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.

Evaluation of two commercial assays for the detection of Chlamydophila abortus antibodies.

Two commercial enzyme-linked immunosorbent assays (ELISA), the CHEKIT-CHLAMYDIA which uses inactivated Chlamydophila psittaci antigen, and the Chlamydophila abortus ELISA produced by the Institut Pourquier which uses a recombinant fragment of the 80-90 kDa protein, were evaluated with the objective to determine whether the new ELISAs would perform as improved alternatives to the complement fixation test (CFT) for the serological diagnosis of ovine enzootic abortion (OEA). The results were compared to those obtained by the CFT and the competitive ELISA (cELISA). The tests were assessed with a panel of 17 serum samples from specific pathogen-free (SPF) lambs experimentally infected with various subtypes of Chlamydophila pecorum, with sera from 45 C. abortus-infected pregnant sheep and from 54 sheep free of OEA. The C. abortus ELISA was identified as being more specific and sensitive than the other tests. The 4 assays were evaluated further with 254 sera from flocks with documented OEA, from flocks with no history of abortion and from animals after abortion of unknown cause. The C. abortus ELISA by the Institut Pourquier identified less OEA-positive sera than the other assays though it identified correctly 9 of 10 OEA-positive flocks. The basis of the discordant results is discussed.

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.

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.

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.

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.

Diversity among abortion strains of Chlamydia psittaci demonstrated by inclusion morphology, polypeptide profiles and monoclonal antibodies.

Twenty eight C. psittaci abortion strains had been previously classified in to 4 immunologically distinct groups on the basis of cross-protection experiments in a mouse model. To identify the molecular basis of their immunological divergence 4 representative strains were investigated by cellular, molecular and immunological techniques. An identical pattern was obtained by Alul digestion of the amplified major outer membrane protein gene (MOMP) by the polymerase chain reaction (PCR) of the 4 strains. However, inclusion morphology and polypeptide profiles clearly distinguished one strain, named LLG, and its homologous strain POS from the other prototypes by the presence of a unique protein at 26.5 kDa and the absence of a polypeptide at 23 kDa. Six out of 10 monoclonal antibodies (mAbs) raised against abortion strains failed to react with inclusions of the 2 strains. All 6 mAbs reacted with the chlamydial outer membrane complex (COMC). Two of these mAbs, one against the MOMP and one against an antigen at 90 kDa, did not react with immunoblots of LLG and POS. The data provide direct demonstration of the existence of strain variation in the field and classify strains LLG and POS as a distinct C. psittaci serotype 1-subtype. The antigenic diversity among abortion strains should be taken into consideration when designing a subunit vaccine.

Epidemiology and diagnosis of rabies in Greece.

During the decade 1951-1960, 8626 rabies cases were reported among domestic animals in Greece and 53 deaths in humans. During the decades 1961-1970, 1971-1980 cases were reduced to 3009 and 242, respectively, while no more cases in man occurred since 1970. Vaccination campaigns of dogs, besides the low prevalence of rabies among wild animals, resulted in its eradication from the country. Immunofluorescence is the method used for diagnosis.

Olivocerebellar connections in sheep studied with the retrograde transport of horseradish peroxidase.

We describe here the morphology of the inferior olive and the localization of labeled cells after HRP injections into various lobules of vermis and hemisphere of the cerebellum of the sheep. The medial part of the caudal half of the medial accessory olive projects to a medial zone in the anterior lobe, the simple lobule, and the lobules VII and VIII. The lateral part of the medial accessory olive projects to more lateral parts of these lobules with the exception of lobule VII. The group beta projects in a differential manner to the lateral parts of the lobules VII and VIII and the medial parts of the lobules IX and X. The dorsomedial cell column projects to lobules VIII, IX, and X; the connections of the dorsal cap are restricted to lobule X. Fibers from the caudal limb of the dorsal accessory olive terminate in the B zone, the simple lobule, and in lobule VIII. The rostral half of the medial accessory olive projects to lobule IX and to the hemisphere. The other projections of the accessory olives and the principal olive to the hemisphere are similar to those reported for the cat. An accessory cell group in the sheep, located between the principal and the dorsal accessory olive, has connections with the caudal vermis and the hemisphere.

Cerebellar cortical afferents from the dorsal column nuclei in sheep, demonstrated with retrograde transport of horseradish peroxidase.

Following horseradish peroxidase injections in the cortex of the sheep cerebellum (except the ventral part of the anterior lobe, the flocculus and ventral paraflocculus), labeled cells were found in nucleus gracilis, medial cuneate and lateral cuneate. The present findings provide evidence that the projection of the dorsal column nuclei to the cerebellum in the sheep is more extensive than has been reported for the common laboratory animals. Projections to the vermis are bilateral, projections to the hemisphere are mainly ipsilateral.

Cerebellar afferents from the vestibular and perihypoglossal nuclei in sheep studied with retrograde transport of horseradish peroxidase.

Cerebellar afferents from the vestibular and perihypoglossal nuclei have been studied in 25 sheep. Following injections of HRP in all cerebellar lobules, with the exceptions of the ventral part of anterior lobe, the ventral paraflocculus and the flocculus, bilateral cerebellar projections from these nuclei with ipsilateral preponderance are evidenced. Experiments in sheep show a wider field of origin and termination of secondary vestibulocerebellar fibers than reported earlier. For the descending and medial vestibular nuclei a topographical organization in their vestibulocerebellar projections is evidenced. Fibers to lobule X mainly originate from their dorsal parts, those to lobule IX from their ventral portion, and fibers to lobule VIII from their caudal pole and the lateral part of the descending nucleus. Cell group x projects to all the cerebellar lobules studied except crus. I. Labeled cells in the lateral vestibular nucleus are few. The superior vestibular nucleus projects mainly to lobule X. Vestibular cells projecting to the hemisphere are located mainly in the cell group x and in the central parts of the descending and medial vestibular nuclei. The interstitial nucleus of the vestibular nerve, the nucleus prepositus hypoglossi and the nucleus intercalatus project to all lobules.

Cerebellar afferents from motor nuclei of cranial nerves, the nucleus of the solitary tract, and nuclei coeruleus and parabrachialis in sheep, demonstrated with retrograde transport of horseradish peroxidase.

Following HRP injections in the cerebellar cortex of the sheep (except the ventral part of the anterior lobe, the flocculus and ventral paraflocculus), labeled cells were evident in motor nuclei of cranial nerves(XII, VII, VI, III, visceromotor nucleus of X and nucleus ambiguus), in the solitary tract nucleus, the nucleus coeruleus and the parabrachial nucleus.

Secondary trigeminocerebellar projections in sheep studied with the horseradish peroxidase tracing method.

Secondary trigeminocerebellar connections have been studied with HRP histochemistry in 25 sheep. The results indicate that almost all of the cerebellar cortex except flocculus, ventral paraflocculus and lobules I-IV receives bilateral (mostly ipsilateral) fibers from the trigeminal nuclei. A topographical organization of trigeminocerebellar fibers is present. The mesencephalic tract nucleus projects to the anterior lobe, the simple lobule (HVI), lobules VI, VIII, and the dorsal paraflocculus. The ventral group of the princeps and spinal tract (mainly IDV) nuclei projects to all lobules studied in vermis and hemispheres. More dorsal parts of these nuclei have a more restricted projection field including the vermal lobules VI, VII, and IX and the hemisphere. Cells within and ventral to the motor nucleus of the trigeminal nerve were found labeled after injections into the anterior lobe, the simple lobule, and lobule IX. Labeled cells in the region of the nucleus ovalis and close to the solitary tract project to the simple and paramedian lobule and lobule IX.

Visual thalamocortical connections in sheep studied by means of the retrograde transport of horseradish-peroxidase.

In order to study the visual thalamocortical connections in the sheep, horseradish peroxidase (0.3--0.5 microliter of a 30% solution) has been injected in the gyri marginalis, ectomarginalis medius pars medialis, ectomarginalis medius pars lateralis and ectosylvius caudalis. The results show that: (1) the dorsal lateral geniculate nucleus (LGNd) projects to the former three gyri. Dorsal parts of the LGNd project to caudal areas, whereas its ventral parts project to rostral areas of these gyri; medial parts of the LGNd project to the gyrus ectomarginalis medius pars lateralis, while lateral parts project to the gyrus marginalis; (2) the medial interlaminar nucleus (MIN) or pars geniculata pulvinaris of Rose ('42b) projects to the caudal part of the gyrus marginalis and to the gyrus ectomarginalis medius pars lateralis; (3) the pulvinar proper of Rose (PUL) projects to the caudal part of the gyrus ectosylvius caudalis whereas the rostral part of this gyrus receives input from the medial geniculate body. In relation to Rose's cytoarchitectonic study of the cortex of sheep ('42a) the present study has shown that the LGNd projects to both the area striata (gyrus marginalis + gyrus ectomarginalis medius pars medialis) and area occipitalis (gyrus ectomarginalis medius pars lateralis) of Rose, that the gyrus marginalis and the area occipitals receive a second projection (from the MIN), and that the PUL projects beyond the area occipitalis to the area parietalis of Rose.