are currently being analyzed and will be used to parameterize a system dynamics model in StellaR (software). A group model building workshop will be held to validate the model structure and behavior with stakeholders. Scenarios of different farm management and policy interventions will be developed with stakeholders and their potential socioeconomic outcomes quantified. One student will conduct research on the interactions of Marek's control strategies with production efficiency and welfare at farm/household level as part of their MPhil research at the University of Ghana. A second student will conduct research on the gender dimensions of Marek's disease management and control among small and medium-size poultry layer farmers in Ghana, as part of their MS research at the Oklahoma State University. Both studies contribute to the aim of understanding socio-economic dimensions of Marek's disease's epidemiology, control, and management in small and medium scale poultry enterprises in a developing country setting. In Objective 4, we will analyze data from the elementary and high school PAVE programs, submit abstract for the elementary PAVE program results to the North American College & Teachers of Agriculture annual meeting (June 2024), submit abstract for the high school PAVE workshop results on the Poultry Science Association annual meeting (July 2024), and prepare and submit manuscripts for both the elementary and high school programs.
Impacts
What was accomplished under these goals?
Objective 1: Determine the influence of imperfect vaccines, host genetics, and viral mutation rate on transmission and evolution to higher virulence (90% completion). Four high-resolution empirical datasets for detailed analyses of the effects of HVT vaccination and host genetics on Marek's disease virus (MDV) transmission and virulence evolution have been generated from four large-scale MDV transmission experiments, carried out by USDA-ADOL. These datasets are unique in their scope to (1) assess both the direct and indirect effects of vaccination or host genetic resistance to Marek's disease (MD) on the virus transmission, and to (2) monitor differences between vaccinated and non-vaccinated birds or birds that are genetically resistant / susceptible to MDV, with regards to changes in the virus sequences and associated changes in virulence over 10 successive generations (passages) of virus transmission between naturally infected shedder birds and naïve contact birds. Experimental protocols have been standardized and refined over successive experiments, and documented in detail. Generation of similar data for infectious bronchitis virus (IBV) is in process and expected to be completed by the end of the current reporting period. Objective 2: Validate viral genome polymorphisms associated with increased virulence and the ability of the virus to escape immune surveillance (50% completion). Using a target capture approach designed and established in Year 1, Freie Universität Berlin has performed targeted sequencing of MDV from various tissue samples obtained from the experiments described above (Objective 1). Sequencing indicates high genetic stability of MDV with few polymorphisms discovered across the MDV genome. In parallel, Freie Universität Berlin has continued to develop targeted long-read sequencing using a CRISPR-Cas9 based enrichment approach combined with Oxford Nanopore sequencing. Using this approach, we are able to enrich and sequence MDV DNA strands of >10 kb with up to 90% reads on target from cell culture and infected tissues. We are currently establishing methods to detect recombination in individual virus genomes. The next generation MDVsequence data from the experimental samples has been generated by USDA and Freie Universitaet Berlin partners in 2023. This sequence data, together with virulence phenotype measurements will be used to examine viral genome polymorphisms (or viral haplotype diversity) associated with virulence, and compared to previous findings. As a precursor, a bioinformatic pipeline for processing Illumina short read sequences was developed at the Roslin Institute to investigate MDV polymorphisms in both consensus sequences and within mutant spectra. Sequencing reads were firstly subjected to reference-based assembly to obtain the consensus, then remapped against this consensus to disclose minor variants. The procedure was subsequently validated using deep-sequencing reads generated from a historical collection of MDV strains. We found evidence of heterogeneous virus populations, including minor haplotypes of known virulence genes and in other genome regions. Objective 3: Build data-informed evolutionary-epidemiological simulation models to develop strategies to control the ecology, evolution and socio-economic burden of MD (50% completion). Statistical models of vaccination effects on transmission and disease outcomes from Experiment 1 data is complete and the first draft of a manuscript has been prepared. This manuscript focuses on the experimental design and statistical modelling techniques as well as the results of full-vaccination, as baseline situation. The statistical analyses of Experiment 1 has shown that vaccination against MD does not block infection and virus transmission but significantly reduces virus shedding, MDincidence and mortality of vaccinated birds. In addition, transmission of vaccinated bird significantly reduces virus shedding, MD incidence and mortality of non-vaccinated Contact birds. The validity of these findings across different MDV vaccine doses and genetic differences in host resistance will be further assessed using the data of all experiments combined, with the use of both statistical and epidemiological models. Statistical path analyses and computational inference algorithms are currently developed to enable accurate estimation of vaccination and host genetic effects on key transmission and virulence parameters from the experimental data and models. Population-based epidemiological ordinary differential equation models have been constructed in R to determine how vaccination or host genetics affect virus transmission and evolution in the experiments of objective 1. The models account for heterogeneity in shedding patterns, taking into account vaccination status and initial exposure due to an environmental reservoir. The models are coded so that they fully represent the experimental design of the experiments carried out in objective 1, with the corresponding movement and infection dynamics in each passage. The models are currently fitted to the data of experiment 1 using model fitting algorithms in R's FME library. Posterior distributions for the epidemiological transition rates give evidence that infection outcome depends on exposure dose and vaccination status of the focal birds and the shedder birds, but appear relatively stable across passages. Parallel to the population-based epidemiological models, individual based epidemiological models with complementary Bayesian inference methods that can account for individual variation in host resistance and infectivity are currently developed. In addition to the epidemiological models described above, phylodynamic inference models are being developed to incorporate the viral genomic data and metrics (e.g. diversity, polymorphisms, specific haplotypes) derived in Objective 2. Objective 4: Disseminate information on MDV and IBV, and the impact of vaccination to poultry producers and the public through training, workshops, online videos, seminars, and various engagement activities (80% completion). In 2023, the primary focus included 1) administering the elementary PAVE modules, 2) developing and recruiting 7th - 12th grade students to the workshops, and 3) administering the workshops. Elementary PAVE: In early 2023 nine teachers received boxes in the mail that contained materials needed to complete the PAVE modules (teacher's facilitator guide, student interactive notebooks, and cards for the in-class assignment). In total 333 students enrolled in the program representing 13 classrooms in both Michigan and Indiana. Each student received their own login so we could track and view program completion. High School PAVE: In summer 2023, we created learning outcomes and activities designed for a 90-minute workshop. Eggsellent Careers: Discovering Animal Health Through Poultry Science included the following student learning outcomes: Compare and contrast blood samples of different species Describe proper technique for running an avian whole-blood plate diagnostic test Create a bio-secure poultry facility addressing the three levels of biosecurity Discuss opportunities in the poultry industry and explore the process of candling eggs and its importance Four stations were created to align with the learning outcomes. These included: Animal Health and Hematology (multiple species blood smears, microscopes identification) Avian Disease Diagnostics (Pullorum-Typhoid testing using the whole blood-plate test) A Birdseye View of Biosecurity (Glo Germ activity) Egg Quality, Marketing, and Careers (exploring egg cartons, candling eggs, career discussion) In total, 64 students participated across four workshop offerings. Students represented California, Indiana, Louisiana, and Arizona. Through our UK partnership, significant progress has also been achieved regarding to modelling the socio-economic aspects of MD in Africa.
Publications
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience:Academic and industry scientists working on animal health, especially with respect to genetics, disease transmission, and evolution. K-12 students and poultry producers. Changes/Problems:The most significant obstacle encountered has been recovering from delays from on-site work restrictions due to COVID-19. What opportunities for training and professional development has the project provided?At USDA-ADOL, we hired two post-undergraduates in March 2021 for training and assistance with Objective 1. Freie Universität Berlin hired a PhD student in December 2021 to work on the sequencing of chicken derived MDV and Infectious Bronchitis Virus (IBV) samples for Objective 2. Purdue University hired a graduate student (PhD) for Objective 4. University of Edinburgh hired 3 post-docs and 1 bio-informatician to obtain training in experimental design and statistical analysis (objective 1), bio-informatics and phylodynamics (Objective 2), and statistical and dynamic modelling and inference (Objective 3). How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In Objective 1, we expect to complete all remaining MDV experiments during the next reporting period and half of the IBV experiments and the corresponding statistical analyses to provide the relevant summary statistics and hypotheses for the models in Objectives 2 and 3. In Objective 2 and 3, we will complete sequencing of viruses generated from Objective 1, analyze polymorphisms, and complete model development and fitting to the experimental data. In Objective 4, we will have students and teachers complete the PAVE program and assessment materials. We will analyze the elementary PAVE program and high school workshop data, prepare learning outcomes and materials for the PAVE summer workshops, develop assessment questions for workshops and submit for IRB approval, recruit high school students for the PAVE summer workshops, and then facilitate the three summer workshops.
Impacts
What was accomplished under these goals?
Objective 1: Determine the influence of imperfect vaccines, host genetics, and viral mutation rate on transmission and evolution to higher virulence (80% completion). Three high-resolution empirical datasets for detailed analyses of the effects of HVT vaccination and host genetics on Marek's disease virus (MDV) transmission and virulence evolution have been generated from three bespoke MDV transmission experiments, carried out by USDA-ADOL. These datasets are unique in their scope to (1) assess both the direct and indirect effects of vaccination or host genetic resistance to MD on the virus transmission, and to (2) monitor differences between vaccinated and non-vaccinated birds or birds that are genetically resistant / susceptible to MDV, with regards to changes in the virus sequences and associated changes in virulence over 10 successive generations (passages) of virus transmission between naturally infected shedder birds and naïve contact birds. Experimental protocols have been standardized and refined over successive experiments, and documented in detail. Specifically, using inbred ADOL 15I5 x 71 antibody negative birds, Experiments 1 and 3 provided data to determine to what extent vaccinations with the full recommended dose or 1/10 of the recommended dose of HVT, respectively, influences MDV transmission and viral genome evolution. Experiment 2 used outbred SPF layers Charles River line 22 and Babcock birds, known for their high and low resistance to MDV, respectively, to examine how host genetic resistance influences MDV transmission and evolutionary dynamics. The transmission design allows for detailed investigation about the direct protective effects that vaccines offer to vaccinated birds as well as the indirect protective effects on vaccinated or non-vaccinated contact birds. Statistical analyses of the experimental data showed that the infection and transmission dynamics of birds that have been inoculated with MDV differ substantially from those of birds that have become naturally infected through contact with infected shedder birds, highlighting the importance of mimicking modes of transmissions representative of field conditions in vaccination and other MDV challenge experiments. Furthermore, experiment 1 demonstrated that HVT vaccination does not prevent MDV transmission within all 10 subsequent passages. However, vaccination with the full recommended HVT dose was found to not only provide direct protection from MD and death to the vaccinated birds, but also indirect protection for non-vaccinated contact birds. Generation of data for infectious bronchitis virus (IBV) has been delayed due to COVID-19 restrictions and the ensuing issues with performing experiments in containment. The IBV vaccination/challenge experiments and serial passages are currently planned for Q3/Q4 of 2023 and Q1/Q2 of 2024. Objective 2: Validate viral genome polymorphisms associated with increased virulence and the ability of the virus to escape immune surveillance (15% completion). Freie Universität Berlin is continuing to develop and implement strategies for the enrichment and subsequent sequencing of MDV DNA from various sample types including blood samples collected during MDV in vivo evolution experiments conducted by ADOL. We have established an Illumina short-read based sequencing technique that reliably enriches whole MDV genomes from samples containing as little as 10 MDV copies. Thus far, no, or only few polymorphisms were discovered in MDV sequences obtained by the serial passaging experiments. In parallel, we have established Oxford Nanopore-based long read sequencing employing a CRISPR-Cas9 based approach for enrichment of long virus DNA fragments. Sequencing MDV fragments of >10 kb obtained from infected chicken embryonic cell cultures with >100-fold enrichment of MDV over host sequences is now routinely possible. We are currently further exploring the use of this technique with samples obtained from infected chickens. The next generation Marek's disease virus and infectious bronchitis virus sequence data from the experimental samples will be generated by USDA and Freie Universitat partners in 2023 Q1. This sequence data, together with virulence phenotype measurements will be used to examine viral genome polymorphisms (or viral haplotype diversity) associated with virulence, and compared to previous findings (Objective 2). Objective 3: Build data-informed evolutionary-epidemiological simulation models to develop strategies to control the ecology, evolution and socio-economic burden of MD (20% completion). Statistical analyses of experiment 1 data supports the hypotheses that MDV infection outcome of individual birds depends strongly on their exposure dose, that the latter decreases at a faster rate over successive generations of transmission in vaccinated or resistant birds and that feather virus load of infected shedder birds is a good indicator for their infectivity and, cumulatively, for the exposure dose of contact birds. The validity of these hypotheses will be further assessed using the data of all experiments combined, with the use of both statistical and epidemiological models. To this purpose, statistical general linear models, as well as dynamic epidemiological and phylogenetic models have been developed to represent the experimental designs and the virus transmission and evolutionary dynamics. Statistical path analyses and computational inference algorithms are currently developed to enable accurate estimation of vaccination and host genetic effects on key transmission and virulence parameters from the experimental data and models. Additionally phylodynamic inference models are being developed to incorporate the viral genomic data and metrics (e.g. diversity, polymorphisms, specific haplotypes) derived in Objective 2. Development of the socio-economic models for Marek's disease by the UK subcontractor ILRI as part of Objective 3 has not yet started because of an unexpected delay in the approval of the project consortium agreement required by ILRI to work on the project, but will commence in 2023. Objective 4: Disseminate information on MDV and IBV, and the impact of vaccination to poultry producers and the public through training, workshops, online videos, seminars, and various engagement activities (40% completion). In 2022, the primary focus continued to be on K-12 curriculum development and recruitment. We worked to integrate suggestions that were made by our expert content reviewers (one poultry professional, one elementary teacher, and one poultry academic) into the five modules. Videos and other materials were collected in Spring and edited in the summer. The modules and the simulation games were finalized in summer 2022. In Fall 2022, we worked with Purdue to prepare the course shell for the program in Purdue's learning management software system. This is the site that teachers and students will visit to gain access to the program. Every student and teacher have their own individual login so we can track and view program completion. In addition to the curriculum development, we developed the assessment materials needed to answer our research questions. In Fall 2022, we received final IRB approval for the research component of the PAVE program and began recruiting teachers to participate. Overall, 401 4th and 5th grade students enrolled in the program (202 from Michigan and 199 from Indiana). Additionally, an interactive notebook and classroom-based project were created. All materials were shipped to teachers with the expectation that the program will begin in Spring 2023.
Publications
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Progress 01/01/21 to 12/31/21
Outputs
Target Audience:Academic and industry scientists working on animal health, especially with respect to genetics, disease transmission and virus evolution. K-12 students and poultry producers. Changes/Problems:The most significant obstacle encountered has been hiring and on-site work restrictions due to COVID-19. What opportunities for training and professional development has the project provided?At USDA-ADOL, we hired two post-undergraduates in March 2021 for training and assistance with Objective 1. Freie Universität Berlin hired a PhD student in December 2021 to work on the sequencing of chicken derived MDV and Infectious Bronchitis Virus (IBV) samples for Objective 2. Purdue University hired a graduate student (PhD) for Objective 4. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In Objective 1, we expect to complete all remaining MDV experiments during the next reporting period. We also expect to generate our first set of IBV data for analysis. In Objective 2 and 3, we will initiate sequencing of viruses generated from Objective 1, analyze polymorphisms, and begin model development. In Objective 4, we expect to collect and edit all videos needed for modules, finalize module creation in Storyline, create and validate assessment materials for project to answer research questions, work with Purdue's Envision Center to create an interactive simulation game that will be embedded in module 2, obtain IRB approval and recruit 500 4th and 5th grade students (Indiana and Michigan) to enroll in the Spring 2023, begin preparing the in-person component of the program that will target high school students
Impacts
What was accomplished under these goals?
Objective 1: Determine the influence of imperfect vaccines, host genetics, and viral mutation rate on transmission and evolution to higher virulence (50% completion). We are using a shedder-sentinel challenge model to naturally passage Marek's disease virus (MDV) through 10 successive groups. Each group consists of 10 birds kept in an individual isolator and replicated at least 3 or 6 times. Viral replication and transmission are assessed by sampling shedder (donor) birds that transmit infectious virions prior to, at, and following co-housing with the contact (recipient) birds. Birds infected in Passage 1 transmit virus to recipients in Passage 2, and so on. There will be 4 large experiments, each consisting of 10 passages. During this first year we have completed the first 2 experiments, focusing on vaccination status and host genetics, respectively. The first experiment passaged virus through vaccinated vs. unvaccinated chickens. The second experiment passaged virus through susceptible vs. resistant maternal antibody-negative commercial specific-pathogen-free (SPF) birds. All 10 serial passages have been completed for both experiments and samples collected. In the first experiment, MD incidence has been maintained at high incidence through passage of the virus through unvaccinated chickens, and at very low incidence in vaccinated chickens. Preliminary results, however, suggest that MDV is being transmitted through serial passage in both vaccinated and unvaccinated chickens. In the second experiment MD incidence was consistently maintained at higher levels across passages in susceptible chickens. Generation of data for infectious bronchitis virus (IBV) has been delayed due to COVID-19 restrictions. Objective 2: Validate viral genome polymorphisms associated with increased virulence and theability of the virus to escape immune surveillance (5% completion). Freie Universität Berlin started the project in the second half of 2021 with establishing the MDV whole genome sequencing described here (https://doi.org/10.1038/s41564-019-0547-x) for samples derived from chicken infected with MDV at USDA/ADOL facilities. To this end, the tiling array employed for enrichment of MDV sequences from highly impure DNA, was modified and tested for its ability to enrich the MDV genome from samples that contain as little as 103 MDV genomes per µg of total DNA. This is important since blood and feather samples will be the major samples type in this project and, depending on the time of sampling, contain relatively little MDV DNA. We confirmed our ability to enrich MDV DNA from these samples using consecutive rounds of enrichment to obtain enough reads to cover the entire genome. In the same time, we are implementing an Oxford-Nanopore based workflow to obtain long MDV DNA reads from samples with greater MDV abundance. This will help to identify physical linkage of mutations on single virus genomes. Obtaining reads >10 kb remains a challenge in the moment, this problem will be subject to further exploration in 2022. Objective 3: Build data-informed evolutionary-epidemiological simulation models to develop strategies to control the ecology, evolution and socio-economic burden of MD (0% completion). Work will begin on Objective 3 once first set of samples from Objective 1 have been processed and sequenced. Objective 4: Disseminate information on MDV and IBV, and the impact of vaccination to poultry producers and the public through training, workshops, online videos, seminars, and various engagement activities (5% completion). The primary focus in 2021 was on K-12 curriculum development. We established that the online curriculum will consist of five online modules designed for students in 4th and 5th grade. The list below highlights the module topics and the learning outcomes for each module. Additionally, we created curriculum for each of the modules. This consisted of designing modules that included interactive and engaging features. Each module was designed to take approximately 30 to 40 minutes. During this process, we were able to identify videos that we will need to collect in year 2. We also collected the information needed to start working with Purdue's Envision Center to create an interactive simulation game in Year 2. Module 1: Introduction to Animal Health: History & Present Define animal health as it relates it to both livestock animals and any animals in your life Identify sick versus healthy animals from descriptions, drawings and videos Define One Health and discuss the connection between the health of people, animals and the environment Module 2: Biosecurity & Marek's Disease Identify Biosecurity Issues and why they are important Relate common health issues/diseases with biosecurity protocols Develop proper practices to keep humans and animals safe Module 3: Immunology: How Animals Fight Disease Explain the animal's defense system and how they fight disease Identify different pathogens and explain how the body protects itself from viruses, bacteria, and fungi Module 4: Vaccination Protocols Describe purpose and use of vaccine Discuss technology used to vaccinate flocks Recognize reasons vaccines can fail Module 5: Animal Health: Opportunities Discover different opportunities in animal science and animal health industry and why they are important Discuss the importance of the poultry industry to Midwest economy (versus States overall).
Publications
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