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.

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.

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.

Polarisation of major histocompatibility complex II host genotype with pathogenesis of European Brown Hare syndrome virus.

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

Serological and molecular investigation into the role of wild birds in the epidemiology of West Nile virus in Greece.

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.