Deer in Pennsylvania woods
Photo courtesy of John Armstrong

Our Research

– CWD –

  • Identifying CWD Biomarkers

    Project Title: Identifying CWD biomarkers that can be used for development of a rapid field CWD diagnostics test in deer; Investigating the microbiome associated with infection of CWD in white-tailed deer feces

    Dr. Anna Kashina, Penn VetPrincipal Investigator: Dr. Anna Kashina   

    Description: CWD belongs to a broader class of prion diseases, caused by accumulation of abnormally misfolded prion proteins. Healthy animals are believed to acquire this disease by oral exposure to infected animal by-products containing misfolded prions, including feces, saliva, urine, and animal remains which are accidentally ingested by the deer in the contaminated environment and are then absorbed through the digestive tract into the body. Once acquired, this disease is lethal, and currently there are no effective therapies and very limited means of diagnostics in live animals.

    Our research is focused on identifying novel CWD biomarkers that would enable high throughput field diagnostics of the disease in animal by-products and live animals, as well as, longer term, development of therapeutic approaches that would prevent disease propagation in the wild. To achieve these goals, we are pursuing the following research directions:

    1.  Identification of microbiomics signatures of CWD.

    Gastrointestinal tract represents the primary route of CWD infection. Studies of the past decade have uncovered that uptake of orally ingested products in the body is closely regulated by the gut microbiota, a multi-species population of symbiotic and pathogenic microorganisms normally residing in the intestines of every animal. Gut microbiotas interact with the host animal via exchange of metabolites, as well as exchange in the extracellular vesicles (exosomes) originating from both the host and the microbes.

    Based on the available evidence, recent papers propose a direct link between the gut microbiome and the pathogenesis and pathology of prion diseases, but this highly promising field is still in its infancy. Gut microbiome and the composition of extracellular vesicles in the digestive tract can be easily analyzed using feces, which contain a representative sample of bacteria from each individual. Feces are commonly found in natural deer habitats and can be collected without disturbing the environment or coming into dangerous contact with infected animals. Thus, identification of potential feces based diagnostic markers of CWD would provide an excellent tool for disease surveillance and control.

    Here, we are conducting a proof-of-concept study to investigate CWD-dependent changes in the gut microbiome as a potential diagnostic marker, as well as an expanded study to use advanced proteomics technologies to identify tissue-based CWD biomarkers in the diseased deer muscle.

    2.  Identification of novel protein biomarkers of CWD.

    Like other prion diseases, CWD pathogenesis arises through changes entirely at the protein level, by misfolding and abnormal accumulation of the native prion proteins in different tissues in the body. While this disease primarily targets the brain, its clinical manifestations are commonly seen in muscle, resulting in muscle wasting and behavioral changes that ultimately lead to death. In addition, other tissues in the body are also affected by prions, which are, e.g., secreted through saliva and bodily fluids. Evidence suggests the presence of prion proteins in the muscle and salivary glands of deer with CWD, but very few studies to date address this possibility. Moreover, no one has ever addressed protein-level changes in the muscle and other tissues during CWD, which may arise as a result of prion accumulation in other tissues in the body.

    Our project is aimed to address this critical gap by analyzing the proteome (total protein composition) of tissue biopsies from deer with mild and severe CWD, compared to healthy controls. We are currently analyzing the muscle, by performing comparative quantification of different proteins in the diseased and healthy deer muscle and analyzing potential modifications of these proteins that can serve as markers of CWD. Our longer term plans involve adding other tissues to this list, starting with those that carry the highest potential of live animal diagnostics.

    Dr. Anna Kashina is a Professor of Biochemistry at the School of Veterinary Medicine, University of Pennsylvania, with a broad research focus on the biology of neurodegeneration through posttranslational protein regulation and cytoskeleton-dependent vesicle transport (including secretion of extracellular vesicles).  

  • Are detection dogs a useful tool to screen White-tailed deer feces for CWD?

    Project Title: A proof of concept: are detection dogs a useful tool to screen White-tailed deer feces for CWD?

    Cindy Otto Penn VetPI: Dr. Cindy Otto (University of Pennsylvania’s Working Dog Center)

    Co-Is: Drs. Lisa Murphy, Julie C. Ellis, Michelle Gibison (WFP)

    Description: Chronic wasting disease (CWD) is a naturally occurring infectious, fatal, transmissible spongiform encephalopathy of cervids. Environmental contamination (and persistence) from excreta (e.g., saliva, urine, and feces) is thought to play a significant role in the rapid proliferation of CWD across North America over the past five decades.

    Currently, disease confirmation relies on postmortem detection of infectious prions in the medial retropharyngeal lymph nodes or obex region of the brain utilizing immunohistochemistry (IHC). Despite the long-standing recognition of CWD and progress made in the understanding the disease, there is still no live-animal test with comparable or greater sensitivity than that of postmortem IHC or ELISA.

    Feces are readily found on landscapes inhabited by cervid species and would be an ideal surveillance sample in areas where CWD incursion is a concern. Use of fecal samples for CWD detection would also preclude the need for physical contact with live, free-ranging animals that require sedation or restraint for traditional sample collection posing risks to both the deer and the investigators.

    Fecal volatile organic compound (VOC) analysis has been explored as a non-invasive method of disease detection. VOCs are organic chemicals that enter a gaseous phase at low temperatures. Animals (including humans) produce VOCs via dietary and metabolic pathways in response to immunologic or inflammatory stimulation, and via host–pathogen interactions.

    Detection dogs have an extremely sensitive sense of smell and can accurately detect low concentrations of VOCs present in the urine, blood, feces, and even exhaled breath of human cancer patients. The use of detection dogs could provide a rapid, non-invasive screening technique to identify CWD-positive or CWD-exposed deer in both captive and wild settings. In this project, we will document the sensitivity and specificity of odor detection dogs to discriminate between feces samples from CWD-positive and CWD-negative deer. We will document this canine ability in both the laboratory and field setting.

  • CWD detection in white-tailed deer and elk using RT-QulC

    Project Title: Investigating the use of third eyelids in white-tailed deer and elk for CWD detection using RT-QuIC

    Michelle Gibison, Wildlife Futures PIs: Drs. Michelle Gibison (WFP), Kevin Brightbill (Pennsylvania Department of Agriculture), Deep Tewari (Animal Diagnostic Laboratory, Pennsylvania Animal Diagnostic Laboratory System, Pennsylvania State University)

    Description: Chronic wasting disease (CWD) is a fatal disease that causes neurological signs in members of the Cervidae (deer) Family. The disease etiology is attributed to prions. CWD was first diagnosed in the United States in captive mule deer by the Colorado Division of Wildlife Foothills Wildlife Research Facility in Fort Collins, Colorado in 1967.

    After CWD-infected wild cervids were discovered in the upper Midwest in 2002, the disease was finally identified as a national priority and listed in 2004 to receive federal funding for research, wildlife disease surveillance and testing, management, and eradication. Many states have ongoing surveillance for CWD in wild and captive cervid populations.

    Approved diagnostic testing for the disease includes immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) on either retropharyngeal lymph nodes or on a section of brainstem called the obex. Given the time and effort required to obtain these diagnostic samples, more readily attainable and potentially antemortem sampling has been explored for cervids such as third eyelid and rectal mucosa-associated lymphoid tissue (RMALT).

    WF-Deer-CWDBoth these tissues show potential as additional diagnostic samples for CWD detection. Recent advances in molecular testing using real-time quaking-induced conversion (RT-QuIC) has been shown to be a more efficient, sensitive, and specific test for several prion diseases including CWD. This research will be used to evaluate and potentially validate the use of third eyelid specimens as a CWD diagnostic sample using RT-QuIC. The results will be compared with currently approved diagnostic methods and RT-QuIC for CWD using retropharyngeal lymph node (RLN) tissue from paired samples obtained from hunter harvested deer in PA.

    Project objectives: Determine Sensitivity and Specificity of third eyelids in wild white-tailed deer with RT-QuIC

    Through a joint partnership among the Pennsylvania Department of Agriculture, Animal Diagnostic Laboratory, Pennsylvania Animal Diagnostic Laboratory System, and the WFP, we are working on developing and validating RT-QuIC as a reliable diagnostic test for CWD, targeting third eyelids as an easily retrievable sample. Diagnostic testing on third eyelids will need to be compared with current validated and accepted ELISA and IHC protocols for tissue samples of retropharyngeal lymph nodes, and obex, when available.

– SARS CoV2 –

  • Detection of SARS-CoV-2 in bat guano

    Project Title:Evaluation of a real-time RT-PCR panel for detection of SARS-CoV-2 in bat guano

    Dr. Eman Anis, Penn Vet PADLSPrincipal Investigator: Dr. Eman Anis

    Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which is an ongoing global health concern.

    Bat-c-Ian-Gereg copy

    The exact source of the virus has not been identified, but it is believed that this novel coronavirus originated in animals; bats in particular have been implicated as the primary reservoir of the virus. SARS-CoV-2 can also be transmitted from humans to other animals, including tigers, cats, and mink.

    Consequently, infected people who work directly with bats could transfer the virus to a wild North American bat, resulting in a new natural reservoir for the virus, and lead to new outbreaks of human disease.

    We evaluated a reverse-transcription real-time PCR panel for detection of SARS-CoV-2 in bat guano. We found the panel to be highly specific for SARS-CoV-2, and able to detect the virus in bat guano samples spiked with SARS-CoV-2 viral RNA.

    Our panel could be utilized by wildlife agencies to test bats in rehabilitation facilities prior to their release to the wild, minimizing the risk of spreading this virus to wild bat populations.

    Photo courtesy of Ian Gereg
  • Screening bat guano for coronaviruses

    Project Title: Screening bat guano for SARS CoV-2 and Coronavirus in native North American bat species.

    Dr. Eman Anis, Penn Vet PADLSPrincipal Investigator: Dr. Eman Anis

    Project Description: Coronaviruses (CoVs) are found in a wide variety of wild and domestic animals in which they can cause respiratory, enteric, hepatic, and neurological diseases of varying severity. While rare, CoVs that infect animals can sometimes spread to humans.

    Severe acute respiratory syndrome (SARS), Middle Eastern respiratory syndrome (MERS), and recently coronavirus disease 19 (COVID-19), which is caused by SARS- coronavirus 2 (SARS-CoV-2), are examples of diseases caused by CoVs that originated in animals and spread to humans.

    SARSCOV2-Bat-Threat-Assessment-Pennsylvania Game CommissionBats have been implicated to be the original source of the three disease pathogens. Bat CoVs are highly prevalent around the world and show great genetic diversity, making up almost 60% of all known Alphacoronavirus and Betacoronavirus species.

    Interestingly, there is far more CoV diversity in Old compared to New-World bat species. Currently, there is limited information about the susceptibility of bats to infection by SARS-COV-2, and it is difficult to assess the risk of transmission from humans to bats when handling them.

    It has been shown that SARS-CoV-2 can be transmitted from humans back to some animals including tigers, cats, dogs and mink. Consequently, infected people who work directly with bats could transfer the virus to bats, resulting in a new natural reservoir for the virus, and leading to new outbreaks of human disease.

    To minimize the risk of spreading SARS-CoV-2 among bats and humans in North America, we propose to:

    1. Test bat samples collected from zoos and wildlife rehabilitation facilities for SARS-COV-2 using a real time RT-PCR panel that we recently evaluated in our lab for the detection of SARS-CoV-2 in bat guano,
    2. Screen the collected bat samples for other CoVs using pancoronavirus primers that target a highly conserved region of CoV to assess the genetic diversity of CoVs circulating in native North American bats (in wildlife rehabilitation facilities) and captive bats in zoos.

– Additional Topics –

  • Exposure to and effects of anticoagulant rodenticides on 3 species of carnivores in Pennsylvania

    Project Title: Exposure to and effects of anticoagulant rodenticides on 3 species of carnivores in Pennsylvania.

    Dr. Julie Ellis, Penn Vet New Bolton Center Wildlife FuturesPrincipal InvestigatorDr. Julie C. Ellis

    Co-Is: Dr. Lisa Murphy (WFP)Dr. Aaron Facka (PA Game Commission), Dr. Jacqueline Frair and Stephanie Cunningham [SUNY ESF Roosevelt Wild Life Station (NY)], Dr. Krysten Schuler and Melissa Fadden (Cornell Wildlife Health Lab at Cornell University College of Veterinary Medicine)  

    Description: Over the last century, a number of anticoagulant rodenticides (ARs) have been developed to reduce rodent populations in proximity to humans. Secondary exposure to rodenticides can result in adverse effects on carnivores including decreased ability to forage, periods of lethargy, or even death through internal hemorrhaging.

    A growing area of concern is also the sublethal effect of chronic exposure to ARs, which combined with other stressors acting on wildlife populations, may pose a serious challenge for long-term population viability. Here, we propose to use fisher, bobcats, and river otters as models for investigating spatial and inter-specific variation in AR exposure across Pennsylvania.

    Fisher CatBuilding upon ongoing research in New York (NY) on AR exposure in carnivores, we will also expand the study across PA and NY to compare spatial patterns of exposure in the three carnivore species in the region, and between fisher populations in areas where they are declining, stable, or increasing, to assess potential population level effects of AR exposure on this species.

    Specifically, we propose to: 1) document the types, prevalence, and concentrations of ARs that fishers, bobcats, and otters are exposed to via liver samples from legally harvested animals; 2) map AR hotspots and investigate spatial drivers of exposure (e.g., land use patterns); 3) within fishers, relate local population trends to exposure profile (i.e., percent of population exposed to ARs). In Pennsylvania, little is known about the overall use, abundance, and distribution of rodenticides or their commensurate prevalence in carnivores. There is essentially no information on differences in AR exposure among species of carnivores, their habitats, or their relationship to human activity. This study will provide important baseline data for PA, and by comparing to NY, we have the unique opportunity to compare across a larger landscape, and assess population-level impacts of AR exposure in fisher in NY and PA.

  • Investigating Red Cell Reservoirs to Detect Emerging Pathogens in Mammalian Hosts

    Project Title: Investigating Red Cell Reservoirs to Detect Emerging Pathogens in Mammalian Hosts

    Dr. Nilam Mangalmurti, Penn MedicinePI: Dr. Nilam Mangalmurti (University of Pennsylvania’s School of Medicine, PennMed)

    Co-PI: Dr. Julie C. Ellis (WFP)

    Co-Is: Greg Turner, Dr. Andrew Di Salvo (Pennsylvania Game Commission), Dr. Eman Anis (WFP), Dr. Audrey R. Odum John (Division of Pediatric Infectious Diseases at Children's Hospital of Philadelphia)

    Description: At nearly 30 trillion cells, red blood cells (RBCs) are the most common circulating cell in the human body and have tremendous capacity to modulate inflammation.

    RBCs perform many non-gas exchanging functions, including chemokine regulation, complement binding, and pathogen immobilization. Interestingly, these immune functions appear to result from the evolutionarily advantageous RBC adaptations that arose in response to malarial pathogens.

    The purpose of this project is to extrapolate our previous investigations in humans to bat and deer, two mammalian orders previously reported to have malarial infection and likely to have erythrocyte immune function. The ultimate goal is to create red cell-based molecular diagnostics to detect pathogenic DNA reservoirs and develop model systems to identify emerging zoonoses.