Impact of carbon neutralization policy on the suitable habitat distribution of the North China leopard.

The Chinese government has introduced a carbon neutral policy to cope with the rapid changes in the global climate. It is not clear what impact this policy will have on wildlife. Therefore, this study analyzed the suitable habitat distribution of China's unique leopard subspecies in northern Shaanxi, and simulated the potential suitable habitat distribution under different carbon emission scenarios at two time points of future carbon peak and carbon neutralization. We found that in the future SSPs 126 scenario, the suitable habitat area and the number of suitable habitat patches of North China leopard will continue to increase. With the increase of carbon emissions, it is expected that the suitable habitat of North China leopard will continue to be fragmented and shifted. When the annual average temperature is lower than 8 °C, the precipitation seasonality is 80-90 mm and the precipitation of the warmest quarter is greater than 260 mm, the probability of occurrence of North China leopard is higher. The increase in carbon emissions will lead to the reduction, migration, and fragmentation of the suitable habitat distribution of the North China leopard. Carbon neutrality policies can protect suitable wild habitats. In the future, the impact of carbon neutrality policies on future wildlife habitat protection should be carried out in depth to effectively promote the construction of wildlife protection projects.

Home range, habitat use and capture-release of translocated leopards in Gir landscape, Gujarat, India.

Understanding the spatial ecology of translocated leopards (Panthera pardus fusca) is crucial for their conservation and the effective assessment of conflict management strategies. We investigated the home range and habitat preferences of five radio-collared leopards (n = 5; 2 males; 3 females) in the Gir landscape. Additionally, we examined the usefulness of the capture-release strategy for these animals. We assessed home range and habitat selection using kernel density estimation (at 95% and 50% levels) and compositional analysis. Our findings revealed that leopards exhibited distinct patterns of movement, often returning to their original capture site or nearby locations or exploring new areas within 3 to 25 days, covering distances ranging from 48 to 260 km. The average home range (95% FK) was estimated at 103.96±36.37 (SE) km2, with a core area usage (50% FK) of 21.38±5.95 km2. Seasonally, we observed the largest home ranges during summer and the smallest during winter. Males exhibited larger home ranges (95% FK, 151±64.28 km2) compared to females (56.18±14.22 km2). The habitat analysis indicated that agricultural areas were consistently preferred in the multi-use landscape at the 2nd order habitat selection level. Additionally, habitat around water bodies was highly favoured at the 3rd order, with distinct variations in habitat selection observed during day and night. This study highlights the significance of riverine and scrubland habitats, as leopards exhibited strong preferences for these habitats within their home ranges. We emphasize the importance of conserving natural habitat patches, particularly those surrounding water bodies. We also report on the characteristics of the capture-release strategy and provide our observations indicating no escalated aggression by leopards' post-release. In conclusion, this study evaluates widely employed approaches to conflict mitigation and suggests the continuous review and assessment of management strategies for mitigating human-leopard conflicts.

Identifying risk zones and landscape features that affect common leopard depredation.

Human-wildlife conflict (HWC) is a pressing issue worldwide but varies by species over time and place. One of the most prevalent forms of HWC in the mid-hills of Nepal is human-common-leopard conflict (HLC). Leopard attacks, especially in forested areas, can severely impact villagers and their livestock. Information on HLC in the Gorkha district was scarce, thus making it an ideal location to identify high-risk zones and landscape variables associated with such events. Registered cases were collected and reviewed from the Division Forest Office (DFO) during 2019-2021. Claims from DFO records were confirmed with herders and villagers via eight focus group discussions. To enhance modeling success, researchers identified a total of 163 leopard attack locations on livestock, ensuring a minimum distance of at least 100 meters between locations. Using maximum entropy (MaxEnt) and considering 13 environmental variables, we mapped common leopard attack risk zones. True Skill Statistics (TSS) and area under receiver-operator curve (AUC) were used to evaluate and validate the Output. Furthermore, 10 replications, 1,000 maximum iterations, and 1000 background points were employed during modeling. The average AUC value for the model, which was 0.726 ± 0.021, revealed good accuracy. The model performed well, as indicated by a TSS value of 0.61 ± 0.03. Of the total research area (27.92 km2), about 74% was designated as a low-risk area, 19% as a medium-risk area, and 7% as a high-risk area. Of the 13 environmental variables, distance to water (25.2%) was the most significant predictor of risk, followed by distance to road (16.2%) and elevation (10.7%). According to response curves, the risk of common leopard is highest in the areas between 1.5 to 2 km distances from the water sources, followed by the closest distance from a road and an elevation of 700 to 800 m. Results suggest that managers and local governments should employ intervention strategies immediately to safeguard rural livelihoods in high-risk areas. Improvements include better design of livestock corrals, insurance, and total compensation of livestock losses. Settlements near roads and water sources should improve the design and construction of pens and cages to prevent livestock loss. More studies on the characteristics of victims are suggested to enhance understanding of common leopard attacks, in addition to landscape variables. Such information can be helpful in formulating the best management practices.

Turning the tide on big cat trade: Expert opinion on trends and conservation lessons from the Republic of Korea.

Unsustainable trade in big cats affects all species in the genus, Panthera, and is one of the foremost threats to their conservation. To provide further insight into the impact of policy interventions intended to address this issue, we examine the case study of the Republic of Korea (South Korea), which in the early 1990s was one of the world's largest importers of tiger (Panthera tigris) bone and a major manufacturer of tiger-derived medicinal products. In 1993, South Korea became a Party to the Convention on International Trade in Endangered Species (CITES) and introduced a ban on commercial trade in CITES Appendix I-listed big cats a year later. We used an expert-based questionnaire survey and an exploration of the CITES trade database to investigate what has since happened to big cat trade in South Korea. Expert opinion suggested that big cat trade has likely substantially reduced since the early 1990s, as a result of the trade ban and broad socioeconomic changes. However, illegal trade has not been eradicated entirely and we were able to confirm that products reportedly derived from big cats were still publicly available for sale on a range of Korean online marketplaces, sometimes openly. The items most commonly reported by respondents from post-1994 trade and supported by expert-led evidence were tiger and leopard (Panthera pardus) skins and tiger bone wine. Although South Korea may provide a useful case study of a historically significant consumer country for tiger which has made strong progress in addressing unsustainable levels of big cat trade within a short period of time, there remains a need to address recalcitrant small-scale, illegal trade. We also recommend further investigation regarding reports of South Korean nationals being involved in illegal trade in tiger-derived products in Southeast Asia.

Species discrimination from hair using ATR-FTIR spectroscopy: Application in wildlife forensics.

Hair is a commonly encountered trace evidence in wildlife crimes involving mammals and can be used for species identification which is essential for subsequent judicial proceedings. This proof of concept study aims, to distinguish the black guard hair of three wild cat species belonging to the genus Panthera i.e. Royal Bengal Tiger (Panthera tigris tigris), Indian Leopard (Panthera pardus fusca), and Snow Leopard (Panthera uncia) using a rapid and non-destructive ATR-FTIR spectroscopic technique in combination with chemometrics. A training dataset including 72 black guard hair samples of three species (24 samples from each species) was used to construct chemometric models. A PLS2-DA model successfully classified these three species into distinct classes with R-Square values of 0.9985 (calibration) and 0.8989 (validation). VIP score was also computed, and a new PLS2DA-V model was constructed using variables with a VIP score ≥ 1. External validation was performed using a validation dataset including 18 black guard hair samples (6 samples per species) to validate the constructed PLS2-DA model. It was observed that PLS2-DA model provides greater accuracy and precision compared to the PLS2DA-V model during cross-validation and external validation. The developed PLS2-DA model was also successful in differentiating human and non-human hair with R-Square values of 0.99 and 0.91 for calibration and validation, respectively. Apart from this, a blind test was also carried out using 10 unknown hair samples which were correctly classified into their respective classes providing 100 % accuracy. This study highlights the advantages of ATR-FTIR spectroscopy associated with PLS-DA for differentiation and identification of the Royal Bengal Tiger, Indian Leopard, and Snow Leopard hairs in a rapid, accurate, eco-friendly, and non-destructive way.

Leopard density and determinants of space use in a farming landscape in South Africa.

Protected areas are traditionally the foundation of conservation strategy, but land not formally protected is of particular importance for the conservation of large carnivores because of their typically wide-ranging nature. In South Africa, leopard (Panthera pardus) population decreases are thought to be occurring in areas of human development and intense negative interactions, but research is biased towards protected areas, with quantitative information on population sizes and trends in non-protected areas severely lacking. Using Spatially Explicit Capture-Recapture and occupancy techniques including 10 environmental and anthropogenic covariates, we analysed camera trap data from commercial farmland in South Africa where negative human-wildlife interactions are reported to be high. Our findings demonstrate that leopards persist at a moderate density (2.21 /100 km2) and exhibit signs of avoidance from areas where lethal control measures are implemented. This suggests leopards have the potential to navigate mixed mosaic landscapes effectively, enhancing their chances of long-term survival and coexistence with humans. Mixed mosaics of agriculture that include crops, game and livestock farming should be encouraged and, providing lethal control is not ubiquitous in the landscape, chains of safer spaces should permit vital landscape connectivity. However, continuing to promote non-lethal mitigation techniques remains vital.

Whole-genome analysis of Escherichia coli isolated from wild Amur tiger (Panthera tigris altaica) and North China leopard (Panthera pardus japonensis).

Background: Escherichia coli is an important intestinal flora, of which pathogenic E. coli is capable of causing many enteric and extra-intestinal diseases. Antibiotics are essential for the treatment of bacterial infections caused by pathogenic E. coli; however, with the widespread use of antibiotics, drug resistance in E. coli has become particularly serious, posing a global threat to human, animal, and environmental health. While the drug resistance and pathogenicity of E. coli carried by tigers and leopards in captivity have been studied intensively in recent years, there is an extreme lack of information on E. coli in these top predators in the wild environment. Methods: Whole genome sequencing data of 32 E. coli strains collected from the feces of wild Amur tiger (Panthera tigris altaica, n = 24) and North China leopard (Panthera pardus japonensis, n = 8) were analyzed in this article. The multi-locus sequence types, serotypes, virulence and resistance genotypes, plasmid replicon types, and core genomic SNPs phylogeny of these isolates were studied. Additionally, antimicrobial susceptibility testing (AST) was performed on these E. coli isolates. Results: Among the E. coli isolates studied, 18 different sequence types were identified, with ST939 (21.9%), ST10 (15.6%), and ST3246 (9.4%) being the most prevalent. A total of 111 virulence genes were detected, averaging about 54 virulence genes per sample. They contribute to invasion, adherence, immune evasion, efflux pump, toxin, motility, stress adaption, and other virulence-related functions of E. coli. Sixty-eight AMR genes and point mutations were identified. Among the detected resistance genes, those belonging to the efflux pump family were the most abundant. Thirty-two E. coli isolates showed the highest rate of resistance to tetracycline (14/32; 43.8%), followed by imipenem (4/32; 12.5%), ciprofloxacin (3/32; 9.4%), doxycycline (2/32; 6.3%), and norfloxacin (1/32; 3.1%). Conclusions: Our results suggest that E. coli isolates carried by wild Amur tigers and North China leopards have potential pathogenicity and drug resistance.

Unravelling the maternal evolutionary history of the African leopard (Panthera pardus pardus).

The African leopard (Panthera pardus pardus) has lost a significant proportion of its historical range, notably in north-western Africa and South Africa. Recent studies have explored the genetic diversity and population structure of African leopards across the continent. A notable genetic observation is the presence of two divergent mitochondrial lineages, PAR-I and PAR-II. Both lineages appeared to be distributed widely, with PAR-II frequently found in southern Africa. Until now, no study has attempted to date the emergence of either lineage, assess haplotype distribution, or explore their evolutionary histories in any detail. To investigate these underappreciated questions, we compiled the largest and most geographically representative leopard data set of the mitochondrial NADH-5 gene to date. We combined samples (n = 33) collected in an altitudinal transect across the Mpumalanga province of South Africa, where two populations of leopard are known to be in genetic contact, with previously published sequences of African leopard (n = 211). We estimate that the maternal PAR-I and PAR-II lineages diverged approximately 0.7051 (0.4477-0.9632) million years ago (Ma). Through spatial and demographic analyses, we show that while PAR-I underwent a mid-Pleistocene population expansion resulting in several closely related haplotypes with little geographic structure across much of its range, PAR-II remained at constant size and may even have declined slightly in the last 0.1 Ma. The higher genetic drift experienced within PAR-II drove a greater degree of structure with little haplotype sharing and unique haplotypes in central Africa, the Cape, KwaZulu-Natal and the South African Highveld. The phylogeographic structure of PAR-II, with its increasing frequency southward and its exclusive occurrence in south-eastern South Africa, suggests that this lineage may have been isolated in South Africa during the mid-Pleistocene. This hypothesis is supported by historical changes in paleoclimate that promoted intense aridification around the Limpopo Basin between 1.0-0.6 Ma, potentially reducing gene flow and promoting genetic drift. Interestingly, we ascertained that the two nuclear DNA populations identified by a previous study as East and West Mpumalanga correspond to PAR-I and PAR-II, respectively, and that they have come into secondary contact in the Lowveld region of South Africa. Our results suggest a subdivision of African leopard mtDNA into two clades, with one occurring almost exclusively in South Africa, and we identify the potential environmental drivers of this observed structure. We caution that our results are based on a single mtDNA locus, but it nevertheless provides a hypothesis that can be further tested with a dense sample of nuclear DNA data, preferably whole genomes. If our interpretation holds true, it would provide the first genetic explanation for the smaller observed size of leopards at the southernmost end of their range in Africa.

NONDOMESTIC FELID ABC BLOOD PHENOTYPING, GENOTYPING, AND CROSSMATCHING.

Based upon previous clinical experience with domestic cats (Felis catus), the ability to assess ABC blood types and blood (in-)compatibilities of nondomestic felids, and adequately consider and plan for blood transfusions, may be important. Although nondomestic felids appear to have an ABC blood group system similar to domestic cats, typing with point-of-care kits and by CMAH genotyping for domestic cats have not been reported. In this study, 162 blood samples from 18 different nondomestic felid species (cheetah [Acinonyx jubatus, n = 42], lion [Panthera leo, n = 33], tiger [Panthera tigris, n = 23], Canada lynx [Lynx canadensis, n = 11], snow leopard [Uncia uncia, n = 10], puma [Puma concolor, n = 7], clouded leopard [Neofelis nebulosa, n = 6], serval [Leptailurus serval, n = 5], jaguar [Panthera onca, n = 5], fishing cat [Prionailurus viverrinus, n = 4], Pallas cat [Felis manul, n = 3], bobcat [Lynx rufus, n = 3], ocelot [Leopardus pardalis, n = 3], black footed cat [Felis nigripes, n = 2], leopard [Panthera pardus, n = 2], African wildcat [Felis lybica, n = 1], caracal [Caracal caracal, n = 1], and sand cat [Felis margarita, n = 1]) were ABC blood typed by laboratory and point-of-care tests, genotyped for four known CMAH variants for type B and type C (AB) phenotypes, and crossmatched with one another and domestic type A cats. Traditional tube typing identified blood type A (n = 106), type B (n = 8), type C (n = 43), and no discernible ABC type (n = 4). Several discrepancies were found between point-of-care and traditional typing test results. None of the tested felids possessed the four CMAH variants responsible for type B and C (AB) in domestic cats. Crossmatch incompatibilities (≥2+ agglutination) were identified within and between nondomestic felid species and beyond ABC incompatibilities. Of 26 crossmatches performed between domestic cats and various nondomestic felids, only 7 (27%) were compatible. In conclusion, point-of-care typing kits and CMAH genotyping, successfully used in domestic cats, may not identify the correct ABC blood type in nondomestic felids. Prior crossmatching is recommended to increase the likelihood of compatible transfusions between any nondomestic felids.

NEW RECORDS OF HELMINTHS OF THE JAGUAR IN MEXICO, WITH AN UPDATED LIST OF SPECIES IN THE AMERICAS.

An inventory of parasites infecting the jaguar (Panthera onca) across its distribution range is relevant for the conservation of this threatened big cat. In this study, we report the occurrence of helminths in a jaguar from Mexico using morphological techniques (cleared and stained mounts and scanning electron microscopy) and partial sequences of the 28S ribosomal RNA (28S rRNA) gene and the cytochrome c oxidase 1 mitochondrial (COI) gene. We also provide an updated list of helminth species reported in jaguars in the Americas. Three helminth taxa are identified in the jaguar examined from Mexico: Toxocara cati, Physaloptera sp., and Taenia sp. The new 28S rRNA sequences of To. cati, Physaloptera sp., and Taenia sp. and the COI sequence of Taenia sp. corroborate the identity of the helminths isolated from this host. One hundred and twenty-nine records of helminths parasitizing jaguars from 49 studies up to May 2023 were identified in the Americas. In most of these studies (73.6%), helminths were identified using coproparasitological techniques. Sixteen helminths (7 nematodes, 5 cestodes, 3 acanthocephalans, and 1 trematode) were identified at the species level in free-ranging and captive jaguars. The study demonstrates the value of an integrative taxonomy approach to increase the accuracy of parasite identification in wildlife, especially when helminth specimens are scarce or poorly fixed.

Preserving the spots: Jaguar (Panthera onca) distribution and priority conservation areas in Colombia.

The jaguar (Panthera onca) is a charismatic species considered Vulnerable in Colombia but yet largely unknown in the country. The species is mostly threatened by the continuous decline in its habitats, mostly derived from deforestation and habitat loss, additional to hunting and conflicts with humans. Thus, the future of jaguars in Colombia depends on protecting and recovering existing habitats. The aims of this study were to 1) evaluate jaguar distribution and identify the remnant patches of habitat in Colombia, 2) define an ecological connectivity network within the country, and 3) propose a priority areas portfolio for the conservation and recovery of jaguars. We used a presence background model for estimating species potential distribution and subsequently identified remaining habitat patches across the country based on land cover and species-specific ecological attributes. We then created an ecological connectivity network based on circuit theory and following a multi-criteria approach identified jaguar priority areas for conservation (JPCA) and recovery (JPRA). Jaguar potential distribution comprises 1103122.43 km2, from which 56.71% maintain suitable patches of potential habitat. We identified 960 corridors between remnant patches of natural or semi-natural vegetation. Based on the criteria, JPCAs with greater importance were identified in each of the five Colombian regions. JPRAs were located mainly towards the Andean and Caribbean regions. These JPCAs and JPRAs could serve as a guide for designing and implementing management strategies for the long-term conservation and recovery of the species in Colombia.

Dietary type (carnivore, herbivore and omnivore) and animal species modulate the nutritional metabolome of terrestrial species.

Ecometabolomics could be implemented as a powerful tool in molecular ecology studies, but it is necessary to know the baseline of certain metabolites and understand how different traits could affect the metabolome of the animals. Therefore, the main objective of this study was to provide values for the nutritional metabolome profile of different diet groups and animal species, as well as to study the differences in the metabolomic profile due to the effect of diet type and species. To achieve this goal, blood samples were taken from healthy animals (n = 43) of different species: lion (Panthera leo), jaguar (Panthera onca), chimpanzee (Pan troglodytes), bison (Bison bison), gazelle (Gazella cuvieri) and fallow deer (Dama dama), and with different types of diet (carnivore, herbivore and omnivore). Each blood sample was analysed to determine nutritional metabolites. The main results this study provides are the nutritional metabolic profile of these animals based on the type of diet and the animal species. A significant effect of the dietary type was found on nutritional metabolite levels, with those metabolites related to protein metabolism (total protein and creatine) being higher in carnivores. There is also an effect of the species on nutritional metabolites, observing a metabolome differentiation between lion and jaguar. In the case of herbivores, bison showed higher levels of uric acid and cholesterol, and lower urea levels than gazelle and fallow deer. More molecular ecology studies are needed to further the knowledge of the metabolism of these animals.

Ancestry dynamics and trait selection in a designer cat breed.

The Bengal cat breed was developed from intercrosses between the Asian leopard cat, Prionailurus bengalensis, and the domestic cat, Felis catus, with a last common ancestor approximately 6 million years ago. Predicted to derive ∼94% of their genome from domestic cats, regions of the leopard cat genome are thought to account for the unique pelage traits and ornate color patterns of the Bengal breed, which are similar to those of ocelots and jaguars. We explore ancestry distribution and selection signatures in the Bengal breed by using reduced representation and whole-genome sequencing from 947 cats. The mean proportion of leopard cat DNA in the Bengal breed is 3.48%, lower than predicted from breed history, and is broadly distributed, covering 93% of the Bengal genome. Overall, leopard cat introgressions do not show strong signatures of selection across the Bengal breed. However, two popular color traits in Bengal cats, charcoal and pheomelanin intensity, are explained by selection of leopard cat genes whose expression is reduced in a domestic cat background, consistent with genetic incompatibility resulting from hybridization. We characterize several selective sweeps in the Bengal genome that harbor candidate genes for pelage and color pattern and that are associated with domestic, rather than leopard, cat haplotypes. We identify the molecular and phenotypic basis of one selective sweep as reduced expression of the Fgfr2 gene, which underlies glitter, a trait desired by breeders that affects hair texture and light reflectivity.

Diversity of selected toll-like receptor genes in cheetahs (Acinonyx jubatus) and African leopards (Panthera pardus pardus).

The anthropogenic impact on wildlife is ever increasing. With shrinking habitats, wild populations are being pushed to co-exist in proximity to humans leading to an increased threat of infectious diseases. Therefore, understanding the immune system of a species is key to assess its resilience in a changing environment. The innate immune system (IIS) is the body's first line of defense against pathogens. High variability in IIS genes, like toll-like receptor (TLR) genes, appears to be associated with resistance to infectious diseases. However, few studies have investigated diversity in TLR genes in vulnerable species for conservation. Large predators are threatened globally including leopards and cheetahs, both listed as 'vulnerable' by IUCN. To examine IIS diversity in these sympatric species, we used next-generation-sequencing to compare selected TLR genes in African leopards and cheetahs. Despite differences, both species show some TLR haplotype similarity. Historic cheetahs from all subspecies exhibit greater genetic diversity than modern Southern African cheetahs. The diversity in investigated TLR genes is lower in modern Southern African cheetahs than in African leopards. Compared to historic cheetah data and other subspecies, a more recent population decline might explain the observed genetic impoverishment of TLR genes in modern Southern African cheetahs. However, this may not yet impact the health of this cheetah subspecies.

Positive selection in gamete interaction proteins in Carnivora.

The absence of robust interspecific isolation barriers among pantherines, including the iconic South American jaguar (Panthera onca), led us to study molecular evolution of typically rapidly evolving reproductive proteins within this subfamily and related groups. In this study, we delved into the evolutionary forces acting on the zona pellucida (ZP) gamete interaction protein family and the sperm-oocyte fusion protein pair IZUMO1-JUNO across the Carnivora order, distinguishing between Caniformia and Feliformia suborders and anticipating few significant diversifying changes in the Pantherinae subfamily. A chromosome-resolved jaguar genome assembly facilitated coding sequences, enabling the reconstruction of protein evolutionary histories. Examining sequence variability across more than 30 Carnivora species revealed that Feliformia exhibited significantly lower diversity compared to its sister taxa, Caniformia. Molecular evolution analyses of ZP2 and ZP3, subunits directly involved in sperm-recognition, unveiled diversifying positive selection in Feliformia, Caniformia and Pantherinae, although no significant changes were linked to sperm binding. Structural cross-linking ZP subunits, ZP4 and ZP1 exhibited lower levels or complete absence of positive selection. Notably, the fusion protein IZUMO1 displayed prominent positive selection signatures and sites in basal lineages of both Caniformia and Feliformia, extending along the Caniformia subtree but absent in Pantherinae. Conversely, JUNO did not exhibit any positive selection signatures across tested lineages and clades. Eight Caniformia-specific positive selected sites in IZUMO1 were detected within two JUNO-interaction clusters. Our findings provide for the first time insights into the evolutionary trajectories of ZP proteins and the IZUMO1-JUNO gamete interaction pair within the Carnivora order.

Mortality associated with SARS-CoV-2 in nondomestic felids.

Between September and November 2021, 5 snow leopards (Panthera uncia) and 1 lion (Panthera leo) were naturally infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) and developed progressive respiratory disease that resulted in death. Severe acute respiratory syndrome coronavirus 2 sequencing identified the delta variant in all cases sequenced, which was the predominant human variant at that time. The time between initial clinical signs and death ranged from 3 to 45 days. Gross lesions in all 6 cats included nasal turbinate hyperemia with purulent discharge and marked pulmonary edema. Ulcerative tracheitis and bronchitis were noted in 4 cases. Histologically, there was necrotizing and ulcerative rhinotracheitis and bronchitis with fibrinocellular exudates and fibrinosuppurative to pyogranulomatous bronchopneumonia. The 4 cats that survived longer than 8 days had fungal abscesses. Concurrent bacteria were noted in 4 cases, including those with more acute disease courses. Severe acute respiratory syndrome coronavirus 2 was detected by in situ hybridization using probes against SARS-CoV-2 spike and nucleocapsid genes and by immunohistochemistry. Viral nucleic acid and protein were variably localized to mucosal and glandular epithelial cells, pneumocytes, macrophages, and fibrinocellular debris. Based on established criteria, SARS-CoV-2 was considered a contributing cause of death in all 6 cats. While mild clinical infections are more common, these findings suggest that some SARS-CoV-2 variants may cause more severe disease and that snow leopards may be more severely affected than other felids.

A cost-effective blood DNA methylation-based age estimation method in domestic cats, Tsushima leopard cats (Prionailurus bengalensis euptilurus) and Panthera species, using targeted bisulphite sequencing and machine learning models.

Individual age can be used to design more efficient and suitable management plans in both in situ and ex situ conservation programmes for targeted wildlife species. DNA methylation is a promising marker of epigenetic ageing that can accurately estimate age from small amounts of biological material, which can be collected in a minimally invasive manner. In this study, we sequenced five targeted genetic regions and used 8-23 selected CpG sites to build age estimation models using machine learning methods at only about $3-7 per sample. Blood samples of seven Felidae species were used, ranging from small to big, and domestic to endangered species: domestic cats (Felis catus, 139 samples), Tsushima leopard cats (Prionailurus bengalensis euptilurus, 84 samples) and five Panthera species (96 samples). The models achieved satisfactory accuracy, with the mean absolute error of the most accurate models recorded at 1.966, 1.348 and 1.552 years in domestic cats, Tsushima leopard cats and Panthera spp. respectively. We developed the models in domestic cats and Tsushima leopard cats, which were applicable to individuals regardless of health conditions; therefore, these models are applicable to samples collected from individuals with diverse characteristics, which is often the case in conservation. We also showed the possibility of developing universal age estimation models for the five Panthera spp. using only two of the five genetic regions. We do not recommend building a common age estimation model for all the target species using our markers, because of the degraded performance of models that included all species.