The establishment of a collaborative surveillance program with indigenous hunters to characterize primate health in Southern Guyana.

The consumption of primates is integral to the traditional subsistence strategies of many Indigenous communities throughout Amazonia. Understanding the overall health of primates harvested for food in the region is critical to Indigenous food security and thus, these communities are highly invested in long-term primate population health. Here, we describe the establishment of a surveillance comanagement program among the Waiwai, an Indigenous community in the Konashen Amerindian Protected Area (KAPA). To assess primate health in the KAPA, hunters performed field necropsies on primates harvested for food and tissues collected from these individuals were analyzed using histopathology. From 2015 to 2019, hunters conducted 127 necropsies across seven species of primates. Of this sample, 82 primates (between 2015 and 2017) were submitted for histopathological screening. Our histopathology data revealed that KAPA primates had little evidence of underlying disease. Of the tissue abnormalities observed, the majority were either due to diet (e.g., hepatocellular pigment), degenerative changes resulting from aging (e.g., interstitial nephritis, myocyte lipofusion), or nonspecific responses to antigenic stimulation (renal and splenic lymphoid hyperplasia). In our sample, 7.32% of individuals had abnormalities that were consistent with a viral etiology, including myocarditis and hepatitis. Internal parasites were observed in 53.66% of individuals and is consistent with what would be expected from a free-ranging primate population. This study represents the importance of baseline data for long-term monitoring of primate populations hunted for food. More broadly, this research begins to close a critical gap in zoonotic disease risk related to primate harvesting in Amazonia, while also demonstrating the benefits of partnering with Indigenous hunters and leveraging hunting practices in disease surveillance and primate population health assessment.

A De Novo Whole Genome Assembly and Annotation of Parelaphostrongylus tenuis.

Parelaphostrongylus tenuis causes ungulate morbidity and mortality in eastern and central North America, but no reference genome sequence exists to facilitate research. Here, we present a P. tenuis genome assembly and annotation, generated with PacBio and Illumina technologies. The assembly is 491 Mbp, with 7285 scaffolds and 185 kb N50.

Great ape health watch: Enhancing surveillance for emerging infectious diseases in great apes.

Infectious diseases have the potential to extirpate populations of great apes. As the interface between humans and great apes expands, zoonoses pose an increasingly severe threat to already endangered great ape populations. Despite recognition of the threat posed by human pathogens to great apes, health monitoring is only conducted for a small fraction of the world's wild great apes (and mostly those that are habituated) meaning that outbreaks of disease often go unrecognized and therefore unmitigated. This lack of surveillance (even in sites where capacity to conduct surveillance is present) is the most significant limiting factor in our ability to quickly detect and respond to emerging infectious diseases in great apes when they first appear. Accordingly, we must create a surveillance system that links disease outbreaks in humans and great apes in time and space, and enables veterinarians, clinicians, conservation managers, national decision makers, and the global health community to respond quickly to these events. Here, we review existing great ape health surveillance programs in African range habitats to identify successes, gaps, and challenges. We use these findings to argue that standardization of surveillance across sites and geographic scales, that monitors primate health in real-time and generates early warnings of disease outbreaks, is an efficient, low-cost step to conserve great ape populations. Such a surveillance program, which we call "Great Ape Health Watch" would lead to long-term improvements in outbreak preparedness, prevention, detection, and response, while generating valuable data for epidemiological research and sustainable conservation planning. Standardized monitoring of great apes would also make it easier to integrate with human surveillance activities. This approach would empower local stakeholders to link wildlife and human health, allowing for near real-time, bidirectional surveillance at the great ape-human interface.

The Gombe Ecosystem Health Project: 16 years of program evolution and lessons learned.

Infectious disease outbreaks pose a significant threat to the conservation of chimpanzees (Pan troglodytes) and all threatened nonhuman primates. Characterizing and mitigating these threats to support the sustainability and welfare of wild populations is of the highest priority. In an attempt to understand and mitigate the risk of disease for the chimpanzees of Gombe National Park, Tanzania, we initiated a long-term health-monitoring program in 2004. While the initial focus was to expand the ongoing behavioral research on chimpanzees to include standardized data on clinical signs of health, it soon became evident that the scope of the project would ideally include diagnostic surveillance of pathogens for all primates (including people) and domestic animals, both within and surrounding the National Park. Integration of these data, along with in-depth post-mortem examinations, have allowed us to establish baseline health indicators to inform outbreak response. Here, we describe the development and expansion of the Gombe Ecosystem Health project, review major findings from the research and summarize the challenges and lessons learned over the past 16 years. We also highlight future directions and present the opportunities and challenges that remain when implementing studies of ecosystem health in a complex, multispecies environment.

BASELINE PHYSIOLOGIC AND HEMATOLOGIC HEALTH IN WILD-CAUGHT MUSKRATS (ONDATRA ZIBETHICUS) FROM A NEAR-PRISTINE ECOSYSTEM IN NORTHERN MINNESOTA.

Muskrat (Ondatra zibethicus) populations show long-term and widespread declines across North America, necessitating research into potential mechanistic explanations, including population health. Previous research established reference hematology values, a proxy of individual health, of muskrats occurring in highly modified ecosystems. However, our knowledge of hematology metrics in muskrat populations occurring in more natural ecosystems is limited. We measured several hematological parameters of wild-caught muskrats (n = 73) in the Greater Voyageurs Ecosystem in northern Minnesota in 2018-2019 to establish baseline muskrat health in a relatively intact, near-pristine ecosystem. Additionally, we measured rectal temperature and heart and respiratory rates and collected whole blood for complete blood cell count assessment. We established baseline physiologic and hematologic reference ranges for the population and describe variations between total white blood cells, nucleated cell differentials, and basic erythron and platelet estimates and demonstrate methods of estimation to be poor proxies for more standardized counting methods. Our results establish a baseline to compare muskrat health assessments for populations affected by landscape change or in decline.

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.

Molecular and Biological Characterization of a Cervidpoxvirus Isolated From Moose with Necrotizing Dermatitis.

Cervidpoxvirus is one of the more recently designated genera within the subfamily Chordopoxvirinae, with Deerpox virus (DPV) as the only recognized species to date. In this study, the authors describe spontaneous disease and infection in the North American moose (Alces americanus) by a novel Cervidpoxvirus, here named Moosepox virus (MPV). Three 4-month-old moose calves developed a multifocal subacute-to-chronic, necrotizing, suppurative-to-granulomatous dermatitis that affected the face and the extremities. Ultrastructurally, all stages of MPV morphogenesis-that is, crescents, spherical immature particles, mature particles, and enveloped mature virus-were observed in skin tissue. In vitro infection with MPV confirmed that its morphogenesis was similar to that of the prototype vaccinia virus. The entire coding region, including 170 putative genes of this MPV, was sequenced and annotated. The sequence length was 164,258 bp with 98.5% nucleotide identity with DPV (strain W-1170-84) based on the whole genome. The genome of the study virus was distinct from that of the reference strain (W-1170-84) in certain genes, including the CD30-like protein (83.9% nucleotide, 81.6% amino acid), the endothelin precursor (73.2% nucleotide including some indels, 51.4% amino acid), and major histocompatibility class (MHC) class I-like protein (81.0% nucleotide, 68.2% amino acid). This study provides biological characterization of a new Cervidpoxvirus attained through in vivo and in vitro ultrastructural analyses. It also demonstrates the importance of whole-genome sequencing in the molecular characterization of poxviruses identified in taxonomically related hosts.

Socioecological correlates of clinical signs in two communities of wild chimpanzees (Pan troglodytes) at Gombe National Park, Tanzania.

Disease and other health hazards pose serious threats to the persistence of wild ape populations. The total chimpanzee population at Gombe National Park, Tanzania, has declined from an estimated 120 to 150 individuals in the 1960's to around 100 individuals by the end of 2013, with death associated with observable signs of disease as the leading cause of mortality. In 2004, we began a non-invasive health-monitoring program in the two habituated communities in the park (Kasekela and Mitumba) with the aim of understanding the prevalence of health issues in the population, and identifying the presence and impacts of various pathogens. Here we present prospectively collected data on clinical signs (observable changes in health) in the chimpanzees of the Kasekela (n = 81) and Mitumba (n = 32) communities over an 8-year period (2005-2012). First, we take a population approach and analyze prevalence of clinical signs in five different categories: gastrointestinal system (diarrhea), body condition (estimated weight loss), respiratory system (coughing, sneezing etc.), wounds/lameness, and dermatologic issues by year, month, and community membership. Mean monthly prevalence of each clinical sign per community varied, but typically affected <10% of observed individuals. Secondly, we analyze the presence of clinical signs in these categories as they relate to individual demographic and social factors (age, sex, and dominance rank) and simian immunodeficiency virus (SIVcpz) infection status. Adults have higher odds of being observed with diarrhea, loss of body condition, and wounds or lameness when compared to immatures, while males have a higher probability of being observed with wounds or lameness than females. In contrast, signs of respiratory illness appear not to be related to chimpanzee-specific factors and skin abnormalities are very rare. For a subset of known-rank individuals, dominance rank predicts the probability of wounding/lameness in adult males, but does not predict any adverse clinical signs in adult females. Instead, adult females with SIVcpz infection are more likely to be observed with diarrhea, a finding that warrants further investigation. Comparable data are needed from other sites to determine whether the prevalence of clinical signs we observe are relatively high or low, as well as to more fully understand the factors influencing health of wild apes at both the population and individual level. Am. J. Primatol. 80:e22562, 2018. © 2016 Wiley Periodicals, Inc.

Factors influencing detection and co-detection of Ranavirus and Batrachochytrium dendrobatidis in Midwestern North American anuran populations.

Amphibian populations are in decline worldwide as they face a barrage of challenges, including infectious diseases caused by ranaviruses and the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). Here we describe seasonal dynamics of Bd and ranavirus detection in free-ranging post-metamorphic wood frogs Lithobates sylvaticus, boreal chorus frogs Pseudacris maculata/triseriata, and gray treefrogs Hyla versicolor/chrysoscelis, sampled over a 3 season gradient in Minnesota (USA) wetlands. We detected Bd in 36% (n = 259) of individuals sampled in 3 wetlands in 2014, and 33% (n = 255) of individuals sampled in 8 wetlands in 2015. We also detected ranavirus in 60% and 18% of individuals sampled in 2014 and 2015, respectively. Ranavirus and Bd were detected concurrently in 26% and 2% of animals sampled in 2014 and 2015, respectively. We report clinical signs and associated infection status of sampled frogs; of the clinical signs observed, skin discoloration was significantly associated with ranavirus infection. Using generalized estimating equations, we found that species, season, wetland, and a species × season interaction term were significant predictors of Bd detection, whereas test year approached significance as a predictor of ranavirus detection. The odds of detecting both pathogens concurrently was significantly influenced by species, season, a species × season interaction term, year, and environmental ammonia. We propose an amphibian health monitoring scheme that couples population size surveys with seasonal molecular surveys of pathogen presence. This information is crucial to monitoring the health of remaining strongholds of healthy amphibian populations, as they face an uncertain future of further anthropogenic change.

SERIAL ULTRASOUND TO ESTIMATE FETAL GROWTH CURVES IN SOUTHERN TAMANDUA (TAMANDUA TETRADACTYLA).

From 2012 to 2015, 16 pregnancies were monitored by ultrasonography in nine tamanduas ( Tamandua tetradactyla ) housed in three zoological facilities. Sonographic measurements were recorded to establish fetal growth curves using thoracic and skull landmarks described for giant anteaters ( Myrmecophaga tridactyla ). All pregnancies resulted in the uncomplicated delivery of healthy offspring, thus gestational development was considered normal. These data may be used as a reference for normal fetal development with potential for estimating parturition date in the absence of breeding data.

Noninvasive test for tuberculosis detection among primates.

Traditional testing methods have limited epidemiologic studies of tuberculosis among free-living primates. PCR amplification of insertion element IS6110 of Mycobacterium tuberculosis from fecal samples was evaluated as a noninvasive screening test for tuberculosis in primates. Active tuberculosis was detected among inoculated macaques and naturally exposed chimpanzees, demonstrating the utility of this test.

FATAL SUPPURATIVE INFLAMMATION IN DAURIAN PIKAS (OCHOTONA DAUURICA) NATURALLY INFECTED WITH CASTELLANIELLA GINSENGISOLI-LIKE BACTERIA.

Fifteen Daurian pika (Ochotona dauurica) were introduced to a zoological collection in December 2011 as founders for a planned breeding colony. Despite breeding success, the colony shrunk over 37 mo to 11 animals. Mortality in 11 of 46 deceased animals, including wild-caught "founders" and captive-born offspring, was associated with a suppurative inflammation and abscess formation of the spleen, skin, peripheral and internal lymph nodes, liver, lungs, kidney, or a combination of organs. Gram-negative, non-fermenting, rod-shaped bacteria were isolated from the abscesses in all cases. Steiner Chapman silver stain identified rod-shaped bacteria in the abscesses of seven animals. The bacteria were not detected with Gram stain, acid-fast stain, or Grocott silver methenamine stain and was not detectable by periodic acid-Schiff reaction. In two cases, including the index case, the bacteria were presumptively identified as Ralstonia pickettii on the basis of conventional biochemical characterization. The bacteria in the other cases were not further classifiable with conventional methods. Matrix-assisted laser desorption/ionization time of flight mass spectrometry and 16s rDNA gene sequencing resulted in identification to the genus level as Castellaniella in 10 of 12 cases. Comparative 16S rDNA gene sequence analysis showed that these isolates and Castellaniella ginsengisoli Strain DCY36T were 99% similar. Castellaniella ginsengisoli, a gram-negative bacterium isolated from soil of a ginseng field in South Korea, has not previously been associated with disease in animals or humans. It is uncertain how the bacterium was introduced to the Daurian pika colony or how it spread.

The risk of tuberculosis transmission to free-ranging great apes.

Pathogen exchange between humans and primates has been facilitated by anthropogenic disturbances, such as changing land use patterns, habitat destruction, and poaching, which decrease population sizes and increase levels of primate-human interaction. As a result, human and domestic animal diseases have become a recognized threat to endangered primate populations. Tuberculosis is a major global human and animal health concern, especially in equatorial Africa where many of the remaining free-living great ape populations exist in proximity with exposed and/or infected human populations and their domestic animals. Increased anthropogenic pressure creates an opportunity for the anthropozoonotic spread of this disease. This review examines current evidence of the risk of tuberculosis transmission to great apes, the benefits and limitations of current detection methods, the impact of current great ape conservation and management strategies on this risk, and the need for an ecosystem health-based approach to mitigating the risks of tuberculosis transmission to great apes.

Uterine adenocarcinoma in a Przewalski's wild horse (Equus ferus przewalskii).

A 25-yr-old, nulliparous, female Przewalski's wild horse (Equus ferus przewalskii) with a history of pituitary pars intermedia dysfunction and recent onset of serosanguinous vaginal discharge was euthanized after a period of lethargy and inappetance. Postmortem examination confirmed an infiltrative uterine adenocarcinoma, which is an uncommon neoplasia in equids. Reproductive disease is significant in this species as they are considered endangered by IUCN. Reproductive soundness and success are paramount to conservation efforts.

Health of wild fish exposed to contaminants of emerging concern in freshwater ecosystems utilized by a Minnesota Tribal community.

Fish serve as indicators of exposure to contaminants of emerging concern (CECs)-chemicals such as pharmaceuticals, hormones, and personal care products-which are often designed to impact vertebrates. To investigate fish health and CECs in situ, we evaluated the health of wild fish exposed to CECs in waterbodies across northeastern Minnesota with varying anthropogenic pressures and CEC exposures: waterbodies with no human development along their shorelines, those with development, and those directly receiving treated wastewater effluent. Then, we compared three approaches to evaluate the health of fish exposed to CECs in their natural environment: a refined fish health assessment index, a histopathological index, and high-throughput (ToxCast) in vitro assays. Lastly, we mapped adverse outcome pathways (AOPs) associated with identified ToxCast assays to determine potential impacts across levels of biological organization within the aquatic system. These approaches were applied to subsistence fish collected from the Grand Portage Indian Reservation and 1854 Ceded Territory in 2017 and 2019. Overall, 24 CECs were detected in fish tissues, with all but one of the sites having at least one detection. The combined implementation of these tools revealed that subsistence fish exposed to CECs had histological and macroscopic tissue and organ abnormalities, although a direct causal link could not be established. The health of fish in undeveloped sites was as poor, or sometimes poorer, than fish in developed and wastewater effluent-impacted sites based on gross and histologic tissue lesions. Adverse outcome pathways revealed potential hazardous pathways of individual CECs to fish. A better understanding of how the health of wild fish harvested for consumption is affected by CECs may help prioritize risk management research efforts and can ultimately be used to guide fishery management and public health decisions. Integr Environ Assess Manag 2024;20:846-863. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

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.

SPECIES IN THE FECES: DNA METABARCODING TO DETECT POTENTIAL GASTROPOD HOSTS OF PARELAPHOSTRONGYLUS TENUIS Consumed By Moose (Alces Alces).

Our understanding of wildlife multihost pathogen transmission systems is often incomplete due to the difficulty of observing contact between hosts. Understanding these interactions can be critical for preventing disease-induced wildlife declines. The proliferation of high-throughput sequencing technologies provides new opportunities to better explore these cryptic interactions. Parelaphostrongylus tenuis, a multihost parasite, is a leading cause of death in some moose (Alces alces) populations threatened by local extinction in the midwestern and northeastern US and southeastern Canada. Moose contract P. tenuis by consuming infected gastropod intermediate hosts, but little is known about which gastropod species moose consume. To gain more insight, we used a genetic metabarcoding approach on 258 georeferenced and temporally stratified moose fecal samples collected May-October 2017-20 from a declining population in the north-central US. We detected moose consumption of three species of gastropods across five positive samples. Two of these (Punctum minutissimum and Helisoma sp.) have been minimally investigated for the ability to host P. tenuis, while one (Zonitoides arboreus) is a well-documented host. Moose consumption of gastropods documented herein occurred in June and September. Our findings prove that moose consume gastropod species known to become infected by P. tenuis and demonstrate that fecal metabarcoding can provide novel insight on interactions between hosts of a multispecies pathogen transmission system. After determining and improving test sensitivity, these methods may also be extended to document important interactions in other multihost disease systems.

Standardization of Data Analysis for RT-QuIC-Based Detection of Chronic Wasting Disease.

Chronic wasting disease (CWD) is a disease affecting cervids and is caused by prions accumulating as pathogenic fibrils in lymphoid tissue and the central nervous system. Approaches for detecting CWD prions historically relied on antibody-based assays. However, recent advancements in protein amplification technology provided the foundation for a new class of CWD diagnostic tools. In particular, real-time quaking-induced conversion (RT-QuIC) has rapidly become a feasible option for CWD diagnosis. Despite its increased usage for CWD-focused research, there lacks a consensus regarding the interpretation of RT-QuIC data for diagnostic purposes. It is imperative then to identify a standardized and replicable method for determining CWD status from RT-QuIC data. Here, we assessed variables that could impact RT-QuIC results and explored the use of maxpoint ratios (maximumRFU/backgroundRFU) to improve the consistency of RT-QuIC analysis. We examined a variety of statistical analyses to retrospectively analyze CWD status based on RT-QuIC and ELISA results from 668 white-tailed deer lymph nodes. Our results revealed an MPR threshold of 2.0 for determining the rate of amyloid formation, and MPR analysis showed excellent agreement with independent ELISA results. These findings suggest that the use of MPR is a statistically viable option for normalizing between RT-QuIC experiments and defining CWD status.

Herd-level risk factors associated with chronic wasting disease-positive herd status in Minnesota, Pennsylvania, and Wisconsin cervid herds.

The objective of this study was to evaluate risks related to introduction of Chronic Wasting Disease (CWD) to farmed cervid herds in Minnesota (MN), Pennsylvania (PA), and Wisconsin (WI). This was the first study to evaluate risk factors related to multiple pathways of CWD transmission to farmed cervid herds. Participating herds in this case-control study included 22 case and 49 control herds identified through participation in the respective mandatory State CWD herd program. Data was voluntarily collected from white-tailed deer (WTD) study herds using a questionnaire and state animal health agency databases. Univariable analyses identified associations between CWD-positive herds and variables representing different transmission pathways, including direct contact with infected farmed cervids (imported from a herd that later tested positive for CWD) odds ratio (OR):7.16, 95 % confidence intervals (CI):1.64-31.21 and indirect contact with infected wild cervids (access of domestic cats to pens or feed storage area) OR:4.07, 95 % CI:1.35-12.26, observed evidence of mammalian scavengers inside or outside of fenceline in the previous 12 months OR:6.55, 95 % CI:1.37-31.32, ≤ 5 km distance to nearest detected CWD-positive wild cervid OR:3.08, 95 % CI:1.01-9.39, forested area crosses the perimeter fenceline OR:3.54, 95 % CI:1.13-11.11, ≤ 0.3 m distance of water source to fenceline OR:4.71, 95 % CI:1.60-13.83, and water source shared with wild cervids (running or standing water) OR:4.17, 95 % CI:1.34-12.92. Three variables from univariable analyses that represented different biological transmission pathways were placed in a Firth's penalized maximum likelihood multivariable logistic regression to evaluate associations between transmission pathway and CWD herd infection status. For the issue of low sample size and overfitting, 95 % CIs for estimated coefficients for the three variables were computed via bootstrapping of 10,000 independent bootstrap samples. The three biological variables were significantly associated with herd CWD infection status: imported cervids from a herd that later tested positive for CWD (OR:5.63; 95 % CI:1.1-28.2), ≤ 0.3 m distance of cervid water source to perimeter fenceline (OR:4.83; 95 % CI:1.5-16.1), and ≤ 5 km distance to nearest detected CWD-positive wild cervid (OR:4.10; 95 % CI:1.1-15.2). The risk factors associated with CWD herd status identified in this study indicated the importance of transmission through direct contact pathways with infected cervid herds (introduction of cervids from herds later identified as CWD-infected) and indirect contact pathways with infected wild cervids that could be related to other animals through the perimeter fence. Further studies are needed to confirm and clarify understanding of indirect pathways to allow development of improved biosecurity practices to prevent CWD introduction to cervid herds.

Refining the moose serum progesterone threshold to diagnose pregnancy.

Pregnancy determination is necessary for sound wildlife management and understanding population dynamics. Pregnancy rates are sensitive to environmental and physiological factors and may indicate the overall trajectory of a population. Pregnancy can be assessed through direct methods (rectal palpation, sonography) or indicated using hormonal assays (serum progesterone or pregnancy-specific protein B, fecal progestogen metabolites). A commonly used threshold of 2 ng/ml of progesterone in serum has been used by moose biologists to indicate pregnancy but has not been rigorously investigated. To refine this threshold, we examined the relationship between progesterone concentrations in serum samples and pregnancy in 87 moose (Alces alces; 64 female, 23 male) captured from 2010 to 2020 in the Grand Portage Indian Reservation in northeastern Minnesota, USA. Pregnancy was confirmed via rectal palpation (n = 25), necropsy (n = 2), calf observation (n = 25) or characteristic pre-calving behavior (n = 6), with a total of 58 females determined pregnant and 6 not pregnant; 23 males were included to increase the non-pregnant sample size. Using receiver operating characteristic analysis, we identified an optimal threshold of 1.115 ng/ml with a specificity of 0.97 (95% confidence interval [CI] = 0.90-1.00) and a sensitivity of 0.98 (95% CI = 0.95-1.00). Progesterone concentrations were significantly higher in cases of pregnant versus non-pregnant cows, but we did not detect a difference between single and twin births. We applied our newly refined threshold to calculate annual pregnancy rates for all female moose (n = 133) captured in Grand Portage from 2010 to 2021. Mean pregnancy rate during this period was 91% and ranged annually from 69.2 to 100%. Developing a reliable method for determining pregnancy status via serum progesterone analyses will allow wildlife managers to assess pregnancy rates of moose without devoting substantial time and resources to palpation and calf monitoring.