Report to Legislature | 2022

Executive Summary

In 2008, cases of limping elk exhibiting characteristic hoof lesions reported to the Washington Department of Fish and Wildlife (WDFW) increased markedly in southwestern Washington. By 2017, and in response to stakeholder concern regarding the intensity and spread of the disease, the Washington State Legislature unanimously passed Senate Bill 5474 to designate Washington State University’s College of Veterinary Medicine (WSU, CVM) as the state lead in developing a program to monitor and assess causes of and potential solutions for elk hoof disease. Funding of $1,169,900 was provided to WSU CVM for the FY2022-2023 biennium. The WSU CVM team dedicated to addressing elk hoof disease in 2022 included one full-time faculty member, one post-doctoral fellow, three PhD students, one master’s student, and one scientific assistant. Additional WSU faculty, staff, and students and non-WSU collaborators also made significant contributions. Research by our team resulted in publication of three scientific articles, which nearly doubled the published scientific literature on elk hoof disease. We re-evaluated the three-year Research Plan that was developed in 2018 and, with minor revisions, extended the scope of implementation through 2024. During the year, we made progress toward addressing the four principal areas of the Research Plan, as well as ancillary areas of inquiry.

• Study disease agents in the laboratory. We used state of the art technology to sequence DNA from bacteria in hoof samples collected from across the northwestern U.S. to determine what types of bacteria were present. The results provide further support that hoof lesions arise from an infectious disease process commonly associated with Treponema or Treponema-like bacteria; however, a few other types of bacteria are also routinely associated with TAHD lesions and are worthy of further investigation. These findings were reported in an article titled “Surveillance for an emergent hoof disease in elk (Cervus elaphus) in the U.S. Pacific West supplemented by 16S rRNA gene amplicon sequencing” and published in the Journal of Wildlife Diseases. We continued sample analyses for a larger study to further understand the importance of Treponema and other types of bacteria using a larger number of samples. Additionally, we implemented pathogen discovery work to construct the genomes of previously undescribed Treponema-like bacteria that may be critical components of TAHD infection.
  
• Conduct regional surveillance and investigate risk factors. Treponeme-associated hoof disease has been confirmed in Washington, Oregon, Idaho, and California. With this baseline distribution documented, in 2022 we focused sample collection primarily on areas (generally at the county level) where TAHD had not been previously detected and to obtain samples for research. Working with state wildlife managers, we collected hooves for research uses from Washington (13 elk) and for disease surveillance and research uses from Oregon (13 elk), Idaho (3 elk), and South Dakota (1 elk). We did not detect TAHD in any new states or in species other than elk.
  
  Mineral levels in hair of elk with and without TAHD were analyzed to determine if mineral deficiency, or toxicity, contributed to risk of disease. Lower selenium levels were associated with presence of TAHD and suggest that more investigation is warranted. An article titled, “Associations between hair trace mineral concentrations and the occurrence of treponeme-associated hoof disease in elk (Cervus canadensis)” was published in the journal BMC Veterinary Research. Additional research is underway using liver samples to further investigate any correlation of mineral levels and TAHD.
  
• Understand social aspects of the disease and communicate findings. We conducted outreach by distributing information via a listserv, website, and the lay and scientific media. Our quarterly newsletter was renamed ElkTracks in 2022. We also provided individual responses to all inquiries received. We collaborated with the Rocky Mountain Elk Foundation and the California Department of Fish and Wildlife to conduct a virtual scientific meeting on TAHD to share information among wildlife managers and health professionals from state, federal, and tribal agencies. We presented scientific findings from WSU’s research program at this scientific meeting as well as at the Wildlife Disease Association, Wildlife Society, and Lameness in Ruminants conferences.
  
• Ancillary projects. Two additional studies were conducted in direct response to stakeholder interest. In the first, we used hunters’ observations reported in harvest data routinely collected by WDFW that were shared with WSU to determine that, as suspected by stakeholders, antler asymmetry occurs at a higher rate in elk with hoof lesions. A manuscript reporting the findings titled “Association of antler asymmetry with hoof disease in elk” was published in the Journal of Wildlife Management. We also collaborated to investigate epigenetic effects (changes in gene activity and expression separate from the DNA sequence) that may influence susceptibility of elk to TAHD. Preliminary results indicate some differences in elk with and without TAHD. Preparation of a manuscript for submission to a scientific journal is underway.

Background and overview

Elk with hoof disease have characteristic ulcers on their feet with associated overgrown, broken, or sloughed hooves. Affected elk are debilitated and, according to preliminary research by WDFW, experience higher mortality which may lead to population level impacts. This disease has the potential to devastate Washington’s elk populations and because of the interaction of wild elk with domestic livestock, it is also of concern to other Washington stakeholders including the livestock industry.

In response to intense stakeholder concern, in 2017 the Washington State Legislature unanimously passed Senate Bill 5474 to designate Washington State University’s College of Veterinary Medicine (WSU, CVM) as the state lead in developing a program to monitor and assess causes of, and potential solutions for, elk hoof disease. A biennial budget was allocated to WSU CVM to address this effort beginning on July 1, 2017. At that time no elk hoof disease program existed at WSU, and a new program was literally created from the ground up, including the construction of an elk research facility. The Legislature continued funding for the FY2022-2023 biennium at a level of $1,169,900.

The following report consists of two sections. The first section, Research and Outreach Accomplishments in 2022, summarizes accomplishments made implementing our research plan in 2022. The second section, our Research Plan, was developed in 2018 and will be implemented through 2024. It defines the research approach that guides our work and is attached as an Addendum at the end of this report for reference.

Research and outreach accomplishments in 2022

General

• Student training. Students contribute to research while gaining education. Current students that are contributing to research described in this report include:
  • Elizabeth Goldsmith, DVM, is a fifth-year combined pathology residency/PhD student. Dr. Goldsmith’s research focuses on pathogen discovery using metagenomics techniques. She successfully completed PhD preliminary examinations in 2022. Anticipated graduation 2025.
  • Holly Drankhan, DVM, is a fourth-year combined pathology residency/PhD student. Dr. Drankhan is developing a reliable disease transmission model using elk at the captive research facility. Anticipated graduation 2025.
  • Zachary Robinson completed a master’s degree program in 2022. Zach grew-up in southwestern Washington observing elk with hoof disease. He studied disease transmission and lesion development in elk at the captive research facility.
  • Steven Winter, MS, began a PhD program in 2021. Steven is studying spatial and temporal distribution and risk factors of TAHD using computer modeling. Anticipated graduation 2024.
  • Thomas LeClair, MS, conducted a summer research project following his first year of veterinary school. Thomas investigated pathogens present in elk hooves with TAHD using shotgun sequencing of bacterial DNA.
  • Jessica Yamauchi conducted a summer research project following her second year of veterinary school. Jessica compiled a dataset on liver mineral levels in elk with and without TAHD.
    
• Post-doctoral fellow. Sushanta Deb, PhD, joined our research lab in fall 2022. Dr. Deb brings extensive experience with computer analysis of complex genetic codes (a field called bioinformatics). He will apply and expand his knowledge to discover and describe bacteria associated with TAHD.
  
• Staffing. A scientific assistant, Charlie Park, manages the laboratory, oversees diagnostic case submissions and processing, and coordinates research animal care. Veterinary and undergraduate students assist with laboratory tasks and animal care.

Study the disease cause(s) and contributing factors in captive elk

• Captive elk facility. We maintained the elk research facility, which was constructed in 2020, and performed upgrades to its laboratory space. One research experiment was completed at the facility in 2022.
  
• Animal care and biosafety. Coordination with the WSU Environmental Health and Safety office and animal care oversight programs contributed to ensuring compliance with applicable standards as well as state and federal regulations. Protocols for holding and conducting research on captive elk were maintained and approved by the WSU Institutional Animal Care and Use Committee (IACUC), a federally mandated oversight group.

Study disease agents in the laboratory

• Analysis of bacterial DNA to support disease surveillance. We continued investigating bacteria that are present in TAHD lesions by analyzing a portion of DNA extracted from foot tissue. This technique (16S amplicon sequencing) allows for identification of bacteria based on their unique DNA sequence in one specific gene. Samples were obtained from both free-ranging elk submitted by wildlife managers for TAHD surveillance and from captive elk experimentally challenged with TAHD. In both cases, results suggested that hoof lesions arise from an infectious disease process commonly associated with Treponema or Treponema-like bacteria; however, a few other types of bacteria, e.g., Mycoplasma, are also routinely associated with TAHD lesions and are under further investigation. Results from this inquiry to identify bacteria present in samples from across the known range of TAHD are reported in a manuscript published in the Journal of Wildlife Diseases (2022; 58:487-499). The importance of public involvement in TAHD surveillance and reporting that allows studies such as this to be conducted was highlighted in the Fall 2022 ElkTracks newsletter.

SURVEILLANCE FOR AN EMERGENT HOOF DISEASE IN ELK (CERVUS ELAPHUS) IN THE U.S. PACIFIC WEST SUPPLEMENTED BY 16S rRNA GENE AMPLICON SEQUENCING

Margaret A. Wild, Kyle R. Taylor, Devendra H. Shah, Kyle Garrison, Kristin Mansfield, Julia Burco, Steven N. Winter, Mark L. Drew, Sushan Han, Robert Bildfell, and Brandon A. Munk

ABSTRACT: A novel hoof disease of elk (Cervus elaphus) was described in southwestern Washington, US, in 2008 and was subsequently diagnosed in an adjacent area in northwestern Oregon in 2014. The disease, currently referred to as treponemes-associated hoof disease (TAHD), is characterized by lesions ranging from mild erosions; to severe ulcers with underrunning of the hoof capsule and heel-sole junction; to overgrown and avulsed hoof capsules. Histologically, lesions exhibit epithelial erosion or ulceration, suppurative inflammation, and the presence of argyrophilic spirochetes. We used data collected by the Washington Department of Fish and Wildlife and Oregon Department of Fish and Wildlife from 2008-2017 as reference for disease distribution. We then conducted enhanced surveillance in 2018-2020 by obtaining 164 submissions from four US Pacific West states. We detected TAHD for the first time in Idaho and northern California, as well as in multiple counties in Washington and Oregon where it had not been previously reported. Given the unexpectedly broad disease distribution, continued surveillance is warranted to determine the full geographic extent of TAHD. Using samples from 22 elk, we investigated 16S rRNA gene amplicon sequencing as a technique that could be used to supplement TAHD surveillance. Operational taxonomic units of the family Spirochaetaceae were identified in 10/12 histologically diagnosed TAHD-positive cases and 2/10 TAHD-negative cases. Phyla Spirochaetae (P<0.008), Fusobacteria (P<0.006), and Tenericutes (P<0.01) were overrepresented in samples from TAHD-positive feet when compared to TAHD-negative elk. A unique spirochete, PT19, was detected in hooves of 11 elk and from at least one elk in each state. Results support the use of 16S rRNA gene amplicon sequencing as a reliable and informative tool to supplement investigations into distribution and etiology of this presumed polybacterial disease.

• Pathogen discovery using bacterial DNA analysis. We continued hoof sample collection for a larger study and began classifying microscopic lesions from over 150 cases representing both TAHD-affected and normal hooves for use in this analysis (Goldsmith PhD project).
  
  Additionally, we began even more in-depth investigation of bacteria than can be achieved with the 16S amplicon sequencing described above. We conducted a pilot study to investigate a technique called shotgun sequencing that has not to our knowledge been previously used to study hoof disease in elk. Shotgun sequencing produces longer DNA sequences that can more specifically identify bacteria at the species-level. Further, the genomes of previously uncharacterized bacteria can be constructed to discover new species or strains of bacteria. This approach is particularly useful in studying diseases like TAHD that are associated with bacteria that are challenging, if not currently impossible, to grow in the laboratory.

Conduct regional surveillance and investigate risk factors

• Spatial distribution and risk. Preliminary analysis examining risk factors for TAHD and its distribution indicated that a more complete and rigorous evaluation was needed to understand this complex disease. We proceeded on investigations to help us understand why the disease occurs where it does and if predisposing factors, like environmental conditions or human alterations, are important in disease occurrence (Winter PhD project).
  
  One proposed factor that may change an elk’s susceptibility to TAHD is the level of important minerals in the body. We analyzed minerals in hair collected opportunistically from feet submitted with and without TAHD to determine if mineral concentration correlated with TAHD status. Mineral levels in hair varied among elk, making interpretation of differences between the groups more challenging. However, a consistent relationship of lower selenium levels with presence of TAHD was found and indicates that more investigation is warranted. We did not find that other minerals thought to be potentially protective against hoof disease, such as copper and zinc, were consistently lower in elk with hoof disease. Similarly, there was no indication that increased levels of toxicants, such as arsenic and chromium, contributed to occurrence of hoof disease. We are currently collaborating with wildlife managers in California to investigate the relationship between TAHD and mineral levels in liver, the ideal tissue for analysis, to compare with our findings using hair. Although we would all like a magic bullet to cure or prevent elk hoof disease, at this point we do not have sufficient evidence to support the benefit of mineral supplementation to free-ranging elk, particularly because congregation around a mineral lick could promote disease transmission. A summary of the study and its application will be the focus of the Winter 2023 ElkTracks newsletter. Results were published in the journal BMC Veterinary Research (2022; 18:446).

ASSOCIATIONS BETWEEN HAIR TRACE MINERAL CONCENTRATIONS AND THE OCCURRENCE OF TREPONEME-ASSOCIATED HOOF DISEASE IN ELK (CERVUS CANADENSIS)

Steven N. Winter, Maria del Pilar Fernandez, Kyle R. Taylor, and Margaret A. Wild

ABSTRACT: Trace minerals are important for animal health. Mineral deficiency or excess can negatively affect immune function, wound healing, and hoof health in domestic livestock, but normal concentrations and health impairment associated with mineral imbalances in wild animals are poorly understood. Treponeme-associated hoof disease (TAHD) is an emerging disease of free-ranging elk (Cervus canadensis) in the U.S. Pacific Northwest. Selenium and copper levels identified in a small number of elk from areas where TAHD is established (i.e., southwestern Washington) suggests a mineral deficiency may increase susceptibility to TAHD. Our objectives were to determine trace mineral concentrations using hair from elk originating in TAHD affected areas of Washington, California, Idaho,
and Oregon and assess their associations with the occurrence of the disease. We identified limited associations between TAHD occurrence and severity with hair mineral concentrations in 72 free-ranging elk, using Firth’s logistic regression and multinomial regression models. We found consistent support for a priori hypotheses that selenium concentration, an important mineral for hoof health, is inversely associated with the occurrence of TAHD. Less consistent support was observed for effects of other minerals previously associated with hoof health (e.g., copper or zinc) or increased disease risk from potential toxicants. Trace mineral analysis of hair is a non-invasive sampling technique that offers feasibility in storage and collection from live animals and carcasses. For some minerals, levels in hair correlate with visceral organs that are challenging to obtain. Our study using hair collected opportunistically
from elk feet submitted for diagnostic investigations provides a modest reference of hair mineral levels in elk from the U.S. Pacific Northwest that may be useful in future determination of reference ranges. Although our results revealed high variability in mineral concentrations between elk, consistent relationship of possibly low selenium levels and TAHD suggest that further investigations are warranted.

Understand social aspects of the disease and communicate findings

• Social science inquiry. We continued collaboration with the WSU Social and Economic Sciences Research Center (SESRC), led by Dr. Lena Le, to draft a scientific manuscript that reports findings from the 2020 survey of public perceptions of elk hoof disease in Washington. We expect that the manuscript will be submitted to a scientific journal in early 2023.

• Stakeholder communication. Informal correspondence continued, with timely responses to all inquiries from stakeholders, legislators, and the media. Articles in the Rocky Mountain Elk Foundation (RMEF) Bugle magazine (see below) prompted inquiries and apparently raised awareness of TAHD. Outreach presentations were also provided at the RMEF Washington Rendezvous and to WSU veterinary, wildlife, and animal science classes.
• Outreach via media. With the support of college communications specialist Charlie Powell, WSU’s work on hoof disease received coverage in local and regional media. Of particular note, an article on antler asymmetry associated with TAHD that originally ran in the Spokane Spokesman-Review was picked up by Yahoo news for broad distribution. Rocky Mountain Elk Foundation Bugle magazine featured an article on WSU research into TAHD in the May-June issue with a follow-up in the November-December issue.
• Website. The elk hoof disease website was maintained and updated to a new format to provide up-to-date information on elk hoof disease and our research.
• Scientific outreach. In February 2022, we collaborated with the RMEF and the California Department of Fish and Wildlife (CDFW) to conduct a virtual scientific meeting on elk hoof disease that was attended by about 70 wildlife managers and health professionals from state, federal, and tribal agencies. This was the first meeting of what is now planned to become an annual event. We presented scientific findings from WSU’s research program at this scientific meeting as well as at the Wildlife Disease Association, Wildlife Society, and Lameness in Ruminants conferences. Additional outreach and collaborative planning meetings were conducted with the U.S. Forest Service and CDFW.

Collaborate with WDFW and Tribes

• Collaborative disease investigation. WSU collaborated with wildlife managers in Washington, Idaho, Oregon, California, South Dakota, and the Northwest Indian Fisheries Commission (NWIFC) to obtain hooves from elk harvested or found recently deceased in locations of interest for disease surveillance and/or for collection of research samples. Experts at WSU assist collaborators in detecting the disease. Findings from all locations help inform our understanding of the disease in Washington and are critical for our research.
• Collaboration with WDFW. In addition to regular communications, we conducted two virtual meetings to share information and plan and coordinate work. WDFW efforts were critical for obtaining fresh elk hooves for research.

Ancillary projects

During the course of any planned research, unexpected new and important questions often arise. Addressing these questions must be prioritized to avoid overextending resources, but in some cases opportunistic projects that can be supported are added to the research program. This is particularly true for projects that are of particular interest to stakeholders.

• Antler asymmetry. During discussions with stakeholders in southwestern Washington, concerns were voiced about antler asymmetry and the potential association with the high prevalence of hoof disease. We used hunter harvest data routinely collected by WDFW between 2016-2018 to investigate whether antler asymmetry occurs at a higher rate in elk with hoof lesions. Results indicate that elk with hoof abnormalities are more likely to exhibit asymmetric antlers. Previous research on deer has documented that trauma to a limb (similar to elk with hoof disease) can result in an antler anomaly in an individual; however, impacts from a transmissible disease affect a much larger proportion of a population. A summary of the study was reported in the Fall 2022 ElkTracks newsletter. Our manuscript was published in the Journal of Wildlife Management (2022; 86: e22245).

ASSOCIATION OF ANTLER ASYMMETRY WITH HOOF DISEASE IN ELK

Margaret A. Wild, Glen A. Sargeant, Kyle Garrison, and Dylan Conradson

ABSTRACT: Treponeme-associated hoof disease (TAHD) is an emergent disease of elk (Cervus canadensis) in the Pacific West of the United States. Although lesions are usually restricted to the feet, anecdotal reports suggested increased prevalence of abnormal antlers in affected elk. We used hunter harvest reports for 1,688 adult male elk harvested in southwestern Washington, USA, 2016–2018 to evaluate anecdotal reports. We used Akaike’s Information Criterion to compare 18 logistic regression models describing the prevalence of asymmetrical antlers, indicated by unequal antler point counts. Our leading model (84% of model weight) described additive effects of TAHD (odds ratio = 1.91; 95% CI = [1.49, 2.44]) and maximum number of antler points. Confidence intervals overlapped zero for all other parameters, which described ecotypic, geographic, and age-related effects. Effects of physical injury on antler development have been described elsewhere; however, injuries leading to instances of antler deformity do not have population-level management implications. In contrast, we describe effects of a transmissible disease that was reported by hunters in > 35% of adult male elk and was associated with an increase of ≥16 percentage points in the prevalence of gross asymmetry. Unequal point counts are quite common in elk with otherwise typical antlers and seem unlikely to attract public notice or be attributed to hoof lesions; thus, we suspect our results and anecdotal reports reflect more prominent deformities that are important to stakeholders who enjoy hunting and wildlife viewing.

• Epigenetic impacts. While we now know that TAHD lesions are caused by bacterial infection, other factors could alter an elk’s susceptibility to developing disease. We are collaborating with researcher Dr. Michael Skinner, a professor in the WSU School of Biological Sciences, to investigate epigenetic transgenerational inheritance of disease (increased susceptibility of future generations due to “ghosts” in their genome). We completed analyses on data from elk with and without TAHD. Preliminary results revealed differences called epimutations (changes in the chemical structure of DNA that regulate genome activity independent of DNA sequence) in elk with TAHD. These changes can be passed to subsequent generations and potentially change their susceptibility to TAHD. We drafted a manuscript that will be finalized for submission and review by a scientific journal in early 2023.

Next steps

Since the passage of Senate Bill 5474, active disease surveillance conducted collaboratively between WSU and state, tribal, and federal wildlife agencies resulted in diagnosis of the disease over a broader geographic area than was previously described. Unfortunately, the disease has expanded from a primarily local concern in southwestern Washington to a statewide issue. Moreover, with continuing cases in Oregon, and detections in Idaho and California, it has emerged as a multi-state regional issue. The broader geographic range amplifies the need for continued
research on this important emerging disease. Moving forward our research will reflect this broader scope.

General

• Research team. We will maintain staff and students that reflect transitions that occurred at the end of 2022. Specifically, a post-doctoral fellow will be funded to increase laboratory capacity for bacterial genome sequencing and the master’s degree student that graduated will not be replaced.
• Legislative reporting. The next report covering the period January-December 2023 will be submitted by February 16, 2024. Legislators and their staff are welcome to contact Dr. Margaret Wild at any time to ask questions or receive additional information as it is developed.
• Implement research plan. The initial research plan we developed (Phase One) covered 2019-2021. In 2022, we reviewed the plan and made minor revisions to extend the scope of the plan through 2024. We will continue to implement this research plan in the coming year.

Study the disease cause(s) and contributing factors in captive elk

• Maintain and procure captive elk for study. Elk that were procured in 2021 and 2022 will be used in continuing studies in 2023. Approximately eight additional elk calves will need to be procured for studies in 2024. We will continue to work with Starkey Experimental Forest in Oregon to obtain elk; however, if chronic wasting disease is detected in Oregon (CWD is nearing the eastern border of both Oregon and Washington), our source of elk may be terminated if Oregon elk are not allowed to enter Washington.
• Environmental transmission trials. We will finalize a manuscript that reports findings of our initial transmission study for publication in a scientific publication. We will complete analyses of samples and data from the study initiated in 2021 to investigate transmission of TAHD to elk naturally exposed to contaminated soil and determine whether results are rigorous enough for development of a draft manuscript for submission to a scientific journal.
• Initiate studies to develop a transmission model in captive elk. We will conduct at least two of the four planned phases of study to develop a reliable TAHD transmission model in captive elk (Drankhan PhD project). The first two phases are: 1) a pilot study to develop a boot that is tolerated by elk and that will contain local infection on the hoof, and 2) to use the developed boot system to expose elk to affected hoof material that will reliably transmit TAHD.

Study disease agents in the laboratory

• Detect pathogens associated with TAHD using bacterial DNA analysis. Two studies focused on DNA sequencing (16S amplicon sequencing) to identify bacteria in TAHD lesions are in progress. In the first study (Goldsmith PhD), we will develop a grading system to classify TAHD lesions based on microscopic changes. The bacterial community associated with each grade will then be determined using DNA sequencing. The second study is led by collaborators with the USDA Agricultural Research Service and was initiated in 2018 using samples from southwestern Washington. We are currently awaiting progress by our collaborator and plan to jointly draft a manuscript to report findings.
• Pathogen discovery. Our initial bacterial identification approach using short DNA sequences (16S amplicon sequencing) routinely identified unique spirochetes (a group of spiral shaped bacteria that includes Treponema species) in TAHD lesions. We will conduct whole genome sequencing of these bacteria in two studies and attempt to determine how they are related to Treponema species that affect livestock. First, in a pilot study, we will use a new pipeline for analysis to construct genomes of uncharacterized spirochetes in a small set of samples collected from elk with TAHD. We will draft a manuscript to report the technique and the newly identified spirochetes in a scientific journal (Deb post-doctoral study). Next, we will apply the technique to a larger set of samples associated with a range of microscopic lesions (Goldsmith PhD and Deb post-doctoral study) to broaden our understanding of these newly described bacteria.
• Compare hoof sampling techniques. We will collect samples and extract DNA for use in a study that will compare three methods of sampling elk feet for bacterial DNA analysis (Drankhan PhD). Samples collected using a punch biopsy, skin scraping, and skin swab will be compared to determine if less invasive techniques (swabbing or scraping) are as reliable as a biopsy in detecting Treponema and characterizing the bacterial community present in TAHD lesions.
• Enrich Treponema for experimental challenges. Our findings to date lend further support for the importance of Treponema or Treponemalike bacteria in TAHD lesions; however, other bacteria are also likely important in disease development. Treponema species and other potentially important species (e.g., Mycoplasma) are notoriously difficult to grow, or culture, in the laboratory. As an alternative to culture, we will develop laboratory methods that are selectively conducive to the growth of potential pathogens. These techniques will be applied in future development of the reliable transmission model (Drankhan PhD).

Conduct regional surveillance and investigate risk factors

• Disease surveillance. Disease surveillance will continue using samples submitted by wildlife agencies. Priority will continue to be on game management units (GMUs) in Washington where suspect cases of the disease have been observed but diagnostic testing has not confirmed TAHD and in new geographic areas (at the GMU or county level) where TAHD has not been previously diagnosed. We will also collaborate to examine hoof samples from other states to determine the geographic extent and study progression of the disease. Additionally, we will work with CDFW on a data-sharing agreement so that we can obtain geographic locations of their confirmed TAHD cases. These locations will be added to the national TAHD distribution map maintained by WSU.
• Spatial epidemiology. We will integrate information from multiple data streams, from both community science and professional sources, to identify patterns in distribution of TAHD in space and over time (Winter PhD). We will draft a manuscript reporting results for submission to a scientific journal.
• Mineral status of elk. We will continue collaboration with CDFW to investigate the correlation of mineral levels in liver with the occurrence of TAHD (Winter PhD). We will conduct analyses and draft a manuscript reporting findings for submission to a scientific journal.

Understand social aspects of the disease and communicate findings

• Social science inquiry. We will finalize the manuscript reporting findings of the 2020 survey of Washington residents and submit our findings to a scientific journal for publication.
• Outreach. Outreach via the listserv, website, media, legislative briefings, and stakeholder meetings will continue. Additionally, presentations to Washington’s legislators and staff can be arranged. Communication with the scientific community will occur through publications and presentations.

Collaborate with WDFW and tribes

• Collaborative disease investigation. We will continue collaboration with WDFW, NWIFC, and other wildlife agencies to obtain hooves from elk harvested or found recently deceased in locations of interest for disease surveillance, for collection of research samples, and in joint research projects. Expertise at WSU assists managers in Washington and across the Northwest in detecting the disease. Findings from all locations help inform our understanding of the disease in Washington and are critical for our research.
• Scientific meeting. We will co-host with RMEF and CDFW the second annual TAHD virtual scientific meeting with state, federal, and tribal wildlife managers and veterinarians to share information, identify knowledge gaps, and solicit input on research priorities.
• Collaboration with WDFW. In addition to regular communications, we will conduct quarterly meetings. We will also continue to collaborate on procuring elk hooves for disease surveillance and research. Additionally, WDFW will contribute important data for spatial epidemiology research.
• Native American Tribes. We will continue collaboration with Native American tribes to share information and obtain hoof samples. We will schedule virtual meetings as requested to promote outreach.

Ancillary projects

• Epigenetic impacts. We will finalize a draft manuscript for submission to a scientific journal. Following peer review and publication, findings will be shared via outreach to stakeholders.
• Investigate new monitoring techniques. We will collaborate with Advanced Telemetry Systems (ATS) to develop an “anklet” monitor that collects and transmits activity data to a receiver. We will test the monitor in captive elk to determine reliability in identifying bedded, standing, and walking behavior. The anklet could be used to detect lameness in captive research elk or potentially free-ranging elk. We will also investigate use of digital thermography to identify early TAHD lesions in captive elk. Thermal imaging may be able to detect areas of higher temperature on the foot that indicate inflammation associated with disease development.

ADDENDUM

RESEARCH PLAN
Phase One: 2019-2024

The first step in establishing the research program was to define research goals and identify key research questions. In alignment with Senate Bill 5474, the goal of WSU elk hoof disease research is to identify the cause(s) of the disease and how to successfully manage it in the wild. Achieving this goal will require an incremental multi-pronged biological and social science research approach implemented over multiple years. The first phase of work addresses foundational questions and was initially conducted with a three-year horizon (2019-2021). Program evaluation led to extension and expansion of the plan to incorporate a six year period (2019-2024). Successive phases of work will build on findings from these initial studies. The four principal areas of inquiry for these studies are:

• Study the disease cause(s) and contributing factors in captive elk. We will use captive elk in a controlled environment to investigate the cause(s) of hoof disease and contributing factors that make elk more or less susceptible.
  
  Need: The definitive cause(s) of hoof disease are not known and are required for effective management as well as to identify risk to other species. Treponema species are associated with hoof lesions; however, it is unknown whether these bacteria are the primary cause of disease, or secondary invaders. Extensive stakeholder concern exists regarding elk exposure to herbicides, fertilizers, and habitat changes as a cause or contributing factor for disease. Controlled studies are needed to investigate the individual and collective impacts of pathogens and other contributing factors to disease.
  
  Approach: Initial work will focus on development of a disease challenge model to determine if the disease 1) is infectious and contagious and 2) can be reliably reproduced in elk following exposure to infectious material. Based on results, modifications to the challenge model will be investigated. For example, addition of contributing factors, such as reduced nutritional status or exposure to herbicides, may be required to reproduce disease.
  
• Study disease agents in the laboratory. We will use state of the art technology to identify pathogens associated with hoof disease.
  
  Need: Many pathogens, including Treponema species, are not easily cultured using standard techniques. Advanced approaches are needed to identify pathogens in samples collected from free-ranging and captive research elk to determine which organisms are, and are not, contributing to disease. This work is needed to guide improvement of methods to isolate the causative agent(s) and develop tests to detect, and potentially treat, them.
  
  Approach: Initial work will use metagenomics (looking at genetic material in a sample to determine which bacteria are present) to identify bacteria associated with hoof disease in general, and at specific points during progression of the disease.
  
• Conduct regional surveillance and investigate risk factors. We will collaborate with WDFW and other wildlife management agencies to collect hoof samples for diagnostic investigation and analyze surveillance data.
  
  Need: Disease surveillance and monitoring is key to documenting where a disease occurs and to estimate prevalence. It provides baseline data to measure changes in the future and can also be used to identify risk factors for disease occurrence.
  
  Approach: In collaboration with wildlife managers, we will collect and perform diagnostic evaluation of hoof samples from across Washington and other states in the Northwest to document where TAHD occurs. Surveillance samples can also be used to address additional research questions. Initially we will focus additional collections from four geographically distinct areas to investigate whether or not the pathogens involved are the same in every area to determine if one disease outbreak is spreading, or if multiple independent outbreaks are occurring. Additionally, we will overlay disease distribution data collected from surveillance efforts with potential risk factors to investigate if disease occurrence is correlated with particular locations or environmental factors.
  
• Understand social aspects of the disease and communicate findings. Implement outreach and education efforts that are grounded in an understanding of stakeholder’s beliefs, values, and concerns about hoof disease and elk management.
  
  Need: Effective outreach and education is an important companion to the implementation of biological research, particularly when addressing wildlife issues with multiple opposing stakeholder perspectives. Information gained from social science inquiry can guide outreach and education efforts and contribute to setting goals for research and management.
  
  Approach: Initial research will be conducted in collaboration with the WSU Social and Economic Sciences Research Center (SESRC). We will use focus groups of interested stakeholders to gather qualitative information regarding public opinion on hoof disease. This information will be used to develop a questionnaire for a statewide survey that will provide statistical representation of public opinion. Additionally, we will conduct program development work to guide outreach and education efforts, while concurrently seeking to increase public awareness through media outlets.

In addition to these WSU research priorities, we will support related WDFW and tribal research and management as requested. This includes providing staff support for field work or diagnostic investigations, providing diagnostic services for hoof samples submitted to the Washington Animal Disease Diagnostic Laboratory (WADDL), and conducting collaborative research. This level of cooperation requires a commitment to communication that will be addressed in part through regularly scheduled quarterly meetings between WSU and WDFW.

Washington State University is recognized as a leader in elk hoof disease research. As a result, wildlife agencies outside of Washington also seek collaborations. Such collaborations provide access to additional datasets and research opportunities and will be pursued when they also contribute to the understanding and management of hoof disease in Washington.