Session Three: Wild Animal Population Concerns

Moderator: Patrice N. Klein, U.S. Forest Service

Patrice N. Klein from the U.S. Forest Service of the U.S. Department of Agriculture (USDA) introduced Session Three. She explained the session will be an overarching discussion on biosafety and biosecurity germane to working in natural wildlife communities with recognition of the One Health concept—that we are all interconnected: humans and animals (both domestic and wild) in our shared environment. She said that understanding the basic principles of biosafety and biosecurity and knowing the hazards present in the natural environment in which one will be working and in the animal population promotes best practices. Klein explained, risk mitigation measures are developed with tools like the National Institute for Occupational Safety and Health’s (NIOSH’s) Hierarchy of Controls or Job Hazard Analysis; and the occupational safety and health (OSH) biosafety principles and practices developed for biomedical laboratories can be extrapolated to natural field settings. She suggested that it is essential for the OSH program and the Institutional Animal Care and Use Committee (IACUC) to be partners and work with the principal investigators (PIs) in determining the best biosecurity and biosafety plans for wildlife research activities. Klein said this session’s objectives were to consider the potential impacts on populations and communities beyond the level of the individual animal that can result from field activities; to recognize biosafety as multi-directional among animals, humans, and the environment; and to discuss the responsibilities and limitations of permitting agencies and IACUCs regarding oversight of potential impacts. She added that the speakers in this session will highlight the dynamic, complex, and often dangerous risks in wildlife research activities; the risk of introduction and spread of wildlife diseases by field researchers and impacts on biodiversity and species conservation; the importance of dialogue among the PI, the IACUC, and the OSH committees; and the need to develop standards of practice around biosecurity and biosafety and animal welfare.

CHALLENGES OF HIGH-RISK FIELDWORK AND WORKING WITH VENOMOUS AND HAZARDOUS SPECIES FOR IACUCs

Christopher L. Parkinson is professor in biological sciences and forestry and environmental conservation at Clemson University and former IACUC chair at the University of Central Florida (UCF). Currently serving as herpetology chair for the American Society of Ichthyologists and Herpetologists (ASIH), Parkinson discussed the challenges of fieldwork, including defining what it means to do fieldwork, discussing how the Animal Welfare Act (AWA) handles programs with field studies, and how to apply risk management practices to non-traditional studies. Parkinson provided examples for these activities from his own research: bringing in endangered frogs to breed in captivity, extracting venom from snakes, working with sea turtles, doing morphometrics on iguanas, and then being in the field and capturing wild animals.

Parkinson submits that “the field” is anywhere not in an office or a laboratory setting. At its root, the difference is a controlled versus a non-controlled environment. Parkinson works in natural settings that most people would consider the field, but the setting may be a university campus, which would not be considered “the field.” He explained that the wild lands on his campus had a great population of eastern diamondback rattlesnakes (Crotalus adamanteus). It was commonplace for him to be called by campus police because there was a rattlesnake in a parking lot, he said.

The UCF Rattlesnake Project is an example of urban ecology, Parkinson added. Another example is the Atlanta Coyote Project, where researchers study coyotes found in Atlanta proper. Similar work is conducted by Jalene LaMontagne’s laboratory at DePaul University in Chicago, where her students work in cemeteries and parks on coyotes. In these examples, the field is downtown suburbia, introducing

different risks compared to those encountered when conducting wildlife research under traditional field conditions.

Turning to how the AWA deals with field studies, Parkinson said that a 2000 AWA amendment included field studies that are conducted on free-living wild animals in their natural habitat and contained a qualifying statement: If the study is not invasive, does not harm the animal, or materially alter the behavior of the animal under study, then the study is exempt from the AWA (USDA National Agricultural Library Digital Collections, n.d.). Some IACUCs at different universities interpret this differently, Parkinson said, with some saying researchers have to submit to an IACUC, while others saying they did not have to submit to an IACUC when following the AWA. The latter interpretation pertains to captive wildlife and wild animals held in captivity for more than 12 hours. Projects that call for animals to be brought into the laboratory for an overnight stay to conduct some work are therefore covered by the AWA. Particularly for venomous and wildlife animals, Parkinson added, there are a patchwork of federal and state laws, policies, and guidelines that must be followed, as well as local laws and ordinances pertaining to venomous snakes that researchers have to follow when carrying out wildlife work.

A field safety program is essential for an IACUC and university setting, Parkinson said, and the same level of planning and discussion that goes into laboratory safety could be considered when planning fieldwork. Field researchers can conduct job safety analyses and identify safety risks and hazards in the workplace, as well as out in the field environment. While many PIs do not discuss evaluating risk with their students, researchers need to evaluate that risk, Parkinson asserted. PIs have a responsibility to determine what can go wrong prior to it going wrong and cannot assume that people in the field fully understand the risks. These risks are not unique to biologists if they are doing fieldwork in an urban setting. A sociologist or an anthropologist has similar risk in their field settings, so scholars could work collaboratively to determine risks. Parkinson has encountered risk, including a mountain lion coming into close proximity to three people that were caught out at night because of a rainstorm in Arizona; being trapped on a beach in Costa Rica by a jaguar; and accidents in all-terrain vehicles (ATVs) and small planes.

He asked How do researchers mitigate these risks? Parkinson said that an obvious first answer is a medical surveillance program. Researchers could prepare health profiles on team members, including making sure members are up to date on vaccines and immunizations. Researchers could always carry emergency medical kits, including medicines to deal with allergic reactions, he said. Moreover, all students could have wilderness first aid training, as well as ATV, utility terrain vehicle (UTV), and/or boat training if needed. Researchers could have a communication plan and a field evacuation plan if something goes wrong while in the field.

One of the easiest aids for communication is a satellite phone, Parkinson said. He has worked with his director of environmental health and safety (EHS) to buy a bank of satellite phones for his team and other members of the university. There can be urban areas where cell phones do not work, in which case satellite phones are helpful. As part of the plan, the researcher pays for the minutes used, although sometimes not all minutes are actually used in the field, Parkinson added. Parkinson next discussed risk mitigation dealing with zoonotic and vector-borne diseases, with the caveat that it is not his area of expertise as shown in Box 3-1. Every year, researchers catch hantaviruses or the plague from small mammal trapping. Rabies can be transmitted easily, he said. Tick-borne disease is probably the most common that researchers have to deal with, especially if they are in the field up in the Northeast. Lyme disease is prevalent in those areas. Mosquito-borne diseases, such as malaria or dengue, are found in Honduras, Costa Rica, and Panama. Mitigating them may include using bug spray and other chemicals. Parkinson said that researchers need to take steps to mitigate and communicate risks to ensure that other researchers and students understand risks.

Other risks come from people, Parkinson said; a December 2021 article published in Buzzfeed News “Welcome to the Jungle” (Jha 2021) described sexual misconduct actions and harassment that has occurred to numerous people at the Smithsonian Tropical Research Institute in Panama. The article brings to light things that happen in the field (Woolston 2022). Parkinson said that none of the trainings he has ever undertaken included this aspect of fieldwork safety, so his trainings for students now discuss the

risks of doing fieldwork when it comes to discrimination and sexual misconduct. He pointed to another article, published in October 2020 by Amelia-Juliette Claire Demery and Monique Avery Pipkin (two Cornell graduate students) in Nature Ecology and Evolution, titled “Safe Fieldwork Strategies for At-Risk Individuals, Their Supervisors and Institutions” (Demery and Pipkin 2021). Parkinson added that researchers can start conversations to identify prejudice. It is known that some individuals, because of the color of their skin, their gender, or their sexual orientation, are more vulnerable to conflict and violence when they are in the field (Building a Better Fieldwork Future n.d.). Everyone deserves to conduct fieldwork safely, and researchers can work to ensure this is possible. Parkinson noted this is rarely included in trainings, including his own, and he encourages his colleagues to raise awareness of this risk.

Turning to non-model animal use under laboratory conditions, Parkinson discussed the many differences in husbandry, housing, feeding, veterinary care, and occupational health and safety in fieldwork, which he said look different when bringing wildlife into the facility (ASIH 2004). This involves planning, including getting the facility personnel, the EHS, and IACUCs involved. Researchers need to write and work on many SOPs, he added. As the chair of the ASIH, Parkinson said he receives many calls and emails from IACUC chairs asking how to work with their wildlife researchers, and his response is to connect them to experts in the field.

Parkinson focused the final portion of his talk to further his point on the high-risk challenges of both field and laboratory work; in this case, on utilizing venomous snakes in research settings. Parkinson has worked in more than 20 countries to do fieldwork, including collecting animals for genomics and venom work, where his group uses a specially designated room. The snakes are never physically handled, instead snake hooks and acrylic tubes are used to move animals around. There are two people in the room at all times; in the field, there are always multiple investigators present. At Clemson University, the animal facility has a special snake room behind multiple card key access doors. Parkinson’s group is responsible for all cleaning and maintenance within the room, including cage cleanings. The snakes are kept in rack cages. The room is an open space room to allow researchers to deal with each individual

snake safely. Each cage is labeled with the IACUC protocol, the PI’s name, the species name, and what anti-venom to use. Different colored cards identify venomous versus non-venomous snakes.

Parkinson next illustrated how his team extracts venom from a snake using an acrylic tube in a procedure known as “tubing,” in which a snake is prodded to go into the acrylic tube for handling. Researchers will almost never touch the head of the snake, unless for some unusual reason a different procedure has to be done. In those cases, animals are anesthetized to reduce that risk. There are several things to keep in mind to handle venomous snakes in captivity as safely as possible. The first suggestion from Parkinson is partnering with a local emergency medicine physician and a snakebite specialist and ensuring those two physicians are on the same page. He next suggests liaising with the local fire and emergency medical services (EMS) and setting up emergency procedures as SOPs. In his case, it turned out many EMS providers are afraid of snakes and refused to come into the room. This required that a designated “meet” site be set up outside the facility at the loading zone. Another suggestion is to set up a snake identification and outreach program to help educate EMS personnel.

As a final consideration, Parkinson suggested that the same level of planning and discussion that goes into a laboratory safety protocol should also be done in a field safety protocol. He added that researchers need to communicate, build partnerships and collaborations, and document everything. It would be beneficial for the investigators, students, department, university, institutional EHS, and the IACUC to work together. Parkinson emphasized that this important research be done safely and with risk mitigation, adding that this entails trainings and conversations to support implementation. In closing, Parkinson stressed that everyone deserves to conduct fieldwork as safely as possible.

STANDARDS OF PRACTICE FACILITATE RESEARCH AND MANAGEMENT OF WHITE-NOSE SYNDROME IN BATS

Jonathan Reichard, a wildlife biologist and FWS assistant coordinator for white-nose syndrome (WNS), presented information on how an FWS international collaborative effort has produced standards of practice that facilitate research and management of WNS in bats. Providing an overview of WNS, Reichard explained that this disease of hibernating bats is caused by the cold-loving fungus Pseudogymnoascus destructans (Pd). Pd is new to North America, where it is spreading rapidly through native bat populations (WNS Response Team 2019). Since 2007, Pd has also been detected among numerous species across Europe and Asia, he said, and molecular analyses point to Europe as the likely origin of the fungal isolate that is now abundant in North America. At least 12 North American bat species are known to be susceptible to this disease when Pd invades their skin when they are in torpor states and conditions are sufficiently cold for this fungus to grow. Although there is variation in impacts among species, for many species WNS is often fatal, so WNS is a major management concern for wildlife agencies across the United States, Canada, and Mexico, Reichard said.

Reichard discussed the 2007 discovery by wildlife biologists and cavers of large numbers of dead bats found in caves and mines during winter near Albany, New York. Sick and dying bats were found with a white fungal growth on their faces and wings, something that had not previously been reported in scientific literature. At the time of its discovery, the cause or causes of bat mortality events were mysterious. As WNS has continued to spread, the dedicated partnership of scientists and managers has made great strides in improving knowledge of the pathogen, hosts, and disease. Progress is made easier by initiatives of this partnership to evaluate high-priority activities, to investigate and manage the disease, and to evaluate the risks such activities may pose to the bats.

Since then, WNS has become a dominant topic at regional bat working group meetings across the country, Reichard said. The FWS convenes annual symposia or workshops to share the latest scientific information and coordinate activities to manage the disease. Because little was known about WNS and its underlying cause, the FWS’s early focus was to develop standard approaches to address critical information needs. Responses to WNS were modeled on existing efforts for chronic wasting disease (cervids) and colony collapse disorder (honey bees), including developing collaborative strategies to slow

or stop the spread of the disease, tracking its causes and impacts, and identifying management strategies for affected bats, Reichard explained.

When WNS was first discovered, it was unknown if it posed a threat to other taxa or people, Reichard added. He said as a result, research conducted through agencies and institutions treated the situation with a high degree of caution. Reichard explained that laboratories working with disease specimens were required to meet the standard Biosafety Level 2 or higher. He added, in caves, many researchers were required to don personal protective equipment: N95 mask particulate respirators or high efficiency particulate air (HEPA) filter masks, face shields, and full body with Tyvek® suits and gloves; and that all of the materials leaving the caves and mines in the WNS-affected area were treated as biohazardous materials. Partnering IACUCs quickly reviewed protocols adapted from other systems to approve studies into this emerging disease, he said.

Reichard said that these systems were effective in permitting important research to proceed. As efforts continued, the WNS response community quickly identified additional needs to refine certain precautionary measures. He pointed out that the first disinfection protocol was developed largely from a human safety perspective because of the initially unknown nature of the pathogen. He said, at the time, disinfection protocols for other wildlife diseases were referenced, such as conjunctivitis in house finches, and identified bleach solutions, quaternary ammonium, and Simple Green® for cleaning gear used around bats. He explained that through the WNS response community, this protocol was quickly adopted by scientists and managers across the continent and the FWS also issued a cave advisory that recommended restricting access to caves and mines used by bats (FWS 2009) with the objective to reduce further disturbance of sick bats and limit interactions between people and the pathogen. These two tools, the decontamination protocol and restricted access to bat hibernacula, remain important management tools in use today, Reichard said.

He explained that in 2011, states, tribes, and federal agencies came together under the formal framework of the WNS National Plan (WNS Response Team 2011). He indicated that this document outlines structure, coordination, and priorities to address the threat of the disease moving forward. He said that the collaborative response team now has partners from more than 150 agencies, organizations, and institutions. Reichard stated that partners in the Northeast United States contribute with the perspective of an endemic wildlife disease in their bat populations, while others in the Western part of the United States contribute with anticipation of how their bat populations will respond when Pd arrives in their regions.

Through the national response, researchers and managers have developed numerous recommendations and practices to guide work on WNS, Reichard said. Many of these efforts fall under a variety of regulations and policies that affect animal welfare. Reichard provided the example of the FWS enforcement of the Endangered Species Act (ESA), where potential impacts to ESA-listed species are evaluated when making grant decisions or issuing permits for WNS research and management activities. He pointed out that these activities are also reviewed for compliance with the National Environmental Policy Act (NEPA) and the National Historic Preservation Act to assess impacts to the environment and cultural resources. State agencies make similar assessments when issuing wildlife permits and conducting work under their own management authorities or Animal Care and Use Committee permissions. Reichard said that the WNS-specific standards of practice, which were developed through the WNS National Plan, provide a baseline by which various partners can adopt WNS-specific measures for biosafety, and readily apply these standards in their research management plans.

Reichard asserted that established practices improved efficiency and consistency to enable great scientific progress for bat conservation. He added that researchers from federal, state, and non-government organizations made quick progress, including satisfying Robert Koch’s postulates to establish the pathogen–disease link between Pd and bats. He said researchers also developed robust diagnostic and surveillance tools to understand physiological responses to infection, as well as the capability to detect Pd at minute levels in the environment and conducted a wide variety of studies to estimate variation in bat susceptibility to the pathogen due to behavior, physiology, and other variables.

Ever-increasing knowledge about WNS and Pd has allowed researchers to refine and improve some of the early precautions developed, Reichard added. For example, with more knowledge about how

bats respond to Pd infection and how Pd invades locations, managing access to caves to protect bats can be conducted more strategically (WNS Response Team 2018). The community has also adapted these recommendations for use in show caves that house bats so that these important commercial and educational resources can continue operating, while also reducing risks to the wildlife. Reichard said the WNS decontamination protocol has also been updated multiple times because early versions used heavy-handed techniques involving bleach and quaternary ammonium. Most recently, the FWS conducted efficacy tests of a variety of disinfecting agents and processes, which identified options for disinfecting a wide range of items used in caves and mines without risking damage to expensive technology or safety equipment. It is now clear that some bats survive the worst outcomes of disease, Reichard said. Researchers have identified other stressors that can be reduced by applying similar standards for protecting bats. To that end, researchers have adapted practices for decontamination and disturbance from other user groups, such as wildlife rehabilitators and wildlife control operators, said Reichard.

Reichard explained that a primary goal of the national response to the WNS is to develop and deploy tools that reduce the prevalence of Pd in the environment, and to bolster bats’ abilities to survive infections. However, there are many steps needed before any effective tool can be safely brought to scale, he said. In 2015, the FWS convened a workshop for specialists who would be involved in testing and using these tools. He said, they outlined key metrics and steps important to the research and development process; and one outcome of this workshop was a path to sequentially advance developing treatments from conceptual plans to laboratory testing in situ and in vivo, and eventually to field testing and broad implementation.

Reichard said that, with these guiding principles in mind, researchers have identified a variety of agents that can kill or stop the growth of Pd or improve bat survival. Multiple tools have been sufficiently tested for efficacy and non-target effects to demonstrate they are ready for adaptive use in the field. Some examples include a Cold War–era bunker modified to be cleanable hibernacula, a spray of polyethylene glycol to hinder the growth of Pd in an abandoned railroad tunnel, and an ongoing cave bat vaccination trial in Texas.

The emergence of SARS-CoV-2 disrupted many activities for managing WNS, Reichard said. Biologists had many questions about the potential impact of this new virus on native species. There was little information about the potential for spillback into native populations or whether animals could become sick or harbor a new reservoir for the virus. With Pd still moving so quickly and dangerously through North American bat populations, there was a serious concern about what added impact SARSCoV-2 might have on bats, Reichard explained. Even with these concerns, researchers knew important work on WNS was still needed. The WNS National Response Team partnership was well positioned to quickly adopt the best available precautionary measures to allow researchers to move forward with bat conservation work despite these uncertainties. In June 2020, scientists from the U.S. Geological Survey (USGS) and the FWS led a rapid risk assessment, which determined that there was a non-negligible risk that bats could become infected with SARS-CoV-2 from sick people conducting bat work, Reichard said. This finding reinforced the need for agencies and researchers to take precautionary measures while conducting work with bats.

An in-depth risk assessment of the potential risks to bats posed by SARS-CoV-2 was conducted (Runge et al. 2020; Cook et al. 2021), which determined that screening field personnel for COVID-19 is an effective way to reduce risks of human to bat transmission, Reichard said. One reason this is important is that bats suffering from WNS have a higher risk of becoming infected with SARS-CoV-2 from a sick person than healthy bats. Over the past 2 years, early action by the WNS partnership to adopt safety measures amid rapidly changing and uncertain risks may have helped to avoid a major setback in bat conservation efforts.

While WNS has decimated several North American bat species in the past 15 years, Reichard said that conservation biologists, working together with shared strategies, have made great progress in managing the disease and the bats affected. Nevertheless, there is a long road ahead for these species to recover, he added. Other threats, such as pollution, habitat modification, intentional harm, climate change, and energy development, also pose threats to bats. Addressing the science and management needs of these

threats will benefit from agreed-upon standards of practice developed to facilitate necessary activities, while also maintaining safety for the animal researchers seeking to conserve bats.

ANIMAL WELFARE CHALLENGES IN RESEARCH ON AMPHIBIAN DISEASE ECOLOGY: IMPACTS ON NATURAL SYSTEMS, BIODIVERSITY, AND BIOSAFETY (PART 1)

Karen Lips, professor at the University of Maryland, discussed how to understand animal welfare challenges in research and education on wildlife, non-model species, and biodiversity. She specifically focused on wild animal populations and how the emergence of a wildlife disease has led to the evolution of new biosecurity efforts in the field.

Lips pointed out that the number of wildlife pathogens and parasites is increasing every year, as is the frequency of those outbreaks. Most IACUC policies and guidelines are not designed for use with wildlife and these pathogens, yet research is important to understand these challenges of emerging infectious diseases. Given the risks to researchers, study organisms, and their environments, Lips said, new policies will be needed that can ensure human safety, support wildlife research, and reduce environmental health risk related to emerging infectious diseases. Lips discussed her work on the amphibian-chytrid pathogen background, how it influences biosecurity protocols, the field biosecurity and safety precautions that have emerged from this new disease, and its relevance for research and conservation.

Lips described two types of fungal pathogens. The first, Batrachochytrium dendrobatidis (Bd), was discovered in 1997. The life cycle of Bd alternates between infections in the skin of amphibians that then releases sperm-like zoospores, which move about in wet or moist environments (Van Rooij et al. 2015). Accordingly, any biosecurity protocol will need to focus on not only the animals involved but also the water, the soil, and other parts of the environment to reduce disease spread, Lips said, because Bd has been found in animal populations around the globe. Lips said that international cooperation is important to establish global biosecurity protocols to prevent its spread to uninfected areas.

The impact of Bd so far has been horrific on the amphibian biodiversity of the world (Fisher et al. 2009; Scheele et al. 2019), Lips said, with at least 500 species in decline because of this fungus. This means that researchers cannot focus on just one or a few species, she said, but that they need to consider all amphibians as potential vectors or reservoirs of the disease. More than 90 species have already gone extinct because of this disease, while an additional 39% of study populations are in decline (Scheele et al. 2019). This reminds researchers that the fungus persists in the environment even when amphibians are rare, she said. As such, good biosecurity protocols will be important everywhere for the foreseeable future.

Bd comes in different genotypes, lineages, or variants, Lips explained. While North America has the common global pandemic lineage, other types are often restricted to one or two other continents. Genetic evidence obtained from animal samples shows that the international pet trade contributes to the global spread of Bd and probably other wildlife diseases (O’Hanlon et al. 2018). Once it has been introduced, this disease can spread on its own without human interference through the contact of frog to frog or frog to infected environment. Take, for example, the epizootic spread of Bd through Costa Rica and Panama in the 1990s (Lips et al. 2008), which was introduced at some point in northern Costa Rica in the late 1980s, spreading on its own through Costa Rica and Panama. This illustrates that there is no way to eliminate it or stop its spread once it has been introduced, Lips said.

The second chytrid that Lips discussed was Batrachochytrium salamandrivorans (Bsal), discovered in 2014 (Fisher 2017). This pathogen infects salamanders (Stegen et al. 2017). Its life cycle is similar to Bd, with the only difference being that Bsal produces encysted fungal spores that float and passively adhere to a passing host (e.g., on the feet of ducks, on the boots of researchers) through water currents or through the spread of infected soil (Van Rooij et al. 2015), which Lips says necessitates that extra attention be paid to this fungus. Luckily, Bsal is restricted to two different parts of the world as of 2016. It is thought to have originated in Asia, she said, but now has been introduced into parts of Europe where it is spreading, causing mortality and population declines in the native amphibians there. This is

especially concerning for North America because the continent is the world’s global biodiversity hot spot for salamanders (Jenkins et al. 2013). Bsal is not yet in North America, but without good biosecurity, she said, it may be only a matter of time before an infected animal, soil, or water spreads Bsal into North America.

Lips said that, while the FWS coordinates biosecurity and safety precaution efforts for WNS in bats (Session Three, Talk 2), there is no research coordination, no official plan, and no enforcement office in the United States or globally for amphibians (Olson et al. 2021a). Lips said that while the USGS has made some effort to develop emergency response and risk mitigation tools, including the development of a Bsal task force ready to step into action, the research community has largely driven the development of biosecurity protocols and practices. They have shared this information internationally and with other stakeholder groups, including industry, pet stores, zoos, and aquariums, Lips added. Furthermore, scientific societies have endorsed and shared these resources by posting information on their websites. There is also a national disease task team in the United States, she said.

Some of the biosecurity protocols that field researchers use today include the decontamination of boots, clothing, equipment, and vehicles, Lips explained. Good cleaning of boots can reduce the spread of these pathogens, as well as others, into uninfected areas, she said. There are also animal health precautions to take when researching emerging infectious diseases (Olson et al. 2021b). Researchers want to reduce the spread of disease among the animals that they handle. This is normally done by changing gloves between handling every individual amphibian. Furthermore, researchers want to make sure that when they do have to restrain animals or keep them housed in bags, buckets, or aquariums, this is done in a way that keeps animals collected from different sites as separate as possible, and that different species are not combined. Lips said that animals that have been kept in captivity should not be put back into the field on the chance that they may have picked up other diseases, which Lips said raises issues for conservation re-introductions or experiments.

Lips discussed some rules for biosecurity when working with amphibian diseases that have developed (Olson et al. 2021b), which she said can also apply to many other wildlife diseases. These rules fall into two general categories: (1) the standard biosecurity employed between sites, including disinfecting gear, equipment, vehicles, and boots; and (2) the enhanced biosecurity employed for working on rare or endangered species or working in areas that are pathogen free. In these cases, researchers still follow the standard biosecurity protocols, and she added, they may decide to dedicate gear to a particular site. For example, Lips’s group assigns nets and boots that may never leave a particular site. Sampling protocols may be designed in a way where researchers begin working in the uninfected sites and only later move into the infected sites to prevent the spread into those pathogen-free areas. Furthermore, researchers may decide to start at the top of the mountain or in the headwater streams, and then move down the mountain or into the lower reaches to help reduce the spread into those more protected areas.

Lips said that researchers should also consider how to extend biosecurity to the public and to other field researchers. She added researchers should share their practices, as well as learn from practices that have been developed by the aquatic invasive and aquatic disease fields. She added, it is not just amphibian researchers that may move disease from site to site, public visitation to national and state parks may do so as well. Lips explained that researchers studying other organisms could also be vectors for disease spread and researchers want to be careful that they are not going to transport pathogens through their movements or the movement of wild animals, water, or soil between sites. She added, do not release captive, pet, or experimental animals that may have picked up diseases while in captivity. Aquatic pathogen and invasive species organizations have done good work in these areas that amphibian researchers can adapt.

Turning to the implications for conservation and for research, Lips said that there is a trade-off between doing the research to address these problems and the fact that research may reduce wild populations even further (Grant et al. 2016). She said, ethical choices related to study species choice and the numbers of individuals that may be available for research should be discussed during IACUC protocol review. She said, furthermore, researchers want to also discuss in detail the source of those experimental animals. Lips added, for example, the question Can the animals be taken from the wild or will

researchers need to use captive populations and/or surplus animals from captive breeding, or other sources? A growing problem is being seen now where posting locality data for rare, endangered, or valuable species is leading to illegal take and over-collecting of those already rare or endangered species.

In summary, Lips said that there is a lot that can be done to co-develop new policies with this broad stakeholder audience. She explained, not only should researchers want to involve university IACUCs, institutional EHS, and biosafety offices but they may also want to involve permitting agencies both in the United States and internationally in collaborating nations. Lips shared that land managers and field station managers have a lot of control over mitigating the spread of disease through communication and regulation. She also said scientific societies have a role to play in endorsing and communicating these new protocols. In closing, Lips recognized these trade-offs between research and conservation, and the value added by being able to work on these new topics needs to be recognized; and researchers could share best practices and policies more broadly, including with non-target researchers and the public.

ANIMAL WELFARE CHALLENGES IN RESEARCH ON AMPHIBIAN DISEASE ECOLOGY: IMPACTS ON NATURAL SYSTEMS, BIODIVERSITY, AND BIOSAFETY (PART 2)

Vance T. Vredenburg, professor at San Francisco State University, delivered a second part of the discussion on Animal Welfare Challenges in Research on Amphibian Disease Ecology: Impacts on Natural Systems, Biodiversity, and Biosafety presented by Lips. Vredenburg focused on discussing and understanding animal welfare challenges in research and education in wildlife and amphibian diseases and emerging infectious diseases that are increasingly common in wildlife. Vredenburg said that most IACUC policy and guidelines are not necessarily designed with wildlife populations in mind, in particular, regarding pathogens and parasites, and that research is needed to understand these challenges.

Vredenburg, who works on amphibians, said that as of February 2022 there are more than 8,400 species of described amphibians in the world. His research subjects include the Chinese salamander that lives in China and Japan; the wolverine frog; and the gastric brooding frog, where the females swallow fertilized eggs and then rear their young in their gastrointestinal tracts. The gastric brooding frogs used to be found in Australia, but are unfortunately now extinct, he said., Compared to the other living vertebrates on Earth, amphibians are in the worst shape; more than 30% are threatened with extinction and maybe about 41% of all known species are declining at least in some of their populations (Collins et al. 2009), and those numbers are likely worse today, Vredenburg said.

Vredenburg asserts amphibians provide a window into what is happening around the globe in terms of potential mass extinction events, such as when the dinosaurs went extinct. The amphibians made it through that and other mass extinction events, he said, but today they are in much worse shape, with populations and species of amphibians that are in trouble all over the planet (Wake and Vredenburg 2008). He provided an example from a study in the Sierra Nevada mountains in California, which is one of the most protected areas in North America and the location of famous national parks like Yosemite, Sequoia, and Kings Canyon. The parks are surrounded by other national forest lands that are wilderness lands, which means that there are no roads, no buildings, and no extraction of resources within those areas, he said. Nevertheless, a majority of the species of amphibians there, which include salamanders, frogs, and toads, are in major trouble despite that habitat protection, Vredenburg said.

In a 2009–2010 story, National Geographic published photos by Joel Sartore depicting the die-off in the study area where Vredenburg was working on a disease called chytridiomycosis, which is caused by a pathogen called Bd. This microscopic fungal pathogen infects the skin of the host, Vredenburg said. It has a free-swimming zoospore that emerges from the host skin cells where the pathogen is growing, which then either re-infects the same host or infects a new host. Due to its asexual reproduction, the fungus can grow incredibly fast and cause mass infections (Van Rooij et al. 2015). The disease greatly reduced the metapopulation of frogs in Kings Canyon National Park (Vredenburg et al. 2007) based on repeated visual counts (more than 900 counts from 1996 through 2008). During that time, skin swabs were collected and used to conduct pathogen assay analysis without harming individuals. Vredenburg’s

research group collected more than 6,000 skin swabs from 2004–2008 on two species: Rana muscosa, also known as the Mountain yellow-legged frog; and Rana sierrae, the Sierra Nevada yellow-legged frog.

Vredenburg presented what happened as Bd spread through these habitats using data from Sixty Lake Basin in Kings Canyon National Park, which contain lakes that drain to the north. These lakes contain the largest metapopulation of frogs for this species anywhere in the world, he said. From 1996 to 2003, there was no record or evidence that the fungal pathogen Bd was there. Vredenburg first detected the pathogen in 2004 in two populations. By 2005 Bd had already spread across a large part of the basin, interestingly moving uphill. In 2006 and 2007 it spread further, and by 2008 Bd had spread across the entire basin and wiped out the majority of the more than 10,000 adult frogs in that area (Vredenburg et al. 2010). Unfortunately, by 2010, only several dozen frogs were left in the entire basin. To put it succinctly, he said, this reduced the largest metapopulation of frogs to a few individuals. The frogs were eventually rescued by the San Francisco Zoo.

Chytridiomycosis is not just affecting frogs in California, said Vredenburg, but it is also affecting frogs and salamanders in Central America, throughout the Andes in South America, parts of Africa, and Australia (Lips et al. 2008; Fisher and Garner 2020). Bsal, which is the second pathogen that causes chytridiomycosis, was discovered to have spread from Asia into Europe, likely through the pet trade. People buy amphibian pets, and unfortunately many of those pets end up being released into the wild, along with all of the flora and fauna that live on and within the animal, creating a dangerous situation. Bsal in particular has not been found in North America, but researchers are worried because North America has more salamanders than anywhere else in the world. A paper Vredenburg published in Science reported areas in North America with the highest potential vulnerability to Bsal based on modeling (Yap et al. 2015). Cities with a high number of live animals in trade are areas of concern, he said.

Scientists are interested in understanding how these individual species react to different pathogens in the laboratory, which is a great setting for these kinds of studies, said Vredenburg. This laboratory work involves taking special and important steps to make sure that pathogens tested in the laboratory do not get out into the wild. For example, Bsal has been tested in several laboratories in the United States, and it has not been found in the wild, so it is an important biosecurity issue. Researchers have to be cautious, but at the same time be able to do tests to figure out what assists the spread of this pathogen, Vredenburg explained. It is one thing to understand the underlying causes of susceptibility, he said, but it is another to know what can be done to mitigate that susceptibility. A 2007 study Vredenburg published in Biological Conservation found that some of the bacteria that live symbiotically on the skin of frogs protects them from the fungus (Woodhams et al. 2007). Vredenburg shared that there is interest now in figuring out if it is possible to bio-augment those bacteria on amphibians to save them in the wild, and there is much research to be done; for example, on coating frogs in the symbiotic bacteria.

Vredenburg said the Bd pathogen has infected more than 500 species across the globe, and it may be closer to 1,000 species. At least 200 species have dramatically decreased in the wild by epizootics. He asked What can be done? For one, researchers can describe what is going on in terms of the skin microbiome. They can also identify strains that may work as tools to protect against infection. There is also a lot of interest now to use zoos and researchers to re-introduce animals into the wild. For example, the San Francisco Zoo and the Oakland Zoo both have breeding programs working with the two species of yellow-legged frogs in California. In one approach under investigation, the frogs are inoculated with the pathogen, following which the infection is cleaned off to give the frogs a fighting chance; the frogs are then re-released into the wild.

This brings up many issues, Vredenburg said. Researchers may need to be able to do these experiments both in the laboratory and in the field, while being careful. It is important for researchers to use as many tools as possible, from population genetics to the microbiology of the symbiotic strains to understanding how multiple pathogens can affect individuals both in the field and in the laboratory. Researchers could continue to think carefully and clearly about what research and conservation actions might mean.

Four different pathogens that have caused die-offs in amphibians are Bd, Bsal, ranavirus, and snake fungal disease, which is an emerging pathogen that kills amphibians and is potentially dangerous for snakes in North America. Scientists may want to re-introduce species to areas affected by those pathogens, and zoo animals and captive populations are an important part of re-introductions, Vredenburg said. In Panama, the Amphibian Ark group is trying to rear and then re-introduce some of these amphibians into the wild. For example, one of the most important group of frogs is the genus Atelopus, which is a type of diurnal toad. There are more than 100 known species in this genus, more than 90 of which have gone either extinct or close to extinction since the Bd epizootics.

Vredenburg closed by suggesting scientists learn from successes in synthetic biology. For example, synthetic biology has helped increase the genetic diversity of surviving individuals of the black-footed ferret, he said. Researchers should leave open possibilities that allow for controlled exploratory science that may potentially assist species that are in trouble, he said, and do not close options. Vredenburg added that researchers should think carefully about biosecurity concerns, which entails ensuring the correct groups are involved in this conversation, including IACUCs, scientists, land managers, and ethicists. The eventual hope, Vredenburg said, is to achieve a situation where both those issues are discussed in terms of who is safe and how they are safe, where people also think carefully about the ethical considerations, and consider how to share and communicate that information to make sure that the best possible decisions are made.

A BRIEF SUMMARY OF THE INHERENT ASPECTS OF RISK MANAGEMENT IN THE CONTEXT OF WILDLIFE ANIMAL USE ACTIVITIES

John A. Bryan II, who works as a freelance wildlife veterinarian, provided a high-level overview of the focal points for his talk: the risk management obligations of animal welfare oversight bodies, the inherent risks of conducting animal use activities involving wildlife in their natural habitats on a macro and micro scale, and, finally, a path forward for how oversight bodies (IACUCs) can achieve competence in the arena of risk management and biosafety concerning wildlife animal use activities (WAUAs). Bryan’s insights are based on his experience working with numerous academic, federal, state, and private-sector agencies and entities involved with wildlife animal welfare, and he also mentioned his service on many IACUCs as the chair, a voting member, and/or as an attending veterinarian. Bryan said that he deliberately does not use the term “field study” for the reasons explained in earlier presentations about the AWA regulations versus the Guide for the Care and Use of Laboratory Animals (the Guide), which employs a more liberal interpretation of the term “field study” to basically describe any kind of wildlife animal use in the field.

Bryan began by pointing out some contradictions in the risk management obligations of animal welfare oversight bodies. He started with the AWA regulations stating that IACUCs are not mandated to oversee occupational health programs (APHIS 1966); however, the Guide is a little different, stating that IACUCs are mandated to oversee occupational health programs (NRC 2011). However, both the AWA regulations and the Guide indicate a duty to assess wildlife animal use protocol (AUP) with due diligence toward risk mitigation and biosafety for humans, target and non-target species, and environment and habitat. Bryan said splitting hairs between the differences in the AWA regulations and the Guide misses the point. Any oversight body, he said, whether registered with the USDA or the National Institutes of Health’s Office of Laboratory Animal Welfare or both must consider in its assessments and deliberations all factors affecting the welfare of the animals under study. In the arena of wildlife oversight, there is little to no difference in the application of these standards among study species, humans, and the environment in which the activity takes place. He said that an IACUC conducting an appropriately informed review of any WAUA will inevitably reach the point where the lines between what is best for humans, the wildlife species under study, and the environment hosting the activity not only become indistinguishable but are inherently interdependent. Bryan said, “oversight of WAUAs demand, on the part of any IACUC, a due diligence effort towards recognizing, understanding, and applying appropriate risk mitigation standards;

standards that by nature respect the innate interplay and interdependence of researchers, wildlife species, and the host environment. In wildlife oversight, there is simply no room for compartmentalizing.”

Bryan moved to discussing the inherent risks of conducting animal use activities involving wildlife in their natural habitats on a macro scale. He discussed risks that represent the larger, broader-scale risks when conducting wildlife use projects in the field: climate, seasonality, target species, non-target species, and equipment. He emphasized that any IACUC should, and even went as far as saying: “shall investigate and weigh in during its deliberations of a wildlife use project.”

With regard to climate, questions include where the activity is taking place—in a desert, alpine, aquatic, urban, tundra, or mountainous area? Is the protocol appropriately designed to proceed safely for both the human team and the target species? What is the landscape and terrain considerations; for example, is the terrain rocky, dangerous, and difficult to navigate? As an example, when studying mountain goats, what are the risk factors for capturing the target animal on rocky or dangerous landscape that might cause the animal to fall? What about animals, such as Florida panthers, which have been chemically immobilized, is there risk of them running off and perhaps drowning in shallow water?

Bryan also said to consider seasonality risks, for instance, whether it is a rainy or dry season and how best to be prepared for that weather. Risks involving target species require consideration of the particular physiology of the animals under study and seasonality. At what time of year is the study being conducted? For example, is it whelping season for wolves or hibernation for bears? There are risks to humans as well to consider in light of this, such as frostbite or overheating.

Bryan explained target species terrain risks also may need to be included. Capture and handling protocols should account for potential risks given difficulties of the terrain, including risks involving non-target species. He added that prompt consideration of any impacts the research protocol may have on the target species will likely include varying degrees of collateral impacts on the immediate environment. Bryan asked Does the AUP address this? Finally, risks involving equipment may entail considerations of potential impact; for example, the risk of lead rounds, sodium pentobarbital, and/or drug-filled stray darts; or even vehicles; for example, spotter aircraft, helicopters, and trucks. His point was to keep risk factors to a minimum for humans, target species, and the environment.

Turning to inherent risks on a micro scale, Bryan focused on smaller-scale, more specific risks and biosafety concerns: disease, trauma, euthanasia and carcass disposition, and exposures (e.g., chemicals, allergens, toxins, poisonous or venomous species). Bryan emphasized that any wildlife AUP may need to account for such risks and also the reviewing IACUC may need to likewise inquire as to the AUP’s ability to address such issues. He touched on some of the general categories of concern:

• Disease is paramount, as a great deal of WAUAs are indeed focused on disease, whether it be at the species level, genus level, or class level, specific to those taxonomic specifications or a zoonotic agent. The risk of an activity exacerbating or facilitating disease transmission via capture and handling must be weighed by any IACUC, Bryan added.
• Trauma via pursuit, capture, handling, and recovery and unintended consequences; for example, predation following extended recovery, Bryan said.
• Euthanasia and carcass disposition are relevant on any scale and often concern risk with firearms and drugs; for example, leaving a carcass in the field following euthanasia by either lead rounds from a firearm or sodium pentobarbital is unacceptable. Bryan stated, “actions such as these have direct deleterious effects on secondary, tertiary scavengers, and to a degree the landscape as well in the case of lead rounds.”
• Chemical exposure, he said, “having the right drugs for the job is imperative.” He added, “however, this means nothing without accompanying due diligence and drug use protocols that address pharmacology issues in the context of overdose, non-target accidental exposure, specifically to humans, and redress, how to deal with these issues as they arise.”

• Seasonality also comes into play because the risks change, Bryan said; for example, the same drug in winter may require somewhat different use protocols if used in summer.
• Allergens and toxins, he said, remember the goal is to think of all of these in terms of humans, target and non-target species, and the environment. Bryan noted, individual animals are capable of having adverse reactions to certain drugs.
• Exposure to poisonous or venomous species (e.g., animal, plant, fungal) at the landscape level, Bryan added.

Finally, Bryan discussed a path forward by providing a few tools that can be employed by animal oversight bodies to achieve an appropriate and a high level of competence and confidence reviewing and assessing wildlife use activities:

• Committee composition: Bryan said that it can be daunting to have wildlife AUPs on the agenda if the IACUC does not see them often. Moreover, if the reviewing committee lacks any expertise in wildlife, Bryan said, then the process risks becoming burdensome, confusing, delayed, and perhaps even insufficiently reviewed for all involved. The simple solution, he said, is to find wildlife experts and put them on the committee. Bryan added that any IACUC that knows it will occasionally see wildlife AUPs would do well to populate the committee with at least one or two wildlife professionals (e.g., biologists, wildlife veterinarians). He said this would go a long way toward boosting the committee confidence and ability in appropriately reviewing such activities.
• Consultation: Bryan explained that even if the IACUC has one member with wildlife experience, it could be helpful to seek additional insight on WAUA reviews from expert consultants. Even though they may not hold voting privileges, they can contribute profoundly to the IACUC’s ability to make an appropriate and informed review. He added that this includes the PI as researchers themselves can be extremely helpful in explaining their work, especially in terms of the aforementioned risks.
• Wildlife-specific AUP submission forms: Bryan noted another potential path forward was that many standardized AUP submission forms lack the suite of appropriate questions concerning WAUAs. He explained that this often results in incomplete assessments, frustration on the parts of both the PI and the IACUC, and prolonged delays. This process has the potential to miss most potential risks and biosafety issues inherent to a given WAUA. He shared that examples of such forms exist and are being used by many IACUCs at state and federal agencies that frequently deal with WAUAs.
• Collaboration and continuing education: Bryan said these are simple, adding that both the AWA regulations and the Guide require training for animal welfare oversight bodies. There are many opportunities for training, as well as continued education on issues specific to WAUAs, especially in the areas of risk management, biosecurity, and safety, said Bryan.

Bryan highlighted his take-home message: WAUAs have dynamic, complex inherent macro- and micro-scale risks that can be dangerous endeavors, regardless of the locale or species. IACUCs and PIs overseeing such projects are obligated to know these risks and account for them in the AUP. This likely requires the development of commensurately in-depth, informed strategy to mitigate biosafety risks to all involved, said Bryan, adding that “anything less is an inadequate effort and may result in tremendous harm to humans, animals, or the environment.”