Antimicrobial resistance in commensal Staphylococcus aureus from wild ungulates is driven by agricultural land cover and livestock farming.

Staphylococcus aureus is a human pathobiont (i.e., a commensal microorganism that is potentially pathogenic under certain conditions), a nosocomial pathogen and a leading cause of morbidity and mortality in humans. S. aureus is also a commensal and pathogen of companion animals and livestock. The dissemination of antimicrobial resistant (AMR) S. aureus, particularly methicillin-resistant (MRSA), has been associated to its ability for establishing new reservoirs, but limited attention has been devoted to the role of the environment. To fill this gap, we aimed to characterize animal carrier status, AMR phenotypes, predominant clonal lineages and their relationship with clinical and food-chain settings, as well as to find predictors of AMR occurrence. Nasal swabs (n = 254) from wild boar (n = 177), red deer (n = 54) and fallow deer (n = 23) hunted in Portugal, during the season 2019/2020, yielded an overall carrier proportion of 35.8%, ranging from 53.7% for red deer and 32.2% for wild boar to 21.7% for fallow deer. MRSA from wild boar and phenotypically linezolid-resistant S. aureus from wild boar and red deer were isolated, indicating that resistance to antimicrobials restricted to clinical practice also occurs in wildlife. The most prevalent genotypes were t11502/ST2678 (29.6%) and t12939/ST2678 (9.4%), previously reported in wild boar from Spain. Clonal lineages reported in humans and livestock, like CC1, CC5 or CC8 (19.1%) and ST425, CC133 or CC398 (23.5%), respectively, were also found. The sequence type ST544, previously restricted to humans, is described in wildlife for the first time. We also identified that land use (agricultural land cover), human driven disturbance (swine abundance) and host-related factors (sex) determine resistance occurrence. These findings suggest that antibiotics used in clinical settings, agriculture and livestock farming, spill over to wildlife, leading to AMR emergence, with potential biological, ecological, and human health effects. This work is one of the most comprehensive surveys in Europe of S. aureus occurrence and determinants among widely distributed wild ungulates.

Sett Use, Density and Breeding Phenology of Badgers in Mediterranean Agro-Sylvo-Pastoral Systems.

Carnivores social organization varies widely, from strongly social to solitary predators. European badgers are facultative social carnivores that also shows a geographical variation in social structure. These patterns derive mainly from central/west European regions, with an under-representation of Mediterranean populations that face different conservation challenges, especially regarding group composition, sett use patterns and breeding phenology. We addressed these traits topics for a population inhabiting a Portuguese agro-silvo-pastoral system. Based on monthly monitoring of 34 setts and continuous camera-trapping surveys of 12, we showed that setts surrounded by diversified vegetation and located in sandy sites are more used, a pattern probably linked to food availability and ease of sett excavation and maintenance, respectively. Badgers followed a general pattern regarding group size (2-4 adults), but showed an intermediate population density (0.49-0.73 badgers/km2), with values higher than those estimated for other Mediterranean environments, but lower than for central-western populations. This, together with the breeding (November/January) and cub emergence (1.8 cubs/sett; March/April) periods, indicates an ecological adaptation to the landscape context, where human-related resources and mild environmental conditions allow badger to reach higher densities than in many southern populations, and to reproduce earlier than their northern counterparts.

Protein metabolism and physical fitness are physiological determinants of body condition in Southern European carnivores.

The physiological significance of biometric body condition indices (bBCI) is poorly understood. We hypothesized that bBCI are composite metrics of nutritional physiology, physical fitness and health. To test this hypothesis, we first compared the performance of eight bBCI, using 434 Southern European carnivores from six species as a model system; and then identified, by non-destructive methods, the hematology and serum biochemistry correlates of three selected bBCI. Fulton's K Index, Major Axis Regression Residuals and Scaled Mass Index were the only bBCI insensitive to the effect of sex and age. The most informative physiological parameters in explaining the variation of these bBCI were the albumin (Effect Size (ES) = - 1.66 to - 1.76), urea (ES = 1.61 to 1.85) and total bilirubin (ES = - 1.62 to - 1.79). Hemoglobin and globulins (positive) and cholesterol (negative) were moderately informative (0.9 <|ES|< 1.5). This study shows that most bBCI do not control for the effect of age and sex in Southern European carnivores. Our results support that bBCI are composite measures of physiologic processes, reflecting a positive gradient from protein-poor to protein-rich diets, accompanied by increased physical fitness. Biometric body condition indices allow the integration of ecologically relevant physiological aspects in an easily obtained metric.

Niche differentiation mechanisms among canopy frugivores and zoochoric trees in the northeastern extreme of the Amazon

Frugivores and zoocoric trees represent an important proportion of tropical rainforest biodiversity. As niche differences favor species coexistence, we aimed to evaluate morphological and temporal niche segregation mechanisms among zoochoric trees and canopy frugivores in a tropical rainforest in the northeastern extreme of the Brazilian Amazon. We tested the effects of fruit morphology, tree size, frugivore body size and time of day on fruit consumption. We recorded the frugivore species that fed on 72 trees (44 species, 22 genera) and whether these frugivores swallowed the seeds. We monitored trees only once from 07:00 to 17:00 h between January and September 2017. We observed fruit consumption in 20 of the 72 trees. Seventy-three frugivore individuals from 22 species visited the trees. Heavier fruits were consumed by larger frugivores, while seed size was inversely correlated with frugivore size. Narrower fruits and fruits with smaller seeds had greater probability of having their seeds ingested, and larger frugivores were more prone to ingest seeds. Trees bearing fruits with smaller seeds were visited by a greater number of frugivores. Taxonomic groups differed in the time of arrival at fruiting trees. None of the evaluated variables (fruit weight and size, and seed size) affected the richness of frugivores that visited the trees. We concluded that, in the studied forest, fruit morphology (weight, size and seed size) is a niche segregation mechanism among zoochoric trees, while body size and time of day are niche segregation mechanisms among frugivores. (AU)

Molecular detection and characterization of Leishmania infantum in free-ranging Egyptian mongoose (Herpestes ichneumon).

Wild mammals are susceptible to infection by Leishmania parasites. Although canine leishmaniasis is widely distributed in mainland Portugal, the sylvatic cycle of the parasite remains poorly understood. In this study, the occurrence of L. infantum in wild carnivores from Portugal was assessed by molecular screening of 132 hunted or accidentally road-killed animals. Spleen samples from Egyptian mongoose, red fox, stone marten, common genet and European badger were tested by amplification of Leishmania kinetoplastid DNA and ITS1. Five egyptian mongoose were confirmed Leishmania DNA-positive by kDNA-PCR. Phylogenetic analysis of a kDNA amplicon sequence clustered the strain with L. infantum sequences from Portugal. These results may suggest that L. infantum strains circulating in wild animals are genetically related with strains from more humanized settings. Exposure of wild carnivores to Leishmania infantum emphasizes the need of systematic studies to clarify the role of several taxa in the eco-epidemiology of leishmaniasis in Portugal, particularly in areas of carnivore species synanthropy and wherein disease control in the domestic population is inefficient or insufficient.

Ecological drivers of Mycobacterium avium subsp. paratuberculosis detection in mongoose (Herpestes ichneumon) using IS900 as proxy.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease or paratuberculosis, a chronic infection affecting domestic ruminants worldwide. Despite sporadic reports of MAP occurrence in non-ruminants, information on the risk factors predisposing for infection is still scarce and evidence of transmission paths linking the livestock-wildlife-environment interfaces also remains lacking. In this study, we predicted that environmental, host-related, land use and human driven disturbance factors would modulate carnivore exposure to MAP. To test these hypotheses, we performed a retrospective survey, based on microbiological and molecular methods, in mainland Portugal including five sympatric species from the Herpestidae, Canidae, Viverridae, and Mustelidae families (n = 202) and examined 16 variables as putative predictors of MAP occurrence. Molecular evidence of MAP using IS900 as proxy was demonstrated in 7.43% (95%CI: 4.55-11.9) of surveyed carnivores, the highest proportions being registered for red fox (Vulpes vulpes) (10%; 95%CI: 4.0-23) and Egyptian mongoose (Herpestes ichneumon) (6.0%; 95%CI: 3.2-11). We demonstrate that important species of the Mediterranean carnivore guild, such as stone marten (Martes foina) and common genet (Genetta genetta), may also be exposed to MAP, being this the first time that occurrence in genet is reported. The high proportion of DNA-positive specimens, concurrent with the apparent lack of gastro-enteric lesions and molecular confirmation of IS900 in feces, argue for the presence of subclinical carriers that occasionally shed bacteria, potentially aiding as source of infection to susceptible species and possibly contributing for environmental contamination. Achievement of MAP isolation would prove beyond any doubt that MAP is present in this wildlife population. Ecological modelling results suggested that the probability of MAP infection using IS900 as proxy in mongoose is positively associated with higher altitude and temperature stability, as well as with lower annual rainfall. Density of livestock farms was found not to be a significant predictor, which may indicate that the livestock-wildlife interface is probably not important as an infection route for mongoose.

Research trends and geographical distribution of mammalian carnivores in Portugal (SW Europe).

Information regarding species' status at a regional scale is instrumental for effective conservation planning. Some regions of southwestern Europe, such as Portugal, albeit included in the Mediterranean biodiversity hotspot, lack a detailed assessment of the distribution patterns of several taxonomic groups, such as carnivores. Moreover, information is scattered, often unreliable and biased towards some species or regions. This study aimed at reviewing the existing knowledge on mammalian terrestrial carnivores in Portugal, to analyse research trends, update the species checklist and assess their historical and current distribution patterns. We conducted a comprehensive review of 755 scientific studies to analyse several publication metrics and compiled 20,189 presence records of all mammalian terrestrial carnivores occurring in Portugal since historical times to evaluate their distribution patterns. Carnivore research in Portugal began in the 18th century, with a recent boost in the mid-1990s, and has been biased towards certain research topics and regionally threatened species. There are 15 extant species in Portugal, with nine occurring across the country, six showing a more limited range, as well as one additional species currently locally extinct (Ursus arctos). Over the last decades, the distribution ranges of seven species apparently remained stable, two expanded, two contracted, and three showed unclear trends. The presence of a new invasive carnivore, the raccoon (Procyon lotor), is also documented here. This study illustrates the relevance of a comprehensive analysis of non-systematic data to assess the historic and current status of mammalian terrestrial carnivores at a national level, and to identify knowledge gaps and research priorities.

Coxiella burnetii DNA detected in domestic ruminants and wildlife from Portugal.

Coxiella burnetii is the etiological agent of Q fever or Coxiellosis, a zoonosis mainly affecting domestic ruminants. Information on the population structure and epidemiology of C. burnetii in animals is scarce in Portugal. Evidence of C. burnetti infection was sought in domestic, wild and captive animals based on the detection of bacterial DNA. Tissue samples from 152 domestic animals (cattle=24, goats=51, sheep=76 and swine=1), 55 wild carnivores (Egyptian mongoose=45, red fox=4, common genet=3, weasel=2 and European badger=1) and 22 zoo animals (antelopes=15, impala=1; rhinoceros=1, deer=2, zebras=2 and giraffe=1) were screened by nested-touchdown PCR. Cloacae swabs from 19 griffon vultures were also analysed. Among the domestic ruminants, goats presented the highest prevalence of infection (23.53%), followed by cattle, (20.83%) and sheep (10.53%). C. burnetii DNA was also detected in five Egyptian mongooses and two antelopes and one giraffe. Using a 6-locus multiple-locus variable-number tandem repeat analysis (MLVA-6) six complete genotypes, T, I and CM and the first reported CN, CO and CP, were identified, respectively, in small ruminants and Egyptian mongooses. Clustering analysis of genotypes exposed four distinct groups, according to detection source, enlightening an apparent association between C. burnetii genotype and host.

Acorn selection by the wood mouse Apodemus sylvaticus: a semi-controlled experiment in a Mediterranean environment.

Fruits are highly important food resources for mammals in Mediterranean Europe, and due to the dominance of oaks (Quercus sp.), acorns are among those used by a vast array of species, including rodents. The metabolic yield of acorn intake may determine a selection pattern: preference for fat, carbohydrate, and consequently energy-rich fruits; or avoidance of fruits containing high concentrations of secondary chemical compounds (e.g., tannic acid). We studied the acorn feeding selection pattern of wood mice (Apodemus sylvaticus) inhabiting a mixed oak woodland, southwest Portugal, using an experiment conducted in an open-air enclosure. We tested which variables associated with the wood mouse (e.g., sex) and acorns (e.g., size and nutrient content) from three oak species (holm Q. rotundifolia, Portuguese Q. faginea and cork Q. suber oak) could be constraining acorn consumption. Our results indicate that wood mice are selecting acorns of the most common oak species (Q. suber), probably due to their previous familiarization with the fruit due to its dominance in the ecosystem but probably also because its chemical characteristics (sugar contents). Rodent gender and acorn morphology (width) are also influential, with females more prone to consume acorns with smaller width, probably due to handling limitation. This selective behaviour may have consequences for dispersion and natural regeneration of the different oak species.

Estimating home-range size: when to include a third dimension?

Most studies dealing with home ranges consider the study areas as if they were totally flat, working only in two dimensions, when in reality they are irregular surfaces displayed in three dimensions. By disregarding the third dimension (i.e., topography), the size of home ranges underestimates the surface actually occupied by the animal, potentially leading to misinterpretations of the animals' ecological needs. We explored the influence of considering the third dimension in the estimation of home-range size by modeling the variation between the planimetric and topographic estimates at several spatial scales. Our results revealed that planimetric approaches underestimate home-range size estimations, which range from nearly zero up to 22%. The difference between planimetric and topographic estimates of home-ranges sizes produced highly robust models using the average slope as the sole independent factor. Moreover, our models suggest that planimetric estimates in areas with an average slope of 16.3° (±0.4) or more will incur in errors ≥5%. Alternatively, the altitudinal range can be used as an indicator of the need to include topography in home-range estimates. Our results confirmed that home-range estimates could be significantly biased when topography is disregarded. We suggest that study areas where home-range studies will be performed should firstly be scoped for its altitudinal range, which can serve as an indicator for the need for posterior use of average slope values to model the surface area used and/or available for the studied animals.

Snapshot of viral infections in wild carnivores reveals ubiquity of parvovirus and susceptibility of Egyptian mongoose to feline panleukopenia virus.

The exposure of wild carnivores to viral pathogens, with emphasis on parvovirus (CPV/FPLV), was assessed based on the molecular screening of tissue samples from 128 hunted or accidentally road-killed animals collected in Portugal from 2008 to 2011, including Egyptian mongoose (Herpestes ichneumon, n = 99), red fox (Vulpes vulpes, n = 19), stone marten (Martes foina, n = 3), common genet (Genetta genetta, n = 3) and Eurasian badger (Meles meles, n = 4). A high prevalence of parvovirus DNA (63%) was detected among all surveyed species, particularly in mongooses (58%) and red foxes (79%), along with the presence of CPV/FPLV circulating antibodies that were identified in 90% of a subset of parvovirus-DNA positive samples. Most specimens were extensively autolysed, restricting macro and microscopic investigations for lesion evaluation. Whenever possible to examine, signs of active disease were not present, supporting the hypothesis that the parvovirus vp2 gene fragments detected by real-time PCR possibly correspond to viral DNA reminiscent from previous infections. The molecular characterization of viruses, based on the analysis of the complete or partial sequence of the vp2 gene, allowed typifying three viral strains of mongoose and four red fox's as feline panleukopenia virus (FPLV) and one stone marten's as newCPV-2b type. The genetic similarity found between the FPLV viruses from free-ranging and captive wild species originated in Portugal and publicly available comparable sequences, suggests a closer genetic relatedness among FPLV circulating in Portugal. Although the clinical and epidemiological significance of infection could not be established, this study evidences that exposure of sympatric wild carnivores to parvovirus is common and geographically widespread, potentially carrying a risk to susceptible populations at the wildlife-domestic interface and to threatened species, such as the wildcat (Felis silvestris) and the critically endangered Iberian lynx (Lynx pardinus).

Use of multiple den sites by Eurasian badgers, Meles meles, in a Mediterranean habitat.

Den sites are a conspicuous feature of Eurasian badgers, Meles meles, and in many environments include large communal burrows used by several group members. In Serra de Grândola, southwest Portugal, nine badgers from three social groups were captured and radio collared from 2000 to 2004. A total of 1,787 locations of badgers in their resting sites were registered along with a brief description of the type of site and weather conditions. Resting sites were grouped according to structure (burrows, shrubs, rocks, hollow trees and man-made structures) and function (main, secondary and occasional). Although main setts were the most frequently used shelter (62.25%), an average of 14 (SD 7.55) resting sites were used in each territory. The pattern of use varied seasonally, showing differences according to sex and social group. Overall, females used more than twice as many occasional resting sites as did males. Generally burrows, predominantly main setts, were most frequently used during winter and autumn, whilst non-burrow shelters were preferred during spring and summer, when the weather was hot, dry and not windy. Proximity to food patches had no apparent influence on the location of resting sites. Our results offered no support for the foraging-related hypotheses that multiple resting sites are a means of conserving energy or of maintaining proximity to rich food patches. We suggest that other factors such as thermoregulation needs, disturbance, and reproductive status, could be influencing the observed pattern of resting-site use by badgers in Serra de Grândola.