Plasmid-Mediated Colistin Resistance Genes mcr-1 and mcr-4 in Multidrug-Resistant Escherichia coli Strains Isolated from a Healthy Pig in Portugal.

Antimicrobial resistance encoded by mobile colistin resistance (mcr) genes is a global and emergent threat. In this study, we report the occurrence of two different populations of colistin-resistant Escherichia coli harboring mcr-1 and mcr-4 variants in the intestinal microbiome of a healthy pig. Following antimicrobial susceptibility determination, the presence of mcr genes in two E. coli strains, isolated according to different selective microbiological procedures, was screened by PCR. Whole-genome sequencing confirmed that both strains were multidrug-resistant; INIAV_002EC was an AmpC producer carrying blaCMY-2, blaTEM-1B, qnrS1, mcr-1.1 genes, and INIAV_001EC carrying blaTEM-1A, tetB, and mcr-4.1 genes, along with mutations in quinolone resistance-determining regions. In addition, both strains harbored sul3, dfrA, and aadA1 determinants. Further genome analysis revealed different plasmid replicons associated with the mcr genes, IncX4 associated with mcr-1.1, and ColE10 with mcr-4.1. In addition, other replicons, including IncFIA, IncI1-Iγ, IncX1, IncY, in INIAV_002EC, and IncX1, IncI1, and p0111, in INIAV_001EC, were identified. Furthermore, both strains belonged to ST215 serotype O68:H12 and ST156 serotype O25:H28, respectively. This finding highlights the pig gut flora as a potential reservoir of mobile colistin resistance genes and reports the presence of the mcr-4.1 gene found for the first time in Portugal.

Emergence of Cfr-Mediated Linezolid Resistance among Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) from Healthy Pigs in Portugal.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST398 is mainly found in Europe and North America, colonizing the nasal cavity of pigs. This study characterized the MRSA isolates recovered from pig nasal swabs (n = 171) by evaluating the antimicrobial susceptibility profile by broth microdilution and characterizing the genetic lineages by spa-typing. Three linezolid-resistant isolates were subjected to Whole-Genome Sequencing (WGS). All strains harbored the mecA gene and were resistant to tetracycline and susceptible to vancomycin. A high frequency of multidrug resistance (97.6%) was evidenced, with 55 different multidrug resistance profiles identified. The MRSA strains were found to belong to 17 spa-types, three being novel. The linezolid-resistant strains appeared to belong to the ST398 type, spa-type t011, and SCCmec_type_Vc and to harbor the cfr, fexA, blaZ, mecA, tetM, and tetK genes. The cfr gene was predicted to be carried in the plasmid, flanked by ISSau9 and the transposon TnpR. MRSA from Portuguese fattening pigs present a high diversity of genetic lineages. The presence of cfr-positive LA-MRSA may represent a risk of transmission to humans, mainly to those in contact with livestock.

Antibiotic Susceptibility Profiles and Resistance Mechanisms to ß-Lactams and Polymyxins of Escherichia coli from Broilers Raised under Intensive and Extensive Production Systems.

The intensive and extensive broiler production systems imply different veterinary interventions, including the use of antimicrobials. This study aimed to compare the antimicrobial susceptibility profiles of Escherichia coli isolated from both systems, characterize resistance mechanisms to ß-lactams and polymyxins, and identify genetic elements such as integrons. E. coli isolates recovered from broiler cecal samples were assayed for antimicrobial susceptibility through the broth microdilution technique. The molecular characterization of acquired resistance mechanisms to ß-lactams and colistin and the detection of integrons was performed by a multiplex PCR. For most antibiotics tested, the prevalence of reduced susceptibility is higher in commensal and extended-spectrum ß-lactamases (ESBL)/AmpC producers from broilers raised in the intensive system, compared with those raised under extensive conditions. SHV-12 was the most common ESBL enzyme found in both production systems. Other ESBL variants such as CTX-M-1, CTX-M-55, CTX-M-14, CTX-M-32, CTX-M-9, TEM-52, and plasmid-encoded AmpC enzyme CMY-2 were also present. MCR-1 was identified in a colistin-resistant isolate from broilers raised under the intensive system. This study highlights the differences in E. coli antibiotic susceptibility from both production types and emphasizes that a great deal of work remains to decrease consumption and antimicrobial resistance levels.

Antimicrobial Susceptibility of Enterococcus Isolates from Cattle and Pigs in Portugal: Linezolid Resistance Genes optrA and poxtA.

Enterococci are part of the commensal gut microbiota of mammals, with Enterococcus faecalis and Enterococcus faecium being the most clinically relevant species. This study assesses the prevalence and diversity of enterococcal species in cattle (n = 201) and pig (n = 249) cecal samples collected in 2017. Antimicrobial susceptibility profiles of E. faecium (n = 48) and E. faecalis (n = 84) were assessed by agar and microdilution methods. Resistance genes were screened through PCR and nine strains were analyzed by Whole Genome Sequencing. A wide range of enterococci species was found colonizing the intestines of pigs and cattle. Overall, the prevalence of resistance to critically important antibiotics was low (except for erythromycin), and no glycopeptide-resistant isolates were identified. Two daptomycin-resistant E. faecalis ST58 and ST93 were found. Linezolid-resistant strains of E. faecalis (n = 3) and E. faecium (n = 1) were detected. Moreover, oxazolidinone resistance determinants optrA (n = 8) and poxtA (n = 2) were found in E. faecalis (ST16, ST58, ST207, ST474, ST1178) and E. faecium (ST22, ST2138). Multiple variants of optrA were found in different genetic contexts, either in the chromosome or plasmids. We highlight the importance of animals as reservoirs of resistance genes to critically important antibiotics.

Prediction scenarios of past, present, and future environmental suitability for the Mediterranean species Arbutus unedo L.

Climate change is a challenge for forests in the coming decades, with a major impact on species adaptation and distribution. The Mediterranean Basin is one of the most vulnerable hotspots for biodiversity conservation under climate change in the world. This research aimed at studying a Mediterranean species well adapted to the region: the Arbutus unedo L. (strawberry tree). The MaxEnt, a presence-only species-distribution software, was used to model A. unedo's environmental suitability. The current species potential distribution was accessed based on actual occurrences and selected environmental variables and subsequently projected for the Last Glacial Maximum (LGM), the Mid-Holocene (MH), and the years 2050 and 2070, considering the two Representative Concentration Pathways: RCP4.5 and RCP8.5. Results from the LGM projection suggest the presence of refugia in the core of the Mediterranean Basin, in particular the Iberian Peninsula (IP). The projections for the MH indicate increasing climatic suitability for the species and an eastward expansion, relatively to LGM. The predicted future environmental changes will most likely act as a catalyst for suitable habitat loss and a range shift towards the North is likely to occur.

Prevalence and Characterization of ESBL/AmpC Producing Escherichia coli from Fresh Meat in Portugal.

The present study aimed to characterize the extended-spectrum ß-lactamases and plasmid-mediated AmpC ß-lactamases (ESBL/PMAß) among Escherichia coli producers isolated from beef, pork, and poultry meat collected at retail, in Portugal. A total of 638 meat samples were collected and inoculated on selective medium for the search of E. coli resistant to 3rd generation cephalosporins. Isolates were characterized by antimicrobial susceptibility testing, molecular assays targeting ESBL/AmpC, plasmid-mediated quinolone resistance (PMQR), and plasmid-mediated colistin resistance (PMCR) encoding genes. The highest frequency of E. coli non-wild type to 3rd generation cephalosporins and fluoroquinolones was observed in broiler meat (30.3% and 93.3%, respectively). Overall, a diversity of acquired resistance mechanisms, were detected: blaESBL [blaCTX-M-1 (n = 19), blaCTX-M-15 (n = 4), blaCTX-M-32 (n = 12), blaCTX-M-55 (n = 8), blaCTX-M-65 (n = 4), blaCTX-M-27 (n = 2), blaCTX-M-9 (n = 1), blaCTX-M-14 (n = 11), blaSHV-12 (n = 27), blaTEM-52 (n = 1)], blaPMAß [blaCMY-2 (n = 8)], PMQR [qnrB (n = 27), qnrS (n = 21) and aac(6')-Ib-type (n = 4)] and PMCR [mcr-1 (n = 8)]. Our study highlights that consumers may be exposed through the food chain to multidrug-resistant E. coli carrying diverse plasmid-mediated antimicrobial resistance genes, posing a great hazard to food safety and a public health risk.

Whole Genome Sequencing Refines Knowledge on the Population Structure of Mycobacterium bovis from a Multi-Host Tuberculosis System.

Classical molecular analyses of Mycobacterium bovis based on spoligotyping and Variable Number Tandem Repeat (MIRU-VNTR) brought the first insights into the epidemiology of animal tuberculosis (TB) in Portugal, showing high genotypic diversity of circulating strains that mostly cluster within the European 2 clonal complex. Previous surveillance provided valuable information on the prevalence and spatial occurrence of TB and highlighted prevalent genotypes in areas where livestock and wild ungulates are sympatric. However, links at the wildlife-livestock interfaces were established mainly via classical genotype associations. Here, we apply whole genome sequencing (WGS) to cattle, red deer and wild boar isolates to reconstruct the M. bovis population structure in a multi-host, multi-region disease system and to explore links at a fine genomic scale between M. bovis from wildlife hosts and cattle. Whole genome sequences of 44 representative M. bovis isolates, obtained between 2003 and 2015 from three TB hotspots, were compared through single nucleotide polymorphism (SNP) variant calling analyses. Consistent with previous results combining classical genotyping with Bayesian population admixture modelling, SNP-based phylogenies support the branching of this M. bovis population into five genetic clades, three with apparent geographic specificities, as well as the establishment of an SNP catalogue specific to each clade, which may be explored in the future as phylogenetic markers. The core genome alignment of SNPs was integrated within a spatiotemporal metadata framework to further structure this M. bovis population by host species and TB hotspots, providing a baseline for network analyses in different epidemiological and disease control contexts. WGS of M. bovis isolates from Portugal is reported for the first time in this pilot study, refining the spatiotemporal context of TB at the wildlife-livestock interface and providing further support to the key role of red deer and wild boar on disease maintenance. The SNP diversity observed within this dataset supports the natural circulation of M. bovis for a long time period, as well as multiple introduction events of the pathogen in this Iberian multi-host system.

Proposal of Epidemiological Cutoff Values for Apramycin 15 µg and Florfenicol 30 µg Disks Applicable to Staphylococcus aureus.

Apramycin and florfenicol are two antimicrobial agents exclusively used in veterinary medicine. Resistance determinants to these antimicrobial agents have been described in several staphylococci, yet no inhibition zone-based epidemiological cutoff (ECOFF) values are available to detect populations harboring resistance mechanisms. In this study, we propose disk diffusion inhibition zone ECOFF values of Staphylococcus aureus for apramycin and florfenicol. The susceptibility to apramycin and florfenicol was evaluated by disk diffusion of five S. aureus collections, comprising 352 isolates of animal (n = 265) and human (n = 87) origin. The aggregated distributions of inhibition zone diameters were analyzed by the normalized resistance interpretation method to obtain normalized wild-type (WT) population distributions and corresponding ECOFF values. The putative WT populations of S. aureus were characterized by an inhibition zone ≥15 mm (ECOFF = 15 mm) for apramycin and ≥21 mm for florfenicol (ECOFF = 21 mm). Five nonwild-type (NWT) isolates were detected for apramycin, all without inhibition zone and harboring the apmA gene, whereas five NWT isolates were identified for florfenicol, all carrying the fexA gene. The proposed ECOFF values for apramycin and florfenicol may be a valuable tool in future antimicrobial resistance monitoring and surveillance studies to identify S. aureus NWT populations toward these antimicrobial agents.

Long-term molecular surveillance provides clues on a cattle origin for Mycobacterium bovis in Portugal.

Animal tuberculosis (TB), caused by Mycobacterium bovis, is maintained in Portugal in a multi-host system, with cattle, red deer and wild boar, playing a central role. However, the ecological processes driving transmission are not understood. The main aim of this study was thus to contribute to the reconstruction of the spatiotemporal history of animal TB and to refine knowledge on M. bovis population structure in order to inform novel intervention strategies. A collection of 948 M. bovis isolates obtained during long-term surveillance (2002-2016, 15 years) of cattle (n = 384), red deer (n = 303) and wild boar (n = 261), from the main TB hotspot areas, was characterized by spoligotyping and 8 to 12-loci MIRU-VNTR. Spoligotyping identified 64 profiles and MIRU-VNTR distinguished 2 to 36 subtypes within each spoligotype, enabling differentiation of mixed or clonal populations. Common genotypic profiles within and among livestock and wildlife in the same spatiotemporal context highlighted epidemiological links across hosts and regions, as for example the SB0119-M205 genotype shared by cattle in Beja district or SB0121-M34 shared by the three hosts in Castelo Branco and Beja districts. These genomic data, together with metadata, were integrated in a Bayesian inference framework, identifying five ancestral M. bovis populations. The phylogeographic segregation of M. bovis in specific areas of Portugal where the disease persists locally is postulated. Concurrently, robust statistics indicates an association of the most probable ancient population with cattle and Beja, providing a clue on the origin of animal TB epidemics. This relationship was further confirmed through a multinomial probability model that assessed the influence of host species on spatiotemporal clustering. Two significant clusters were identified, one that persisted between 2004 and 2010, in Beja district, with Barrancos county at the centre, overlapping the central TB core area of the Iberian Peninsula, and highlighting a significant higher risk associated to cattle. The second cluster was predominant in the 2012-2016 period, holding the county Rosmaninhal at the centre, in Castelo Branco district, for which wild boar contributed the most in relative risk. These results provide novel quantitative insights beyond empirical perceptions, that may inform adaptive TB control choices in different regions.

The Gut Microbiota of the Egyptian Mongoose as an Early Warning Indicator of Ecosystem Health in Portugal.

The Egyptian mongoose is a carnivore mammal species that in the last decades experienced a tremendous expansion in Iberia, particularly in Portugal, mainly due to its remarkable ecological plasticity in response to land-use changes. However, this species may have a disruptive role on native communities in areas where it has recently arrived due to predation and the potential introduction of novel pathogens. We report reference information on the cultivable gut microbial landscape of widely distributed Egyptian mongoose populations (Herpestes ichneumon, n = 53) and related antimicrobial tolerance across environmental gradients. The panel of isolated species is consistent with the typical protein-based diet of a carnivore: Firmicutes predominate (89% of individuals), while Clostridiales, Enterobacteriales, and Lactobacillales are the major classes. Forty-one individuals (77.4%) harbour Clostridium spp. A spatial influence on mongooses' microbiota is confirmed by nonmetric multidimensional scaling analysis, with a significant contribution of municipality to their microbiota composition. Antimicrobial susceptibility testing of mongoose commensal bacteria to 28 compounds evidences xenobiotic tolerance of Escherichia coli (E. coli), enterococci, Salmonella Spartel and Mbandaka serotypes and Pseudomonas bacteria, among others. The common isolation of antimicrobial tolerant microbiota from the mongoose's gut suggests this species is exposed to anthropogenic influence and is affected by forestry and agricultural-related practices, reflecting its easy adaptation to ecological gradients across agroecosystems. We thus propose regular microbial and phenotypic resistance profiling of widely distributed mongooses as a sentinel tool for xenobiotics' lifecycle and ecosystem health in Portugal.

Polyclonal infection as a new scenario in Mycobacterium caprae epidemiology.

Portugal is one of the European Union countries with an ongoing eradication program for bovine tuberculosis (TB), which does not include systematic goat testing. However, surveillance in small ruminants is increasingly important, since goat and sheep can harbour Mycobacterium caprae and be an infection source to cattle with impact in the success of bovine TB control. Furthermore, the information regarding the epidemiology and biology of M. caprae is quite limited comparing to the cognate bovine-adapted ecotype, M. bovis. In this work, we applied spoligotyping and MIRU-VNTR (Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats) to M. caprae obtained between 2003 and 2014 from 55 animal hosts, including goat (n = 29), cattle (n = 21), sheep (n = 1) and wild boar (n = 4) from Portugal. The molecular analysis revealed a unique spoligotyping pattern (SB0157) and 24 MIRU types. Genotyping of serial M. caprae from herds with recurrent outbreaks enabled further discrimination of epidemiologically related isolates, supporting a clonal structure in Portugal and denoting the emergence of clonal diversity at the herd level, more apparent for MIRU4. Results suggest a founder effect and adaptive genotypic divergence, paving the way for sympatric speciation. Double allele findings at MIRU4 in over 20 % of infected animals indicates that co-infection and in vivo microevolution may be frequent in the goat-adapted ecotype. While polyclonal infection appears common in M. caprae epidemiology, the functional significance of subtle genotypic variations remains to be disclosed, namely at the interface with the host, to expand knowledge on the epidemiology and biology of this neglected ecotype.

Oral Health Knowledge, Perceptions, and Habits of Adolescents from Portugal, Romania, and Sweden: A Comparative Study.

AIMS: The study aimed to assess and compare the self-reported oral health knowledge, habits, and perceptions of adolescents in Portugal, Romania, and Sweden and the factors that may affect them. MATERIALS AND METHODS: An oral health questionnaire with 25 questions was put together in English language and translated into Portuguese, Romanian, and Swedish language to investigate and compare oral health-related knowledge, habits, and perceptions of adolescents from the different countries. A voluntary sample of 879 teenagers completed it, mean aged 14.9 (±1.83) years, from Romania (n = 455), Portugal (n = 200), and Sweden (n = 224) as part of the EuHyDens project. The survey took place between November 2015 and June 2016. RESULTS: It showed some differences between the countries studied but more similarities between Portuguese and Romanian adolescents as related to perceptions of oral health and the use of oral services. Sweden and Portugal are more similar regarding oral health habits (toothbrushing and diet) as compared to Romanian adolescents. Portuguese adolescents have a higher oral health-related knowledge. CONCLUSION: Assessment of knowledge, perceptions, and habits of a population is essential for the adequate understanding of the oral health-care needs of the society. From the data collected, several differences were found. These differences can be used for tailor interventions to minimize inequalities between countries.

The Dental Hygienists in Sweden and Portugal: A Comparative Study.

AIMS AND OBJECTIVES: The aim of the present study was to identify the dental hygienists (DHs)' working areas and compare these findings between Sweden and Portugal. MATERIALS AND METHODS: A web-based questionnaire was sent by E-mail to all 2943 members of the Swedish DH Association and 381 members of the Portuguese DH Association. The questionnaire contained questions about; number of years of work, which patient groups they worked with, if they had got sufficient knowledge from the education and what different tasks they worked with. Independent sample t-test, relative risk, and logistic regression analysis were used as statistical methods. RESULTS: The response rate for Sweden was 31% (n = 923) and the corresponding figure for Portugal was 53.5% (n = 204). The most common tasks for the DHs, in both countries, were to diagnose and treat patients with periodontitis and to diagnose and perform caries prevention. In Portugal, 86.5% worked with orthodontic patients compared to 32.3% in Sweden (P = 0.001). The logistic regression analysis showed that there was a 25% (P = 0.02) higher probability that Swedish DHs took more X-rays and 38% (P = 0.042) probability that they had more communications skills compared to the Portuguese DHs. CONCLUSION: The DH profession in Sweden and Portugal was overall quite similar. Despite a few local differences, both the academic education and scope of practice corresponded well between the countries. The few discrepancies could be explained by differences in legislations in each country.

Self-reported oral health-related habits, attitudes and knowledge in adults from Portugal, Romania and Sweden-A comparative study.

OBJECTIVE: The aim of the present study was to compare the self-reported oral health knowledge, habits and attitudes of adults in Portugal, Romania and Sweden, each benefitting from a different oral health system. METHODS: A cross-sectional study was conducted on 1081 adults in the three countries, using an adapted version of the WHO oral health questionnaire. This survey took place between November 2015 and June 2016. RESULTS: The mean age of the respondents was 46.85 with 95% CI (45.84, 47.86). Over 85% of the Portuguese and the Swedish, but only 55% of the Romanians, reported having 20 or more teeth left (P < 0.001). Most Swedes visit the dental office regularly, but only 50.5% of the Portuguese and 20.6% of the Romanians do so (P < 0.001). Interdental cleaning aids and fluoridated toothpaste were used the most in Sweden and the least in Romania. Daily consumption of sweets was lowest for Portugal and highest for Romania (P < 0.001), and daily intake of fresh fruits and vegetables was lowest for Sweden (P < 0.001). CONCLUSIONS: There is a difference between the three countries regarding oral health, diet, dental attendance and oral health-related quality of life, with Romania being in most need of improvement in these areas. The differences could be explained by the distinct healthcare systems adopted by the countries.

Revealing mcr-1-positive ESBL-producing Escherichia coli strains among Enterobacteriaceae from food-producing animals (bovine, swine and poultry) and meat (bovine and swine), Portugal, 2010-2015.

We screened 1840 Enterobacteriaceae isolates from food-producing animals, meat, meat products and animal feed, for the detection of plasmid-mediated colistin resistance, during 2010-2015. The mcr-1 gene was detected in 8.0% (97/1206) Escherichia coli and in 0.47% (3/634) Salmonella enterica isolates, with a high number of mcr-1 positive E. coli isolates (45.7%) being extended-spectrum ß-lactamase or plasmid-mediated AmpC ß-lactamase co-producers. No mcr-2 gene was detected. Our findings highlight the spread of mcr-1 genes within a wide-ranging sample of food-producing animals and meat, in Portugal.

Bioclimatic modeling in the Last Glacial Maximum, Mid-Holocene and facing future climatic changes in the strawberry tree (Arbutus unedo L.).

Increasing forest wildfires in Portugal remain a growing concern since forests in the Mediterranean region are vulnerable to recent global warming and reduction of precipitation. Therefore, a long-term negative effect is expected on the vegetation, with increasing drought and areas burnt by fires. The strawberry tree (Arbutus unedo L.) is particularly used in Portugal to produce a spirit by processing its fruits and is the main income for forestry owners. Other applications are possible due to the fruit and leaves' anti-oxidant properties and bioactive compounds production, with a potential for clinical and food uses. It is a sclerophyllous plant, dry-adapted and fire resistant, enduring the Mediterranean climate, and recently considered as a possibility for afforestation, to intensify forest discontinuity where pines and eucalypts monoculture dominate the region. To improve our knowledge about the species' spatial distribution we used 318 plots (the centroid of a 1 km2 square grid) measuring the species presence and nine environmental attributes. The seven bioclimatic variables most impacting on the species distribution and two topographic features, slope and altitude, were used. The past, current and future climate data were obtained through WorldClim. Finally, the vulnerability of the strawberry tree to the effects of global climate change was examined in the face of two emission scenarios (RCP 4.5 and 8.5), to predict distribution changes in the years 2050 and 2070, using a species distribution models (MaxEnt). The reduction of suitable habitat for this species is significant in the southern regions, considering the future scenarios of global warming. Central and northern mountainous regions are putative predicted refuges for this species. Forest policy and management should reflect the impact of climate change on the usable areas for forestry, particularly considering species adapted to the Mediterranean regions and wildfires, such as the strawberry tree. The distribution of the species in the Last Glacial Maximum (LGM) and Mid-Holocene (MH) agrees with previous genetic and paleontological studies in the region, which support putative refuges for the species. Two in the southern and coastal-central regions, since the LGM, and one in the east-central mountainous region, considered as cryptic refugia.

New insights into resistance to colistin and third-generation cephalosporins of Escherichia coli in poultry, Portugal: Novel blaCTX-M-166 and blaESAC genes.

The increasing incidence of intestinal colonization with extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae and Gram negative organisms that has been observed in food animals such as poultry, cattle and pigs, are suggestive that animals, food and environment are potential sources of ESBL-producing bacteria. Hence, the aim of this study was to characterized commensal E. coli obtained from healthy broiler and turkey flocks at slaughter for the presence of penicillinases-, ESBL-, extended-spectrum AmpC (ESAC)-, plasmid-mediated quinolone resistance- and MCR-encoding genes. Study of clonal relatedness showed genetic diversity among CTX-M-type, SHV-12 and TEM-52 producing isolates with human isolates of the same type, was also assessed. We detected that eleven (5.4%, 11/202) and forty-five (2.2%, 45/185) E. coli isolates from broilers and turkeys, respectively, carried blaESBL or blaESAC genes and two isolates from turkeys carried mcr-1 gene. A new variant blaCTX-M-166 was reported in a multidrug resistant isolate from a broiler flock. Overall, we detected a diversity of resistance mechanisms among E. coli from food-producing animals, all of them with high importance at a public health level.

Exposure of Threatened Accipitridae to Mycobacterium bovis Calls for Active Surveillance.

Anthropogenic activities have cumulatively led to the dramatic decline of world populations of vultures that currently face serious survival challenges in several regions of the world. In Portugal, the three resident species qualify as endangered and are under conservation efforts, mainly in the central east and south-east regions, where habitat protection and artificial feeding stations were implemented. Concurrently, the areas under protection are highly affected by tuberculosis (TB) in cattle and wild ungulates, whose potentially infected carcasses may naturally or artificially be used as feed by local vultures. In this work, we opportunistically surveyed populations of Eurasian griffon (Gyps fulvus) and Eurasian black vulture (Aegypius monachus) for the presence of Mycobacterium bovis. Nine pathogenic mycobacteria, including one M. bovis isolate, were cultured from the oropharynx of nine of the surveyed vultures (n = 55), sampled in recovery centres or in artificial feeding stations. Genotyping of the M. bovis strain indicated spoligotype SB0121, the most frequent type in Portugal, and a unique MIRU-VNTR profile that differed in two loci from the profiles of SB0121 bovine and deer strains from the same geographical area. The M. bovis-positive griffon exhibited poor clinical condition when admitted to the recovery centre; however, clinical evidence of TB was not present. Although the significance of M. bovis isolation in this vulture specimen could not be ascertained and despite the accepted notion that vultures are naturally resistant to microbial pathogens, the sanitary follow-up of Accipitridae vulture populations in TB-hotspot areas is essential to safeguard ongoing conservation efforts and also to evaluate the suitability of standing legislation on deliberate supplementary feeding schemes for menaced birds of prey.