Occurrence and Characteristics of ESBL- and Carbapenemase- Producing Escherichia coli from Wild and Feral Birds in Greece

Wild and feral birds are known to be involved in the maintenance and dissemination of clinically-important antimicrobial-resistant pathogens, such as extended-spectrum β-lactamase (ESBL) and carbapenemase-producing Enterobacteriaceae. The aim of our study was to evaluate the presence of ESBL- and carbapenemase-producing Escherichia coli among wild and feral birds from Greece and to describe their antimicrobial resistance characteristics. In this context, fecal samples of 362 birds were collected and cultured. Subsequently, the antimicrobial resistance pheno- and geno-type of all the obtained E. coli isolates were determined. A total of 12 multidrug-resistant (MDR), ESBL-producing E. coli were recovered from eight different wild bird species. Eleven of these isolates carried a bla CTX-M-1 group gene alone or in combination with bla TEM and one carried only bla TEM . AmpC, fluoroquinolone, trimethoprim/sulfamethoxazole, aminoglycoside and macrolide resistance genes were also detected. Additionally, one carbapenemase-producing E. coli was identified, harboring bla NDM along with a combination of additional resistance genes. This report describes the occurrence of ESBL- and carbapenemase-producing E. coli among wild avian species in Greece, emphasizing the importance of incorporating wild birds in the assessment of AMR circulation in non-clinical settings

Polarisation of Major Histocompatibility Complex II Host Genotype with Pathogenesis of European Brown Hare Syndrome Virus.

A study was conducted in order to determine the occurrence of European Brown Hare Syndrome virus (EBHSV) in Denmark and possible relation between disease pathogenesis and Major Histocompatibility Complex (MHC) host genotype. Liver samples were examined from 170 brown hares (hunted, found sick or dead), collected between 2004 and 2009. Macroscopical and histopathological findings consistent with EBHS were detected in 24 (14.1%) hares; 35 (20.6%) had liver lesions not typical of the syndrome, 50 (29.4%) had lesions in other tissues and 61 (35.9%) had no lesions. Sixty five (38.2%) of 170 samples were found to be EBHSV-positive (RT-PCR, VP60 gene). In order to investigate associations between viral pathogenesis and host genotype, variation within the exon 2 DQA gene of MHC was assessed. DQA exon 2 analysis revealed the occurrence of seven different alleles in Denmark. Consistent with other populations examined so far in Europe, observed heterozygosity of DQA (H o = 0.1180) was lower than expected (H e = 0.5835). The overall variation for both nucleotide and amino acid differences (2.9% and 14.9%, respectively) were lower in Denmark than those assessed in other European countries (8.3% and 16.9%, respectively). Within the peptide binding region codons the number of nonsynonymous substitutions (dN) was much higher than synonymous substitutions (dS), which would be expected for MHC alleles under balancing selection. Allele frequencies did not significantly differ between EBHSV-positive and -negative hares. However, allele Leeu-DQA*30 was detected in significantly higher (P = 0.000006) frequency among the positive hares found dead with severe histopathological lesions than among those found sick or apparently healthy. In contrast, the latter group was characterized by a higher frequency of the allele Leeu-DQA*14 as well as the proportion of heterozygous individuals (P = 0.000006 and P = 0.027). These data reveal a polarisation between EBHSV pathogenesis and MHC class II genotype within the European brown hare in Denmark

Development of a multiplex bead assay for simultaneous serodiagnosis of antibodies against Mycobacterium bovis, Brucella suis, and Trichinella spiralis in wild boar

This article belongs to the Special Issue Farm Animal and Wildlife Zoonotic Microorganisms.The aim of this study was to evaluate the diagnostic performance of a multiplex bead assay for the simultaneous detection of antibodies against Mycobacterium bovis, Brucella suis, and Trichinella spiralis. Sera from Eurasian wild boar of known serological status for TB (64 seropositive, 106 seronegative), Brucella (30 seropositive, 39 seronegative), and Trichinella (21 seropositive, 97 seronegative) were used for the development and evaluation of the assay. Magnetic beads coated with recombinant MPB83 antigen (TB), a whole-cell B. suis 1330 antigen, and an E/S T. spiralis antigen were used for the detection of specific antibodies using Bio-Rad Bio-Plex technology. The sensitivities (Se) and specificities (Sp) of the multiplex assay were, for M. bovis, 0.98 and 0.86; for B. suis, 1.00 and 0.97; and for T. spiralis, 0.90 and 0.99 (Se and Sp, respectively). The results show the diagnostic potential of this assay for the simultaneous detection of antibodies against M. bovis, B. suis, and T. spiralis in wild boar.We thankfully acknowledge the financial support of the European Union Seventh Framework Programme (2007–2013) under grant agreement no. 222633 (WildTech) titled “Novel Technologies for Surveillance of Emerging and Re-emerging Infections of Wildlife”.Peer reviewe

Use of wild bird surveillance, human case data and GIS spatial analysis for predicting spatial distributions of West Nile Virus in Greece

West Nile Virus (WNV) is the causative agent of a vector-borne, zoonotic disease with a worldwide distribution. Recent expansion and introduction of WNV into new areas, including southern Europe, has been associated with severe disease in humans and equids, and has increased concerns regarding the need to prevent and control future WNV outbreaks. Since 2010, 524 confirmed human cases of the disease have been reported in Greece with greater than 10% mortality. Infected mosquitoes, wild birds, equids, and chickens have been detected and associated with human disease. The aim of our study was to establish a monitoring system with wild birds and reported human cases data using Geographical Information System (GIS). Potential distribution of WNV was modelled by combining wild bird serological surveillance data with environmental factors (e.g. elevation, slope, land use, vegetation density, temperature, precipitation indices, and population density). Local factors including areas of low altitude and proximity to water were important predictors of appearance of both human and wild bird cases (Odds Ratio = 1,001 95%CI = 0,723–1,386). Using GIS analysis, the identified risk factors were applied across Greece identifying the northern part of Greece (Macedonia, Thrace) western Greece and a number of Greek islands as being at highest risk of future outbreaks. The results of the analysis were evaluated and confirmed using the 161 reported human cases of the 2012 outbreak predicting correctly (Odds = 130/31 = 4,194 95%CI = 2,841–6,189) and more areas were identified for potential dispersion in the following years. Our approach verified that WNV risk can be modelled in a fast cost-effective way indicating high risk areas where prevention measures should be implemented in order to reduce the disease incidence

Ecology, conservation and management of large mammals and their habitats in a highway corridor: case study of the Egnatia Highway and the brown bear, wild boar and roe deer in NE Pindos mountains Greece

Protection of biodiversity is the subject of much debate worldwide. The fragmentation of habitats has also been recognized internationally as one of the most important issues that threaten the conservation of biodiversity. For Europe it has been estimated that the average area of undisturbed land not contiguous segmented by major roads ranges from 20 Km2 to 600 Km2 Belgium to Finland with an average for the EU the 130 Km2. However in Europe, the highest biodiversity values lies with the Mediterranean and distinguish between the Balkans and particularly in Greece and in proportion to its size, is characterized by particularly high levels of biodiversity of animal species. Among the animal species characteristic of Greece is the brown bear belonging to large carnivores while herbivores from large mammals, are the roe deer and wild boar. In particular, both the brown bear as for the roe deer and wild boar, our knowledge about the ecology and biology in Greece is incomplete. The purpose of this dissertation focuses on ecology, conservation and management of three large mammals and their habitats in the area of construction and operation of a closed motorway in NE Pindos. For this purpose, field data collected for the three species under study in the period 2005-2011 while the bear specifically collected data and satellite telemetry for 20 bears the period 2007-2010. The aim of the first chapter was to examine the spatiotemporal interactions and social behavior among males and females in a population bears NE Pindos. The aim of the second chapter was to estimate the size of home ranges per season and the exploration of movement and activity patterns in 20 brown bear using satellite telemetry during 2007-2010. The aim of the third chapter was to estimate the permeability of the Egnatia highway for 20 brown bears and to study the effect of environmental variables on the selection of spots of crossings. The aim of the fourth chapter was the use of ecological models in combination with GIS to predict suitable habitat for brown bear, roe deer and wild boar. Data from GPS-GSM telemetry showed that home ranges were large percentage of overlap even for areas with high intensity of use. The home ranges of the animals that were studied separately in each season showed significant differences supporting the existence of a different standard of behavior between genders. The bears crossed Egnatia highway showing preference to areas far away from settlements and livestock facilities, gentle slopes, with a high percentage of forest cover and with greater proportion of food available. Mixed forests, oak forests of the important factors determining the habitat suitability of all the studied speciesΗ προστασία της βιοποικιλότητας αποτελεί αντικείμενο μεγάλης συζήτησης παγκοσμίως. Η κατάτμηση ενδιαιτημάτων έχει επίσης αναγνωριστεί διεθνώς ως ένα από τα πιο σημαντικά ζητήματα που απειλούν τη διατήρηση της βιοποικιλότητας. Για την Ευρώπη έχει υπολογιστεί ότι η μέση έκταση αδιατάρακτης συνεχόμενης γης που δεν κατακερματίζεται από σημαντικούς οδικούς άξονες κυμαίνεται από 20 Km2 στο Βέλγιο έως 600 Km2 στην Φινλανδία με ένα μέσο όρο για την Ε.Ε. τα 130 Km2. Παρόλα αυτά στην Ευρώπη, τις υψηλότερες τιμές βιοποικιλότητας φέρουν οι περιοχές της Μεσογείου και μεταξύ αυτών ξεχωρίζουν τα βαλκάνια και ιδιαίτερα η Ελλάδα ενώ αναλογικά με το μέγεθός της, χαρακτηρίζεται από ιδιαίτερα υψηλά επίπεδα βιοποικιλότητας ζωικών ειδών. Ανάμεσα στα χαρακτηριστικά ζωικά είδη της Ελλάδας είναι και η καφέ αρκούδα που ανήκει στα μεγάλα σαρκοφάγα ενώ από τα μεγάλα φυτοφάγα θηλαστικά, είναι το ζαρκάδι και ο αγριόχοιρος. Ειδικότερα, τόσο για την καφέ αρκούδα όσο για το ζαρκάδι και τον αγριόχοιρο οι γνώσεις μας γύρω από την οικολογία και τη βιολογία τους στον Ελλαδικό χώρο είναι ελλιπείς.Ο σκοπός της διατριβής αφορά στην οικολογία, διατήρηση και διαχείριση τριών μεγάλων θηλαστικών και των ενδιαιτημάτων τους στην περιοχή κατασκευής και λειτουργίας ενός κλειστού αυτοκινητοδρόμου στη ΒΑ Πίνδο. Για το σκοπό αυτό συλλέχθηκαν δεδομένα πεδίου για τα τρία υπό μελέτη είδη την περίοδο 2005-2011 ενώ για την αρκούδα ειδικότερα συλλέχθηκαν και δεδομένα δορυφορικής τηλεμετρίας για 20 άτομα αρκούδας την περίοδο 2007-2010. Στόχος του 1ου κεφαλαίου ήταν να εξετάσει τις χωροχρονικές αλληλεπιδράσεις και την κοινωνική συμπεριφορά ανάμεσα σε αρσενικά και θηλυκά άτομα. Στόχος του 2ου κεφαλαίου ήταν η εκτίμηση του μεγέθους των ζωτικών χώρων ανά εποχή και η διερεύνηση των προτύπων μετακινήσεων και δραστηριότητας σε 20 άτομα καφέ αρκούδας με τη χρήση δορυφορικής τηλεμετρίας την περίοδο 2007-2010. Στόχος του 3ου κεφαλαίου ήταν η εκτίμηση της διαπερατότητας της Εγνατίας Οδού για 20 άτομα αρκούδας αλλά και η μελέτη της επίδρασης των περιβαλλοντικών μεταβλητών στην επιλογή των θέσεων διέλευσης πριν, κατά και μετά την κατσκευή του οδικού άξονα στη ΒΑ Πίνδο. Στόχος του 4ου κεφαλαίου ήταν η χρήση οικολογικών μοντέλων σε συνδυασμό με τα GIS για την πρόβλεψη των κατάλληλων ενδιαιτημάτων για τα τρία υπό μελέτη είδη στην περιοχή διέλευσης της Εγνατίας Οδού. Από τα στοιχεία της δορυφορικής παρακολούθησης προέκυψε ότι οι ζωτικοί χώροι είχαν μεγάλο ποσοστό επικάλυψης ακόμα και για τις περιοχές με υψηλή ένταση χρήσης. Οι ζωτικοί χώροι των αρκούδων που μελετήθηκαν ξεχωριστά ανά επoχή εμφάνισαν σημαντική διαφοροποίηση υποστηρίζοντας την ύπαρξη ενός διαφορετικού προτύπου συμπεριφοράς ανάμεσα στο φύλο. Oι αρκούδες διέσχισαν τον άξονα της Εγνατίας Οδού δείχνοντας προτίμηση σε περιοχές με ποσοστό δασοκάλυψης, ήπιες κλίσεις και αυξημένα τροφικά διαθέσιμα. Τα μικτά δάση, τα δρυοδάση και οι αγροδασικές εκτάσεις είναι από τους σημαντικούς παράγοντες που καθορίζουν την εξάπλωση όλων των υπό μελέτη ειδών

Risk and Environmental Factors Associated with the Presence of Canine Parvovirus Type 2 in Diarrheic Dogs from Thessaly, Central Greece

Canine parvovirus type 2 (CPV-2) primarily infects dogs, which are the main host reservoir, causing severe gastrointestinal disease associated with immunosuppression. The present study was conducted in Thessaly, Greece and aimed to identify risk and environmental factors associated with CPV-2 infection in diarrheic dogs. Fecal samples were collected from 116 dogs presenting diarrhea and were tested by polymerase chain reaction (PCR) for the presence of CPV-2 DNA. Supplementary data regarding clinical symptoms, individual features, management factors and medical history were also gathered for each animal during clinical evaluation. Sixty-eight diarrheic dogs were found to be positive for the virus DNA in their feces. Statistical analysis revealed that CPV-2 DNA was less likely to be detected in senior dogs, while working dogs, namely hounds and shepherds, had higher odds to be positive for the virus. Livestock density and land uses, specifically the categories of discontinuous urban fabric and of human population density, were identified as significant environmental parameters associated with CPV-2 infection by using Geographical Information System (GIS) together with the Ecological Niche Model (ENM). This is the first description of the environmental variables associated with the presence of CPV-2 DNA in dogs’ feces in Greece

Indication of West Nile Virus (WNV) Lineage 2 Overwintering among Wild Birds in the Regions of Peloponnese and Western Greece

West Nile virus (WNV), a zoonotic mosquito-borne virus, has recently caused human outbreaks in Europe, including Greece. Its transmission cycle in nature includes wild birds as amplifying hosts and ornithophilic mosquito vectors. The aim of this study was to assess WNV circulation among wild birds from two regions of Greece, Peloponnese and Western Greece, during 2022. To this end, a total of 511 birds belonging to 37 different species were sampled and molecularly screened. WNV RNA was detected from February to November in a total of 71 wild birds of nine species originating from both investigated regions. The first eight positive samples were sequenced on a part of NS3 and, according to the phylogenetic analysis, they belonged to evolutionary lineage 2 and presented similarity to previous outbreak-causing Greek strains (Argolis 2017, Macedonia 2010 and 2012). It was more likely to identify a PCR positive bird as the population density and the distance from water sources decreased. The present report provides evidence of WNV occurrence in both Peloponnese and Western Greece during 2022 and underlines its possible overwintering, highlighting the need for avian species surveillance to be conducted annually and throughout the year. Magpies are proposed as sentinels for WNV monitoring

Antimicrobial Resistance Genes in ESBL-Producing Escherichia coli Isolates from Animals in Greece

The prevalence of multidrug resistant, extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is increasing worldwide. The present study aimed to provide an overview of the multidrug resistance phenotype and genotype of ESBL-producing Escherichia coli (E. coli) isolates of livestock and wild bird origin in Greece. Nineteen phenotypically confirmed ESBL-producing E. coli strains isolated from fecal samples of cattle (n = 7), pigs (n = 11) and a Eurasian magpie that presented resistance to at least one class of non β-lactam antibiotics, were selected and genotypically characterized. A DNA-microarray based assay was used, which allows the detection of various genes associated with antimicrobial resistance. All isolates harbored blaCTX-M-1/15, while blaTEM was co-detected in 13 of them. The AmpC gene blaMIR was additionally detected in one strain. Resistance genes were also reported for aminoglycosides in all 19 isolates, for quinolones in 6, for sulfonamides in 17, for trimethoprim in 14, and for macrolides in 8. The intI1 and/or tnpISEcp1 genes, associated with mobile genetic elements, were identified in all but two isolates. This report describes the first detection of multidrug resistance genes among ESBL-producing E. coli strains retrieved from feces of cattle, pigs, and a wild bird in Greece, underlining their dissemination in diverse ecosystems and emphasizing the need for a One-Health approach when addressing the issue of antimicrobial resistance