Endoparasites of the Iberian wolf (Canis lupus signatus) and mesocarnivores in Central Portugal.

At the end of the nineteenth century, massive population declines were observed in carnivores due to the emergence of infectious diseases. This study aims to investigate, by means of coprological analysis, the prevalence and intensity of the parasites that infect the endangered Iberian wolf Canis lupus signatus and two mesocarnivores (the red fox Vulpes vulpes and the stone marten Martes foina) in Central Portugal. In total, 67.2% of the samples screened were infected; Toxascaris leonina (40.6%) was the parasite with the highest prevalence, followed by Ancylostomatidae and Eimeria spp. (28.1%). Eimeria spp. was found in stone marten with the highest infection rate (37,800 OPG), followed by T. leonina (10,100 EPG) in a red fox sample. Moderate to high levels of parasitic infections were identified in 73.3% of red foxes from the western area. Our results highlight the possibility of cross-infection among these carnivore species and cross-contamination in the wildlife-livestock-human interface.

A walk on the wild side: Wild ungulates as potential reservoirs of multi-drug resistant bacteria and genes, including Escherichia coli harbouring CTX-M beta-lactamases.

Extended-spectrum ß-lactamases (ESBL)-producing Enterobacterales have been classified as critical priority pathogens by the World Health Organization (WHO). ESBL are universally distributed and, in 2006, were firstly reported on a wild animal. Understanding the relative contributions of wild animals to ESBL circulation in the environment is urgently needed. In this work, we have conducted a nationwide study in Portugal to investigate the occurrence of bacteria carrying clinically significant antimicrobial resistance genes (ARG), using widely distributed wild ungulates as model species. A total of 151 antimicrobial resistant-Enterobacterales isolates were detected from 181 wild ungulates: 50% (44/88) of isolates from wild boar (Sus scrofa), 40.3% (25/62) from red deer (Cervus elaphus), 41.4% (12/29) from fallow deer (Dama dama) and 100% (2/2) from mouflon (Ovis aries subsp. musimon). Selected isolates showed a diversified resistance profile, with particularly high values corresponding to ampicillin (71.5%) and tetracycline (63.6%). Enterobacterales strains carried blaTEM, tetA, tetB, sul2, sul1 or dfrA1 ARG genes. They also carried blaCTX-M-type genes, which are prevalent in human infections, namely CTX-M-14, CTX-M-15 and CTX-M-98. Strikingly, this is the first report of CTX-M-98 in wildlife. Almost 40% (n = 59) of Enterobacterales were multi-drug resistant. The diversity of plasmids carried by ESBL isolates was remarkable, including IncF, K and P. This study highlights the potential role of wild ungulates as environmental reservoirs of CTX-M ESBL-producing E. coli and in the spill-over of AMR bacteria and their determinants. Our findings suggest that wild ungulates are useful as strategic sentinel species of AMR in terrestrial environments, especially in response to potential sources of anthropogenic pollution, providing early warning of potential risks to human, animal and environmental health.

A high-risk carbapenem-resistant Pseudomonas aeruginosa clone detected in red deer (Cervus elaphus) from Portugal.

Pseudomonas aeruginosa is a ubiquitous bacterium, successfully exploiting a variety of environmental niches due to its remarkable metabolic versatility. The World Health Organization classifies P. aeruginosa as a "priority pathogen" due to its a great ability to overcome the action of antimicrobials, including carbapenems. Hitherto, most studies have focused on clinical settings from humans, but much less on animal and environmental settings, particularly on wildlife. In this work, we report the isolation of a carbapenem-resistant Pseudomonas aeruginosa strain recovered from the faeces of a red deer adult female sampled in a humanized area. This isolate was obtained during a nationwide survey on antimicrobial resistance in wildlife aimed to determine the occurrence of carbapenem-resistant bacteria among 181 widely distributed wild ungulates. This P. aeruginosa isolate was found to be a high-risk clone, belonging to the sequence type (ST) 274. The genomic analysis of P. aeruginosa isolate UP4, classified this isolate as belonging to serogroup O3, which was also found to harbour the genes blaPAO, blaPDC-24, blaOXA-486 (encoding resistance to beta-lactams), aph(3')-IIb (aminoglycosides resistance), fosA (fosfomycin resistance) and catB7 (chloramphenicol resistance). Antimicrobial susceptibility screening, according to EUCAST, showed resistance to imipenem and intermediate resistance to meropenem and doripenem. To our knowledge, this is the first description of carbapenem-resistant P. aeruginosa in deer in Europe. Our results highlight the importance of wild ungulates either as victims of human activity or amplifiers of AMR, either way with potential impacts on animal, human and ecosystem health, since excretion of AMR bacteria might directly or indirectly contaminate other animals and the surrounding environment, perpetuating the spill-over and chain dissemination of AMR determinants.

Mapping the scientific knowledge of antimicrobial resistance in food-producing animals.

Antimicrobial resistance (AMR) can be highlighted as one of the most significant health concerns among the last decades, for which antimicrobial drug use in food-producing animals has contributed as one of the major drivers. Food-producing animals are one of the most important and rapidly expanding commercial agricultural sectors worldwide but there is currently limited knowledge on the temporal and geographical distribution of scientific research on antimicrobial resistance in food-producing animals. We provide a global overview of the spatial and temporal trends of scientific knowledge on AMR in food-producing animals. Peer-reviewed papers of AMR on food-producing animals were retrieved from the Web of Science, systemized and dissected. The final validated dataset contained 1341 occurrences observations covering the 1957-2018 period. There has been a shift of research efforts, both geographically and temporally, emphasizing regional differences in food animal production and changing practices in the food production industry. It becomes evident that many regions have been poorly surveyed, wherein intensified sampling and testing efforts should be most valuable. This systematization of knowledge will be crucial in helping to determine how to optimally allocate limited resources available for AMR monitor and control, aiding in the prediction where the threat of new resistant infections will be greatest. AMR research in food-producing animals in developing countries is markedly growing, reflecting changes in food animals production systems but also posing a particularly significant threat, not only due to intensive animal production, but also exacerbated by poor sanitation. We highlight that the use of antibiotics in food producing animals is pervasive, calling for urgent action. These findings raise the possibility to finetuning key priorities on AMR global issues.

Emergence of colistin resistance genes (mcr-1) in Escherichia coli among widely distributed wild ungulates.

The environment is considered a major reservoir of antimicrobial resistant microorganisms (AMR) and antimicrobial resistance genes (ARG). Colistin, a "last resort" antibiotic, is used for the treatment of severe infections caused by multidrug-resistant Gram-negative bacteria. The global dissemination of mobile colistin resistance genes (mcr) in natural and non-natural environments is a major setback in the fight against antimicrobial resistance. Hitherto, there is a limited number of studies screening this resistance determinant in bacteria from wildlife. In this study, we describe for the first time the detection of plasmid-mediated colistin resistance in Escherichia coli from wild ungulates in Portugal, which are also widely distributed across Europe. This information is critical to identify the importance of ungulates in the dissemination of resistant bacteria, and their corresponding genes, across the environment. Here, 151 resistant-Enterobacteriaceae isolated from 181 samples collected from different wild ungulate species throughout Portugal were screened for mcr genes. Four mcr-1-positive Escherichia coli were detected from four fallow deer individuals that were sampled in the same hunting ground. These four isolates harboured mcr-1-related IncP plasmids belonging to sequencing types ST155, ST533 and ST345 (n = 2), suggesting bacterial and/or plasmid circulation. All mcr-1-positive E. coli also showed other resistance phenotypes, including MDR, including the B1 commensal phylogenetic profile. All mcr-1-positive E. coli show additional resistance phenotypes, including MDR, including the B1 commensal phylogenetic profile. Our findings are upsetting, highlighting the global dissemination of colistin resistance genes in the whole ecosystem, which, under the One Health framework, emphasizes the urgent need for effective implementation of AMR surveillance and control in the human-animal-environment interfaces.

Temporal and geographical research trends of antimicrobial resistance in wildlife - A bibliometric analysis.

Antimicrobial resistance (AMR) is a complex and global problem. Despite the growing literature on AMR in the medical and veterinary settings, there is still a lack of knowledge on the wildlife compartment. The main aim of this study was to report the global trends in AMR research in wildlife, through a bibliometric study of articles found in the Web of Science database. Search terms were "ANTIMICROBIAL" OR "ANTIBIOTIC" AND "RESISTANT" OR "RESISTANCE" and "WILDLIFE" "MAMMAL" "BIRD" "REPTILE" "FERAL" "FREE RANGE". A total of 219 articles were obtained, published between 1979 and 2019. A rising interest in the last decades towards this topic becomes evident. During this period, the scientific literature was distributed among several scientific areas, however it became more multidisciplinary in the last years, focusing on the "One Health" paradigm. There was a geographical bias in the research outputs: most published documents were from the United States, followed by Spain, Portugal and the United Kingdom. The most productive institutions in terms of publication number were located in Portugal and Spain. An important level of international collaboration was identified. An analysis of the main keywords showed an overall dominance of "AMR", "E. coli", "genes", "prevalence", "bacteria", "Salmonella spp." and "wild birds". This is the first study providing a global overview of the spatial and temporal trends of research related to AMR in wildlife. Given the growth tendency over the last years, it is envisaged that scientific production will expand in the future. In addition to offering a broad view of the existing research trends, this study identifies research gaps both in terms of geographical incidence and in relation to unexplored subtopics. Unearthing scientific areas that should be explored in the future is key to designing new strategic research agendas in AMR research in wildlife and to inform funding programs.

The Multiple Origins of Roe Deer Populations in Western Iberia and Their Relevance for Conservation.

The roe deer (Capreolus capreolus) is native and widespread in Europe and its phylogeography has been clarified in the last decades. Southern peninsulas are considered as reservoirs of genetic diversity and the source for the recolonization of Europe after the last glacial maximum. Even though roe deer populations have been genetically characterized, there is a major knowledge gap about the populations at the western edge of its distribution. To fill this caveat, and based on mitochondrial and nuclear DNA data, we aim to: (i) characterize the genetic diversity and structure of roe deer in western Iberia; (ii) clarify the origins and phylogeographical affinities of these populations, namely the relict population from Peneda Gerês National Park (PNPG, Portugal) and the likely allochthonous populations from central and south (CS) Portugal; (iii) discuss the implications of our findings for the management and conservation of the roe deer. Three major genetic clusters were inferred based on nuclear genotypes and were structured in a similar way as the three major mtDNA clades present in Iberia. Patterns inferred with nuclear markers confirmed PNPG as a relict population. Roe deer from CS Portugal share haplotypes with Central Europe rather than with other western Iberian populations, confirming its mainly allochthonous origin. Our results highlight western Iberia as a diversity hotspot for roe deer. We highlight the role of intraspecific genetic diversity as a source of resilience against ongoing global changes; the need for transboundary management and the importance of genetic data to inform management and conservation. When considered, repopulation or translocation measures should follow the IUCN Law of Reintroductions and meticulously conducted in order to preserve the genetic heritage of the species.

What does the wolf eat? Assessing the diet of the endangered Iberian wolf (Canis lupus signatus) in northeast Portugal.

The Iberian wolf (Canis lupus signatus) is a top predator that inhabits the Iberian Peninsula. In Portugal, its numbers and distribution declined throughout the 20th century, due to human persecution, habitat degradation and prey decline, which have led to higher predation rates of livestock in the remaining packs. In Montesinho Natural Park (northeast Portugal), wild ungulate populations have been increasing in the last years, which may have led wolf to predate upon them. In order to assess Iberian wolf diet in this area, 85 wolf scats were collected from transects distributed throughout the study area in two periods between November 2017 and August 2019. Scat analysis indicated a high predation on wild ungulates, where the frequency of occurrence showed that roe deer was the most consumed prey (44%), followed by red deer (26%) and wild boar (24%). Domestic/wild cat (6%), domestic goat and stone marten (5%) were consumed in lower quantities. It was found a higher selection towards roe deer (D = 0.71) and this was the only prey item which was significantly dependent of the season of the year (χ2 = 16.95, df = 3, p < 0.001). This is the first study in Portugal where was recorded that wolves feed mainly on wild ungulates. We conclude that lower livestock predation may be correlated with higher wild ungulates densities in our study area, as well as suitable husbandry practices, leading to a shift on Iberian wolf diet from mainly livestock on previous studies to wild ungulates.

Wild boar as a reservoir of antimicrobial resistance.

Antimicrobial resistance (AMR) has been recognized as an emerging and growing problem worldwide. Knowledge concerning AMR bacteria circulating in wildlife is currently limited, although it could provide important insights into AMR emergence and persistence. Across Europe, wild boar (Sus scrofa) populations have dramatically increased their distribution and number over the last decades. In the context of AMR dynamics, wild boar is a perfect model species to unveil the emergence, spread and persistence of AMR at the human-livestock-wildlife interface. Here, we summarize the current knowledge on the importance of wild boar as a reservoir of antimicrobial resistant bacteria, and its possible use as sentinel species for surveillance. Analyses of available data showed a rising interest on this topic in the last years, highlighting the growing concern on wild boar potential role as AMR facilitator and it is foreseen that the importance of antimicrobial resistance research in wild boar will continue to increase in years to come. Available studieshave been focused on specific bacterial species, particularlyE. coli, Salmonellaspp. andEnterococcusspp., bioindicators of AMR, and have been mainly conducted in three countries: Spain, Portugal and Germany.Strikingly, AMR surveillance in wild boar is uneven and still poorly allocated as many wild boar high-density countries do not yet have publications on the topic.Overall, accumulated data showed thatwild boar are carriers of antimicrobial resistant bacteria, withvariation in the prevalence of bacterial species and thepercentage of resistance to different antibiotics. Thelack of harmonized sampling and testing protocols makes it difficult to compare AMR in wild boar.The need for the establishment of standardised protocols keen to provide quantitative comparable data is highlighted. We finally suggest the long-term monitoring of wild boar as a sentinel species for AMR surveillance in order to inform public policies on this topic.

Helminth parasites of stone marten (Martes foina) in central Portugal

The stone marten (Martes foina) is one of the most common species of marten in Europe. Due to increased urbanization and forest degradation, it is now often found in urban habitats, which increased the possibility of parasites transmission between wildlife-domestic animals and humans. Our preliminary study of this mustelid in Portugal, aimed to assess prevalence of helminths. Six helminth parasites were found (Crenosoma vulpis, Angiostrongylus sp., Toxocara sp., Toxascaris leonina, Ancylostomatidae and Strongyloides sp.). To our knowledge, this is the first report of Toxascaris leonina in this host species.

Parasite species of the endangered Iberian wolf (Canis lupus signatus) and a sympatric widespread carnivore.

Parasites have a profound impact on wildlife population dynamics. However, until some years ago, studies on the occurrence and prevalence of wildlife parasites were neglected comparatively with the studies on humans and domestic animals. In this study, we determined the parasite prevalence of two sympatric wild canids: the endangered Iberian wolf (Canis lupus signatus) and the widespread red fox (Vulpes vulpes), in central Portugal. From November 2014 to July 2015, fresh fecal samples from both species were collected monthly in several transects distributed throughout the study area. All samples were submitted to several coprological techniques. In total, 6 helminth parasites (Crenosoma vulpis, Angiostrongylus vasorum, Toxocara canis, Trichuris vulpis, Ancylostomatidae, Toxascaris leonina), and a protozoa (Balantidium coli) were identified based on size and morphology. The red fox was infected by seven different parasites while the Iberian wolf was infected by four. All parasites present in wolf were also present in the red fox. C. vulpis had the higher prevalence in red fox, while Ancylostomatidae were the most prevalent parasites in wolf. To our knowledge, this is the first study in this isolated subpopulation of the Iberian wolf. Our results show that both carnivores carry parasites that are of concern as they are pathogenic to humans and other wild and domestic animals. We suggest that surveillance programs must also include monitoring protocols of wildlife; particularly endangered species.

To Eat or Not To Eat? The Diet of the Endangered Iberian Wolf (Canis lupus signatus) in a Human-Dominated Landscape in Central Portugal.

Livestock predation by large carnivores and their persecution by local communities are major conservation concerns. In order to prevent speculations and reduce conflicts, it is crucial to get detailed and accurate data on predators' dietary ecology, which is particularly important in human dominated landscapes where livestock densities are high. This is the case of the endangered Iberian wolf in Portugal, an endemic subspecies of the Iberian Peninsula, which has seen its population distribution and abundance decline throughout the 20th century. Accordingly, the diet of the Iberian wolf was analyzed, using scat analysis, in a humanized landscape in central Portugal. From 2011 to 2014, a total of 295 wolf scats were collected from transects distributed throughout the study area, prospected on a monthly basis. Scat analysis indicated a high dependence of Iberian wolf on livestock. Domestic goat predominated the diet (62% of the scats), followed by cow (20%) and sheep (13%); the only wild ungulate present in the scat analysis was the wild boar (4% of the scats). Our results show that even though livestock constitute most part of wolves diet, different livestock species may represent different predation opportunities. We conclude that the high levels of livestock consumption may be a result of low diversity and density of wild ungulates that settles livestock as the only abundant prey for wolves. Our findings help on the understanding of the Iberian wolf feeding ecology and have implications for conflict management strategies. Finally, management implications are discussed and solutions are recommended.

Living on the edge: roe deer (Capreolus capreolus) density in the margins of its geographical range.

Over the last decades roe deer (Capreolus capreolus) populations have increased in number and distribution throughout Europe. Such increases have profound impacts on ecosystems, both positive and negative. Therefore monitoring roe deer populations is essential for the appropriate management of this species, in order to achieve a balance between conservation and mitigation of the negative impacts. Despite being required for an effective management plan, the study of roe deer ecology in Portugal is at an early stage, and hence there is still a complete lack of knowledge of roe deer density within its known range. Distance sampling of pellet groups coupled with production and decay rates for pellet groups provided density estimates for roe deer in northeastern Portugal (Lombada National Hunting Area--LNHA, Serra de Montesinho--SM and Serra da Nogueira--SN; LNHA and SM located in Montesinho Natural Park). The estimated roe deer density using a stratified detection function was 1.23/100 ha for LNHA, 4.87/100 ha for SM and 4.25/100 ha in SN, with 95% confidence intervals (CI) of 0.68 to 2.21, 3.08 to 7.71 and 2.25 to 8.03, respectively. For the entire area, the estimated density was about 3.51/100 ha (95% CI - 2.26-5.45). This method can provide estimates of roe deer density, which will ultimately support management decisions. However, effective monitoring should be based on long-term studies that are able to detect population fluctuations. This study represents the initial phase of roe deer monitoring at the edge of its European range and intends to fill the gap in this species ecology, as the gathering of similar data over a number of years will provide the basis for stronger inferences. Monitoring should be continued, although the study area should be increased to evaluate the accuracy of estimates and assess the impact of management actions.