Phenotypic Investigation and Detection of Biofilm-Associated Genes in Acinetobacter baumannii Isolates, Obtained from Companion Animals.

Bacteria of the genus Acinetobacter, especially Acinetobacter baumannii (Ab), have emerged as pathogens of companion animals during the last two decades and are commonly associated with hospitalization and multidrug resistance. A critical factor for the distribution of relevant strains in healthcare facilities, including veterinary facilities, is their adherence to both biotic and abiotic surfaces and the production of biofilms. A group of 41 A. baumannii isolates obtained from canine and feline clinical samples in Greece was subjected to phenotypic investigation of their ability to produce biofilms using the tissue culture plate (TCP) method. All of them (100%) produced biofilms, while 23 isolates (56.1%) were classified as strong producers, 11 (26.8%) as moderate producers, and 7 (17.1%) as weak producers. A correlation between the MDR and XDR phenotypes and weak or moderate biofilm production was identified. Moreover, the presence of four biofilm-associated genes bap, blaPER, ompA, and csuE was examined by PCR, and they were detected in 100%, 65.9%, 97.6%, and 95.1% of the strains respectively. All isolates carried at least two of the investigated genes, whereas most of the strong biofilm producers carried all four genes. In conclusion, the spread and persistence of biofilm-producing Ab strains in veterinary facilities is a matter of concern, since they are regularly obtained from infected animals, indicating their potential as challenging pathogens for veterinarians due to multidrug resistance and tolerance in conventional eradication measures. Furthermore, considering that companion animals can act as reservoirs of relevant strains, public health concerns emerge.

Phenotypic Investigation of Florfenicol Resistance and Molecular Detection of floR Gene in Canine and Feline MDR Enterobacterales.

Florfenicol is a promising antibiotic for use in companion animals, especially as an alternative agent for infections caused by MDR bacteria. However, the emergence of resistant strains could hinder this potential. In this study, florfenicol resistance was investigated in a total of 246 MDR Enterobacterales obtained from canine and feline clinical samples in Greece over a two-year period (October 2020 to December 2022); a total of 44 (17,9%) florfenicol-resistant strains were recognized and further investigated. Most of these isolates originated from urine (41.9%) and soft tissue (37.2%) samples; E. coli (n = 14) and Enterobacter cloacae (n = 12) were the predominant species. The strains were examined for the presence of specific florfenicol-related resistance genes floR and cfr. In the majority of the isolates (31/44, 70.5%), the floR gene was detected, whereas none carried cfr. This finding creates concerns of co-acquisition of plasmid-mediated florfenicol-specific ARGs through horizontal transfer, along with several other resistance genes. The florfenicol resistance rates in MDR isolates seem relatively low but considerable for a second-line antibiotic; thus, in order to evaluate the potential of florfenicol to constitute an alternative antibiotic in companion animals, continuous monitoring of antibiotic resistance profiles is needed in order to investigate the distribution of florfenicol resistance under pressure of administration of commonly used agents.

Coagulase-Negative Staphylococci as an Etiologic Agent of Ovine Mastitis, with a Focus on Subclinical Forms.

The objective of this review is to investigate the distribution and the characteristics of coagulase-negative Staphylococci (CoNS) implicated in ovine mastitis, and especially in subclinical cases, in order to provide a global perspective of the current research data and analyze specific critical aspects of the issue. PRISMA guidelines were implemented in the search of the last 20 years of the related literature in two databases. In total, 139 studies were included in this review. Relevant data were tracked down, assembled, and compared. Regarding the geographical distribution, most studies originated from Europe (68), followed by South America (33). Lacaune was the most examined breed, while S. epidermidis was the predominantly identified species, representing approximately 39% of the obtained isolates. Antibiotic resistance in the relevant bacteria was documented mostly for Penicillin (32.8%) and Amoxicillin (32.1%), while biofilm- and toxin-associated genes were encountered in variable rates because significant inequalities were observed between different articles. Significantly higher rates of antimicrobial resistance were detected in Asia and South America compared to Europe. Finally, the diagnostic procedures carried out in the respective studies were evaluated. Conventional culture and biochemical tests were mostly performed for simple strain identification; therefore, further molecular investigation of isolates should be pursued in future studies, as this will provide important data regarding specific aspects of the implication of CoNS in ovine mastitis.

Antimicrobial Susceptibility Profiles of Acinetobacter baumannii Strains, Isolated from Clinical Cases of Companion Animals in Greece.

Acinetobacter baumannii-calcoaceticus (Abc) Complex bacteria are troublesome nosocomial pathogens in human medicine, especially during the last 30 years. Recent research in veterinary medicine also supports its emergence as an animal pathogen. However, relevant data are limited. In this study, we obtained 41 A. baumannii isolates from clinical samples of canine and feline origin collected in veterinary clinics in Greece between 2020 and 2023. Biochemical identification, antimicrobial susceptibility testing, molecular identification and statistical analysis were performed. Most of the samples were of soft tissue and urine origin, while polymicrobial infections were recorded in 29 cases. Minocycline was the most effective in vitro antibiotic, whereas high resistance rates were detected for almost all the agents tested. Notably, 20 isolates were carbapenem resistant and 19 extensively drug resistant (XDR). This is the first report of canine and feline infections caused by Abc in Greece. The results create concerns regarding the capability of the respective bacteria to cause difficult-to-treat infections in pets and persist in veterinary facilities through hospitalized animals, contaminated equipment, and surfaces. Moreover, the prevalence of highly resistant strains in companion animals constitutes a public health issue since they could act as a reservoir, contributing to the spread of epidemic clones in a community.

Building the National Antimicrobial Resistance Surveillance Network in Animals in Greece: A "One Health" Approach.

It is widely accepted that, in order to prevent and control antimicrobial resistance (AMR), surveillance systems across human, animal and environmental sectors need to be integrated, in a One Health approach. Currently, in Europe, there are surveillance networks established only for the human and food sector and, until now, there has been no organized effort to monitor AMR in bacterial pathogens derived from diseased animals in Europe. Since 2017, efforts to fill this gap have taken place by the European Antimicrobial Resistance Surveillance network in a veterinary medicine (EARS-Vet) initiative, included in the EU Joint Action on AMR and Healthcare-Associated Infections (EU-JAMRAI). EARS-Vet is designed to complement and integrate with existing European monitoring systems for AMR as well as constitute a European network of national monitoring systems. As Greece has no national AMR surveillance system for pathogens of animal origin currently in place, in the context of the development of EARS-Vet, an initiative took place for the organization of such a system by competent agencies and other stakeholders. In this article, the steps to organize a first AMR national surveillance network in Greece are presented and a Strengths, Weaknesses, Opportunities, and Threats (SWOT) analysis is performed to present main characteristics of the approach implemented.

Fosfomycin Resistance in Bacteria Isolated from Companion Animals (Dogs and Cats).

Fosfomycin is an old antibacterial agent, which is currently used mainly in human medicine, in uncomplicated Urinary Tract Infections (UTIs). The purpose of this review is to investigate the presence and the characteristics of Fosfomycin resistance in bacteria isolated from canine or feline samples, estimate the possible causes of the dissemination of associated strains in pets, and underline the requirements of prospective relevant studies. Preferred Reporting Items for Systematic Reviews (PRISMA) guidelines were used for the search of current literature in two databases. A total of 33 articles were finally included in the review. Relevant data were tracked down, assembled, and compared. Referring to the geographical distribution, Northeast Asia was the main area of origin of the studies. E. coli was the predominant species detected, followed by other Enterobacteriaceae, Staphylococci, and Pseudomonas spp. FosA and fosA3 were the more frequently encountered Antimicrobial Resistance Genes (ARGs) in the related Gram-negative isolates, while fosB was regularly encountered in Gram-positive ones. The majority of the strains were multidrug-resistant (MDR) and co-carried resistance genes against several classes of antibiotics and especially ß-Lactams, such as blaCTX-M and mecA. These results demonstrate the fact that the cause of the spreading of Fosfomycin-resistant bacteria among pets could be the extended use of other antibacterial agents, that promote the prevalence of MDR, epidemic strains among an animal population. Through the circulation of these strains into a community, a public health issue could arise. Further research is essential though, for the comprehensive consideration of the issue, as the current data are limited.

Current Data on Rickettsia felis Occurrence in Vectors, Human and Animal Hosts in Europe: A Scoping Review.

Rickettsia felis is an emerging pathogen with increasing reports of human cases and detection in arthropod and animal host species worldwide. In this scoping review we record the newest data reported for R. felis in Europe: the vector and host species found to be infected, and the geographical distribution and prevalence of R. felis infection in vectors and hosts. A total of 15 European countries reported the occurrence of R. felis in hosts and vectors during 2017−2022. The vectors found to be infected by R. felis were flea, tick and mite species; Ctenocephalides felis and Ixodes ricinus were the dominant ones. The hosts found to be infected and/or exposed to R. felis were humans, cats and small mammals. Physicians should be aware of the epidemiology and include illness caused by R. felis in the differential diagnosis of febrile disease. Veterinarians should keep training pet owners on the need for effective year-round arthropod control on their pets, especially for fleas.

Preliminary Results on the Comparative Evaluation of Alkaline Phosphatase Commercial Tests Efficiency in Non-Cow Milk Pasteurization.

The demand for non-cow milk and the products derived from it, is constantly increasing; thus, correct and effective pasteurization becomes necessary. Typical practices for evaluating milk pasteurization are mainly based on the thermal inactivation of an endogenous enzyme, alkaline phosphatase (ALP). The ALP tests, originally designed and applied to pasteurized cow milk, are often used to control pasteurization in non-cow milk, without sufficient data on their suitability; EFSA calls on the scientific world for collecting more information on the subject. In this study, the pertinent details of the ALP assay for non-cow milk products are summarized, and a comparison is performed regarding the evaluation of the adequacy of commercially available tests for the determination of ALP activity in non-cow milk. At the same time, raw and pasteurized non-cow milk was analyzed microbiologically using standard ISO methods and MALDI-TOF MS in order to confirm the thermal effect on common microorganisms. In these preliminary results, various ALP tests do not appear to be fully reliable as indicators for the pasteurization of some types of non-cow milk such as camel and donkey milk or even goat and sheep milk, using the EFSA proposed limits. ALP commercial kits may not be suitable as pasteurization indicators for various types on non-cow milk, and alternatives should be investigated.

Development of a Multiplex Bead Assay to Detect Serological Responses to Brucella Species in Domestic Pigs and Wild Boar with the Potential to Overcome Cross-Reactivity with Yersinia enterocolitica O:9.

The aim of this study was to develop a multiplex bead assay using a Brucella rLPS antigen, a Brucella suis smooth antigen, and a Yersinia enterocolitica O:9 antigen that not only discriminates Brucella-infected from Brucella-uninfected pigs and wild boar, but also overcomes the cross reactivity with Y. enterocolitica O:9. Sera from 126 domestic pigs were tested: 29 pigs were Brucella infected, 80 were non-infected and 17 were confirmed to be false positive serological reactors (FPSR). Sera from 49 wild boar were tested: 18 were positive and 31 were negative. Using the rLPS antigen, 26/29 Brucella-infected domestic pigs and 15/18 seropositive wild boar were positive, while 75/80 non-Brucella infected domestic pigs, all FPSR, and all seronegative wild boar were negative. Using the smooth B. suis 1330 antigen, all Brucella-infected domestic pigs, 9/17 FPSR and all seropositive wild boar were positive, while all non-infected pigs and 30/31 seronegative wild boar were negative. The ratio of the readouts from the smooth B. suis antigen and Y. enterocolitica O:9 antigen enabled discriminating all Brucella infected individuals from the FPSR domestic pigs. These results demonstrate the potential of this assay for use in the surveillance of brucellosis, overcoming the cross-reactivity with Y. enterocolitica.

Colistin Resistant mcr Genes Prevalence in Livestock Animals (Swine, Bovine, Poultry) from a Multinational Perspective. A Systematic Review.

The objective of this review is to collect and present the results of relevant studies on an international level, on the subject of colistin resistance due to mcr genes prevalence in livestock animals. After a literature search, and using PRISMA guidelines principles, a total of 40 swine, 16 bovine and 31 poultry studies were collected concerning mcr-1 gene; five swine, three bovine and three poultry studies referred to mcr-2 gene; eight swine, one bovine, two poultry studies were about mcr-3 gene; six swine, one bovine and one poultry manuscript studied mcr-4 gene; five swine manuscripts studied mcr-5 gene; one swine manuscript was about mcr-6, mcr-7, mcr-8, mcr-9 genes and one poultry study about mcr-10 gene was found. Information about colistin resistance in bacteria derived from animals and animal product foods is still considered limited and that should be continually enhanced; most of the information about clinical isolates are relative to enteropathogens Escherichia coli and Salmonella spp. This review demonstrates the widespread dispersion of mcr genes to livestock animals, indicating the need to further increase measures to control this important threat for public health issue.

Association of Melatonin Administration in Pregnant Ewes with Growth, Redox Status and Immunity of Their Offspring.

In this study, the effects of melatonin treatment on growth, redox status and immunity in prenatally stressed newborn lambs were evaluated. Thirty-seven newborn lambs were allocated into two groups (melatonin-MEL and control-CON), based on whether their mothers were treated with melatonin implants or not, respectively. All pregnant ewes were exposed to heat stress. The body weight of lambs was recorded at birth (L0), and then on days 15 (L15) and 40 (L40). Redox biomarkers [total antioxidant capacity (TAC), glutathione (GSH), thiobarbituric acid reactive substances (TBARS)] were assayed in blood samples collected from lambs on days L0, L1, L2, L5, L10 and L40. Chemical analysis and antioxidant capacity were evaluated in colostrum and milk samples collected at the same time points with blood samples. Cytokines (IL-1ß, IL-6, IL-10, IFN-γ) and immunoglobulin (IgG) were assayed in blood and colostrum samples collected from ewes on days L0 and L1, and in lambs' blood on days L0, L1 and L2. The results revealed that body weight gain of newborn lambs did not differ between the two groups (p > 0.05). Better redox status was found in MEL lambs until L2, as well as higher antioxidant capacity in the colostrum of MEL ewes compared to CON ones on day L0 (p < 0.05). In MEL ewes' colostrum, higher protein content was measured on day L0 and higher fat content on L1 compared to CON group (p < 0.05). The highest level of IL-6 was found in MEL ewes on L1, with a concomitant increase of IL-10 level in MEL lambs in comparison to CON lambs on L2. Moreover, CON colostrum resulted in a higher level of IL-10 within time, coupled with an increased level of IgG found in lambs' plasma on L2 (p = 0.04). This study indicated that melatonin could be administered as antioxidant and immune-modulatory regime in prenatally stressed offspring in order to cope with the crucial first days of their life. This effect of melatonin was also amplified by crosstalk between IL-6, IL-10 and IgG production, resulting in an improved quality of produced milk.

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.

Development of a Multiplex Bead Assay for Simultaneous Serodiagnosis of Antibodies against Mycobacterium bovis, Brucella suis, and Trichinella spiralis in Wild Boar.

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.

European Brown hare (Lepus europaeus) as a source of emerging and re-emerging pathogens of Public Health importance: A review.

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.

Antimicrobial practices among small animal veterinarians in Greece: a survey.

BACKGROUND: The inappropriate use of antibiotics is a major issue in clinical practice in Greece with serious implications for public health and animal health. The purpose of the present study was to provide a first insight into the use of antibiotics by small animal practitioners in Greece and assess their compliance with general rules for the rational use of antibiotics. This is the first survey of its kind in Greece. METHODS: A questionnaire was designed to collect basic information on the use of antibiotics by pet veterinarians. The questionnaire was sent to a total of 70 veterinarians mainly operating in the region of Attica, a region that comprises almost 50% of the Greek population and where veterinarians are engaged solely in small animal practice. The questionnaire consisted of 37 closed questions dealing with various aspects on the use of antibiotics. RESULTS: The majority of practitioners report cases where the pet owner initiated antibiotic treatment without veterinary prescription. Almost every clinician reported owner-compliance challenges. Regarding microbiological analysis, 73% of respondents initiate empirical treatment while waiting for laboratory results or use antibiogram only when the treatment is unsuccessful. Eighty-eight per cent declared to use antimicrobials postoperatively in clean surgical procedures. Different types of antimicrobials and treatment durations than the ones proposed by guidelines on rational use of antibiotics are preferred for various organ systems e.g. in urinary and gastrointestinal infections. CONCLUSIONS: Our findings suggest the need for guidelines on antibiotic use in small animal practice in Greece, and the deployment of systematic surveillance on antimicrobials use and resistance to inform the initial choice of antibiotics upon local antimicrobial resistance profiles. Targeting the other end of the problem, pet owners, our findings indicate the need to educate them on the rational use of antibiotics and, critically, stop antibiotic availability without prescription.

Investigation of the Role of the European Brown Hare in the Epidemiology of Bacterial Zoonotic Pathogens: A Serological and Molecular Survey in Greece.

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 during 2-year hunting periods in northern and central Greece. Serum samples were examined for the presence of IgG antibodies by using an immune fluorescence test and/or an enzyme-linked immunosorbent assay. PCR was used to detect Bartonella spp DNA in blood samples and Brucella spp, C. burnetii, and F. tularensis DNA in liver samples. Antibodies against Bartonella spp were detected in 12 hares (12/105); whereas none of the hares examined was seropositive for Brucella spp, C. burnetii, and F. tularensis. The presence of Bartonella spp, Brucella spp, C. burnetii, and F. tularensis DNA was not detected in the samples examined. This study did not provide any evidence that the European Brown hare is involved in the epidemiology of Brucella spp, C. burnetii, and F. tularensis in Greece. However, our results suggest that this species is exposed to Bartonella spp, which gives the impetus for further investigation of its role as another host of this bacterium.

Phylogenetic Analysis of Bird-Virulent West Nile Virus Strain, 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.

Serological and molecular investigation of selected parasitic pathogens in European brown hare (Lepus europaeus) in Greece: inferring the ecological niche of Toxoplasma gondii and Leishmania infantum in hares.

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.

Mammals and habitat disturbance: the case of brown hare and wildfire.

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.

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.