George Valiakos

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

BackgroundA 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.FindingsSerum 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.ConclusionsThis 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.

A serosurvey for selected pathogens in Greek European wild boar

Objectives Serum samples, collected from 94 European wild boar (Sus scrofa) during the hunting seasons 2006 -2010 from different regions of Greece, were examined in order to estimate the role of these wildlife species as reservoir of pathogens important for livestock and/or public health. Materials and Methods The assays used for this purpose were commercial indirect ELISA for the detection of antibodies against porcine circovirus type 2 (PCV-2), porcine reproductive and respiratory syndrome (virus) (PRRSV), Aujeszkys disease virus (ADV), influenza A (IA) virus, Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae, Salmonella species, Trichinella species and indirect immunofluorescence antibody test for the detection of antibodies against Toxoplasma gondii and Neospora caninum. Results Antibodies against PCV-2, PRRSV, ADV, IA virus,A. pleuropneumoniae, M. hyopneumoniae,Salmonella species, Trichinella species, T. gondii and N. caninum were detected in 19.1 per cent, 12.8 per cent, 35.1 per cent, 1.1 per cent, 57.4 per cent, 0 per cent, 4.3 per cent, 6.4 per cent, 5.2 per cent and 1.1 per cent of the samples, respectively. Cluster analysis revealed a hot spot of seropositivity near Bulgarian border; seropositivity to ADV was more common among female animals. Conclusions These results indicate exposure of wild boar to most of the above-mentioned pathogens, raising concern about the possibility that these species may pose a significant health risk for livestock and/or humans.

West Nile Virus State of the Art Report of MALWEST Project

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.

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.

West Nile Virus: Basic Principles, Replication Mechanism, Immune Response and Important Genetic Determinants of Virulence

© 2013 Valiakos et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. West Nile Virus: Basic Principles, Replication Mechanism, Immune Response and Important Genetic Determinants of Virulence

Exposure of Eurasian magpies and turtle doves to West Nile virus during a major human outbreak, Greece, 2011

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.

Abiotic factors and autumn migration phenology of Woodcock (Scolopax rusticola Linnaeus, 1758, Charadriiformes: Scolopacidae) in a Mediterranean area

Abstract This research examines the relationship of abiotic factors to the autumn number increase of Eurasian woodcocks in a wintering area. Data on the number of woodcocks spotted per day using dogs for 12 years in an area of Macedonia are presented. From these records, we attempt to correlate the increase in number of woodcocks with moon phase, photoperiod, temperature, precipitation, visibility, wind intensity and barometric pressure. Meteorological data were obtained from five meteorological stations located in the study area, breeding areas and along the migration route. Descriptive and applied statistics [Factor Analysis, analysis of variance (ANOVA) and Binary Logistic Regression] were used for the analysis. Woodcock numbers increase significantly in October and November. Barometric pressure, wind and precipitation, as measured by the nearer climatic stations to the study area, influence the increase in number of woodcocks. The between-years analysis showed that woodcocks’ arrival was delayed in warmer autumns. Correlating the numerical increase of woodcocks with abiotic factors is a complicated phenomenon, but the constructed model provides evidence for some patterns and it facilitates the design of a future study.

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

Abstract 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.

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.

Dissemination of intestinal pathogens between lambs and puppies in sheep farms

Abstract 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 48h 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.