V. Psychas

Persistence of encephalomyocarditis virus (EMCV) infection in piglets

Six piglets that had survived experimental infection with encephalomyocarditis virus (EMCV) were treated with dexamethasone for a period of 5 days. The virus had not been detected in excretions of putative carriers for a period of 13-20 days before the treatment. All piglets showed a rise in cardiac isoenzyme (CK-MB) activity, from the first day of treatment, suggesting myocardial damage. Antibody titres against EMCV remained stable or slightly decreased during treatment. EMCV was isolated from blood, nasal and faecal samples from all piglets on days 2 and 3 after initiation of treatment and from various tissues of three piglets. Four contact piglets, that were housed together with the dexamethasone-treated piglets, became infected, indicating that EMCV was shed by treated piglets. It is suggested that recovered pigs may play an important role in the dissemination of EMCV.

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, Peyers 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 Peyers 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.

Assessment of bluetongue viraemia in sheep by real-time PCR and correlation with viral infectivity.

Inoculation of embryonated chicken eggs is the standard method for the titration of infectious Bluetongue virus (BTV). Here, six RNA extraction methods coupled with optimised dsRNA denaturation and real-time RT-PCR were evaluated for the quantitation of BTV in blood samples from experimentally infected sheep and results were correlated to infectious virus titres. An exogenous dsRNA internal control (IC) from the closely related Epizootic hemorrhagic disease virus (EHDV) was used to assess the efficiency of BTV genome extraction, dsRNA denaturation, RT, and PCR amplification. Recovery rates of IC and BTV dsRNA copies from extracted blood samples were highly correlated. Adjustment of BTV concentrations according to the IC recovery reduced variation in sample analyses among the different extraction methods and improved the accuracy of BTV quantitation. The EID(50)/ml titre, determined in blood samples from sheep infected experimentally with BTV-1 or BTV-9, correlated highly with the assessed concentration of BTV dsRNA copies. However, this correlation was consistent only during the first 28 days post-infection. The optimised extraction methods and quantitative RT-PCR could be useful for experimental studies of BTV transmission, pathogenesis and vaccine efficacy, or adapted further for the detection and quantitation of EHDV, African horse sickness virus and other dsRNA viruses.

A comparative study of the pathogenic properties and transmissibility of a Greek and a Belgian encephalomyocarditis virus (EMCV) for piglets

Thirteen susceptible piglets, aged 40 days, were divided into two groups and were experimentally infected either with a Greek (myocardial) or a Belgian (reproductive) encephalomyocarditis virus (EMCV) strain (total dose 5 x 10(6) TCID50, intramuscularly and intranasally). Six piglets were placed in the same rooms, 24 h later, as contact controls. The following criteria were studied: ante mortem: clinical signs, serum cardiac isoenzyme activities (CK-MB and LD-1), viraemia, nasal and faecal virus excretion and serological response. Post mortem (after death or euthanasia): gross lesions, virus isolation from tissues, RT-PCR, as well as histopathological and immunohistochemical findings. The Greek strain was more pathogenic, producing mortality, with high cardiac isoenzyme activities and pronounced macroscopic myocardium lesions. The Belgian strain was able to induce mild heart lesions, as detected only by cardiac isoenzyme activity and histopathologically. All contact pigs were infected, within the first 1-2 days of their introduction, that coincided with the period of viral excretion by the experimentally infected pigs (up to the 3rd day post infection). Disease was mild, with no mortality.

Equine infectious anemia in mules: virus isolation and pathogenicity studies

There appears to be a lack of information concerning responses of mules to natural infection or experimental inoculation with equine infectious anemia virus (EIAV). In the present study EIAV was isolated from mules, for the first time, and its pathogenicity in naturally infected and experimentally inoculated animals was investigated. Two naturally infected (A and B) and three EIAV free mules (C, D and E) were used for this purpose. Mule A developed clinical signs, whereas mule B remained asymptomatic until the end of the study. Mules C and D were each inoculated with 10ml of blood from mule A and developed signs of the disease; they were euthanatized or died at day 22 and 25 post-inoculation, respectively. Mule E served as a negative control. The virus was isolated from the plasma samples of mules with clinical signs of the disease (A, C and D), but not from the asymptomatic mule B. Both proviral DNA and viral RNA were amplified from blood and tissues of the infected animals by nested polymerase chain reaction (nPCR). Antibodies were not detected in the two experimentally infected mules until their natural death or euthanasia. Clinicopathological and laboratory findings showed that, in mules, EIAV produced clinical signs similar to those observed in horses and ponies. Nested PCR proved to be a rapid, sensitive and specific diagnostic method for the detection of EIAV, regardless of the disease stage.

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.

European Brown Hare Syndrome in Wild European Brown Hares from Greece

From 1999 to mid-2003, 97 European brown hares (Lepus europaeus) found dead throughout Greece were examined by necropsy, histopathology, and reverse-transcription polymerase chain reaction (RT-PCR) for the presence of European brown hare syndrome (EBHS) and EBHS virus (EBHSV), respectively. Hare losses were sporadic, starting in the cold season and lasting for many months (December to May). The most prominent gross lesions were observed in the liver and included swelling and discoloration; congestion and hemorrhages were present mainly in lungs and tracheal mucosa. Necropsy findings were suggestive of EBHS, which was confirmed by histopathology and RT-PCR. This study documents, for the first time, EBHS in Greece.

DETECTION AND GENETIC CHARACTERIZATION OF PORCINE CIRCOVIRUS 2 ISOLATES FROM THE FIRST CASES OF POSTWEANING MULTISYSTEMIC AND WASTING SYNDROME IN WILD BOARS IN GREECE

In 2002, postweaning multisystemic wasting syndrome (PMWS) was diagnosed in a European female wild boar (Sus scrofa), based on the detection of porcine circovirus 2 (PCV2) DNA in various organs, including the uterus, and on histopathologic lesions. This is the first detection of PCV2 DNA in the uterus of a wild boar. Three years later (2005), a wild boar <6–8 mo of age was found moribund. It presented wasting and dyspnea and finally died. PCV2 DNA was detected in tissue samples, and histopathologic lesions consistent with PMWS were observed. Both wild boars were from neighboring hunting areas in central Greece. Two PCV2 strains from the wild boars were genetically characterized and compared to other reported PCV2 sequences from wild boars and domestic pigs. The PCV-2 sequences from the wild boars in this study were closely related to each other and were grouped with two isolates from wild boars from Hungary. The phylogenetic analysis revealed that the virus might be transmitted between hunting areas. In addition, PCV2 may spread from domestic pigs to wild boars and vice versa.

Congenital nutritional myodegeneration (white muscle disease) in a red deer (Cervus elaphus) calf

Abstract CASE HISTORY A 5-day-old red deer calf was submitted with tachypnoea and dyspnoea, and was reluctant to move. CLINICAL FINDINGS: Muscular damage was established via elevated creatinine phosphokinase (CPK) activities (5,000 U/L), while concentrations of Se in whole blood were low (24.8 nmol/L). The animal died despite treatment with penicillin-streptomycin and 0.1 mg/kg Se/vitamin E administered by S/C injection. DIAGNOSIS: Necropsy and histological examination of cardiac and skeletal muscle confirmed the presumptive diagnosis of congenital white muscle disease (WMD). Prophylactic administration of a Se/vitamin E commercial preparation (as above) to another calf born in the same herd one month later was associated with good health and apparently normal growth and development. CLINICAL RELEVANCE: Congenital WMD due to Se deficiency can be fatal in red deer calves. However, prophylactic administration of Se and vitamin E to neonatal calves may be beneficial for neonatal red deer calves.

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.