Emergence of Equine West Nile Encephalitis in Central Macedonia, Greece, 2010.

During the summer of 2010, an outbreak of West Nile virus (WNV) infections attributed to a lineage 2 WNV strain was reported among humans and horses in Central Macedonia, Northern Greece. Here, the clinical and laboratory investigation of horses that showed severe neurological signs due to WNV infection is being described. Specifically, between August and September 2010, 17 horses with neurological signs were detected. WNV infection was confirmed in all 17 clinical cases by applying laboratory testing. The duration of WNV-specific IgM antibodies in sera obtained from seven of the clinically affected horses was relatively short (10-60 days; mean 44 days). In the regional unit of Thessaloniki, (i) seroprevalence of WNV and fatality rate in horses were high (33% and 30%, respectively), and (ii) the ratio of neurological manifestations-to-infections for this virus strain was high (19%). These observations indicate that the strain responsible for the massive human epidemic of 2010 in Greece was also highly pathogenic for horses. This is the first time that WNV infection has been documented in horses with clinical manifestations in Greece. WNV infection should be included in the differential diagnosis of horses with encephalitis in Greece.

Α case of multiple bilateral testicular capsule mast cell tumours in a dog.

A 5-year-old intact male German Shepherd dog was referred with a diagnosis of leishmaniasis. Several testicular masses were palpated during the physical examination, while the diagnostic screening yielded no remarkable findings. Fine needle aspiration cytology of the masses revealed the presence of intermediately differentiated mast cell tumours. Scrotal ablation and orchiectomy were performed as a definitive treatment option. The pathological examination of the surgical specimens confirmed the diagnosis of grade II mast cell tumours and showed that they were all confined to the testicular capsule. At 7 months post-admission, the dog exhibited neither postsurgical complications nor metastatic foci and was, therefore, given a favourable prognosis. Despite their exceptionally rare occurrence, mast cell tumours should be considered for the differential diagnosis of testicular tumours.

Immunohistochemical expression of dog TERT in canine testicular tumours in relation to PCNA, ki67 and p53 expression.

The objective of the study was to determine the immunohistochemical expression of canine TERT in canine testicular tumours comparing two different antibodies for TERT, and to correlate them with well established markers specific to dividing cells such as PCNA and ki67, and with expression of the p53 tumour suppressor gene. The study included 36 cases of canine testicular tumours, which were categorized as 12 Sertoli Cell Tumours (SCT), 20 seminomas, 3 interstitial cell tumours and 1 mixed germ cell-sex cord stromal tumour (MT). Two antibodies for hTERT were examined; a highly specific TERT antibody, RCK-hTERT, was evaluated for the first time. Immunodetection of RCK-hTERT was observed in 31% of tumours examined (6/20 Seminomas, 4/12 SCT, 1/3 interstitial cell tumour and 0/1 mixed germ cell-sex cord stromal tumour), while the NCL-hTERT in 67% of them (15/20 Seminomas, 6/12 SCT, 3/3 interstitial cell tumour and 0/1 ΜΤ). PCNA immunoreactivity was detected in all cases. Regarding ki67, 3 SCT, 12 seminomas and all interstitial cell tumours showed clear immunoreaction. p53 immunoreactivity was detected in 6 SCT, 15 seminomas and all interstitial cell tumours. The immunohistochemical expression of both TERT antibodies are discussed and compared in order to clarify their potential usefulness in canine testicular malignancies in relation to the expression of well known cell cycle markers. Our results indicate that TERT and PCNA are useful proliferation markers but not helpful to evaluate prognosis. Instead of that ki67 and p53 could be used for predicting aggressiveness in this group of tumours.

Atypical PrPsc distribution in goats naturally affected with scrapie.

The brain and spinal cord of 48 goats from two Greek herds in which scrapie had been reported were examined. All animals were symptomless at the time of euthanasia. Notably, no lesions were observed either at the level of the obex or at other regions of the brain and spinal cord. Immunohistochemical examination revealed PrPsc labelling of the linear and fine punctuate types, mainly in the cerebral cortices, of 36 goats. Twenty-seven of them were negative by ELISA (designed to detect proteinase-resistant PrP) at the level of the obex but positive in a pooled brain sample, and the majority carried PrP genotypes associated with scrapie susceptibility. Surprisingly, in 16 of the 27 animals, PrPsc deposits were detected only in the rostral parts of the brain. In addition, nine animals which were ELISA-positive at the level of the obex exhibited positive immunoreactivity, but not in the dorsal vagal nucleus. The findings indicate that this unusual scrapie type may have been underdiagnosed previously and may be of importance in scrapie surveillance programmes.

A deletion in the R region of long terminal repeats in small ruminant lentiviruses is associated with decreased pathology in the lung.

A particular variant of the maedi visna virus (MVV) that although present in blood causes no clinical signs in infected sheep has been described. This variant carries a 13-14 nucleotide deletion in the R region of the proviral long terminal repeats. The hypothesis that this specific deletion may be associated with low pathogenicity has been investigated by comparing the distribution of proviral sequences, the histopathological lesions and the expression of viral proteins in the brain, lungs and udders of sheep naturally infected with viral strains carrying the deletion. Provirus could be demonstrated in most of the tissues examined from sheep infected with either type of virus, and the tissue-derived virus carried the typical deletion in the study flock animals. Histopathological analysis revealed that the lungs were significantly less affected in the animals infected with virus carrying the deletion. Concomitantly, viral expression was significantly reduced in the lungs of these animals. The findings suggest that the reduced pathogenicity of MVV with the specific deletion in the R region is not due to a restriction in the availability of specific tissues to infection, but is associated with a reduced capacity for viral expression in the lungs.

A clinical case of chicken infectious anemia disease and virus DNA detection in naturally infected broilers in Greece.

In this study, chicken infectious anemia virus (CIAV) DNA was detected from 12-day-old broilers. Clinical history showed that the clinical features were diarrhea, blue wing disease, depression, and death. Necropsy findings were pale liver, severe atrophy of bursa of Fabricius and thymus, and discoloration of the bone marrow as well as hemorrhages subcutaneously and a few in skeletal muscles. The majority of the necropsied broilers had developed gangrenous dermatitis. Histopathology showed hypoplasia of bone marrow and depletion of lymphocytes in spleen, bursa, and subcapsular thymic cortex. Karyorrhexis of lymphocytes was scattered in the thymic cortex and most pronounced in the bursal follicles. Eosinophilic intranuclear inclusion bodies were mainly located in lymphocytes of thymus, with a few in hemopoietic cells of bone marrow. CIAV DNA was detected by polymerase chain reaction from bursa, thymus, and bone marrow. A virus strain was detected and genetically characterized in 639 base pairs of VP1 gene. Phylogenetic analysis revealed that the Greek isolate was clustered together with isolates from Alabama, China, Slovenia, and Bangladesh.

Detection of maedi-visna virus in the liver and heart of naturally infected sheep.

Maedi-visna virus (MVV) in sheep, which infects mainly cells of the monocyte/macrophage lineage, produces changes in the lung, mammary gland, brain and joints. In this study, however, the liver and heart of six naturally infected sheep were examined for the presence of the virus. MVV proviral DNA was demonstrated by polymerase chain reaction (PCR) analysis, and immunohistochemical examination revealed viral antigens in the cytoplasm of hepatocytes and cardiac myocytes. Although histopathological examination showed mild to moderate, chronic lymphocytic cholangiohepatitis and myocarditis and the presence of small lymphoid aggregates, the typical maedi lymphoproliferative lesions (lymphoid follicle-like structures of considerable size with germinal centres) were not seen in the liver and heart. These novel findings suggest that, although the macrophage is the main cell for productive viral replication, the liver and heart represent additional MVV targets.

Histopathological and immunohistochemical features of natural goat scrapie.

Histopathological and immunohistochemical examinations were performed on the brain and spinal cord of 37 goats from two Greek herds in which scrapie had been reported. Of the 37 animals, 18 were from a herd consisting only of goats and 19 were from a herd of goats mixed with sheep. The goats studied were grouped on the basis of the presence or absence of clinical signs. Distinctive lesions and PrP(sc) (PrP, prion protein) deposition were found in the central nervous system (CNS) of eight clinically affected animals and six symptomless animals. The lesion profile and PrP(sc) distribution varied both between and within groups, variation being particularly pronounced in the symptomless goats. The results concerning the latter group suggested a poor correlation between the intensity of lesions, the amount of PrP(sc) in the CNS, and the manifestation of clinical signs. Immunohistochemical examination revealed 10 different PrP(sc) types, four of which are reported for the first time in goats. All scrapie-affected animals carried the VV(21)II(142)HH(143)RR(154) genotype, with the exception of two goats that carried the HR(143) dimorphism and had detectable PrP(sc) deposits. The results suggest that the histopathological and immunohistochemical profile of the natural disease in goats is influenced by the PrP genotype and age of the animals but may not be directly associated with the presence or otherwise of clinical signs.

Pathogenesis of experimental encephalomyocarditis: a histopathological, immunohistochemical and virological study in mice.

Mice (n=20) aged 8 weeks were infected, either by oronasal inoculation or by contact, with one of two different myocardial strains of encephalomyocarditis virus (EMCV), namely, the Greek strain 424/90 and the Belgian strain B279/95. The animals were killed at 18-59 days post-infection (dpi), except for two mice that died at 6 and 32 dpi, and samples of brain, heart, pancreas, kidney, Peyer's patches, spleen, lung and thymus were processed for virological, histopathological and immunohistochemical examination. Apart from the two deaths, the experimental infection was inapparent, but virus was invariably recovered from faeces and several organs. The main histopathological lesions were focal interstitial pancreatitis, depletion of thymus and Peyer's patches, and interstitial pneumonia. Additionally, in the two mice that died, multifocal interstitial myocarditis was observed. EMCV antigen was detected in the cytoplasm of pancreatic acinar cells and in macrophages of the lung and the thymus. Antigen was also detected in the cytoplasm of cardiac muscle cells from three animals, including the two that died. The results support the role of mice, in addition to rats, as reservoir hosts in the epidemiology of EMCV infections on pig farms.

Detection of maedi-visna virus in the kidneys of naturally infected sheep.

Infections with maedi-visna virus (MVV) cause progressive inflammation in different organs, mainly the lung, mammary gland, brain and joints. The aim of the present study was to investigate whether the kidney represents a viral target in natural MVV infection. For this, kidney samples from 13 sheep naturally infected with MVV were examined by histology, polymerase chain reaction (PCR), and immunohistochemistry. The kidneys of nine animals showed membranoproliferative glomerulonephritis and interstitial nephritis. The inflammatory infiltrate consisted of lymphocytes, plasma cells and macrophages. Interestingly, lymphoid follicles resembling those known to occur in other MVV-infected tissues were observed. Lung tissue from the same animals had typical MVV lesions, such as lymphofollicular hyperplasia and interstitial pneumonia. Maedi-visna proviral DNA sequences were detected in renal and lung tissue samples from these nine sheep by PCR, and the specificity of the amplified products was further verified by DNA sequencing. Moreover, MVV-specific immunohistochemistry revealed viral antigen in affected kidneys and lungs. These results suggest that the kidney may be a common target in natural MVV infection, and raise the issue of the role of this organ in the disease.

A novel deletion in the LTR region of a Greek small ruminant lentivirus may be associated with low pathogenicity.

Greek small ruminant lentivirus (SRLV) strains remain relatively uncharacterized at the molecular level, despite the fact that lentiviral diseases of small ruminants are known to be widespread in the country. In the present study, we investigated the sequence diversity of the LTR region in Greek SRLV strains from sheep with and without disease symptoms, since sequence differences within this genomic area have been shown to lead to SRLVs with distinct replication rates. The AP-4 and AML (vis) motifs and the TATA-box were highly conserved among Greek strains, whereas the two AP-1 sites exhibited some substitutions. Pairwise comparisons with reference strains revealed that Greek LTR sequences were closer to the ovine strains (25.7% average divergence) rather than the caprine strain CAEV (59.1% average divergence). The most striking difference observed between the two groups of animals was a 13-14 nucleotide deletion in the strains obtained from the asymptomatic sheep. The deletion was located within the R region of LTR, which was also found to be much less homologous (39.6% average divergence) than the U3 and U5. Taken together, our data suggest that the R region of LTR may be involved in virus transcriptional activation. Furthermore, a specific deletion within this region may, at least in part, be associated with low pathogenicity of some SRLV strains.

Chronic progressive autonomic dysfunction in a dog.

A 3.5-year-old intact male American Pit Bull was presented because of urinary incontinence and dysuria. Constipation, followed by diarrhoea, ocular disturbances and finally regurgitation developed over the next 4 years. Autonomic dysfunction was evidenced by clinical presentation, as well as positive ophthalmic pilocarpine test and subnormal Schirmer tear test. Diagnosis, however, was established through histopathological and immunohistochemical examination. Lesions attributable to inflammatory degenerative neuropathy of the autonomic ganglia, which represents one of the various types of human autonomic failure, were detected.

Pathogenesis of experimental encephalomyocarditis: a histopathological, immunohistochemical and virological study in rats.

Rats (n=40) aged 8 weeks were infected, either by oronasal inoculation or by contact, with one of two different myocardial strains of encephalomyocarditis virus (EMCV), namely, the Greek strain 424/90 and the Belgian strain B279/95. The animals were killed at 11-62 days post-infection (dpi) and samples of brain, heart, pancreas, kidney, Peyer's patches, spleen, lung and thymus were processed for virological, histopathological and immunohistochemical evaluation. This experimental infection was inapparent, but virus was isolated from faeces and several organs of all animals. The main histopathological changes were focal interstitial pancreatitis, degeneration and necrosis of pancreatic acinar cells, depletion of thymus and Peyer's patches, and interstitial pneumonia. EMCV antigen was detected in the cytoplasm of cardiac muscle cells, pancreatic acinar cells and hepatic epithelial cells, and in macrophages of the spleen, lung and thymus. In the heart (the target organ of EMCV in pigs), the presence of EMCV in cardiac muscle cells without lesions lends support to the hypothesis that the rat is a natural reservoir host species of EMCV. The persistence of virus in the macrophages of the thymus may represent a mechanism of perpetuation and reactivation, under immunosuppressive conditions, of the infection.

Outbreak of avian mycobacteriosis in a flock of two-year-old domestic pigeons (Columba livia f. domestica).

This report describes an outbreak of avian mycobacteriosis in a flock of 100 two-yr-old pigeons. Over a 6-mo period, the sick pigeons showed cachexia followed by death. In Columbiformes classic tubercles rarely develop, but in these affected pigeons granulomatous nodular lesions of various sizes, containing numerous acid-fast bacilli, were found in the internal organs. The lesions were observed in the liver, spleen, intestine, bone marrow, ovary, and oviduct. Despite their breeding age, atrophy was also found in the ovary and oviduct. Microorganisms belonging to Mycobacterium avium complex were identified in the affected tissues by polymerase chain reaction.

First partial characterisation of small ruminant lentiviruses from Greece.

Small ruminant lentivirus (SRLV) infections are widespread in Greece, but SRLVs have never been isolated and characterized. In this study, we present the sequence of a 574-nucleotide (191-amino acid) region of the gag gene of SRLV strains from four sheep and one goat from a single geographic area of Greece. All five sequences appeared to be closely related at both nucleotide (2.1-14.2% variation) and deduced amino acid (1.6-4.2% variation) level. Greek SRLV strains were closer to ovine prototypic strains (average divergence 16.8%) than to the caprine strain CAEV-Co (21% divergence). By amino acid composition, the Greek SRLVs were on the average more than twice as distant from CAEV-Co as from other ovine strains. Phylogenetic analysis suggested that Greek strains segregate into a unique group, separate from, but related to, other ovine prototype sequences.

Immunohistochemical investigation of amyloid beta-protein (Abeta) in the brain of aged cats.

To clarify the immunohistochemical features of amyloid deposits and cerebral amyloid angiopathy (CAA), the distribution of the amyloid beta-protein subtypes Abeta40, Abeta42, Abeta43 and Abeta precursor protein (APP) were examined in the brains of fourteen aged cats (7.5-21 year-old). Two types of plaques were detected. The first type was characterized by Ass positive antigenic material and detected in the cortical layers of the frontal and parietal lobes of all examined cats. The second type was characterized by diffuse positive immune staining representing diffuse plaques, which were detected only in the very aged cats (17-21 years old) and distributed throughout the cortical layers of the parietal lobes. Vascular amyloid and the amyloid deposits were strongly positive-stained with the antibody Abeta42. APP was exhibited in neurons and axons while the staining was stronger in the very aged cats (17-21 years old). Our findings suggest that the feline forms a spontaneous model for understanding the early changes of normal brain aging and the early stage of amyloid beta-protein deposition.

Obstructive, granulomatous tracheitis caused by Onchocerca sp. in a dog.

A 3-year-old spayed female dog was admitted with a history of episodes of dyspnoea and coughing. Severe tracheal stenosis was found on radiography. The dog died during an episode of refractory dyspnoea. Necropsy revealed an obstruction of the thoracic part of the trachea because of a chronic granulomatous inflammation protruding into the tracheal lumen. Histological examination revealed nematodes, which were identified as Onchocerca sp. according to their morphological characteristics. In contrast to the common ocular manifestation in dogs, obstructive tracheitis caused by Onchocerca infection has not been reported before.

Vertebral telangiectatic osteosarcoma in a dog.

Telangiectatic osteosarcoma (TOS) affecting the seventh cervical vertebra (C7) was diagnosed in a 4-year-old male dog with signs of locomotor dysfunction. Bone lysis and an extradural-extramedullary mass were observed in radiographs and occipital myelograms. The diagnosis was confirmed by histopathologic and immunohistochemical examination of the mass. The tumor was composed of large blood-filled cavities lined by anaplastic stromal cells (malignant osteoblasts). Around the cavities were characteristic tumor giant cells ("bizarre cells"). Immunohistochemically the tumor cells were positive for proliferating cell nuclear antigen. The lining cells of the cysts were negative for von Willebrand factor. The histologic findings in this case of TOS involving C7 were similar to those seen in other cases of TOS in dogs and in people. Immunohistochemistry was a useful tool for assessing malignancy and for ruling out other differential diagnoses.

Pathogenesis of encephalomyocarditis virus (EMCV) infection in piglets during the viraemia phase: a histopathological, immunohistochemical and virological study.

Six piglets aged 20 days were inoculated oranasally with 5 ml of a suspension (10(6) TCID(50)/ml) of a Greek myocardial strain of encephalomyocarditis virus (EMCV). The animals either died (n=2) or were killed for examination on days 1,2 or 3 post-inoculation (pi). EMCV was isolated from virtually all organs examined (heart, tonsils, palatine glands, pancreas, spleen, small intestine and mesenteric lymph nodes). Histopathologically, interstitial myocarditis, necrosis of cardiac muscle cells and Purkinje fibres, and necrotizing tonsillitis were detected in all inoculated piglets. Focal interstitial pancreatitis, necrosis of pancreatic acinar cells and Langerhans islet cells, and necrosis of germinal centre lymphocytes of the lymph nodes and Peyer's patches were detected in two piglets that died or were killed on day 3 pi. Immunohistochemically, viral antigen was detected in epithelial cells of all organs examined, including the tonsils, palatine glands, pancreatic interlobular ducts and small intestine. This suggests that EMCV is epitheliotropic, in addition to its known myocardial tropism. The frequent presence of intracytoplasmic EMCV in macrophages of the tonsils and spleen supports the hypothesis that macrophages play a role in viral replication and dissemination in the body.

Effect of the compound L-mimosine in an in vivo model of chronic granuloma formation induced by potassium permanganate (KMNO4).

The plant amino acid L-mimosine has recently been suggested to inhibit cells at a regulatory step in late G1 phase before establishment of active DNA replication forks. In addition, L-mimosine is an extremely effective inhibitor of DNA replication in chromosomes of mammalian nuclei. In this work, the effect of L-mimosine on chronic inflammation induced by dorsal injections of 0.2 ml of a 1:40 saturated crystal solution of potassium permanganate in mice, was studied. Seven days afterwards, all mice developed a subcutaneous granulomatous tissue indicative of chronic inflammatory response at the site of infection. The intraperitoneal administration of L-mimosine (200 microg/dose) to the potassium permanganate treated mice for 5 consecutive days (the first at the same time of inoculation of the KMnO4), produced a significant decrease in size and weight of the granuloma when compared to mice not treated with L-mimosine (controls). In addition, in all mice treated with L-mimosine, there was a strong inhibition of tumor necrosis factor alpha that was revealed in the serum (P<0.05) and in the minced granulomas. Interleukin-6 was not detected in the serum of treated and untreated mice. These findings show for the first time, that L-mimosine may have an anti-inflammatory effect on chronic inflammation and an inhibitory effect on tumor necrosis factor alpha and interleukin-6 generation in supernatant fluids of minced granulomas.