Prevalence of tick-borne pathogens in ticks collected from the wild mountain ungulates mouflon and chamois in 4 regions of France.

Ticks are major vectors of various pathogens of health importance, such as bacteria, viruses and parasites. The problems associated with ticks and vector-borne pathogens are increasing in mountain areas, particularly in connection with global climate change. We collected ticks (n = 2,081) from chamois and mouflon in 4 mountainous areas of France. We identified 6 tick species: Ixodes ricinus, Rhipicephalus bursa, Rh. sanguineus s.l., Haemaphysalis sulcata, H. punctata and Dermacentor marginatus. We observed a strong variation in tick species composition among the study sites, linked in particular to the climate of the sites. We then analysed 791 ticks for DNA of vector-borne pathogens: Babesia/Theileria spp., Borrelia burgdorferi s.l., Anaplasma phagocytophilum, A. marginale, A. ovis, and Rickettsia of the spotted fever group (SFG). Theileria ovis was detected only in Corsica in Rh. bursa. Babesia venatorum (2 sites), Borrelia burgdorferi s.l. (B. afzelii and B. garinii; 2 sites) and Anaplasma phagocytophilum (3 sites) were detected in I. ricinus. Anaplasma ovis was detected at one site in I. ricinus and Rh. sanguineus s.l. SFG Rickettsia were detected at all the study sites: R. monacensis and R. helvetica in I. ricinus at the 3 sites where this tick is present; R. massiliae in Rh. sanguineus s.l. (1 site); and R. hoogstraalii and Candidatus R. barbariae in Rh. bursa in Corsica. These results show that there is a risk of tick-borne diseases for humans and domestic and wild animals frequenting these mountain areas.

Title: Prévalence d'agents pathogènes vectorisés chez des tiques collectées chez des ongulés sauvages (mouflons, chamois) dans 4 zones montagneuses en France. Abstract: Les tiques sont des vecteurs majeurs de différents agents pathogènes d'importance sanitaire, tels que des bactéries, des virus et des parasites. Les problématiques liées aux tiques et aux pathogènes vectorisés augmentent en zones de montagne, en lien notamment avec le réchauffement climatique. Nous avons collecté des tiques (n = 2 081) sur des chamois et des mouflons dans 4 zones montagneuses en France. Six espèces ont été identifiées : Ixodes ricinus, Rhipicephalus bursa, Rh. sanguineus s.l., Haemaphysalis sulcata, H. punctata et Dermacentor marginatus. Nous avons observé une forte variation de la composition en espèces de tiques entre les sites d'étude, en lien notamment avec le climat des sites. Nous avons ensuite recherché les ADN d'agents pathogènes vectorisés sur 791 tiques : Babesia/Theileria spp, Borrelia burgdorferi s.l., Anaplasma phagocytophilum, A. marginale, A. ovis, et de Rickettsia du groupe des fièvres boutonneuses (SFG). Theileria ovis a été détecté uniquement en Corse chez Rh. bursa. Babesia venatorum (2 sites), Borrelia burgdorferi s.l. (B. afzelii and B. garinii; 2 sites) et Anaplasma phagocytophilum (3 sites) ont été détectés chez I. ricinus. Anaplasma ovis a été détecté dans un site chez I. ricinus et Rh. sanguineus s.l.. Les Rickettsia SFG ont été détectées dans tous les sites d'étude : Rickettsia monacensis et R. helvetica chez I. ricinus dans les 3 sites où cette tique est présente; R. massiliae chez Rh. sanguineus s.l. (1 site); et R. hoogstraalii et Candidatus R. barbariae chez Rh. bursa en Corse. Ces résultats montrent un risque de transmission de maladies par les tiques pour les personnes et les animaux domestiques et sauvages fréquentant ces zones de montagne.

Projection of current and future distribution of adaptive genetic units in an alpine ungulate.

Climate projections predict major changes in alpine environments by the end of the 21st century. To avoid climate-induced maladaptation and extinction, many animal populations will either need to move to more suitable habitats or adapt in situ to novel conditions. Since populations of a species exhibit genetic variation related to local adaptation, it is important to incorporate this variation into predictive models to help assess the ability of the species to survive climate change. Here, we evaluate how the adaptive genetic variation of a mountain ungulate-the Northern chamois (Rupicapra rupicapra)-could be impacted by future global warming. Based on genotype-environment association analyses of 429 chamois using a ddRAD sequencing approach, we identified genetic variation associated with climatic gradients across the European Alps. We then delineated adaptive genetic units and projected the optimal distribution of these adaptive groups in the future. Our results suggest the presence of local adaptation to climate in Northern chamois with similar genetic adaptive responses in geographically distant but climatically similar populations. Furthermore, our results predict that future climatic changes will modify the Northern chamois adaptive landscape considerably, with various degrees of maladaptation risk.

Immunolocalisation of aquaporins 3, 7, 9 and 10 in the epididymis of three wild ruminant species (Iberian ibex, mouflon and chamois) and sperm cryoresistance.

CONTEXT: In the epididymis, epithelial cells manage changes in the luminal environment for proper sperm maturation. Moreover, aquaglyceroporins, a subgroup of aquaporins (AQP), modulate the transport of water, glycerol and other small molecules in epithelial cells. AIMS: We aim to characterise the lining epithelium, quantify its cell composition and immunolocalise the aquaglyceroporins AQP3, AQP7, AQP9 and AQP10 alongside the epididymal ductus of three wild ruminant species, and to determine if species-specific differences could be associated with cauda sperm cryoresistance variations. METHODS: Epididymides from Iberian ibex (n =5), mouflon (n =5) and chamois (n =6) were obtained. Cauda spermatozoa were collected and sperm parameters were analysed before and after freezing. Histology and immunohistochemistry of AQP3, 7, 9, 10 and T-CD3 were performed in the caput, corpus and cauda epididymal regions. KEY RESULTS: This work first describes the lining epithelium in Iberian ibex, mouflon and chamois epididymis along the three anatomical regions, consisting of principal, basal, apical, clear and halo cells. However, the percentage of each cell type differed in ibex compared to mouflon and chamois. The positive T-CD3 immunolabeling of all the halo cells confirmed their T-lymphocyte nature. Aquaglyceroporin expression patterns were similar among species, except for differences in AQP7 and AQP10 immunolocalisation in ibex. Species-specific differences in epididymal sperm cryoresistance were confirmed. CONCLUSIONS: The epididymal epithelium of the three wild ruminants differ in their relative number of cell types and AQP immunolocalisation, which ultimately appears to affect cauda epidydimal spermatozoa cryoresistance. IMPLICATIONS: Our study provides information on the relevance of the quantitative composition and AQP pattern expression in epididymal lining epithelium on sperm cryoresistance.

New Potential Role of European Mouflon (Ovis aries musimon) in the Epidemiology of Border Disease in the Pyrenees.

Since 2001, high-mortality outbreaks of border disease (BD) have negatively affected populations of Pyrenean chamois (Rupicapra pyrenaica pyrenaica). Studies in the affected areas determined that sympatric wild ruminants did not seem to have an epidemiologic role in the circulation of border disease virus (BDV). However, the recent increase in European mouflon (Ovis aries musimon) densities might enhance the risk of pathogen transmission among chamois and mouflons. We conducted a serologic and virologic investigation of BDV in European mouflon from the Spanish Pyrenees, with the aim of determining potential changes in the role of this species in BDV epidemiology. From 2018 to 2022, we detected antibodies against BDV in 31/185 (16.7%) animals but did not detect BDV RNA in any spleen sample (0/65). These results indicate that BDV infection is occurring in these mouflon populations to a greater extent than previously described, which could shift the current understanding of BD epidemiology in the Pyrenees and cause an unpredictable effect on both chamois and mouflon populations. Further studies on the molecular identification of BDV in mouflon and chamois are required to better understand the contribution of mouflon in the epidemiology of BD.

Sexual segregation results in pronounced sex-specific density gradients in the mountain ungulate, Rupicapra rupicapra.

Sex-specific differences in habitat selection and space use are common in ungulates. Yet, it is largely unknown how this behavioral dimorphism, ultimately leading to sexual segregation, translates to population-level patterns and density gradients across landscapes. Alpine chamois (Rupicapra rupicapra r.) predominantly occupy habitat above tree line, yet especially males may also take advantage of forested habitats. To estimate male and female chamois density and determinants thereof, we applied Bayesian spatial capture-recapture (SCR) models in two contrasting study areas in the Alps, Germany, during autumn. We fitted SCR models to non-invasive individual encounter data derived from genotyped feces. Sex-specific densities were modeled as a function of terrain ruggedness, forest canopy cover, proportion of barren ground, and site severity. We detected pronounced differences in male and female density patterns, driven primarily by terrain ruggedness, rather than by sex-specific effects of canopy cover. The positive effect of ruggedness on density was weaker for males which translated into a higher proportion of males occupying less variable terrain, frequently located in forests, compared to females. By estimating sex-specific variation in both detection probabilities and density, we were able to quantify and map how individual behavioral differences scale up and shape spatial patterns in population density.

Occurrence and limited zoonotic potential of Cryptosporidium spp., Giardia duodenalis, and Balantioides coli infections in free-ranging and farmed wild ungulates in Spain.

Little information is currently available on the occurrence and molecular diversity of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Balantioides coli in wild ungulates and the role of these host species as potential sources of environmental contamination and consequent human infections. The presence of these three pathogens was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were retrospectively collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from the five Spanish bioregions. Overall infection rates were 3.0% (42/1382; 95% CI: 2.1-3.9%) for Cryptosporidium spp., 5.4% (74/1382; 95% CI: 4.2-6.5%) for G. duodenalis, and 0.7% (9/1382; 95% CI: 0.3-1.2%) for B. coli. Cryptosporidium infection was detected in roe deer (7.5%), wild boar (7.0%) and red deer (1.5%), and G. duodenalis in southern chamois (12.9%), mouflon (10.0%), Iberian wild goat (9.0%), roe deer (7.5%), wild boar (5.6%), fallow deer (5.2%) and red deer (3.8%). Balantioides coli was only detected in wild boar (2.5%, 9/359). Sequence analyses revealed the presence of six distinct Cryptosporidium species: C. ryanae in red deer, roe deer, and wild boar; C. parvum in red deer and wild boar; C. ubiquitum in roe deer; C. scrofarum in wild boar; C. canis in roe deer; and C. suis in red deer. Zoonotic assemblages A and B were detected in wild boar and red deer, respectively. Ungulate-adapted assemblage E was identified in mouflon, red deer, and southern chamois. Attempts to genotype samples positive for B. coli failed. Sporadic infections by canine- or swine-adapted species may be indicative of potential cross-species transmission, although spurious infections cannot be ruled out. Molecular evidence gathered is consistent with parasite mild infections and limited environmental contamination with (oo)cysts. Free-ranging wild ungulate species would not presumably play a significant role as source of human infections by these pathogens. Wild ruminants do not seem to be susceptible hosts for B. coli.

Age-specific survival of territorial and non-territorial male chamois.

How alternative reproductive tactics (ARTs) are maintained in wildlife populations is one of the major questions in evolutionary biology. As a dominant status, territoriality is typically linked to increased mating opportunities, and one explanation why this behaviour coexists with other tactics is that dominance implies survival costs. Such a trade-off may occur in the Northern chamois Rupicapra rupicapra, as reproductive advantages of territorial males over non-territorial males could be counterbalanced by a reduction in survival mediated through energy expenditure, stress and parasitic infections, ultimately favouring ART coexistence. Here, we analysed age-dependent survival probabilities of territorial (n = 15) and non-territorial (n = 16) adult chamois using information collected over 12 years between 2010 and 2021 in the Gran Paradiso National Park (Western Italian Alps). Survival rates were estimated with a CMR approach using Burnham's joint modelling of live encounter and dead recovery data. The model selection procedure, based on AICc value minimisation, supported a linear decrease of survival with age but the results did not match our predictions, as territorial chamois did not have lower survival rates than non-territorial chamois. In contrast, territorial males appeared to enjoy reproductive success at lower survival costs. This, in turn, supports the role of other factors, such as snow-dependent environmental stochasticity, in the maintenance of ARTs in chamois populations. The limited sample size, however, calls for caution in interpretation, and long-term studies of lifetime reproductive success and survival are necessary to clarify the mechanisms underlying the expression and coexistence of different reproductive behaviours in this species.

Subsistence of early anatomically modern humans in Europe as evidenced in the Protoaurignacian occupations of Fumane Cave, Italy.

Documenting the subsistence strategies developed by early modern humans is relevant for understanding the success of their dispersal throughout Eurasia. Today, we know that there was not a single colonization event and that the process was progressive while coping with the MIS3 abrupt climatic oscillations. Modern humans expanded into the continent by adapting to different topographic situations and by exploiting resources in diverse ecological niches. The northern part of Italy is one of the first European regions where early modern humans are documented. Here, we present the subsistence regimen adopted by the Protoaurignacian groups in two different levels in Fumane Cave based on archaeozoological data. New radiocarbon dates confirm an overlap between Uluzzian and Protoaurignacian occupations, around 42 and 41,000 cal BP, and reveal that modern humans occupied the cave from GI10 to GS9, the last level coinciding with the Heinrich Event 4. The data indicate seasonal site occupations during late spring/summer and that prey exploitation was focused mostly on ibex and chamois, killed in nearby areas. The whole faunal assemblage suggests the presence of early modern humans in a cold environment with mostly open landscapes and patchy woodlands. The estimation of net primary productivity (NPP) in Fumane, compared with other contemporaneous Italian sites, reflects how the NPP fluctuations in the Prealpine area, where Fumane is located, affected the biotic resources in contrast to known Mediterranean sites. From a pan-European perspective, the spatiotemporal fluctuation of the NPP versus the subsistence strategies adopted by Protoaurignacian groups in the continent supports rapid Homo sapiens dispersal and resilience in a mosaic of environments that were affected by significant climate changes.

Whole genome sequence-based characterisation of Shiga toxin-producing Escherichia coli isolated from game meat originating from several European countries.

Game meat is becoming increasingly popular but may be contaminated with pathogenic bacteria such as Shiga toxin-producing Escherichia coli (STEC). STEC cause gastrointestinal illnesses including diarrhoea, haemorrhagic colitis (HC), and the haemolytic uremic syndrome (HUS). The aim of this study was to assess the occurrence of STEC in 92 meat samples from chamois (n = 2), red deer (n = 27), roe deer (n = 38), and wild boar (n = 25), from Switzerland and other European countries. After enrichment, Shiga-toxin encoding genes (stx) were detected by PCR in 78 (84%) of the samples and STEC were isolated from 23 (25%) of the same samples. Nine different serotypes and eight different sequence types (STs) were found, with O146:H28 ST738 (n = 10) and O110:H31 ST812 (n = 5) predominating. None of the STEC belonged to the so-called top-five serogroups O26, O103, O111, O145, and O157. Subtyping of stx identified stx1c (n = 9), stx2a (n = 1), stx2b (n = 19), stx2e (n = 2), and stx2g (n = 1). Additional virulence factors (VFs) comprised ehx (n = 12), iha (n = 21), sta1 (n = 1), and subAB (n = 19). None of the isolates contained the eae gene. Twenty-one STEC contained VFs associated with extra-intestinal pathogenic E. coli (ExPEC). Overall, the pathogenic potential of STEC in game meat is moderate, though the isolation of one STEC strain carrying stx2a, and of STEC/ExPEC hybrids suggests a role of game meat as a potential source of STEC infections in humans. Therefore, detailed knowledge of the safe handling and preparation of game meat is needed to prevent foodborne infections.

Whole Genome Sequencing of Escherichia coli and Enterococcus spp. in wildlife-livestock interface: a pilot study.

OBJECTIVES: This pilot study provides a multidisciplinary investigation to monitor livestock-wildlife interface. Ecological data, microbiological investigations, and whole genome sequencing were used to characterize eight bacterial isolates obtained from sympatric domestic and wild ruminants in Maiella National Park (Italy) in terms of genetic patterns of antimicrobial resistance. METHODS: Using selective culturing of fresh fecal samples of monitored and georeferenced populations of Apennine chamois, goats, red deer, and sheep, Escherichia coli, Enterococcus faecium, and Enterococcus faecalis isolates were isolated and subjected to minimum inhibitory concentration determination and whole genome sequencing. RESULTS: The analyzed isolates showed phenotypic and genotypic resistance to tetracycline and critically important antibiotics such as linezolid and carbapenems. Virulence genes related to biofilm regulation and Shiga toxins were also detected. Furthermore, serotypes related to nosocomial infections, harbouring plasmids recognized as important mobile resistance gene transmitters, were identified. CONCLUSIONS: This multidisciplinary pilot study represents a promising initial step to identify the environmental drivers and the transmission routes of antimicrobial resistance and virulence factors, providing new data on bacteria from rare and endangered species such as Apennine chamois.

Pilot research on gastrointestinal parasites of the Tatra chamois (Rupicapra rupicapra tatrica).

INTRODUCTION AND OBJECTIVE: The Tatra chamois (Rupicapra rupicapra tatrica) is a significant representative of the High Tatra Mountains endemic fauna species. In terms of health hazards for these animals, an important role is played by parasitic infections that can lead to a significant depletion of the entire population. OBJECTIVE: The aim of the study was to describe the occurrence of gastrointestinal parasites of Tatra chamois in the current environmental and climatic conditions of the High Tatra Mountains. MATERIAL AND METHODS: During the pilot project in 2014 - 2017, a total of 494 chamois faecal samples were collected from the Slovak High Tatra and 114 samples in the Polish part of the mountain and examined using standard coprological methods. RESULTS: The results revealed that the overall positivity for gastrointestinal parasites in chamois of the Slovak High Tatra reached 74.7%. Most frequent were protozoa - Eimeria spp. (42.7%), helminths were represented by Moniezia spp. tapeworms (23.5%), eggs of GIS-strongylids (7.1%), and sporadically Capillaria spp. (1.4%). The chamois from the Polish Tatra Mts. were infected with Eimeria spp. (43.9%), GIT-strongylids (9.6%), and Moniezia spp. (6.1%). Parasitic infection was determined in 59.6 % of faecal samples from the Polish part of the mountains. Statistical analyses revealed a significant difference in Moniezia spp. occurrence in different Slovak Tatra Mts. Regions, as well as between Slovak and Polish Tatra Mts. CONCLUSIONS: Initial research on the gastrointestinal parasites of the Tatra chamois revealed one indisputable finding - a relatively high prevalence of the genus Moniezia, which is closely linked to the climate and microclimate conditions of the mountains. Further intensive research on parasite composition and distribution in Tatra chamois is needed in broader temporal, ecological, and zoological contexts.

Comparative analysis of two genomes of Chlamydia pecorum isolates from an Alpine chamois and a water buffalo.

BACKGROUND: To date, whole genome sequencing has been performed mainly for isolates of Chlamydia trachomatis, C. pneumoniae, C. psittaci and C. abortus, but only a few isolates of C. pecorum have been entirely sequenced and this makes it difficult to understand its diversity and population structure. In this study the genome of two C. pecorum strains isolated from the lung of an Alpine chamois affected with pneumonia (isolate PV7855) and the brain of a water buffalo affected with meningoencephalomyelitis (isolate PV6959), were completely sequenced with MiSeq system (Illumina) and analyzed in their most polymorphic regions. RESULTS: The genome length and GC content of the two isolates were found to be consistent with other C. pecorum isolates and the gene content of polymorphic membrane proteins and plasticity zone was found to be very similar. Some differences were observed in the phospholipase genes for both isolates and in the number of genes in the plasticity zone, such as the presence of some hypothetical proteins in PV6959, not present in any other genomes analyzed in this study. Interestingly, PV6959 possesses an extra pmp and has an incomplete tryptophan biosynthesis operon. Plasmids were detected in both isolates. CONCLUSIONS: Genome sequencing of the two C. pecorum strains did not reveal differences in length and GC content despite the origin from different animal species with different clinical disease. In the plasticity zone, the differences in the genes pattern might be related to the onset of specific symptoms or infection of specific hosts. The absence of a tryptophan biosynthesis pathway in PV6959 may suggest a strict relationship between C. pecorum and its host.

Detection and characterization of bopiviruses in domestic and wild ruminants.

Highly divergent picornaviruses (PVs) classified in the genus Bopivirus have been recently discovered on faecal samples from sheep and goats in Hungary and from fallow and red deer in Australia. In this study, we investigated the epidemiology of these novel viruses in domestic and wild ruminants from Northwestern Italian Alps by testing archival faecal samples collected from 128 sheep, 167 goats, 61 red deer (Cervus elaphus), 77 roe deer (Capreolus capreolus), 43 chamois (Rupicapra rupicapra) and 32 Alpine ibex (Capra ibex). Bopivirus RNA was detected in a total of 19 animals, including 14 sheep (10.9%), 2 red deer (3.3%), 1 roe deer (1.3%), 1 chamois (2.3 %) and 1 Alpine ibex (3.3 %), but not in goats. Upon sequence analysis of the 3DRdRp region, the sequences generated from chamois, roe deer, Alpine ibex and ovine faecal samples showed the highest nucleotide identity (96.8-100%) to bopiviruses detected in goats and sheep from Hungarian farms, whereas strains found in red deer displayed the closest relatedness (90.8%-91.2%) to bopiviruses identified in fallow and red deer in Australia. The nearly complete genome sequence of strains 12/2020/ITA (ON497046) and 14-73/2020/ITA (ON497047) detected in an Alpine ibex and in a sheep, respectively, was determined by combining a modified 3'-RACE protocol with Oxford Nanopore Technologies sequencing platform. On phylogenetic analysis based on the complete polyprotein, both strains segregated into the candidate species Bopivirus B along with ovine and caprine strains detected in Hungary (90.0-94.6% nucleotide and 94.6-98.0% amino acid identities). The findings of this study expand the host range of these novel viruses and hint to a possible virus circulation between domestic ruminants and wild animals.

Interacting lethal and nonlethal human activities shape complex risk tolerance behaviors in a mountain herbivore.

Animals perceive human activities as risky and generally respond with fear-induced proactive behaviors to buffer the circadian patterns of lethal and nonlethal disturbances, such as diel migrations (DMs) between risky places during safe nighttime and safer places during risky daytime. However, such responses potentially incur costs through movement or reduced foraging time, so individuals should adjust their tolerance when human activities are harmless, through habituation. Yet this is a challenging cognitive task when lethal and nonlethal risks co-occur, forming complex landscapes of fear. The consequences of this human-induced complexity have, however, rarely been assessed. We studied the individual DM dynamics of chamois (Rupicapra rupicapra rupicapra), 89 GPS-tracked individual-years, from/to trails in the French Alps in areas with co-occurring lethal (hunting) and nonlethal (hiking and skiing) disturbances, with different intensities across seasons. We developed a conceptual framework relying on the risk-disturbance hypothesis and habituation to predict tolerance adjustments of chamois under various disturbance contexts and across contrasted seasonal periods. Based on spatial and statistical analyses combining periodograms and multinomial logistic models, we found that DM in relation to distance to a trail was a consistent response by chamois (~85% of individuals) to avoid human disturbance during daytime, especially during the hiking and hunting periods. Such behavior revealed a low tolerance of most chamois to human activities, although there was considerable interindividual heterogeneity in DM. Interestingly, there was an increased tolerance among the most disturbed diel migrants, potentially through habituation, with chamois performing shorter DMs in areas highly disturbed by hikers. Crucially, chamois that were most human-habituated during the hiking period remained more tolerant in the subsequent harvesting period, which could increase their risk of being harvested. In contrast, individuals less tolerant to hiking performed longer DMs when hunting risk increased, and compared to hiking, hunting exacerbated the threshold distance to trails triggering DMs. No carryover effect of hunting beyond the hunting period was observed. In conclusion, complex human-induced landscapes of fear with co-occurring disturbances by nature-based tourism and hunting may shape unexpected patterns of tolerance to human activities, whereby animal tolerance could become potentially deleterious for individual survival.

Temporal stability of fecal cortisol metabolites in mountain-dwelling ungulates.

Fecal cortisol metabolites (FCMs) are widely used to track stress responses in wildlife and captive species. Rules of thumb suggest that samples should be collected as soon as possible after defecation, to avoid decay of FCMs. To date, however, only a few studies investigated the stability of defecated FCMs over time, and most of them were conducted in controlled laboratory conditions. Here, we investigated the stability of FCMs over seven consecutive days, in two mountain-dwelling ungulates, under natural environmental conditions using a semi-experimental approach. Fecal samples from Northern chamois Rupicapra rupicapra (n = 24) and red deer Cervus elaphus (n = 22) were collected in summer of 2020 within the Stelvio National Park, Italy, and placed in an open area above 2000 m a.s.l. For the next 7 days, we collected a portion of each sample, and all sub-samples were analyzed with an 11-oxoetiocholanolone enzyme immunoassay. Exposure, temperature, and precipitation were fitted as covariates in non-linear generalized mixed models to assess FCM variation over time, and competing models were selected using AICc. For chamois, the best model included only time as a predictor, while for red deer, it included time, precipitation, and exposure. For both species, FCM values decreased rapidly from the first days after deposition until the fourth day. For red deer, in northern-exposed samples, FCM values decreased slower than in south-exposed ones; furthermore, FCM values increased with increasing precipitation. Our results offer a solid methodological basis to wildlife researchers and practitioners interested in the investigation of the ecological factors affecting stress variation in wildlife and support the recommendation to collect samples as fresh as possible, to avoid misleading inference. Further studies are necessary to evaluate the stability of FCMs when other enzyme immunoassays are used.

Positive effect of spring advance on the diet quality of an alpine herbivore.

Changes in vegetation phenology related to global warming are having alarming effects on the life history traits of many herbivore species. Such changes are particularly critical in alpine ecosystems, where strong climate limitations on plant growth make seasonal synchronization imperative for the growth, reproduction and survival of herbivores. However, despite the pivotal role of resource-use strategies on the performances of such species, few studies have explicitly assessed the mechanistic impact of climate change on their diets. We aimed to fill this gap by studying the effect of spring onset on the dietary composition and quality of a medium-size alpine herbivore while considering density-dependent processes and age- and sex-specific differences in foraging behavior. Using an exceptional, long-term (24 years) direct individual-based dietary monitoring of a Pyrenean chamois population (Rupicapra pyrenaica pyrenaica), we showed that ongoing earlier onsets of spring are leading to an earlier access to high-quality forage and therefore a higher diet quality at a fixed date, without apparent changes in diet composition. We also showed that at high densities, intraspecific competition reduced diet quality by driving animals to feed more on woody plants and less on nutritious forbs and graminoids. By assessing the mechanistic effects of global warming on the dietary patterns of species at the center of trophic networks, this study is an essential step for predictive models aiming at understanding the ongoing ecosystem consequences of the global climatic crisis.

Forests buffer the climate-induced decline of body mass in a mountain herbivore.

Climate change is known to affect key life-history traits, such as body mass, reproduction, and survival in many species. Animal populations inhabiting mountain habitats are adapted to extreme seasonal environmental conditions but are also expected to be especially vulnerable to climate change. Studies on mountain ungulates typically focus on populations or sections of populations living above the tree line, whereas populations inhabiting forested habitats are largely understudied. Here, we investigate whether forested areas can mitigate the impact of climatic change on life-history traits by evaluating the interactive effects of temperature and habitat characteristics on body mass variation in the Alpine chamois Rupicapra rupicapra rupicapra. We examined data of 20,573 yearling chamois collected from 1993 to 2019 in 28 mountain ranges in the Austrian Eastern Alps, characterized by different proportion of forest cover. Our results show that the temporal decline of chamois body mass is less pronounced in areas with greater proportion of forest cover. For chamois living in forest habitats only, no significant temporal change in body mass was detected. Variation in body mass was affected by the interaction between density and snow cover, as well as by the interaction between spring temperatures and forest cover, supporting the role of forests as thermal buffer against the effects of increasing temperatures on life-history traits in a mountain ungulate. In turn, this study suggests a buffering effect of forests against climate change impacts. Assessments of the consequences of climate change on the life-history traits and population dynamics of mountain-dwelling species should thus consider the plasticity of the species with respect to the use and availability of different habitat types.

Temporal and seasonal changes in mercury accumulation in Tatra chamois from West Carpathians.

The main aim of this study was to determine the concentration of total mercury (T-Hg) in different tissues, hair, and faeces from a long-lived animal that actively lives in the subalpine and alpine zone within temperate climate zone throughout the year. Levels of T-Hg in samples from naturally deceased Tatra chamois (n = 72) from the Tatra Mountains (Slovakia) were determined using direct mercury analyses on the basis of dry weight. The mercury concentrations in hair samples were compared over the last three decades. Seasonal changes in mercury concentration and differences in the amounts of mercury in various tissues, hairs, and faeces between age and sex groups were also studied. The highest mercury concentrations in organs were found in the kidneys of Tatra chamois, with mean values of 0.45 ± 0.07 µg/g (dry weight) for adults (n = 18) and 0.39 ± 0.12 µg/g (dry weight) for juveniles (n = 6). These values are considerably high compared to expected Hg levels in wild ungulates from foothill and mountain areas in the temperate zone. Juveniles had higher levels of T-Hg in their muscles (0.011 ± 0.001 µg/g dry weight, n = 10) and faeces (0.189 ± 0.025 µg/g dry weight, n = 7) when compared to adults that had 0.007 ± 0.001 µg/g dry weight of T-Hg in muscles (n = 29) and 0.113 ± 0.015 µg/g dry of T-Hg weight in faeces (n = 19). T-Hg concentrations in individual tissues (heart, kidney, liver, lungs, spleen, tongue, muscle, bone), hairs, and faeces were not significantly different between males and females. Mercury levels in the hair of Tatra chamois have increased significantly since the 1990s (median value of T-Hg: 0.025 µg/g dry weight) with the highest values presenting during the 2000s (0.029 µg/g dry weight). Since 2010 (0.016 µg/g dry weight), levels have declined when compared to those observed during the 1990s and 2000s. Therefore, we can conclude that T-Hg deposition in alpine areas of the Tatra Mountains shows a declining trend since the 2010s. T-Hg concentrations in the heart, muscle, and hair were higher in summer compared to winter. Seasonal changes in mercury concentrations are likely most related to the seasonal availability of food, but may also be related to moulting periods, and this correlation must be explored further.

Developmental validation of Oxford Nanopore Technology MinION sequence data and the NGSpeciesID bioinformatic pipeline for forensic genetic species identification.

Species identification of non-human biological evidence through DNA nucleotide sequencing is routinely used for forensic genetic analysis to support law enforcement. The gold standard for forensic genetics is conventional Sanger sequencing; however, this is gradually being replaced by high-throughput sequencing (HTS) approaches which can generate millions of individual reads in a single experiment. HTS sequencing, which now dominates molecular biology research, has already been demonstrated for use in a number of forensic genetic analysis applications, including species identification. However, the generation of HTS data to date requires expensive equipment and is cost-effective only when large numbers of samples are analysed simultaneously. The Oxford Nanopore Technologies (ONT) MinION™ is an affordable and small footprint DNA sequencing device with the potential to quickly deliver reliable and cost effective data. However, there has been no formal validation of forensic species identification using high-throughput (deep read) sequence data from the MinION making it currently impractical for many wildlife forensic end-users. Here, we present a MinION deep read sequence data validation study for species identification. First, we tested whether the clustering-based bioinformatics pipeline NGSpeciesID can be used to generate an accurate consensus sequence for species identification. Second, we systematically evaluated the read variation distribution around the generated consensus sequences to understand what confidence we have in the accuracy of the resulting consensus sequence and to determine how to interpret individual sample results. Finally, we investigated the impact of differences between the MinION consensus and Sanger control sequences on correct species identification to understand the ability and accuracy of the MinION consensus sequence to differentiate the true species from the next most similar species. This validation study establishes that ONT MinION sequence data used in conjunction with the NGSpeciesID pipeline can produce consensus DNA sequences of sufficient accuracy for forensic genetic species identification.

Babesia spp. and Anaplasma phagocytophilum in free-ranging wild ungulates in central Austria.

Free-ranging wild ungulates are widespread in Austria, and act as hosts (i.e. feeding hosts) for ticks, including Ixodes ricinus, and as reservoir hosts for pathogens transmitted by I. ricinus. Due to climate change, the abundance of I. ricinus might be increasing, which could potentially lead to higher prevalences of tick-borne pathogens, such as Babesia spp. and Anaplasma phagocytophilum, some known for their zoonotic potential. Human babesiosis is classified as an emerging zoonosis, but sufficient data of these parasites in central Austria is lacking. In order to assess the abundance of vector-borne pathogens, blood of roe deer (Capreolus capreolus; n = 137), red deer (Cervus elaphus; n = 37), mouflons (Ovis gmelini; n = 2) and chamois (Rupicapra rupicapra; n = 1), was collected and tested for pathogen DNA in two different sampling sites in central Austria. DNA of tick-borne pathogens was detected in 15.5 % (n = 27) of these animals. Babesia capreoli (n = 22 in roe deer; n = 1 in mouflon), Babesia divergens (n = 1, in red deer), and Anaplasma phagocytophilum (n = 4, in roe deer) were detected. DNA sequencing of the 18S rRNA gene of two C. capreolus samples from Upper Austria featured another new genotype of Babesia, which differs in one nucleotide position to B. divergens and B. capreoli, and is intermediate between the main genotypes of B. capreoli and B. divergens within the partial gene sequence analyzed. This study thus confirms that B. capreoli, B. divergens, and A. phagocytophilum are present in free-ranging ungulates in central Austria. Further testing over a longer period is recommended in order to assess the impact of climate change on the prevalence of blood parasites in central Austria.