Molecular detection and characterization of Blastocystis in herbivore livestock species in Portugal.

Blastocystis is a ubiquitous intestinal protist in humans and animals worldwide. The traditional livestock free-roaming raising system in rural communities increases the risk of infection with contact with a wider range of pathogens transmitted via the faecal-oral route associated with that wildlife-livestock-human interface. However, no studies have been conducted to determine the occurrence and subtype distribution of Blastocystis in livestock in Portugal. Here, we collected 180 faecal samples from herbivore livestock (cattle, goats, horses, and sheep) in different regions of the country to investigate Blastocystis prevalence and subtype diversity using PCR and next-generation amplicon sequencing. Blastocystis was present in 40.6% (73/180; 95% CI: 33.31-48.11) of the samples (goats, 81.0%; sheep, 60.9%; cattle, 32.2%). None of the horse samples were Blastocystis-positive. Eighteen subtypes were detected (ST1-ST3, ST5-ST7, ST10, ST13, ST14, ST21, ST23-ST26, ST30, ST42-ST44). Mixed infections were detected in 97.3% of the Blastocystis-positive samples. Potentially zoonotic subtypes were identified in 75.0%, 96.4%, and 100% of the Blastocystis-positive specimens collected from cattle, sheep, and goats, respectively. These results demonstrate that cattle, sheep, and goats harbour a high diversity of Blastocystis subtypes in the study regions. Importantly, our data provide novel molecular evidence strongly suggesting that some Blastocystis STs/ST subgroups may have differential host specificity.

Occurrence and molecular characterization of Enterocytozoon bieneusi in wild and domestic animal species in Portugal.

The phylum Microsporidia encompasses a diverse group of obligate, intracellular, and spore-forming organisms able to infect a wide range of animal hosts. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and animals. Little is known about the presence and epidemiology of E. bieneusi in wildlife. We investigated E. bieneusi occurrence and genetic diversity in wild and domestic mammals, through molecular-detection methods, from different regions across Portugal. A total of 756 samples were collected from 288, 242, and 226 wild carnivores, wild ungulates, and domestic animals, respectively. Overall, eight specimens were E. bieneusi-positive (1.1%, 8/756) obtained from five wild (Iberian lynx, Iberian wolf, red fox, stone marten, and wild boar) and one domestic (sheep) host. Nucleotide sequence analysis identified four genotypes of E. bieneusi, Type IV, Wildboar3, BEB6, and PtEbIX. Three of those genotypes belong to Groups 1 (Type IV and Wildboar3) and 2 (BEB6), which are known to contain genotypes capable of infecting a variety of hosts, including humans, highlighting their public health importance. PtEbIX belongs to the dog-specific Group 11. This study represents the first, largest, and most comprehensive molecular-based epidemiology survey carried out in Portugal in wild and domestic animals to date and the first worldwide identification of E. bieneusi in wolf species. Our study showed that wild carnivores and ungulates may act as reservoirs of zoonotic genotypes of E. bieneusi, establishing their role in maintaining the sylvatic cycle of this parasite while representing a potential source of infection for humans and domestic animals.

The identification of human-pathogenic genotypes of fungi-related Enterocytozoon bieneusi in wild carnivores and ungulates in Portugal suggests cross-species infection events and overlapping of the sylvatic and domestic transmission cycles, demonstrating a potential transmission risk to humans.

Survey of Zoonotic Diarrheagenic Protist and Hepatitis E Virus in Wild Boar (Sus scrofa) of Portugal.

Enteropathogenic parasites and viruses have been frequently reported in swine and can infect a wide range of mammals, including humans. Among the wide variety of parasites infecting swine, diarrhoeagenic protists are among those that cause significant morbidity. Hepatitis E virus (HEV) has also been reported both in domestic pigs and wild boar and is known to have an important public health significance. These agents share the fecal−oral transmission route, but data on their fecal shedding and circulation pathways are still lacking or incomplete. Hence, the aim of the present study was to characterize the presence of microeukaryotes and HEV in the wild boar of Portugal. Wild boar stool samples (n = 144) were obtained during the official hunting seasons (October to February) in 2018/2019, 2019/2020, and 2021/2022 and tested for Cryptosporidium spp., Balantioides coli, Giardia duodenalis, Blastocystis sp., Enterocytozoon bieneusi and HEV by molecular assays, followed by sequencing and phylogenetic analysis. We have detected Cryptosporidium scrofarum (1.4%, 95% CI: 0.2−4.9), B. coli (14.6%, 95% CI: 9.2−21.4), Blastocystis ST5 (29.2%, 95% CI: 21.9−37.2) and HEV genotype 3 (2.8%, 95% CI: 0.7−6.9; subgenotypes 3e and 3m). Co-infections were observed in thirteen animals where two were positive for both HEV and B. coli, one was positive for both C. scrofarum and Blastocystis ST5, and ten were positive for both B. coli and Blastocystis ST5. Giardia duodenalis and E. bieneusi were not detected in the surveyed wild boar population. As far as we know, this is the first report describing protist infections by Cryptosporidium spp., B. coli, and Blastocystis sp., as well as the first identification of the emerging HEV genotype 3m in wild boar of Portugal. The present work shows that potentially zoonotic protozoa and HEV are circulating in wild boar populations in Portugal. Awareness and epidemic-surveillance network implementation measures targeting wild boar are needed to prevent the spread of these pathogenic agents to humans.

Balantioides coli Fecal Excretion in Hunted Wild Cervids (Cervus elaphus and Dama dama) from Portugal.

Balantioides coli is a zoonotic enteric protozoan parasite of public veterinary health relevance and a concern in animal production and food safety. While wild cervids are recognized reservoirs for several zoonotic pathogens, little is known about the occurrence of B. coli in deer species, especially in Europe. To fill this gap, a total of 130 fecal samples from legally hunted red deer (Cervus elaphus, n = 95) and fallow deer (Dama dama, n = 35) were passively collected during two hunting seasons (October to February; 2018-2019 and 2019-2020) in Portugal. After assessment by PCR assay targeting the complete ITS1-5.8s-rRNA-ITS2 region and the 3' end of the ssu-rRNA gene of the parasite, a prevalence of 4.2% (4/95, 95% CI: 0.2-8.3) in red deer and of 5.7% (2/35, 95% CI: 0.0-13.4) in fallow deer was found. Sequence and phylogenetic analyses allowed the identification of B. coli genetic variants A (in two red deer) and B (in two red deer and two fallow deer). This is the first molecular-based description of B. coli in European deer species, whose population have increased in density and geographical range in recent years. Continued monitoring of wild ungulates as potential vectors of parasitic infection diseases of zoonotic nature is crucial to safeguard public health and food safety.

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.

Epidemiology of Porcine Circovirus Type 2 Circulating in Wild Boars of Portugal during the 2018-2020 Hunting Seasons Suggests the Emergence of Genotype 2d.

Porcine circovirus type 2 (PCV-2) is associated with several syndromes affecting swine, also known as porcine-circovirus-associated diseases, of which post-weaning multi-systemic wasting syndrome stands out due to its high economic impact on swine production. Recent data suggest the increasing circulation of the PCV-2d genotype in several countries worldwide. To provide updated data on PCV-2 genotypes currently circulating in swine in Portugal, we screened wild boar stools collected from several districts across Portugal, during the 2018-2020 hunting seasons, for PCV-2 and genetically characterized detected strains. From a total of 76 stool samples of wild boar tested by PCR for the partial PCV-2 ORF2 gene, two sequences were obtained (2/76; 2.6%, 95% confidence interval: 0.032-9.18). Bidirectional sequencing showed that the sequences were 100% identical and both of the PCV-2d genotype, showing for the first time the presence of this genotype in Portugal. Monitoring wild PCV-2 reservoirs is important for both veterinary public health and economic reasons, since PCV-2 infection has a strong economic impact on the swine industry.

Emergence and Spread of Cephalosporinases in Wildlife: A Review.

In the last decade, detection of antibiotic resistant bacteria from wildlife has received increasing interest, due to the potential risk posed by those bacteria to wild animals, livestock or humans at the interface with wildlife, and due to the ensuing contamination of the environment. According to World Health Organization, cephalosporins are critically important antibiotics to human health. However, acquired resistance to ß-lactams is widely distributed and is mainly mediated by extended-spectrum beta-lactamase and AmpC beta-lactamases, such as cephalosporinases. This work thus aimed to compile and analyse the information available on the emergence and dissemination of cephalosporinases in wildlife worldwide. Results suggest a serious scenario, with reporting of cephalosporinases in 46 countries from all continents (52% in Europe), across 188 host species, mainly birds and mammals, especially gulls and ungulates. The most widely reported cephalosporinases, CTX-M-1, CTX-M-14, CTX-M-15 and CMY-2, were also the most common in wild animals, in agreement with their ubiquity in human settings, including their association to high-risk clones of Escherichia coli (E. coli), such as the worldwide distributed CTX-M-15/ST131 E. coli. Altogether, our findings show that anthropogenic activities affect the whole ecosystem and that public policies promoting animal and environmental surveillance, as well as mitigation measures to avoid antimicrobial misuse and AMR spread, are urgently needed to be out in practise.

Parasites of the Reintroduced Iberian Lynx (Lynx pardinus) and Sympatric Mesocarnivores in Extremadura, Spain.

The Iberian lynx (Lynx pardinus) is one of the most endangered felid species in the world. Conservation efforts have increased its population size and distribution and reinforced their genetic diversity through captive breeding and reintroduction programmes. Among several threats that the Iberian lynx faces, infectious and parasitic diseases have underlined effects on the health of their newly reintroduced populations, being essential to identify the primary sources of these agents and assess populations health status. To achieve this, 79 fresh faecal samples from Iberian lynx and sympatric mesocarnivores were collected in the reintroduction area of Extremadura, Spain. Samples were submitted to copromicroscopic analyses to assess parasite diversity, prevalence, and mean intensity of parasite burden. Overall, 19 (24.1%, ±15.1-35.0) samples were positive for at least one enteric parasite species. Parasite diversity and prevalence were higher in the Iberian lynx (43.8%) compared with the others mesocarnivores under study (e.g., the red fox Vulpes vulpes and the Egyptian mongoose Herpestes ichneumon). Ancylostomatidae and Toxocara cati were the most prevalent (15.6%) parasites. Obtained results revealed that Iberian lynx role as predator control might have reduced parasite cross-transmission between this felid and mesocarnivores due to their decreasing abundances. Surveillance programs must include regular monitoring of this endangered felid, comprising mesocarnivores, but also domestic/feral and wild cat communities.

Dear deer? Maybe for now. People's perception on red deer (Cervus elaphus) populations in Portugal.

The human dimension of wildlife is a subject of increasing interest, especially considering the potential impact of people's perceptions on decision-making concerning wildlife management and thus on species' distribution and abundance. This is particularly important for species that inhabit human-dominated landscapes, where conflicts are likely to arise. These conflicts typically emerge between different human collectives when their interests collide and thus an assessment of their perceptions is valuable. Throughout Europe, ungulates have expanded, and Portugal is no exception. The expansion of red deer (Cervus elaphus) comes with benefits (e.g. hunting opportunities), but also with costs (e.g. vehicle collision or damage to crops), that can shape people's perceptions of these populations. To assess perceptions of red deer populations in continental Portugal, we developed a questionnaire survey with three interest groups: general public, farmers and hunters (total n = 1532). Our results show that perceptions about red deer were generally positive with a high acknowledgement of deer benefits, which we link to a broad level of sympathy towards this species. In addition, farmers showed a higher concern with crop damage caused by deer than non-farmers. Nonetheless, a general lack of knowledge regarding wildlife was present, particularly among younger people, who are potentially more apart and disconnected from nature. Now is the time to implement preventive and mitigation measures - e.g. fencing, reduction of population density - which can be a small contribution to address this problem. The focus should be put on people's acceptance of the required strategies to manage deer populations (which can also include unpopular practices, such as hunting). For that, an evaluation of people's attitudes towards deer populations and awareness of associated problems is essential. People's opinions, as well as the success of monitoring and management strategies, should be evaluated through multi-disciplinary teams, that include natural and social scientists, to ensure their success.

Assessment of antibiotic resistance of Escherichia coli isolates and screening of Salmonella spp. in wild ungulates from Portugal.

Antibiotic resistance is an emerging global problem. Wild animals are rarely exposed to antibiotics and therefore low levels of antibiotic resistance are expected. However, the growing interactions of these animals with humans and livestock may have a huge impact on their bacterial flora. This study aimed to assess the levels of antibiotic resistance in Escherichia coli isolated from widespread wild ungulates in Portugal. The interpretation of inhibition zone diameters was performed according to clinical breakpoints and epidemiological cut-offs, determined with the normalized resistance interpretation (NRI) method. For clinical breakpoints, 16% of the isolates were resistant to at least one antibiotic, including ampicillin (10%), tetracycline (9%), streptomycin (5%) co-trimoxazole (4%), amoxicillin/clavulanic acid (1%) and cefoxitin (1%). The levels of resistance detected in E. coli strains isolated from wild boar were statistically different for ampicillin and co-trimoxasol. According to NRI cut-offs, 10% of the population showed a non-wild-type phenotype against at least one antibiotic, also including tetracycline (9%), co-trimoxazole (6%), streptomycin (4%), ampicillin (2%) and amoxicillin/clavulanic acid (1%). Considering this parameter of comparison, no statistically different levels of resistance were identified between E. coli recovered from the three wild ungulates. Screening of Salmonella spp., which can be potentially pathogenic, was also performed, revealing that its prevalence was very low (1.5%). The study demonstrated that wild ungulates from Portugal are also reservoirs of antibiotic-resistant bacteria.