MOLECULAR AND MORPHOLOGICAL CHARACTERIZATION OF SARCOCYSTIS INFECTIONS IN THE MUSCLES OF GRAY WOLVES (CANIS LUPUS) FROM MINNESOTA SUGGEST THEY MAY SERVE AS RESERVOIRS FOR INFECTION IN DOMESTICATED DOGS.

ABSTRACT: Sarcocystis infections were found for the first time in the muscles of 3 of 3 gray wolves (Canis lupus) from Minnesota. Two kinds (thin-walled and thick-walled) of sarcocysts were detected, based on the appearance of the sarcocyst wall. In wolf 1, sarcocysts were thin-walled (<0.5 µm), and without any visible protrusions. Ultrastructurally, the sarcocyst wall was type 1a and identical to Sarcocystis svanai of the domestic dog (Canis familiaris). The second kind of sarcocyst, with a relatively thicker (>1 µm) sarcocyst wall, was detected in wolves 2 and 3. Ultrastructurally, the sarcocyst wall had undulating, pleomorphic villar protrusion of type 9c; these sarcocysts were identical to Sarcocystis caninum from the domestic dog. Molecularly, the 2 Sarcocystis species were characterized using 18S, 28S, COI, ITS-1, and rpoB genetic markers. All these markers showed 100% identity to either of the 2 species previously described from the domestic dog. The thick-walled sarococyst corresponded to Sarcocystis caninum, whereas the thin-walled sarcocyst corresponded to Sarcocystis svanai.

Predicting chronic wasting disease in white-tailed deer at the county scale using machine learning.

Continued spread of chronic wasting disease (CWD) through wild cervid herds negatively impacts populations, erodes wildlife conservation, drains resource dollars, and challenges wildlife management agencies. Risk factors for CWD have been investigated at state scales, but a regional model to predict locations of new infections can guide increasingly efficient surveillance efforts. We predicted CWD incidence by county using CWD surveillance data depicting white-tailed deer (Odocoileus virginianus) in 16 eastern and midwestern US states. We predicted the binary outcome of CWD-status using four machine learning models, utilized five-fold cross-validation and grid search to pinpoint the best model, then compared model predictions against the subsequent year of surveillance data. Cross validation revealed that the Light Boosting Gradient model was the most reliable predictor given the regional data. The predictive model could be helpful for surveillance planning. Predictions of false positives emphasize areas that warrant targeted CWD surveillance because of similar conditions with counties known to harbor CWD. However, disagreements in positives and negatives between the CWD Prediction Web App predictions and the on-the-ground surveillance data one year later underscore the need for state wildlife agency professionals to use a layered modeling approach to ensure robust surveillance planning. The CWD Prediction Web App is at https://cwd-predict.streamlit.app/ .

Prion forensics: a multidisciplinary approach to investigate CWD at an illegal deer carcass disposal site.

Infectious prions are resistant to degradation and remain infectious in the environment for several years. Chronic wasting disease (CWD) has been detected in cervids inhabiting North America, the Nordic countries, and South Korea. CWD-prion spread is partially attributed to carcass transport and disposal. We employed a forensic approach to investigate an illegal carcass dump site connected with a CWD-positive herd. We integrated anatomic, genetic, and prion amplification methods to discover CWD-positive remains from six white-tailed deer (Odocoileus virginianus) and, using microsatellite markers, confirmed a portion originated from the CWD-infected herd. This approach provides a foundation for future studies of carcass prion transmission risk.

Lesions and viral antigen distribution in bald eagles, red-tailed hawks, and great horned owls naturally infected with H5N1 clade 2.3.4.4b highly pathogenic avian influenza virus.

An epidemic of highly pathogenic avian influenza (HPAI) began in North America in the winter of 2021. The introduced Eurasian H5N1 clade 2.3.4.4b virus subsequently reassorted with North American avian influenza strains. This postmortem study describes the lesions and influenza A virus antigen distribution in 3 species of raptors, including bald eagles (Haliaeetus leucocephalus, n = 6), red-tailed hawks (Buteo jamaicensis, n = 9), and great horned owls (Bubo virginianus, n = 8), naturally infected with this virus strain based on positive reverse transcriptase polymerase chain reaction and sequencing results from oropharyngeal swabs. The birds presented with severe neurologic signs and either died or were euthanized because of the severity of their clinical signs and suspected influenza virus infection. Gross lesions were uncommon and included forebrain hemorrhages in 2 eagles, myocarditis in 1 hawk, and multifocal pancreatic necrosis in 3 owls. Histological lesions were common and included encephalitis, myocarditis, multifocal pancreas necrosis, multifocal adrenal necrosis, histiocytic splenitis, and anterior uveitis in decreasing frequency. Influenza A viral antigen was detected in brain, heart, pancreas, adrenal gland, kidney, spleen, liver, and eye. In conclusion, bald eagles, red-tailed hawks, and great horned owls infected with the HPAI clade 2.3.4.4b virus strain and showing neurological signs of illness may develop severe or fatal disease with histologically detectable lesions in the brain that are frequently positive for viral antigen.

Assessment of Real-Time Quaking-Induced Conversion (RT-QuIC) Assay, Immunohistochemistry and ELISA for Detection of Chronic Wasting Disease under Field Conditions in White-Tailed Deer: A Bayesian Approach.

Chronic wasting disease (CWD) is a transmissible prion disease of the cervidae family. ELISA and IHC tests performed postmortem on the medial retropharyngeal lymph nodes (RPLN) or obex are considered diagnostic gold standards for prion detection. However, differences in CWD transmission, stage of infection, pathogenesis, and strain can limit performance. To overcome these uncertainties, we used Bayesian statistics to assess the accuracy of RT-QuIC, an increasingly used prion amplification assay, to diagnose CWD on tonsil (TLN), parotid (PLN) and submandibular lymph nodes (SMLN), and ELISA/IHC on RPLN of white-tailed deer (WTD) sampled from Minnesota. Dichotomous RT-QuIC and ELISA/IHC results from wild (n = 61) and captive (n = 46) WTD were analyzed with two-dependent-test, one-population models. RT-QuIC performed on TLN and SMLN of the wild WTD population had similar sensitivity (median range (MR): 92.2-95.1) to ELISA/IHC on RPLN (MR: 91.1-92.3). Slightly lower (4-7%) sensitivity estimates were obtained from farmed animal and PLN models. RT-QuIC specificity estimates were high (MR: 94.5-98.5%) and similar to ELISA/IHC estimates (MR: 95.7-97.6%) in all models. This study offers new insights on RT-QuIC and ELISA/IHC performance at the population level and under field conditions, an important step in CWD diagnosis and management.

WEST NILE VIRUS EXPOSURE AND INFECTION AMONG HUNTER-HARVESTED RUFFED GROUSE (BONASA UMBELLUS) COHORTS IN A STABLE POPULATION.

West Nile virus (WNV) was introduced to North America two decades ago, but for many species, including Ruffed Grouse (Bonasa umbellus), the effects of WNV on individuals and populations remain poorly understood. Recent studies suggest the effect of WNV on Ruffed Grouse might vary among geographic regions, depending on habitat conditions. We studied WNV in Minnesota, US, during 2018-19, in a region known to have abundant Ruffed Grouse habitat and a population cycling around a stable long-term average. We worked with cooperating hunters to collect hearts, feathers, and blood on filter strips from birds harvested in the fall to examine exposure to the virus. We detected antibodies to WNV or a flavivirus (probably WNV) in 12.5% and 12.3% of birds in 2018 and 2019, respectively. However, we did not isolate the virus from any heart samples, indicating that exposed birds were not experiencing an active infection of WNV at the time of harvest. Our findings indicate that, although Minnesota Ruffed Grouse are exposed to WNV, some birds mount a successful immune response and survive. However, our sampling approach did not account for birds that might have become infected over the summer and died, so it is unknown how much WNV mortality occurred before the fall harvest. Birds lost to WNV over the summer could reduce the number of birds that hunters see in the fall, thus reducing the quality of their hunting experiences. Management options for mitigating WNV impacts and other stressors consist primarily of providing high-quality Ruffed Grouse habitat that produces birds in good condition that are more likely to recover from infection.

COMPARISON OF CHRONIC WASTING DISEASE DETECTION METHODS AND PROCEDURES: IMPLICATIONS FOR FREE-RANGING WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS) SURVEILLANCE AND MANAGEMENT.

Throughout North America, chronic wasting disease (CWD) has emerged as perhaps the greatest threat to wild cervid populations, including white-tailed deer (WTD; Odocoileus virginianus). White-tailed deer are the most sought-after big game species across North America with populations of various subspecies in nearly all Canadian provinces, the contiguous US, and Mexico. Documented CWD cases have dramatically increased across the WTD range since the mid-1990s, including in Minnesota, US. CWD surveillance in free-ranging WTD and other cervid populations mainly depends upon immunodetection methods such as immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) on medial retropharyngeal lymph nodes and obex. More recent technologies centered on prion protein amplification methods of detection have shown promise as more sensitive and rapid CWD diagnostic tools. Here, we used blinded samples to test the efficacy of real-time quaking-induced conversion (RT-QuIC) in comparison to ELISA for screening tissues collected in 2019 from WTD in southeastern Minnesota, where CWD has been routinely detected since 2016. Our results support previous findings that RT-QuIC is a more sensitive tool for CWD detection than current antibody-based methods. Additionally, a CWD testing protocol that includes multiple lymphoid tissues (e.g., medial retropharyngeal lymph node, parotid lymph node, and palatine tonsil) per animal can effectively identify a greater number of CWD detections in a WTD population than a single sample type (e.g., medial retropharyngeal lymph nodes). These results show that the variability of CWD pathogenesis, sampling protocol, and testing platform must be considered for the effective detection and management of CWD throughout North America.

RT-QuIC detection of CWD prion seeding activity in white-tailed deer muscle tissues.

Chronic wasting disease (CWD) is a prion disease circulating in wild and farmed cervid populations throughout North America (United States and Canada), Europe (Finland, Norway, Sweden), and South Korea. CWD is a long-term threat to all cervid populations and to cervid hunting heritage, with the potential to cause substantial economic losses across multiple sectors. In North America, hunting and farming industries focused on the processing and consumption of white-tailed deer (WTD) venison are particularly vulnerable to CWD prion contamination, as millions of WTD are consumed annually. Real-time quaking-induced conversion (RT-QuIC) is a highly sensitive assay amplifying misfolded CWD prions in vitro and has facilitated CWD prion detection in a variety of tissues and excreta. To date, no study has comprehensively examined CWD prion content across bulk skeletal muscle tissues harvested from individual CWD infected WTD. Here, we use RT-QuIC to characterize prion-seeding activity in a variety of skeletal muscles from both wild and farmed CWD-positive WTD. We successfully detected CWD prions in muscles commonly used for consumption (e.g., backstrap, tenderloin, etc.) as well as within tongue and neck samples of WTD. Our results suggest that CWD prions are distributed across the skeletal muscles of infected WTD. We posit that RT-QuIC will be a useful tool for monitoring CWD prions in venison and that the method (with additional protocol optimization and high-throughput functionality) could be used to reduce and/or prevent CWD prions from entering animal and human food chains.

Assessing Lead Exposure in Free-Ranging Gray Wolves (Canis lupus) in Minnesota, USA.

The gray wolf (Canis lupus) is both an apex predator and a scavenger in Minnesota, US. Monitoring the health of Minnesota's gray wolf population is an important component of wolf management. Concern regarding whether wolves are being exposed to lead through scavenging viscera of hunter-harvested cervids left on the landscape, led to our study to determine lead-exposure rates. In fall 2012, livers from 147 hunter-harvested wolves (89 females, 58 males) were screened for lead and 20 other elements by inductively coupled plasma-atomic emission spectroscopy. Ten wolves (6.8%) were exposed to lead; only one had high enough exposure (6.14 ppm) to suggest lead toxicosis. Lead exposure varied by time of harvest, with nearly all lead-exposed wolves taken in the late hunting and trapping season (from 24 November 2012 to 31 January 2013), compared with the earlier hunting-only season (3-18 November 2012). Further, eight of 10 lead-exposed wolves were taken from deer-permit areas that harvested >1 deer/km2; only two of 10 were taken where deer harvest was less. This suggests the availability of viscera on the landscape may influence exposure risk of lead to wolves. More research is needed to determine baseline levels for toxic concentrations of lead in gray wolves and to determine clinical signs of lead poisoning in wild canids.

EVALUATING THE THRESHOLD DENSITY HYPOTHESIS FOR MOOSE (ALCES ALCES), WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS), AND PARELAPHOSTRONGYLUS TENUIS.

Despite the importance of the Parelaphostrongylus tenuis infection for moose (Alces alces) and white-tailed deer (Odocoileus virginianus) management, only one peer-reviewed study has evaluated the relationship between deer and moose densities and the potential for parasite-mediated competition between the species. Using polynomial-regression modeling, that study identified a deer-density threshold above which moose populations declined; however, the nature of the data and apparent outliers suggests the approach used to develop that threshold may not have been appropriate. We used the data from the original study to test whether alternative models, including linear models and negative binomial models would be less sensitive to outliers and could better explain that relationship. We found no evidence that moose density decreases as deer density increases. We concluded that, although the proposed moose-deer-P. tenuis relationship could be partially density dependent, additional factors, such as frequency dependence of disease transmission, gastropod abundance, and shared use of resources by moose and deer should also be considered.

Eastern Equine Encephalitis Virus in Visibly Affected Ruffed Grouse (Bonasa umbellus) in Michigan, Minnesota, and Wisconsin, USA.

Eastern equine encephalitis virus (EEEV) infects many avian species but has rarely been described in Ruffed Grouse (Bonasa umbellus). Between September and December 2019, 40 Ruffed Grouse, most in poor physical condition, were submitted to the Michigan, Wisconsin, and Minnesota (US) Departments of Natural Resources; eight were positive for EEEV.

Microbial associations and spatial proximity predict North American moose (Alces alces) gastrointestinal community composition.

Microbial communities are increasingly recognized as crucial for animal health. However, our understanding of how microbial communities are structured across wildlife populations is poor. Mechanisms such as interspecific associations are important in structuring free-living communities, but we still lack an understanding of how important interspecific associations are in structuring gut microbial communities in comparison with other factors such as host characteristics or spatial proximity of hosts. Here, we ask how gut microbial communities are structured in a population of North American moose Alces alces. We identify key microbial interspecific associations within the moose gut and quantify how important they are relative to key host characteristics, such as body condition, for predicting microbial community composition. We sampled gut microbial communities from 55 moose in a population experiencing decline due to a myriad of factors, including pathogens and malnutrition. We examined microbial community dynamics in this population utilizing novel graphical network models that can explicitly incorporate spatial information. We found that interspecific associations were the most important mechanism structuring gut microbial communities in moose and detected both positive and negative associations. Models only accounting for associations between microbes had higher predictive value compared to models including moose sex, evidence of previous pathogen exposure or body condition. Adding spatial information on moose location further strengthened our model and allowed us to predict microbe occurrences with ~90% accuracy. Collectively, our results suggest that microbial interspecific associations coupled with host spatial proximity are vital in shaping gut microbial communities in a large herbivore. In this case, previous pathogen exposure and moose body condition were not as important in predicting gut microbial community composition. The approach applied here can be used to quantify interspecific associations and gain a more nuanced understanding of the spatial and host factors shaping microbial communities in non-model hosts.

Emergence of the arterial worm Elaeophora schneideri in moose (Alces alces) and tabanid fly vectors in northeastern Minnesota, USA.

BACKGROUND: Moose (Alces alces) are a culturally and economically valued species in Minnesota. However, the moose population has experienced a sudden, marked decline in their range, including extirpation in the northwest and a 66% decline in the last decade in the northeast portions of the state. Although the exact cause of this decline is unclear, parasitic metastrongylid and filarioid nematode infections are known causes of morbidity and mortality in moose across North America. METHODS: To determine if these parasitic nematodes could be contributing to the Minnesota moose population decline, we molecularly examined banked tissues obtained from moose that died of known and unknown causes for the presence of nematode DNA. Extracted brain DNA of 34 individual moose was amplified utilizing primers targeting the 18S rRNA gene and internal transcribed spacer regions of nematodes. RESULTS: DNA sequencing revealed that PCR products obtained from 15 (44.1%) of the moose were 99% identical to Parelaphostrongylus tenuis, a metastrongylid known to cause neurological disease and death. Additionally, brain tissue from 20 (58.8%) individuals yielded sequences that most closely aligned with Elaeophora schneideri, a parasite associated with neurological impairment but previously unreported in Minnesota. Setaria yehi, a common filarioid parasite of deer, was also detected in the brain tissue of 5 (14.7%) moose. Molecular screening of 618 captured tabanid flies from four trapping sites revealed E. schneideri was present (6%) in the Minnesota environment and transmission could occur locally. Prevalence rates among the flies ranged between 0-100% per trapping site, with Chrysops spp. and Hybomitra spp. implicated as the vectors. CONCLUSIONS: Ultimately, these data confirm that P. tenuis is widespread in the Minnesota moose population and raises the question of the significance of E. schneideri as a contributing factor to morbidity and mortality in moose.

A TECHNIQUE FOR DEPLOYMENT OF RUMEN BOLUS TRANSMITTERS IN FREE-RANGING MOOSE ( ALCES ALCES).

Recent uses for rumen boluses, such as mortality implant transmitters (MITs), in wildlife have made it necessary to adapt deployment techniques developed for livestock. In 29 and 30 attempts to place MITs in Minnesota free-ranging moose ( Alces alces) in 2013 and 2014, respectively, success was achieved 83% and 63% of the time. In 2014, new methods for MIT deployment were evaluated in captive moose in Alaska. Mandible measurements provided guidance for selection of an appropriate-sized bolus applicator. A Schulze mouth gag was used to aid insertion of the applicator, and canola oil was used to lubricate the bolus to facilitate swallowing. Time to first swallow and time to continuous swallow following sedative reversal was measured to gauge appropriate timing for bolus administration. Using the adapted technique with trained personnel, success rates for MIT deployment were 100% (10/10) for captive moose and 88% (21/24) for free-ranging moose in Minnesota in 2015.

Evaluation of pathogen-specific biomarkers for the diagnosis of tuberculosis in white-tailed deer (Odocoileus virginianus).

OBJECTIVE To develop a noninvasive biomarker-based detection system specific for Mycobacterium bovis for monitoring infection in wild animals. SAMPLE Serum samples from 8 experimentally infected yearling white-tailed deer (Odocoileus virginianus) and 3 age-matched control deer and from 393 Minnesota Department of Natural Resources hunter-harvested white-tailed deer in northwest Minnesota. PROCEDURES 8 yearling deer were inoculated with 2 × 108 CFUs of virulent M bovis strain 1315 (day 0), and sera were obtained on days 0, 19, 48, and 60; sera were obtained from 3 uninoculated control deer on those same days. Sera from these deer and 9 M bovis-positive hunter-harvested deer were tested for 3 Mycobacterium-specific biomarkers (MB1895c, MB2515c, and polyketide synthase 5) by use of an indirect ELISA. That same ELISA was used to test sera obtained from 384 exposed noninfected deer in northwest Minnesota from 2007 through 2010, concurrent with an outbreak of tuberculosis involving cattle and deer in that region. RESULTS ELISA results revealed that tuberculosis infection could be detected as early as 48 days after inoculation in experimentally infected deer. Results for 384 deer sera revealed that prevalence of tuberculosis decreased over the 4-year period. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that the prevalence of tuberculosis in Minnesota deer decreased after 2009 but tuberculosis may have persisted (as subclinical disease) at extremely low levels, as indicated by the presence of low concentrations of circulating biomarkers. Biomarker-based diagnostic tests may offer a specific approach for early identification of M bovis infection.

Surveillance for Highly Pathogenic Avian Influenza in Wild Turkeys ( Meleagris gallopavo ) of Minnesota, USA during 2015 Outbreaks in Domestic Poultry.

An outbreak of a novel reassortant of highly pathogenic avian influenza A (H5N2) virus (HPAIV) decimated domestic turkeys ( Meleagris gallopavo ) from March through mid-June, 2015 in the state of Minnesota, US. In response, as part of broader surveillance efforts in wild birds, we designed a pilot effort to sample and test hunter-harvested Wild Turkeys ( Meleagris gallopavo ) for HPAIV in Minnesota counties with known infected poultry facilities. We also collected opportunistic samples from dead Wild Turkeys or live Wild Turkeys showing neurologic signs (morbidity and mortality samples) reported by the public or state agency personnel. Cloacal and tracheal samples were collected from each bird and screened for avian influenza virus (AIV) RNA by real-time reverse transcription PCR. From 15 April to 28 May 2015, we sampled 84 hunter-harvested male Wild Turkeys in 11 Minnesota counties. From 7 April 2015 through 11 April 2016, we sampled an additional 23 Wild Turkeys in 17 Minnesota counties. We did not detect type A influenza or HPAIV from any samples, and concluded, at the 95% confidence level, that apparent shedding prevalence in male Wild Turkeys in central Minnesota was between 0% and 2.9% over the sampling period. The susceptibility of wild turkeys to HPAIV is unclear, but regular harvest seasons make this wild gallinaceous bird readily available for future AIV testing.

A SEROSURVEY OF DISEASES OF FREE-RANGING GRAY WOLVES (CANIS LUPUS) IN MINNESOTA, USA.

We tested serum samples from 387 free-ranging wolves ( Canis lupus ) from 2007 to 2013 for exposure to eight canid pathogens to establish baseline data on disease prevalence and spatial distribution in Minnesota's wolf population. We found high exposure to canine adenoviruses 1 and 2 (88% adults, 45% pups), canine parvovirus (82% adults, 24% pups), and Lyme disease (76% adults, 39% pups). Sixty-six percent of adults and 36% of pups exhibited exposure to the protozoan parasite Neospora caninum . Exposure to arboviruses was confirmed, including West Nile virus (37% adults, 18% pups) and eastern equine encephalitis (3% adults). Exposure rates were lower for canine distemper (19% adults, 5% pups) and heartworm (7% adults, 3% pups). Significant spatial trends were observed in wolves exposed to canine parvovirus and Lyme disease. Serologic data do not confirm clinical disease, but better understanding of disease ecology of wolves can provide valuable insight into wildlife population dynamics and improve management of these species.

New roles in hemicellulosic sugar fermentation for the uncultivated Bacteroidetes family BS11.

Ruminants have co-evolved with their gastrointestinal microbial communities that digest plant materials to provide energy for the host. Some arctic and boreal ruminants have already shown to be vulnerable to dietary shifts caused by changing climate, yet we know little about the metabolic capacity of the ruminant microbiome in these animals. Here, we use meta-omics approaches to sample rumen fluid microbial communities from Alaskan moose foraging along a seasonal lignocellulose gradient. Winter diets with increased hemicellulose and lignin strongly enriched for BS11, a Bacteroidetes family lacking cultivated or genomically sampled representatives. We show that BS11 are cosmopolitan host-associated bacteria prevalent in gastrointestinal tracts of ruminants and other mammals. Metagenomic reconstruction yielded the first four BS11 genomes; phylogenetically resolving two genera within this previously taxonomically undefined family. Genome-enabled metabolic analyses uncovered multiple pathways for fermenting hemicellulose monomeric sugars to short-chain fatty acids (SCFA), metabolites vital for ruminant energy. Active hemicellulosic sugar fermentation and SCFA production was validated by shotgun proteomics and rumen metabolites, illuminating the role BS11 have in carbon transformations within the rumen. Our results also highlight the currently unknown metabolic potential residing in the rumen that may be vital for sustaining host energy in response to a changing vegetative environment.