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Toxoplasmosis, due to Toxoplasma gondii, is a parasitic disease with global importance. Among livestock, chronic T. gondii infection has been reported in higher rates in pigs and small ruminants, b...

In human‐dominated landscapes, connectivity is crucial for maintaining demographically stable mammalian populations. Here, we provide a comprehensive noninvasive genetic study for the brown bear population in the Hellenic Peninsula. We analyze its population structuring and connectivity, estimate its population size throughout its distribution, and describe its phylogeography in detail for the first time. Our results, based on 150 multilocus genotypes and on 244‐bp sequences of the mtDNA control region, show the population is comprised by three highly differentiated genetic clusters, consistent with geographical populations of Pindos, Peristeri, and Rhodope. By detecting two male bears with Rhodopean ancestry in the western demes, we provide strong evidence for the ongoing genetic connectivity of the geographically fragmented eastern and western distributions, which suggests connectivity of the larger East Balkan and Pindos‐Dinara populations. Total effective population size (Ne) was estimated to be 199 individuals, and total combined population size (NC) was 499, with each cluster showing a relatively high level of genetic variability, suggesting that migration has been sufficient to counteract genetic erosion. The mtNDA results were congruent with the microsatellite data, and the three genetic clusters were matched predominantly with an equal number of mtDNA haplotypes that belong to the brown bear Western mitochondrial lineage (Clade 1), with two haplotypes being globally new and endemic. The detection of a fourth haplotype that belongs to the Eastern lineage (Clade 3a1) in three bears from the western distribution places the southernmost secondary contact zone between the Eastern and Western lineages in Greece and generates new hypotheses about postglacial maxima migration routes. This work indicates that the genetic composition and diversity of Europe's low‐latitude fringe population are the outcome of ancient and historical events and highlight its importance for the connectivity and long‐term persistence of the species in the Balkans.

The aim of this study was to investigate the occurrence of Bartonella spp, Brucella spp, Coxiella burnetii, and Francisella tularensis in European Brown hares (Lepus europaeus) hunter harvested dur...

European brown hare (Lepus europaeus, EBH) is probably the most important game animal in Europe throughout its historical distribution. The decline in its populations across its geographic range in Europe have been attributed to factors such as reproductive rate and the ability for adaptation, climate, feed availability, predators, anthropogenic factors and diseases. Apart from common diseases of hares with a high impact on their mortality such as European Brown hare Syndrome, EBH has been involved in the epidemiology of pathogens with zoonotic potential. In this work, the role of EBH as a source of Crimean–Congo haemorrhagic fever virus (CCHFV), Hepatitis E virus (HEV), Yersinia spp., Brucella spp., Francisella tularensis, Toxoplasma gondii and Leishmania infantum is discussed. Hares may significantly contribute to the epidemiology of important emerging zoonotic pathogens through maintenance of high endemicity levels as in the case of CCHFV, as a reservoir of important pathogens such as Yersinia spp., B. suis, F. tularensis and L. infantum and as a potential source of T. gondii for other animals, especially for carnivores but also for humans. However, EBH may also be a host of minor importance as in the case of HEV. The continuous surveillance of hare populations will enable the collection of information on the population health status and the pathogens currently circulating in the area posing risk for wildlife, domestic animals and humans. The possible live animal translocations of infected hares, the fact that this species acts as a host of vectors (fleas, ticks, mosquitoes and sandflies) and the prey of carnivores and omnivores that travel in great distances getting into contact with domestic animals and humans, further highlights the need to be included in surveillance studies. Besides, the hunter‐harvested EBH population is an excellent indicator for recent pathogen transmission due to its short lifespan., The present review provides an overview of the role of European Brown Hare as a source of Crimean–Congo haemorrhagic fever virus (CCHFV), Hepatitis E virus (HEV), Yersinia spp., Brucella spp., Francisella tularensis, Toxoplasma gondii and Leishmania infantum aiming to highlight the contribution of this species to the epidemiology of important emerging zoonotic pathogens.

Η 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.

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.

We report the full polyprotein genomic sequence of a West Nile virus strain isolated from Eurasian magpies dying with neurologic signs in Greece. Our findings demonstrate the local genetic evolution of the West Nile virus strain responsible for a human disease outbreak in the country that began in 2010.

The occurrence of infection or exposure to Toxoplasma gondii, Neospora caninum, and Leishmania infantum was investigated in European brown hares (Lepus europaeus, EBH) hunter-harvested over two consecutive hunting seasons in northern and central Greece. Geographical information system was used along with the ecological niche model to define the geographical distribution of seropositive hares relative to environmental parameters and to identify high-risk areas for hare exposure. Molecular analysis showed that 3.8% and 9.6% of the examined hares were infected with N. caninum and L. infantum, respectively, while, 5.7%, 0.95%, and 12.4% of the hares tested positive for the presence of antibodies against T. gondii, N. caninum, and L. infantum respectively. None of the examined hares was polymerase chain reaction-positive for T. gondii. Mixed exposure against both T. gondii and L. infantum was found in 2.9% of the hares examined. Rainfall indices and land uses significantly influenced the exposure of hares to T. gondii and L. infantum. This is the first molecular and serological survey of protozoan pathogens in EBH in Greece. Furthermore, we report the environmental parameters related to hare seropositivity and present a risk map for hare exposure to T. gondii and L. infantum in northern and central Greece. The ecological niches of T. gondii and L. infantum in the hares presented herein could be applied to other regions with similar environmental and climatic conditions.

Understanding the feeding habits of wolves is essential for designing and implementing fundamental management processes across the range of the species. This is even more important within human-dominated areas, such as southern Europe, and more especially Greece. In this context, we analyzed 123 scat samples, collected between 2010 and 2012, from a mixed agricultural, forested and human-dominated area, centered on the municipality of Domokos in central continental Greece. We used standard laboratory procedures for scat analysis and calculated percentages of frequency of occurrence (FO%), average volume (AV%) and biomass index (BM%) to assess diet composition, and estimated prey selectivity. Domestic prey composed the bulk of wolf diet (FO%=73.5, AV%=84.8, BM%=97.2), wild ungulates were almost absent (FO%=0.5, AV%=0.8, BM%=1.2), whereas grass consumption was high in our area (FO%=19.5, AV%=11.0). The high dependence on livestock corroborates previous studies from Greece and other countries in southern Europe. Goat (FO%=46.0, AV%=61.2, BM%=64.9) was the main prey and was strongly selected, with sheep (FO%=11.5, AV%=9.0, BM%=11.2), pig carrion and cattle ranking behind (FO%=11.5, AV%=10.1, BM%=8.7 and FO%=4.5, AV%=4.5, BM%=12.4, respectively). No differences across seasons were detected, except from pig carrion, which increased during winter. The preference for goats is probably associated with its grazing behavior. High livestock consumption generally results in increased human-wolf conflict. Thus, substantial improvement of husbandry practices and restoration of wild ungulate populations are recommended to facilitate wolf-human coexistence in Greece.

Objective was to recognise areas potentially of high risk for increased frequency of subclinical mastitis in ewes. Milk samples were collected, for bacteriological and cytological examination, from 2198 clinically healthy ewes in 111 farms in all administrative regions of Greece. Data on farms were located in the field using hand-held Global Positioning System Garmin units. Collected data were analysed by an Ecological Niche Model under the framework of a geographic information system. Two separate analyses were performed: one for subclinical mastitis independently of causal agent (prevalence in population sampled: 0.260) and one for subclinical mastitis caused specifically by slime-producing staphylococci (prevalence in population sampled: 0.153). A model was constructed in which sheep farms were divided into two clusters, according to prevalence of subclinical mastitis: farms in the upper three quartiles of prevalence were used as occurrence points for the Ecological niche modelling procedure (‘infected farms’); farms in the lower quartile of prevalence within each category were (pseudo)negative points. Significant differences in environmental parametres prevailing in locations of farms into the study, were identified for up to 13 parametres between locations of farms according to management system applied in farms. When farms in each management system were considered separately, differences became evident between farms in each management system, as well as between the two infections. The factor with the highest relative contribution in the analyses was the distance from other sheep farms; other factors also of importance in the predictive models were the altitude, the maximum temperature of warmest month and the total precipitation of driest month. Verification of the model revealed that ≥ 0.760 of infected farms’ were located in areas predicted as high risk for prevalence of subclinical mastitis or slime staphylococcal subclinical mastitis. The paper describes for the first time potential association of mastitis with environmental factors and presents predictive models for mastitis in ewes taking into account environmental parametres.

Resistance mediated by β-lactamases is a globally spread menace. The aim of the present study was to determine the occurrence of Escherichia coli producing plasmid-encoded AmpC β-lactamases (pAmpC) in animals. Fecal samples from chickens (n = 159), cattle (n = 104), pigs (n = 214), and various wild bird species (n = 168), collected from different Greek regions during 2018–2020, were screened for the presence of pAmpC-encoding genes. Thirteen E. coli displaying resistance to third-generation cephalosporins and a positive AmpC confirmation test were detected. blaCMY-2 was the sole pAmpC gene identified in 12 chickens’ and 1 wild bird (Eurasian magpie) isolates and was in all cases linked to an upstream ISEcp1-like element. The isolates were classified into five different sequence types: ST131, ST117, ST155, ST429, and ST1415. Four chickens’ stains were assigned to ST131, while five chickens’ strains and the one from the Eurasian magpie belonged to ST117. Seven pAmpC isolates co-harbored genes conferring resistance to tetracyclines (tetM, tetB, tetC, tetD), 3 carried sulfonamide resistance genes (sulI and sulII), and 10 displayed mutations in the quinolone resistance-determining regions of gyrA (S83L+D87N) and parC (S80I+E84V). This report provides evidence of pAmpC dissemination, describing for the first time the presence of CMY-2 in chickens and wild birds from Greece.

The objective of this work was to develop, use and present a detailed questionnaire for the evaluation of health management in dairy small ruminants, it includes 442 questions organised in seven sections: general, infrastructure, animals, production characteristics, health management, nutrition, human resources. Consistency of replies was evaluated in 27 farmers, interviewed twice. Inconsistent replies were given by all farmers to 30 different questions (Cronbach&rsquo, s coefficient alpha: 0.987). Then, interviews were performed in 444 farms around Greece. Mean duration of an interview was 63.6 min. Clarifications were requested by 273 farmers to 22 different questions (maximum per farmer: 8). The experience of the investigator, the primary language of farmers and asking clarifications by the farmers affected the duration of the interview. The questionnaire can be used for research work in the field, to record details in the farms under study. In accord with the needs of a particular study, it can be modified, by adding more specific questions or omitting others deemed of less importance. Moreover, it can also be used for routine monitoring purposes, as a useful means to record and maintain details of farms during clinical work. To the best of our knowledge, the questionnaire is the most extensive and detailed one available internationally for dairy small ruminants.

Understanding of the ecology of infected animals facilitates disease risk assessment and is also crucial for wildlife conservation. Relatively little is known about the spatial distribution of infected wild mammals in relation to environmental factors. In neighboring Mediterranean ecosystems 250 European brown hares (Lepus europaeus) were collected and examined with RT-PCR to detect European Brown Hare Syndrome Virus (EBHSV). Multivariate statistics and Geographical Information System (GIS) analysis were applied to estimate spatial patterns of biotic and abiotic factors and human activities as determinants of EBHSV positivity. Hare population abundance was estimated using faeces counts and belt drive censuses. The study showed that EBHSV infected hares had widespread distribution even in isolated areas. However, EBHSV infection prevalence was higher in areas with higher hare abundance, closer to paved road networks and at lower altitudes. The risk map revealed the potential distribution of EBHSV-infected hares. This study shows that host abundance and landscape influence the ecology of the disease, a finding that should be taken into account in future studies. The management of harvest and restocking of hares is also discussed for population conservation.

Seroprevalence of Coxiella burnetii in sheep and goats in central Greece was investigated. Objectives were: (i) to describe spatial distribution of sheep and goat exposure to C. burnetii in the study area, (ii) to identify environmental parametres related to C. burnetii exposure and (iii) to recognise high risk areas for sheep and goat exposure to C. burnetii . Blood samples were collected from sheep (n = 320) or goats (n = 80) in 20 farms, i.e. from 20 animals in each farm. Samples were examined for presence of antibodies against C. burnetii by using a commercial iELISA kit. Data on farms were located in the field using hand-held Global Positioning System Garmin units. Collected data were analysed by an Ecological Niche Model under the framework of a geographic information system. Nine sheep flocks and 2 goat herds were found to be ‘ C. burnetii positive’ (55%) with C. burnetii antibodies detected in total in 46 sheep and 12 goats (14.5%). ‘ C. burnetii positive’ farms were located in mean altitude of 282 m and mean distance from settlements of 919 m. The environmental variable with highest gain when used alone was animal density; the variable that decreased gain the most when omitted, was distance from farms. As areas with probability of >70% for exposure to C. burnetii according to the MaxEnt model, were recognised the central plain of Karditsa and Trikala regional units, located in low altitude zones and in irrigated and cultivated agricultural areas and pastures; in total, 37 human settlements are located in high-risk areas.

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”.

Highlights • An extensive outbreak of bluetongue occurred in Greece in 2014. • Increased morbidity and fatality rates were recorded in sheep. • Clinical anaemia, a rarely seen clinical sign, was recorded in affected sheep. • Virus was detected in wild cervids., Objective of this paper was to review relevant work and to present a general account of the bluetongue outbreak, which occurred in Greece in 2014. In total, 2895 outbreaks of the disease have been reported by the veterinary authorities of Greece; sheep, goats and cattle were affected with officially reported morbidity rates of 11.0%, 2.0% and 3.5%, respectively. No vaccinations were allowed and conservative measures were implemented to attempt to limit the disease, which at the end had expanded throughout the country. In field investigations, a significantly higher bluetongue morbidity rate (27.5%) in sheep has been reported. During that work, clinical anaemia was encountered, which was characterised as macrocytic, hypochromic, regenerative and non-haemolytic. Other investigations, which are reviewed in this paper, have described an outbreak of Citrobacter freundii-associated enteritis in newborn kids, offspring of goats subclinically infected with Bluetongue virus, increased rate of early embryonic deaths, reduced conception rates, increased incidence risk of mastitis and reduced milk yield in herds of subclinically-infected cattle and detection of the virus from hunter-harvested tissue samples of roe-deer. In 2015, vaccines against the disease have been licenced; vaccinations started in May 2015. Then, in 2015, only one outbreak of the disease was confirmed, which could have been the result of a combination of reasons acting concurrently to prevent further cases.

SUMMARYThessaly, Central Greece, is an endemic area for leishmaniasis with higher incidence rate during the last years. We herein investigated the geographical distribution of human leishmaniasis cases and Leishmania infected dogs in relation to environmental parameters to identify high-risk areas. All the human leishmaniasis cases (n = 82) reported to Hellenic Centre for Disease Control and Prevention from 2007 to 2014 and 85 Leishmania polymerase chain reaction positive dogs were included in this study. To analyse the data geographical information system (GIS) together with the Ecological Niche Model (ENM) were used. The most important findings of the study were: (i) Central plain of Thessaly together with the coast line and the western and eastern lowlands were identified as high-risk geographical areas. (ii) The highest percentage of the high-risk areas was found in low altitude (

Ecosystem disturbances, such as wildfires, are driving forces that determine ecology and conservation measures. Species respond differentially to wildfires, having diverse post-fire population evolution. This study reports, for first time, the responses of brown hare (Lepus europaeus Pallas, 1778) to wildfires. Hare relative abundance, age ratio, diet quality, body condition, and diseases were studied. Fire influence on vegetation was calculated at a micro-scale level. Hare abundance was lower the first year after wildfires in burned relative to unburned areas. The reverse was found in the second year when hare abundance was higher in burned areas. Hare abundance in burned areas was also higher in the third and fourth years. In the fifth and sixth years after wildfire no significant difference was found in abundance. At a micro-scale level, higher numbers of hare feces were counted in places with greater wildfire influence on vegetation. Age ratio analysis revealed more juveniles in burned areas, but the same number of neonates in burned and unburned areas, indicating lower mortality of juveniles in burned areas. Reduced predation in burned areas provides the most plausible explanation for our findings.

Objectives of the work presented herewith were to investigate association of prevalence of subclinical mastitis with environmental (climatic and topographic) factors and to identify factors potentially predisposing ewes to the disease. Milk samples were collected from 2198 sheep in 111 farms, in all 13 administrative regions of Greece, for bacteriological and cytological examination. Data on farm location were collected in the field using hand-held Global Positioning System Garmin units. The geo-references were resolved to specific farm level. Prevalence of subclinical mastitis was 0.260. Main aetiological agents were staphylococci (Staphylococcus aureus and coagulase-negative species), which accounted for 0.699 of all isolates recovered. In a multivariable mixed-effects analysis, the two environmental variables found to be associated with increased prevalence of subclinical mastitis were the minimum temperature of coldest month (coefficient: -0.084 ± 0.033, P = 0.014) and the mean temperature for 30 days prior to sampling date (coefficient: 0.031±0.014, P = 0.029).

In human‐dominated landscapes, connectivity is crucial for maintaining demographically stable mammalian populations. Here, we provide a comprehensive noninvasive genetic study for the brown bear population in the Hellenic Peninsula. We analyze its population structuring and connectivity, estimate its population size throughout its distribution, and describe its phylogeography in detail for the first time. Our results, based on 150 multilocus genotypes and on 244‐bp sequences of the mtDNA control region, show the population is comprised by three highly differentiated genetic clusters, consistent with geographical populations of Pindos, Peristeri, and Rhodope. By detecting two male bears with Rhodopean ancestry in the western demes, we provide strong evidence for the ongoing genetic connectivity of the geographically fragmented eastern and western distributions, which suggests connectivity of the larger East Balkan and Pindos‐Dinara populations. Total effective population size (N e) was estimated to be 199 individuals, and total combined population size (N C) was 499, with each cluster showing a relatively high level of genetic variability, suggesting that migration has been sufficient to counteract genetic erosion. The mtNDA results were congruent with the microsatellite data, and the three genetic clusters were matched predominantly with an equal number of mtDNA haplotypes that belong to the brown bear Western mitochondrial lineage (Clade 1), with two haplotypes being globally new and endemic. The detection of a fourth haplotype that belongs to the Eastern lineage (Clade 3a1) in three bears from the western distribution places the southernmost secondary contact zone between the Eastern and Western lineages in Greece and generates new hypotheses about postglacial maxima migration routes. This work indicates that the genetic composition and diversity of Europe's low‐latitude fringe population are the outcome of ancient and historical events and highlight its importance for the connectivity and long‐term persistence of the species in the Balkans., Using microsatellites and mtDNA, we reveal the genetic structure, diversity, population size of the brown bear in Greece, and its detailed phylogeography for the first time, revealing a new matrilinear contact zone

Epidemiological studies of disease ecology typically ignore the influence of host sex, age, nutritional condition and immunocompetence factors. However, this can lead to shortcomings and incorrect conclusions regarding the mechanisms of pathogen transmission and prevalence in wild animals. In many European countries, European Brown Hare Syndrome Virus (EBHSV) causes a disease lethal to brown hares Lepus europaeus. Associations of EBHSV infection, sex, age, body condition and spleen mass were studied in hares collected, from the prefectures of Chalkidiki and Thessaloniki, Hellas, during the hunting season. Data showed a sex-biased prevalence with twice as many males infected with EBHSV than females (P = 0.061), indicating a sexual dimorphism in disease exposure or susceptibility. EBHSV infection was not related to hare body condition (P = 0.853). Adults had significantly greater spleen mass than young hares (P = 0.003), indicating past infections. Significantly greater spleen mass was found in hares positive for EBHSV (P = 0.010) and a negative relation was found between spleen mass and body condition (P = 0.038), indicating energy consumption for immune defense investment. The present study is one of the few that examine the relevance of individual risk factors in understanding patterns of viral infections in natural populations of wild animals.

Bluetongue is a constant threat to ruminants, affecting primarily the sheep industry. Bluetongue Virus, the causative agent, has a worldwide distribution and a large number of antigenically different serotypes (at least 26), with vector-borne transmission. All these make effective control measures difficult to implement. Expansion of the virus to Northern Europe changed the epidemiology of the disease and highlighted the existence of hitherto unknown vectors and susceptible hosts. A possible epidemiological role of wildlife as hosts of the virus must be considered, complicating the control strategies. In this review, facts regarding the disease in Europe and its impact in wildlife are presented; concerns are also discussed regarding implication of wild ruminants in surveillance of the disease and formulation of novel control strategies. Further, preliminary results of a study into the presence of Bluetongue Virus in cervids in Greece, performed during the outbreak currently (2014–15) prevailing in the country, have indicated (by using molecular techniques) presence of the virus in 3 of 19 samples from roe deer in areas adjoining domestic ruminant farms. It is concluded that wild ruminants should be included in surveillance programs and in strategies for controlling Bluetongue Virus infections in a region.

In this study, we compare the degree of antibiotic resistance between the E. coli strains isolated from swine faecal and farm workers. Resistance in nine widely used antibiotics was assessed by the determination of Minimum Inhibitory Concentration. Sixty-four out of 72 E. coli isolates derived from pigs and sixty out of 72 derived from farm workers showed resistance to at least one antibiotic. High resistance in tetracycline, ampicillin and amoxicillin was detected in both human and swine E. coli isolates, showing similar resistance patterns. This finding indicates that the use of antibiotics in pig industry and the increased antibiotic resistance of animal E. coli isolates, also affects the resistance of isolates collected from farm workers, at least in the area of study.

Wildlife managers are challenged with the task of deciding whether a management measure is appropriate or not, and furthermore they have to convince others about the merits of their decision. Population decline of some hare species (genus Lepus) has resulted in their Red Listing and conservation measures are being undertaken. Release or restocking is a frequent measure in some countries, and thousands of hares are released annually, mainly for hunting purposes. These hares can be obtained by either intensive or extensive rearing or translocation of the wild animals. Each method entails difficulties and different survival rates in the wild. Survival rates in the wild are low for hares intensively reared in cages but are higher for hares reared extensively in enclosures and wild translocated hares. The benefits of the hare release practice are significantly lower than the action's implementation cost. Hare releases have not increased significantly the wild hare population or the hunting harvest in areas where the practice has been applied. The risk of genetic and evolutionary degradation and pathogen transmission is possible in wild populations. The need for wise management of this practice is evident and the term 'Permitted Transferring Units' should be introduced to denote regions where hares should not be transferred for rearing and release.

During the last three years Greece is experiencing the emergence of West Nile virus (WNV) epidemics. Within this framework, an integrated surveillance and control programme (MALWEST project) with thirteen associate partners was launched aiming to investigate the disease and suggest appropriate interventions. One out of seven work packages of the project is dedicated to the State of the Art report for WNV. Three expert working groups on humans, animals and mosquitoes were established. Medical databases (PubMed, Scopus) were searched together with websites: e.g., WHO, CDC, ECDC. In total, 1,092 relevant articles were initially identified and 258 of them were finally included as references regarding the current knowledge about WNV, along with 36 additional sources (conference papers, reports, book chapters). The review is divided in three sections according to the fields of interest: (1) WNV in humans (epidemiology, molecular characteristics, transmission, diagnosis, treatment, prevention, surveillance); (2) WNV in animals (epidemiological and transmission characteristics concerning birds, horses, reptiles and other animal species) and (3) WNV in mosquitoes (control, surveillance). Finally, some examples of integrated surveillance programmes are presented. The introduction and establishment of the disease in Greece and other European countries further emphasizes the need for thorough research and broadening of our knowledge on this viral pathogen.

SUMMARYThe objectives of this work were (i) geographical analysis of the 2012–2014 outbreak of rabies in Greece using GIS and (ii) comparative analysis of animal cases with data of potential human exposure to rabies together with environmental data, in order to provide information for risk assessment, effective monitoring and control. Most animal cases (40/48) involved red foxes, while domestic animals were also diagnosed with rabies. Overall, 80% of the cases were diagnosed in central northern Greece; 75% of the cases were diagnosed in low altitudes (2. Rabies re-occurrence in Greece emphasizes the need for ongoing surveillance in cross-border areas and in areas with intense human activity.

Highlights • Rotavirus was detected in samples from lambs with diarrhoea. • Salmonella enterica subsp. diarizonae was isolated in samples from lambs with diarrhoea. • Antibiotic resistance was detected in >60% of Escherichia coli strains isolated in the study. • Escherichia coli with identical antibacterial resistance were detected in lambs and puppies., Objectives of the present work were (i) to confirm pathogens implicated in cases of diarrhoea in newborn and young lambs in sheep farms in Greece and (ii) to investigate a possible relation in dissemination of pathogens between lambs and dogs present in the farm. Work was carried out in 22 sheep farms, with (i) flock size over 150 animals, (ii) presence of clinical signs of diarrhoea in lambs in the flock and (iii) close and continuous contact and movement of shepherd dogs within the animal shed of each farm. Faecal sample collection from lambs was performed within 48 h of onset of clinical signs and prior to administration of any antimicrobial or antiparasitic medication to lambs. Faecal samples were also collected from puppies in the farm. In total, samples were collected from 126 lambs and 58 puppies. Samples were processed by using established techniques for isolation of bacteria, detection of viruses and observation of protozoan oocycts. Escherichia coli isolates obtained during the study, were tested for antimicrobial resistance against a variety of antimicrobial agents. In total, 236 bacterial isolates were recovered from faecal samples of lambs and 165 isolates from faecal samples of puppies. E. coli was the most frequently isolated microorganism: 104 isolates from lambs and 109 isolates from puppies were recovered. Other bacteria isolated were Enterobacter spp., Proteus spp., Klebsiella spp., (lambs and puppies), Clostridium perfringens, Citrobacter freundi, Salmonella enterica subsp. diarizonae (only lambs) and Streptococcus spp. (only puppies). Group A Rotavirus was detected in samples from lambs (2.5%) and Parvovirus in samples from puppies (5%). Cryptosporidium spp. oocysts were observed in samples from lambs and puppies. This is the first report of isolation of S. enterica subsp. diarizonae and of detection of Rotavirus from lambs in Greece. Rates of E. coli isolates from puppies resistant to antimicrobial agents were, in general, smaller than respective rates in isolates from lambs. Two pairs of isolates from the same farm (one from a lamb and one from a puppy) with identical patterns of resistance to antimicrobial agents were detected, which provides some evidence in support of a hypothesis that members of each pair might possibly have been spread from one animal species to the other.

A major number of West Nile virus (WNV) infections in humans occurred in 2010 in northern Greece, with 262 laboratory confirmed cases. In 2011, fewer cases were reported, but the pattern was more dispersed throughout the Greek mainland. Isolated strains were similar to lineage 2 strains detected in previous years in Austria and Hungary from birds of prey. We conducted a serological surveillance study on hunter-harvested wild birds, to determine possible exposure of avian species during the current outbreak. Serum samples from a total of 113 Eurasian magpies and 85 turtle doves (abundant resident and migratory avian species, respectively, with potential roles in WNV epidemiology) were tested. These birds were hunter-harvested during 2011 from various prefectures both affected and not affected by the WNV outbreak in Greece. Sera were tested for the presence of WNV IgG antibodies by indirect immunofluorescence assay (IFA). Verification of positive results by a micro-virus neutralization test (VNT) was also performed. A total of 23 out of 113 (20.4%) Eurasian magpies and 6/85 (7.1%) turtle doves were found positive. Results showed association of human cases with wild birds’ exposure to the virus; no avian sera were found positive in prefectures not affected by the WNV outbreak. In contrast, positive avian sera were found in every prefecture that human WNV cases occurred in 2011. High seroprevalence in Eurasian magpies suggests high activity of WNV in the areas. Findings of past exposure of migratory birds like turtle doves to WNV upon their arrival in resting areas in Greece suggest various avian species with similar migration traits as target species for viral isolation studies, as they can be considered candidates for the introduction of WNV lineage 2 in Greece from Central Europe.

The brown bear has proved a useful model for studying Late Quaternary mammalian phylogeography. However, information is lacking from northern continental Eurasia, which constitutes a large part of the species' current distribution. We analysed mitochondrial DNA sequences (totalling 1943 bp) from 205 bears from northeast Europe and Russia in order to characterize the maternal phylogeography of bears in this region. We also estimated the formation times of the sampled brown bear lineages and those of its extinct relative, the cave bear. Four closely related haplogroups belonging to a single mitochondrial subclade were identified in northern continental Eurasia. Several haplotypes were found throughout the whole study area, while one haplogroup was restricted to Kamchatka. The haplotype network, estimated divergence times and various statistical tests indicated that bears in northern continental Eurasia recently underwent a sudden expansion, preceded by a severe bottleneck. This brown bear population was therefore most likely founded by a small number of bears that were restricted to a single refuge area during the last glacial maximum. This pattern has been described previously for other mammal species and as such may represent one general model for the phylogeography of Eurasian mammals. Bayesian divergence time estimates are presented for different brown and cave bear clades. Moreover, our results demonstrate the extent of substitution rate variation occurring throughout the phylogenetic tree, highlighting the need for appropriate calibration when estimating divergence times.

Although the existence of a sylvatic transmission cycle of Leishmania spp., independent from the domestic cycle, has been proposed, data are scarce on Leishmania infection in wild mammals in Greece. In this study, we aimed to investigate the presence of Leishmania infection in the European brown hare in Greece, to infer the phylogenetic position of the Leishmania parasites detected in hares in Greece, and to identify any possible correlation between Leishmania infection in hares with environmental parameters, using the geographical information system (GIS). Spleen samples from 166 hares were tested by internal transcribed spacer-1 (ITS-1)-nested PCR for the detection of Leishmania DNA. Phylogenetic analysis was performed on Leishmania sequences from hares in Greece in conjunction with Leishmania sequences from dogs in Greece and 46 Leishmania sequences retrieved from GenBank. The Leishmania DNA prevalence in hares was found to be 23.49 % (95 % confidence interval (CI) 17.27-30.69). The phylogenetic analysis confirmed that the Leishmania sequences from hares in Greece belong in the Leishmania donovani complex. The widespread Leishmania infection in hares should be taken into consideration because under specific circumstances, this species can act as a reservoir host. This study suggests that the role of wild animals, including hares, in the epidemiology of Leishmania spp. in Greece deserves further elucidation.

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.

Background A West Nile virus (WNV) disease outbreak occurred in 2010 in northern Greece with a total of 262 laboratory-confirmed human cases and 35 deaths. A serological and molecular surveillance was conducted on samples of hunter-harvested wild birds prior to and during the outbreak. Findings Serum and tissue samples from 295 resident and migratory wild birds, hunter-harvested during the 2009–2010 and 2010–2011 hunting seasons at the epicenter of the outbreak in northern Greece, were tested for the presence of WNV-specific antibodies by immunofluorescence assay and virus neutralization test. WNV neutralizing antibodies were detected in 53 avian samples. Fourteen positive sera were obtained from birds hunter-harvested up to 8 months prior to the human outbreak. Specific genetic determinants of virulence (His249Pro NS3 mutation, E-glycosylation motif) were recognized in a WNV lineage 2 strain isolated from a hunter-harvested Eurasian magpie and a nucleotide mismatch was revealed between this strain and a mosquito WNV strain isolated one month earlier in the same area. Conclusions This is the first report regarding exposure of wild birds to WNV prior to the 2010 outbreak, in Greece. Results provide evidence of the implication of wild birds in a local enzootic cycle that could allow maintenance and amplification of the virus before and during the outbreak. Findings of past exposure of migratory birds to WNV upon their arrival in Greece during autumn migration, suggest avian species with similar migration traits as candidates for the introduction of WNV into Greece. The possibility that an endemic circulation of WNV could have caused the outbreak, after an amplification cycle due to favorable conditions cannot be excluded.

The brown bear has proved a useful model for studying Late Quaternary mammalian phylogeography. However, information is lacking from northern continental Eurasia, which constitutes a large part of the species' current distribution. We analysed mitochondrial DNA sequences (totalling 1943 bp) from 205 bears from northeast Europe and Russia in order to characterize the maternal phylogeography of bears in this region. We also estimated the formation times of the sampled brown bear lineages and those of its extinct relative, the cave bear. Four closely related haplogroups belonging to a single mitochondrial subclade were identified in northern continental Eurasia. Several haplotypes were found throughout the whole study area, while one haplogroup was restricted to Kamchatka. The haplotype network, estimated divergence times and various statistical tests indicated that bears in northern continental Eurasia recently underwent a sudden expansion, preceded by a severe bottleneck. This brown bear population was therefore most likely founded by a small number of bears that were restricted to a single refuge area during the last glacial maximum. This pattern has been described previously for other mammal species and as such may represent one general model for the phylogeography of Eurasian mammals. Bayesian divergence time estimates are presented for different brown and cave bear clades. Moreover, our results demonstrate the extent of substitution rate variation occurring throughout the phylogenetic tree, highlighting the need for appropriate calibration when estimating divergence times.