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.

Acute non-A, non-B, non-C hepatitis differences and similarities between hepatitis E virus infection and autoimmune hepatitis, with phylogenetic analysis of hepatitis E virus in humans and wild boars.

Background: Hepatitis E virus (HEV) infection incidence is increasing in Europe, accounting for the majority of acute hepatitis cases. We investigated the prevalence and clinical characteristics of acute hepatitis E (AHE) in patients with acute non-A/B/C hepatitis from central Greece, their differences from acute autoimmune hepatitis (AIH) patients and the molecular similarity of human strains to local HEV strains in wild boars. Methods: Sera from 20 patients with non-A/B/C acute hepatitis (2015-2017) were tested prospectively for anti-HEV IgM, IgG antibodies and HEV-RNA. Sera from patients diagnosed with acute AIH (2000-2015; n=56) were tested retrospectively. Liver tissue samples from 40 wild boars were tested for HEV-RNA. Positive wild boar and patients' samples were sequenced and phylogenetically analyzed. Results: Twelve of the 76 (16%) patients were diagnosed with AHE: HEV-RNA 11.5x104 (38.7-39.7x106) IU/mL; 11/20 (55%) acute non-A/B/C hepatitis and 1/56 (2%) AIH patients. Patients with AHE were older than those without, predominately men, with higher alanine aminotransferase but lower IgG levels (P=0.005 and P=0.002, respectively), and had high titers of smooth muscle antibodies. Liver biopsies, performed in 6/12 patients with HEV infection, revealed histology compatible with AIH. HEV strains from both patients and wild boars belonged to genotype 3. Conclusions: Approximately one sixth of patients with acute non-A/B/C hepatitis had autochthonous HEV infection with AIH features. Therefore, a careful workup to exclude HEV should be carried out in all acute hepatitis cases before a definite diagnosis of AIH is established. Wild boars seem to be an important reservoir of HEV in Greece.

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.

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.

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 ß-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 blaCTX-M-1 group gene alone or in combination with blaTEM and one carried only blaTEM. AmpC, fluoroquinolone, trimethoprim/sulfamethoxazole, aminoglycoside and macrolide resistance genes were also detected. Additionally, one carbapenemase-producing E. coli was identified, harboring blaNDM 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.

Cross-Sectional Survey of Antibiotic Resistance in Extended Spectrum ß-Lactamase-Producing Enterobacteriaceae Isolated from Pigs in Greece.

This study aimed to estimate the prevalence of extended-spectrum ß-lactamase-producing (ESBL) bacteria in swine. Thus, 214 fecal samples were collected from suckling and weaned piglets from 34 farms in Greece (out of an overall population of about 14,300 sows). A subset of 78 (36.5%) ESBL producers were identified as E. coli (69/78, 88.5%), K. pneumoniae spp. pneumoniae (3.8%), P. mirabilis (5.1%), E. cloacae complex (1.3%) and S. enterica spp. diarizonae (1.3%). Resistance to at least one class of non-ß-lactam antibiotics was detected in 78 isolates. Among the E. coli strains, resistance was identified with regard to aminoglycosides (n = 31), fluoroquinolones (n = 49), tetracycline (n = 26) and trimethoprim/sulfamethoxazole (n = 46). Of the three K. pneumoniae spp. pneumoniae, two displayed resistances to aminoglycosides and all were resistant to fluoroquinolones, tetracyclines and trimethoprim/sulfamethoxazole. As for the four P. mirabilis isolates, three had a resistant phenotype for aminoglycosides and all were resistant to imipenem, fluoroquinolones, tetracyclines and trimethoprim/sulfamethoxazole. Molecular characterization of the isolates revealed the presence of CTX-M, SHV and TEM genes, as well as of genes conferring resistance to fluoroquinolones, aminoglycosides, sulfonamides, trimethoprim, macrolides and colistin. High levels of antimicrobial resistance (AMR) were demonstrated in Greek swine herds posing a concern for the efficacy of treatments at the farm level as well as for public health.

Pollution Indicators and HAB-Associated Halophilic Bacteria Alongside Harmful Cyanobacteria in the Largest Mussel Cultivation Area in Greece.

Taking into consideration the essential contribution of Mytilus galloprovincialis farming, it is of rising importance to add knowledge regarding bacterial species occurrence in water samples from aquaculture zones from the point of view of both the organism and public health. In the present study, we investigated the bacterial community existing in water samples from six Mytilus galloprovincialis aquaculture areas in the Thermaikos gulf, northern Greece, that may provoke toxicity in aquatic organisms and humans and may indicate environmental pollution in mussel production as well as algal blooms. Bacterial species were identified molecularly by sequencing of a partial 16s rRNA segment and were analyzed phylogenetically for the confirmation of the bacterial taxonomy. The results obtained revealed the presence of four bacterial genera (Halomonas sp., Planococcus sp., Sulfitobacter sp., and Synechocystis sp.). Members of the Halomonas and Sulfitobacter genera have been isolated from highly polluted sites, Planococcus bacteria have been identified in samples derived directly from plastic debris, and Synechocystis bacteria are in line with microcystin detection. In this context, the monitoring of the bacteria community in mussel aquaculture water samples from the Thermaikos gulf, the largest mussel cultivation area in Greece, represents an indicator of water pollution, microplastics presence, algal blooms, and toxin presence.

ESBL-Producing Moellerella wisconsensis-The Contribution of Wild Birds in the Dissemination of a Zoonotic Pathogen.

Moellerella wisconsensis is an Enterobacteriaceae with unclarified dispersion and pathogenicity. During an ongoing investigation about antimicrobial resistance in Greece, the occurrence of M. wisconsensis was evaluated among wild birds and humans. A total of 445 wild bird and 2000 human fecal samples were collected and screened for the presence of the organism. Subsequently, all M. wisconsensis strains were phenotypically and molecularly characterized regarding their antimicrobial resistance characteristics. Four M. wisconsensis were isolated from a common pheasant (Phasianus colchicus), two Eurasian magpies (Pica pica) and a great white-fronted goose (Anser albifrons). Among these four strains, the three latter presented resistance to 3rd generation cephalosporins, were phenotypically confirmed to produce ESBLs and were found to harbor blaCTX-M-1. The three ESBL isolates additionally exhibited resistance to tetracyclines, while resistance to aminoglycosides was detected in two of them and to trimethoprim/sulfamethoxazole in one. No Moellerella wisconsensis strains were retrieved from the human samples tested. This is the first report that provides evidence of M. wisconsensis dissemination among wild birds in Greece, describing CTX-M-1 production in multidrug resistant wild birds' isolates of this bacterial species.

Plethora of Resistance Genes in Carbapenem-Resistant Gram-Negative Bacteria in Greece: No End to a Continuous Genetic Evolution.

Carbapenem-resistant Gram-negative bacteria are a public health threat that requires urgent action. The fact that these pathogens commonly also harbor resistance mechanisms for several other antimicrobial classes further reduces patient treatment options. The present study aimed to provide information regarding the multidrug resistance genetic background of carbapenem-resistant Gram-negative bacteria in Central Greece. Strains from a tertiary care hospital, collected during routine practice, were characterized using a DNA microarray-based assay. Various different resistance determinants for carbapenems, other beta-lactams, aminoglycosides, quinolones, trimethoprim, sulfonamides and macrolides were detected among isolates of the same sequence type. Eighteen different multidrug resistance genomic profiles were identified among the twenty-four K. pneumoniae ST258, seven different profiles among the eight K. pneumoniae ST11, four profiles among the six A. baumannii ST409 and two among the three K. oxytoca. This report describes the multidrug resistance genomic background of carbapenem-resistant Gram-negative bacteria from a tertiary care hospital in Central Greece, providing evidence of their continuous genetic evolution.

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.

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

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

Poultry and Wild Birds as a Reservoir of CMY-2 Producing Escherichia coli: The First Large-Scale Study in Greece.

Resistance mediated by ß-lactamases is a globally spread menace. The aim of the present study was to determine the occurrence of Escherichia coli producing plasmid-encoded AmpC ß-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.

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.

Detection and genetic characterization of porcine circovirus 2 isolates from the first cases of postweaning multisystemic and wasting syndrome in wild boars in Greece.

In 2002, postweaning multisystemic wasting syndrome (PMWS) was diagnosed in a European female wild boar (Sus scrofa), based on the detection of porcine circovirus 2 (PCV2) DNA in various organs, including the uterus, and on histopathologic lesions. This is the first detection of PCV2 DNA in the uterus of a wild boar. Three years later (2005), a wild boar < 6-8 mo of age was found moribund. It presented wasting and dyspnea and finally died. PCV2 DNA was detected in tissue samples, and histopathologic lesions consistent with PMWS were observed. Both wild boars were from neighboring hunting areas in central Greece. Two PCV2 strains from the wild boars were genetically characterized and compared to other reported PCV2 sequences from wild boars and domestic pigs. The PCV-2 sequences from the wild boars in this study were closely related to each other and were grouped with two isolates from wild boars from Hungary. The phylogenetic analysis revealed that the virus might be transmitted between hunting areas. In addition, PCV2 may spread from domestic pigs to wild boars and vice versa.

European brown hare syndrome in wild European brown hares from Greece.

From 1999 to mid-2003, 97 European brown hares (Lepus europaeus) found dead throughout Greece were examined by necropsy, histopathology, and reverse-transcription polymerase chain reaction (RT-PCR) for the presence of European brown hare syndrome (EBHS) and EBHS virus (EBHSV), respectively. Hare losses were sporadic, starting in the cold season and lasting for many months (December to May). The most prominent gross lesions were observed in the liver and included swelling and discoloration; congestion and hemorrhages were present mainly in lungs and tracheal mucosa. Necropsy findings were suggestive of EBHS, which was confirmed by histopathology and RT-PCR. This study documents, for the first time, EBHS in Greece.