Epizootiological investigation of the most important infectious equine diseases in Greece.

During the period 2001 to 2008, a total of 7,872 equine sera were tested at the Centre of Veterinary Institutes of Athens. Antibodies against seven infectious diseases of equids were determined: equine infectious anaemia (EIA), African horse sickness (AHS), equine viral arteritis (EVA), West Nile encephalitis (WNE), glanders, piroplasmosis and dourine. Tests for the four viral diseases found 4.5% seropositivity for EIA, 0% for AHS, 3.3% for EVA and 4% for WNE. All sera tested for glanders antibodies were negative. Tests for piroplasmosis detected antibodies against T. equi and B. caballi in 12.9% and 1.3% of the sera, respectively. No sample tested positive for dourine. The results of this epidemiological survey provide strong evidence that Greece is free from the diseases of AHS, glanders and dourine.

The epidemiology of sheep pox in Greece from 1987 to 2007.

The authors reviewthe epidemiology of sheep pox outbreaks in Greece between 1987 and 2007. It is believed that sheep pox is introduced into Greece principally from neighbouring countries to the east, and is associated with the movements of infected sheep flocks close to the border and contacts between humans and animals. Disease foci have appeared in several central and north-eastern areas of the country. Between 1982 and 1986, Greece remained free of sheep pox but, in 1987, the disease appeared on the island of Lesvos and, in 1988, outbreaks were seen in the prefecture of Evros. In 1994, a further outbreak occurred in Evros. Over the next four years, more outbreaks occurred in Evros and Thessaloniki (1995); Larissa, Xanthi, Rhodopi, Kavala, Magnissia, Evros and the island of Lesvos (1996); Kavala, Magnissia, Halkidiki, Evros and Rhodopi (1997). In 1998, there were fewer cases of sheep pox, with outbreaks only in the prefecture of Evros. Two years later, a further outbreak was reported in Evros (2000), while the most recent outbreak occurred on the island of Lesvos in January 2007.

Differential effect of the shape of calcium alginate matrices on the physiology of immobilized neuroblastoma N2a and Vero cells: a comparative study.

In order to investigate the effect of cell immobilization in calcium alginate gels on cell physiology, we immobilized Vero or N2a neuroblastoma cells in gels shaped either as spherical beads or as thin membrane layers. Throughout a culture period of 4 weeks cell viability, RNA and cytoplasmic calcium concentration and glutathione accumulation were assayed by fluorescence microscopy after provision of an appropriate dye. Non-elaborate culture conditions were applied throughout the experimental period in order to evaluate cell viability under less than optimal storage conditions. Vero cell proliferation was observed only in spherical beads, while N2a cell proliferation was observed in both configurations until the third week of culture. Increased [Ca2+]cyt could be associated with cell proliferation only when cells were immobilized in spherical beads, while a considerable decrease in the biosynthesis of reduced glutathione and RNA was observed in cells immobilized in thin membrane layers. The observed effects of the shape of the immobilization matrix may be due to differences in external mass transfer resistance. Therefore, depending on cell type, cell proliferation could have been promoted by either increased (Vero) or decreased (N2a) nutrient and oxygen flow to immobilized cells. The results of the present study could contribute to an improvement of immobilized cell sensor storability.

Development of a novel, multi-analyte biosensor system for assaying cell division: identification of cell proliferation/death precursor events.

A novel, miniaturized biosensor system was created by combining the electrophysiological response of immobilized cells with superoxide-sensing technology, optical and fluorescence microscopy. Vero cells were immobilized in a calcium alginate matrix (at a density of 1.7 x 10(6) cells ml(-1)). A 0.5 cm x 0.5 cm piece of cell-containing gel matrix was aseptically adhered on a glass microscope slide with a microfabricated gold electrode array, sealed with a cover slip and provided with Dulbecco's medium +10% (v/v) fetal calf serum every day by means of a capillary feeding tube. During a culture period of 7 days, the membrane potential of immobilized cells was continuously monitored, while cell division was assayed with an optical microscope. In addition, daily measurements of immobilized cell membrane potential, viability, RNA and calcium concentration, radical oxygen species (ROS) and glutathione accumulation, were conducted by fluorescence microscopy after provision of an appropriate dye. Superoxide accumulation was assayed by covering the electrodes with superoxide dismutase (SOD). Maximum cell membrane potential values and superoxide production were observed upon initiation of cell division. Using the novel biosensor, we were able to correlate seven different cell physiological parameters to each other and formulate a model for ROS-mediated signaling function on cell division and death. In addition, we were able to predict cell proliferation or death by comparing the relative response of the electrophysiological and superoxide sensor during the culture period.

Study on the mechanism of Bioelectric Recognition Assay: evidence for immobilized cell membrane interactions with viral fragments.

The Bioelectric Recognition Assay (BERA) is a whole-cell based biosensing system that detects the electric response of cultured cells, suspended in a gel matrix, to various ligands, which bind to the cell and/or affect its physiology. Previous studies have demonstrated the potential application of this method for rapid, inexpensive detection of viruses in a crude sample. However, the understanding, so far, of the fundamental processes that take place during cell-virus interactions within the probe has been rather limited. In the present study, we combined electrophysiological and fluorescence microscopical assays, so that we can prove that animal and plant cells immobilized in BERA sensors respond to different viruses primarily by changing their membrane potential. The response of immobilized cells against different viruses did not depend on the virus ability to penetrate the cell, but was modified after binding each virus to a virus-specific antibody or removal of its coat protein after treatment with a protease. Consequently, we were able to assay the presence of a virus in its complete form or fragments thereof. Combination of immunological recognition with the electrophysiological response of immobilized cells allows for a considerable increase of the specificity of the BERA biosensory assay. In addition, rather than simply detect the presence of a protein or genomic sequence, the method can help gain information on the bioactivity of a virus.

Detection of sheep poxvirus in skin biopsy samples by a multiplex polymerase chain reaction.

The development of a multiplex polymerase chain reaction (PCR) method with amplification of capripoxvirus in a single-step procedure from skin biopsies using three primer pairs, two specific for capripoxvirus and one specific for alpha-tubulin is described. A sensitive multiplex PCR was achieved by optimization of parameters such as the primer concentrations, magnesium and dNTPs concentrations. False negative results that sometimes arise due to inhibitors of DNA amplification may be avoided by the inclusion in the assay of alpha-tubulin primers. The results reported on 42 skin biopsies from sheep suspected to have poxvirus infection, indicated that the assay could monitor simultaneously DNA extraction from skin biopsy samples and allow improved detection of capripoxvirus within 24 h of specimen receipt in the laboratory.

Sheep poxvirus identification by PCR in cell cultures.

A simple, rapid and specific diagnostic polymerase chain reaction (PCR) method was developed for sheep poxvirus identification. The primers used were from the sequenced genomes of the capripox viruses KS-1 and InS-1. Six different sheep pox isolates were tested against two orf (parapox) and three animal herpesviruses as controls. Material from uninfected cell cultures was also used as control. The sensitivity of the PCR was approximately equivalent with each of the two primers and for the six sheep pox isolates. All the negative control virus DNAs were negative and differed clearly from those of the sheep pox strains.

Bluetongue virus diagnosis of clinical cases by a duplex reverse transcription-PCR: a comparison with conventional methods.

A duplex reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of bluetongue virus (BTV) in clinical samples was developed. This assay, which detects the highly conserved S10 region of BTV, was assessed for sensitivity and application as a rapid and dependable diagnostic tool by comparison with standard assays of virus detection, such as virus isolation in embryonated chicken eggs and cell culture. Simultaneous detection of BTV and host beta-actin RNAs minimizes the possibility of false negative results. The sensitivity of the assay was found to be equal to five cell culture infectious dose (CCID(50)) units and its specificity was confirmed as no RT-PCR product was detected with RNAs from two closely related orbiviruses, i.e. epizootic haemorrhagic disease virus (serotypes 1, 2 and 318) and African horse sickness virus, serotype 9, or RNAs from uninfected BHK-21 cells and blood samples from uninfected sheep or goats. In this study, 36 blood samples from naturally infected mixed flocks of sheep and goats were examined. Seventeen animals were identified as BTV-positive by RT-PCR, whereas only 13 were found positive by virus isolation in embryonated chicken eggs and nine by cell culture assays. These results indicate that the duplex RT-PCR could be a useful technique for monitoring BTV infection in the field.

Duration of bluetongue viraemia and serological responses in experimentally infected European breeds of sheep and goats.

The duration of viraemia and the serological responses were studied in two breeds of sheep and two breeds of goats, experimentally infected with bluetongue (BT) virus serotype 4. Viraemia, detectable by cell culture and embryonated chicken egg inoculation, lasted from the third to sixth day until the 27th-54th day post infection (p.i.). Significant differences between sheep and goats were not recorded. Lesbos sheep and goats together appeared to have significantly longer viraemias (n = 9, mean 41.3 days) than east-Friesian sheep and Saanen goats (n = 10, mean 30.4 days, p = 0.0039). Serological response was studied by competitive ELISA (c-ELISA) and agar gel immunodiffusion (AGID) tests. The c-ELISA was more sensitive in detecting BT virus antibodies in all animals than the AGID tests. No significant differences were observed between sheep and goats or between breeds. The epidemiological significance of subclinical infection and the extended BT virus viraemias in Lesbos sheep and goats, in relation to the maintenance of the virus and to overwintering is discussed.

Molecular epidemiology of bluetongue virus serotype 1 isolated in 2006 from Algeria.

This study reports on an outbreak of disease that occurred in central Algeria during July 2006. Sheep in the affected area presented clinical signs typical of bluetongue (BT) disease. A total of 5245 sheep in the affected region were considered to be susceptible, with 263 cases and thirty-six deaths. Bluetongue virus (BTV) serotype 1 was isolated and identified as the causative agent. Segments 2, 7 and 10 of this virus were sequenced and compared with other isolates from Morocco, Italy, Portugal and France showing that they all belong to a 'western' BTV group/topotype and collectively represent a western Mediterranean lineage of BTV-1.

RNA segment 9 exists as a duplex concatemer in an Australian strain of epizootic haemorrhagic disease virus (EHDV): Genetic analysis and evidence for the presence of concatemers as a normal feature of orbivirus replication.

This paper reports a concatemeric RNA in a strain of epizootic haemorrhagic disease virus (EHDV) serotype 5. Sequencing showed that the concatemeric RNA contains two identical full-length copies of genome segment 9, arranged in series, which has apparently replaced the monomeric form of the segment. In vitro translation demonstrated that the concatemeric RNA can act as a viable template for VP6 translation, but that no double-sized protein is produced. Studies were also performed to assess whether mutations might be easily introduced into the second copy (which might indicate some potential evolutionary significance of a concatemeric RNA segment), however multiple (n=40) passages generated no changes in the sequence of either the upstream or downstream segments. Further, we present results that demonstrate the presence of concatemers or partial gene duplications in multiple segments of different orbiviruses (in tissue culture and purified virus), suggesting their generation is likely to be a normal feature of orbivirus replication.

Emergence of bluetongue serotypes in Europe, part 2: the occurrence of a BTV-11 strain in Belgium.

An EDTA-blood sample from a cow without clinical signs, which gave early birth to a newborn calf that died soon after delivery, was shown to be positive for bluetongue virus (BTV)-RNA using a group-specific real-time RT-PCR (RT-qPCR). In-house serotype-specific RT-qPCR assays for bluetongue virus serotype 1 (BTV-1), -6 and -8 all gave negative results. Subsequent assays were carried out using conventional (gel-based) RT-PCR primers for all 25 BTV serotypes and only two primer sets, both specific for BTV-11, gave bands of the expected size. The cDNAs generated were sequenced and comparisons of the genome segment 2 sequence with that of the modified 'live' vaccine strain of BTV-11 from South Africa showed 100% identity. A survey of all ruminants in a 1-km area around the first positive farm using a BTV-11 serotype-specific RT-qPCR revealed five other holdings with in total nine BTV-11 positive animals. A cross-sectional monitoring of dairy cattle in Belgium showed an overall prevalence of 3.8% on herd level and 0.2% on animal level. A BTV-11 has been introduced into the Belgian cattle herd during the 2008 vector season. The source of the infection and the way by which the virus was introduced are unknown.

Quantifying the wind dispersal of Culicoides species in Greece and Bulgaria.

This paper tests the hypothesis that Culicoides (Diptera: Ceratopogonidae) species can be propagated by wind over long distances. Movement patterns of midges were inferred indirectly from patterns of the spread of bluetongue outbreaks between farms (using outbreak data from 1999-2001 for Greece, Bulgaria and Turkey) and then matched to concurrent wind patterns. The general methodology was to determine wind trajectories to and from each outbreak site based on the horizontal and vertical wind components of the European ReAnalysis-40 (ERA-40) dataset from the European centre for medium-range weather forecast (ECMWF). Forward trajectories (downwind or where the windvectors pointed to) and backward trajectories (upwind or where the wind-vectors originated from) were calculated for each outbreak for the period from one week before to one week after it had been recorded. These wind trajectories were then compared with the general outbreak patterns taking into consideration the different serotypes involved. It was found that the wind trajectories could be matched to the temporal distribution of the outbreak cases. Furthermore, the spread of the infected vector via the calculated wind trajectories was corroborated by molecular evidence. The conclusion is that the methodology presented is appropriate for quantifying the risk of spread of infected Culicoides midges by wind and that this approach could form an important component of a regional early-warning system for bluetongue.

VP2-segment sequence analysis of some isolates of bluetongue virus recovered in the Mediterranean basin during the 1998-2003 outbreak.

The complete nucleotide sequences of the VP2 segments of bluetongue virus (BTV) isolates recovered from Italy, Greece and Israel, from 1998 to 2003, were determined. Phylogenetic analysis of these sequences, those from related viruses and the South African vaccine strains, were used to determine the probable geographic origin of BTV incursions into Italy. Results indicated that viruses from each of the four serotypes isolated in Italy (2, 4, 9 and 16) possibly had a different origin. Analysis of the bluetongue virus serotype 2 (BTV-2) isolates gave evidence that this serotype probably moved from Tunisia. BTV-4 results showed probable incursion from the southwest and not from Greece or Israel. BTV-9 isolates clearly have an eastern origin (most probably Greece), whereas BTV-16 isolates are indistinguishable from the BTV-16 live attenuated vaccine strain. The phylogenetic findings were supported by polyacrylamide gel electrophoresis (PAGE) analysis of the complete amplified genome of each isolate except for BTV-16 Italian field isolate, which showed a slightly different PAGE profile. A combination of the complete VP2 sequencing and PAGE analysis of complete genomes, allowed not only phylogenetic analysis, but also vaccine detection and assessment of reassortment events.

Overview of bluetongue in Greece.

The history and epizootiology of bluetongue (BT) in Greece are described in detail. Three major epidemics of BT occurred in Greece, the first in 1979, due to bluetongue virus (BTV) serotype 4, in 1998-1999 due mainly to BTV-4 and BTV-9 and less to BTV-16 and in 2001 due mainly to BTV-1. The evolution of the disease, surveillance and control measures are presented, as well as the distribution of the BTV serotypes isolated.

Sheep poxvirus identification from clinical specimens by PCR, cell culture, immunofluorescence and agar gel immunoprecipitation assay.

Some 40 clinical specimens of skin lesions from sheep pox suspected cases were investigated by four different diagnostic assays: PCR, virus isolation in lamb testis cell cultures, direct immunofluorescent assay (DIFA) and antigen detecting agar gel immune precipitation test (AGIPT). All the specimens were positive by PCR and virus isolation, 29 were positive by DIFA and 16 by AGIPT. Using virus isolation on cell cultures as the gold standard, the PCR sensitivity was 100%, while that of DIFA and AGIPT was 73% and 40%, respectively. Skin samples with orf lesions or normal skin biopsies were PCR-negative. Cross-reactions with orf virus were observed in three samples only in the AGIPT assay. The PCR described combines high specificity and sensitivity with speed. PCR was therefore shown to be the method of choice for sheep poxvirus diagnosis directly from clinical specimens.

S10 segment sequence analysis of some Greek bluetongue virus strains.

Sequence analyses of the non-structural protein gene NS3/NS3A of eight Greek bluetongue (BT) virus (BTV) field isolates from the 1979 and 1999-2001 epizootics provide preliminary molecular data on the epidemiology of BT in Greece. These isolates from infected sheep belonged to serotypes BTV-1, BTV-4, BTV-9 and BTV-16. Phylogenetic analysis of the NS3/NS3A gene segregated these Greek isolates of BTV into two monophyletic groups. The first group was formed by all isolates of BTV-4; all were identical in their sequences, regardless of the area and year of isolation in Greece, and clustered with strains from Tunisia and Corsica. The isolates of BTV-1, BTV-9 and BTV-16 segregated into a second monophyletic group and clustered with Asian strains, showing a high homology (97-99%). From an epidemiological point of view, these preliminary results infer that one group of isolates is Mediterranean, whilst the second appears to be of Asian origin.

VP2 gene sequence analysis of some isolates of bluetongue virus recovered in the Mediterranean Basin during the 1998-2002 outbreak.

Since 1998, five serotypes of bluetongue virus (BTV), BTV-1, BTV-2, BTV-4, BTV-9 and BTV-16, have been reported in countries surrounding the Mediterranean Basin. Preliminary data on the sequencing analysis of the VP2-genes of BTV isolates recovered during the 1998-2002 epizootic of BT in Italy, Greece and Israel were studied. The VP2-genes of the Italian BTV-2 and BTV-9, Greek BTV-4 and BTV-9, Israeli BTV-4 and BTV-16 and South African BTV-2, BTV-4, BTV-9 and BTV-16, together with those of their corresponding South African serotype reference and vaccine strains, were cloned and the sequences of their terminal ends determined. These sequences, as well as those of all BTV VP2-gene sequences currently available on GenBank, were used to compile a phylogenetic tree to determine the probable geographic origins of the BTV incursions into Europe. The Italian isolates included in this study were from different regions, animal hosts and years (2000-2002). The results demonstrated that sequencing of the terminal end of the VP2-gene of BTV can be used for topotyping. According to the phylogenetic analysis, the Italian BTV-2 and BTV-9 isolates were stable across all species, irrespective of geographic origin and year of isolation. The sequencing data of the Italian isolates were identical to those of a BTV-2 isolate from Corsica. There was 97% homology between the Italian and Corsican BTV-2 isolates and the BTV-2 vaccine and reference isolates from South Africa. Italian BTV-9 isolates were also identical to the Greek BTV-9 isolates (99% homology). Surprisingly these BTV-9 isolates had only 67% homology with the reference BTV-9 isolate from South Africa. Conversely, BTV-9 field isolates from Australia and elsewhere in Europe had 89% homology with the Italian isolate at the nucleic acid level. Greek and Israeli BTV-4 isolates were almost identical (98% homology) and shared a 90% homology with the BTV-4 South African reference and vaccine strains. Israeli BTV-16 and South African BTV-16 reference strains were also similar. From these results, it may be concluded that Italian and Corsican BTV-2, Israeli and Greek BTV-4, and South African and Israeli BTV-16 had a common origin. The Greek BTV-9 isolate had more than 99% homology with the isolates from Italy, indicating these isolates to have had a common origin. The European BTV-9 isolates, grouped as 'eastern isolates', were more similar to the Australian isolates than to the South African reference strains.

Bluetongue laboratory diagnosis: a ring test to evaluate serological results using a competitive ELISA kit.

The occurrence of bluetongue (BT) in Italy prompted an increase in disease surveillance. Thus a competitive enzyme-linked immunosorbent assay (c-ELISA) to detect immunoglobulins to BT virus (BTV) was developed and distributed amongst 27 laboratories comprising the Italian veterinary diagnostic laboratories network to screen field sera. This ring test enabled comparison of the results and the evaluation of the reproducibility of the method. The c-ELISA developed by the National Reference Centre for Exotic Diseases (c-ELISA-IZSA&M) was compared also against a commercially available c-ELISA. In addition, results obtained by the Centre of Athens Veterinary Institutions are presented.