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 and conservation of the black - crowned night heron, Nycticorax nycticorax nycticorax L., 1758, at the Kerkini reservoir, Macedonia, Greece

The present thesis concerns the ecology of the Black-crowned Night heron(Nycticorax nycticorax). It was worked out through the years 1995-1996 at the Kerkini reservoir of Serres. An artificial lake constructed in order to regulate the floodwater supply of the Strymon River, to control sediment deposition and to provide an irrigation reservoir for the Serres agricultural plain. At Kerkini there can be found one of the largest heronries of the Mediterranean that is facing serious problems mainly due to fluctuation of the water level. Up until 1995 heron breeding came about in two colonies (one in the old riverbed and one in the new riverbed of the Strymon) in which apart from the Black-crowned Night heron, nest Pygmy cormorants, Little egrets, Squacco herons, Spoonbills etc. In 1996 the heronry found on the west bank of the old riverbed was abandoned.Black-crowned Night herons arrive at the wetland in the second third of Marchand roost in the riverside forest, in places that will later on make up the nest sites. The number of breeding pairs varied from 900 to 980 during the years that the study lasted. The laying period begins by the end of March until the middle of April during which the ground has been flooded by water to depths 30 - 50 cm. The nests are built within dense tree vegetation (salix sp.) at a mean height of 2,58 - 3,38 above ground. The mean clutch size varied from 3,56 in 1995 to 3,61 in 1996. Egg losses in A colony in 1995 was 53,19% of egg total while in Β colony the respective percentage varied from 5,20% (1995) to 9,83% (1996). The major cause of egg losses was nestflooding. The mean number of nestlings having survived until the age of 18 days was 2,44 in 1995 and 3,23 in 1996, while the losses in A colony in 1995 was 36,92% and in Β colony they varied from 5,20% (1995) to 14,74 (1996). The nestling losses were mainly due to nest floodings. The lake's water level during 1995 increased gradually up to 36,33 m a.s.I. (27/05) which resulted in a great number of nest floodings. On the contrary in 1996 the lake's water level increased suddenly the first half of April reaching 35,75 m a.s.I. on 15/04 and therefore the Black-crowned Night herons built nests higher up and so had greater breeding success.The first hatched nestlings grew up better than the next. Bill length is suggested as the most suitable measurement to estimate the age of the Black-crowned Nightheron. Black-crowned Night herons feed mainly at night time except during breeding season whereas they feed throughout the daytime. They wait for quite some time hiding in the vegetation or in the lower branches of trees over the water, seeking out their prey while making few attempts to capture it. They hardly move at all and rarely hunt their prey on foot. During breeding season the major foraging sites of the Blackcrowned Night heron are along the banks of the Strymon up to the Vironia bridge and the irrigation channels east of Kerkini and south of water supply Y2, Important foraging sites mainly during summer months are ponds formed by receding water levels and also the banks of the river.The Black-crowned Night heron's diet consists of fish (86,47%) with a predominance of Bleak (Albumus alburnus) (71,47%). Amphibians and insects contribute less to its diet. During breeding season the birds spent most of the day (except hours 12:00 - 18:00) searching for food.Η παρούσα διατριβή αφορά την οικολογία ενός είδους ερωδιού, του νυχτοκόρακα (Nycticorax nycticorax), που εκπονήθηκε τα έτη 1995 - 1996στη λίμνη Κερκίνη του Ν. Σερρών, μία τεχνητή λίμνη που δημιουργήθηκε γιατην αντιμετώπιση των πλημμυρικών παροχών του Στρυμόνα, τη συγκράτησητων φερτών υλών και την άρδευση της πεδιάδας των Σερρών. Στην Κερκίνη βρίσκεται μια από τις μεγαλύτερες αποικίες ερωδιών της Μεσογείου που αντιμετωπίζει σοβαρά προβλήματα που οφείλονται κυρίως στην αυξομείωση της στάθμης του νερού. Έως το 1995 η αναπαραγωγή των ερωδιών εξελισσόταν σε δύο αποικίες (μία στην παλιά κοίτη και μία στην νέα κοίτη του Στρυμόνα) στις οποίες εκτός από τους νυχτοκόρακες φωλιάζουν κορμοράνοι, λαγγόνες, λευκοτσικνίαδες, κρυπτοτσικνιάδες, χουλιαρομύτες κ.λπ. Το 1996 η αποικία που βρισκόταν στην δυτική όχθη της παλαιάς κοίτης του ποταμού εγκαταλείφθηκε.Οι νυχτοκόρακες φθάνουν στον υγρότοπο το δεύτερο δεκαήμερο του Μαρτίου και κουρνιάζουν στο παραποτάμιο δάσος, στις θέσεις που αργότερα θα αποτελέσουν τους χώρους φωλεοποίησης. Μικρός αριθμός ατόμων ξεχειμωνιάζει στην ευρύτερη περιοχή του υγροτόπου. Ο αριθμός των αναπαραγόμενων ζευγαριών κυμάνθηκε, κατά τα δύο έτη που διήρκεσε η έρευνα, από 900 έως 980. Η ωοτοκία ξεκινάει από το τέλος Μαρτίου έως μέσα Απριλίου κι ενώ το έδαφος έχει κατακλυστεί από νερό βάθους 30 - 50 cm. Οι φωλιές κατασκευάζονται στο εσωτερικό της κόμης των δένδρων (salix sp.) σε μέσο ύψος 2,58 - 3,38 m από την επιφάνεια του εδάφους.Ο μέσος αριθμός αβγών ανά φωλιά κυμάνθηκε από 3,56 το 1995 έως3,61 το 1996. Οι απώλειες των αβγών στην αποικία Α το 1995 ήταν 53,19 %επί του συνόλου των αβγών ενώ στην αποικία Β το αντίστοιχο ποσοστό κυμάνθηκε από 5,20% (1995) έως 9,83% (1996). Κυριότερη αιτία απώλειας αβγών ήταν η κατάκλιση των φωλιών.Ο μέσος αριθμός πτερωθέντων νεοσσών ήταν 2,44 το 1995 και 3,23 το1996, ενώ οι απώλειες στην αποικία Α το 1995 ήταν 36,92% και στην αποικία Β κυμάνθηκαν από 5,20% (1995) έως 14,74% (1996). Οι απώλειες νεοσσών οφείλονταν κατά κύριο λόγο στην κατάκλιση των φωλιών.Η στάθμη της λίμνης κατά το 1995 αυξάνονταν σταθερά μέχρι τα 36,33m a.s.Ι. (27/05) με αποτέλεσμα να κατακλυστεί μεγάλος αριθμός φωλιών.Αντίθετα το 1996 η στάθμη της λίμνης αυξήθηκε απότομα το πρώτο δεκαπενθήμερο του Απριλίου φτάνοντας τα 35,75 m a.s.Ι, στις 15/4, οπότε οι νυχτοκόρακες κατασκεύασαν, αργότερα, τις φωλιές τους πιο ψηλά κι έτσι είχαν μεγαλύτερη επιτυχία αναπαραγωγής.Οι πρώτοι σε σειρά εκκόλαψης νεοσσοί αυξάνονταν καλύτερα από τους επόμενους, ενώ το μήκος του ράμφους προτείνεται ως η καταλληλότερη μέτρηση για την εκτίμηση της ηλικίας νεοσσών του νυχτοκόρακα.Οι νυχτοκόρακες τρέφονται κυρίως την νύχτα εκτός της αναπαραγωγικής περιόδου που τρέφονται καθόλη τη διάρκεια της ημέρας.Περιμένουν αρκετή ώρα κρυμμένοι στη βλάστηση ή στα χαμηλά κλαδιά των δέντρων, πάνω από το νερό, αναζητώντας τα θύματα τους ενώ κάνουν λίγες προσπάθειες σύλληψης τους. Μετακινούνται ελάχιστα και σπάνια κυνηγούν τα θύματα τους περπατώντας. Κατά την αναπαραγωγική περίοδο οι πιο κατάλληλες περιοχές διατροφής του νυχτοκόρακα είναι οι όχθες του Στρυμόνα μέχρι το ύψος της γέφυρας της Βυρώνειας και οι αρδευτικές διώρυγες ανατολικά της Κερκίνης και νότια της υδροληψία Υ2. Καλές περιοχές διατροφής, κυρίως κατά τους καλοκαιρινούς μήνες, είναι οι μικρές λίμνες που σχηματίζονται από την υποχώρηση της στάθμης του νερού καθώς και οι όχθες της λίμνης.Η τροφή του νυχτοκόρακα αποτελείται κατά 86,47% από ψάρια με κυρίαρχο είδος το σίρκο (Albumus alburnus) (71,47%). Μικρότερη συμμετοχή στο διαιτολόγιο του έχουν τα αμφίβια και τα έντομα. Κατά τη διάρκεια της αναπαραγωγικής περιόδου τα πουλιά αφιερώνουν το μεγαλύτερο τμήμα της ημέρας (πλην των ωρών 12:00 - 18:00) αναζητώντας την τροφή τους

Winter diet of the Marsh Harrier Circus aeruginosus (Aves, Accipitriformes) in the Evros Delta (Greece)

The diet of the Marsh Harrier (Circus aeruginosus) was studied with the analysis of pellets collected in the Evros Delta. In total, 141 prey items were identified in 86 pellets. In terms of numbers, the diet consisted of 66.7% mammals, 27.7% birds and 5.7% insects. Considering biomass, birds were the most important prey, while mammals made up 36.6% of the diet. The most important species, in terms of biomass, were Sibling Vole (Microtus levis) -31.1%-,Teal (Anas crecca) -17.7%-, Moorhen (Gallinula chloropus) and Black-headed Gull (Chroicocephalus ridibundus) -11.8% each-. The mean estimated prey weight was 36 g., ranging from 1 to 300 g

Islands and hybrid zones: combining the knowledge from “Natural Laboratories” to explain phylogeographic patterns of the European brown hare

Abstract Background The aim of the study was to use hybrid populations as well as island populations of the European brown hare (Lepus europaeus) to explore the effect of evolutionary events, such as the post-deglaciation translocations, spontaneous and human-mediated, local adaptation and the genetic drift in the shaping of the phylogeographic patterns of the species. For this purpose, we used molecular markers, both nuclear and mitochondrial, that are indicative for local adaptation as well as neutral markers to elucidate the patterns of population differentiation based on geographic isolation and the clade of origin. To broaden our analysis, we included data from our previous studies concerning mainland populations, to explore the genetic differentiation in the base of the geographic origin (mainland/island) of the populations. Results Our results suggest that local adaptation shapes the differentiation in both genomes, favoring specific alleles in nuclear genes (e.g. DQA) or haplotypes in mtDNA (e.g. Control Region, CR). mtDNA variation was found to be in a higher level and was able to give a phylogeographic signal for the populations. Furthermore, the degree of variation was influenced not only by the geographic origin, but also by the clade of origin, since specific island populations of Anatolian origin showed a greater degree of variation compared to specific mainland populations of the European clade. Concerning the hybrid population, we confirmed the existence of both clades in the territory and we provided a possible explanation for the lack of introgression between the clades. Conclusion Our results indicate that the Quaternary’s climatic oscillations played a major role in the shaping of the phylogeographic patterns of the species, by isolating populations in the distinct refugia, where they adapted and differentiate in allopatry, leading to genome incompatibilities observed nowadays

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

West Nile Virus Occurrence and Ecological Niche Modeling in Wild Bird Species and Mosquito Vectors: An Active Surveillance Program in the Peloponnese Region of Greece

West Nile Virus (WNV) is maintained in nature in a bird-mosquito cycle and human infections follow a seasonal pattern, favored by climatic conditions. Peloponnese Region, located in Southern Greece, initiated an active WNV surveillance program to protect public health during 2019–2020. The project included monitoring of avian hosts and mosquito vectors, while sampling locations were prioritized after consideration of WNV circulation in birds, mosquitos and humans during previous seasons. Biological materials were collected from 493 wild birds of 25 species and 678 mosquito pools, which were molecularly screened for WNV presence. In this case, 14 environmental variables were associated with WNV detection in wild birds and mosquitos by using two separate MaxEnt models. Viral RNA was not detected in the target species during 2019, although in 2020, it was reported on 46 wild birds of ten species and 22 mosquito pools (Culex pipiens and Aedes albopictus). Altitude and land uses were significant predictors for both models and in fact, suitable conditions for virus occurrence were identified in low altitude zones. Bird- and mosquito-based surveillance systems yielded similar results and allowed for targeted vector control applications in cases of increased virus activity. Human cases were not reported on Peloponnese in 2020