Detection of classical and atypical/Nor98 scrapie by the paraffin-embedded tissue blot method.

The paraffin-embedded tissue (PET) blot method was used to investigate sections of the central nervous system and lymphatic tissues from 24 cases of classical scrapie and 25 cases of atypical/Nor98 scrapie in sheep and four healthy control sheep. The PET blot detected deposits of PrP(Sc) in the brain tissue of all 49 sheep with scrapie but no PrP(Sc) labelling could be detected in the control sheep. By contrast, not all the atypical/Nor98 scrapie cases were detectable by immunohistochemistry. The high sensitivity of the PET blot method made it possible to observe that in some atypical/Nor98 cases, deposits of PrP(Sc) may be restricted to supratentorial brain structures and that the diagnosis may be missed when only testing the obex area, where deposits are common in classical scrapie, and the cerebellar structures, where deposits are considered to be common in atypical/Nor98 cases.

Recognition of the Nor98 variant of scrapie in the Swedish sheep population.

Within the framework of the active surveillance for transmissible spongiform encephalopathies in sheep in Sweden, 4 cases of the atypical form of scrapie, Nor98, were identified during 2003. Nor98 is a recently recognized and poorly understood variant of scrapie, first described in Norway. The cases were positive by the rapid test (enzyme-linked immunosorbent assay). Immunohistochemical staining showed diffuse thin-granular staining of the cerebellar cortex. Western immunoblotting analysis of specimens of brain stem and cerebellum showed a light band of approximately 12 kDa. Typical scrapie was ruled out based on the confirmatory testing. The affected ewes were from 4 different flocks. They were between 7 and 9 years old. Two were of the ARQ/ARQ genotype, 1 ARR/ARQ, and 1 ARR/AHQ. Two ewes had shown ataxia, and the other 2 had no clinical signs. Whole-flock slaughter was applied, and testing of the flock mates did not reveal additional cases. Nor98 differs from typical scrapie in its epidemiology, frequency of genotypes of sheep affected, clinical signs, microscopic lesions, distribution of scrapie prion protein in the brain, and characteristics of the immunostaining and immunoblotting profiles.

Cases of scrapie with unusual features in Norway and designation of a new type, Nor98.

Five cases of scrapie with unusual features have been diagnosed in Norway since 1998. The affected sheep showed neurological signs dominated by ataxia, and had the PrP genotypes homozygous A136 H154 Q171/ A136H154Q171 or heterozygous A136H154Q171/A136R154Q171, which are rarely associated with scrapie. Brain histopathology revealed neuropil vacuolisation essentially in the cerebellar and cerebral cortices; vacuolation was less prominent in the brainstem, and no lesions were observed at the level of the obex. The deposits of PrPSc were mainly in the cortex of the cerebellum and cerebrum, and no PrPSC was detectable by immunohistochemistry and ELISA in the lymphoid tissues investigated. Western blot analysis showed that the glycotype was different from other known scrapie strains and from the BSE strain. From a diagnostic point of view, these features indicate that this type of scrapie, designated Nor98, could have been overlooked and may be of significance for sampling in scrapie surveillance programmes.

Prion protein gene polymorphisms in sheep with natural scrapie and healthy controls in Norway.

Two-hundred and forty healthy sheep and 32 cases of natural scrapie in Norway were analysed for disease-linked polymorphisms in the prion protein (PrP) gene. Scrapie was strongly associated with the presence of a valine polymorphism at codon 136 (V136), as 68.8% of the cases were homozygous (VV136) and 15.6% were valine/alanine heterozygous (VA136). All cases were homozygous arginine/arginine at codon 154 (RR154), except two which were homozygous histidine/histidine (HH154). All cases except two were homozygous glutamine/glutamine at codon 171 (QQ171), the two exceptions being heterozygous glutamine/arginine (QR171). More than 80% of all scrapie cases in Norway have occurred in a Cheviot-related crossbred type of sheep called Rygja. This type of sheep, which is largely restricted to the south-western coast, carries the V136 allele at a higher frequency than do other breeds of Norwegian sheep. Polymorphisms at codons 138 and 151 are also described.

Toxic nephrosis in moose in Norway.

During the summers 1995/96, toxic nephrosis was diagnosed in nine free-living moose (Alces alces) from Aust-Agder County in southern Norway. Histopathological kidney lesions included tubular degeneration and necrosis, tubular regeneration, and interstitial fibrosis. The disease was probably caused by ingestion of the toxic plants, bog asphodel (Narthecium ossifragum) or oak (Quercus spp.).

Nephrotoxicity in goats caused by dosing with a water extract from the stems of Narthecium ossifragum plants.

Seven goats were given a single dose of an aqueous extract derived from 30 g (wet weight) of Narthecium ossifragum per kg liveweight. Their serum creatinine and urea concentrations increased to day 5 but then fell to normal by day 10. Serum magnesium increased to day 4 and decreased to normal by day 9. Their serum calcium concentration was lower than normal on days 4, 5 and 6. Histopathological examination of the kidneys of goats killed or found dead 2, 4, 6, 8, 11 or 16 days after dosing revealed tubular epithelial cell degeneration and necrosis. Regeneration of the tubular epithelium and signs of interstitial fibroplast proliferation and fibrosis could be seen in animals killed on days 8, 11, 16 and 42. No signs of liver damage were observed in 3 goats dosed with the insoluble plant material from 40 g (wet weight) Narthecium ossifragum per kg liveweight. The total dose was divided into three doses, which were given intraruminally within 7 h. The activities of aspartate aminotransferase, gamma-glutamyltransferase and glutamate dehydrogenase remained within the normal range in all 10 goats after dosing.

Further studies on the presence, qualities and effects of the toxic principles from Narthecium ossifragum plants.

One calf was dosed during one day with an aqueous extract from 3.0 kg (wet weight) of Narthecium ossifragum and another was dosed on the same day with the insoluble plant residue. The concentrations of serum creatinine and magnesium increased only in the calf dosed with the aqueous extract, while the activity of glutamate dehydrogenase increased only in the serum of the calf dosed with the plant residue, so differentiating the nephrotoxic and hepatotoxic principles as water-soluble and water-insoluble compounds, respectively. One calf was dosed with 30 g (wet weight) N. ossifragum flower stems per kg live weight during one day and another was dosed with 30 g (wet weight) N. ossifragum leaves per kg live weight on the same day. The serum creatinine and urea concentrations and also the activities of glutamate dehydrogenase, aspartate aminotransferase and gamma-glutamyltransferase in the serum increased in the calf dosed with the flower stems, whereas there was only a slight temporary increase in the creatinine concentration in serum from the calf dosed with the leaves. However, histopathological examination of the kidneys of the calf dosed with the flower stems revealed severe tubular necrosis and degeneration. It therefore appears that both the toxic principles are present in the flower stems of N. ossifragum rather than in its leaves. The serum creatinine concentration was significantly increased in a non-ruminating calf dosed with an aqueous extract from 32 g (wet weight) N. ossifragum per kg liveweight during one day, showing the intrinsic nephrotoxicity of the plant.

Nephrotoxicity of Narthecium ossifragum in cattle in Norway.

During the summer of 1992 renal failure was diagnosed in 232 grazing cattle in 85 herds on the west coast of Norway. The salient clinical signs were depression, anorexia and melaena or fresh blood in the faeces; diarrhoea was also commonly observed. The serum concentrations of creatinine, urea, magnesium and phosphorus, and the activities of glutamate dehydrogenase, aspartate aminotransferase and creatine kinase were above normal and the serum calcium concentration was below normal. Post mortem examinations consistently revealed renal tubular necrosis. In some cases there was liver necrosis and also erosions at the base of the tongue, in the oesophagus and in the jejunum and colon. The toxicity was probably caused by the plant Narthecium ossifragum (bog asphodel).

Nephrotoxicity and hepatotoxicity in calves apparently caused by experimental feeding with Narthecium ossifragum.

Seven calves were fed a mixture of bog plants containing 15 g (wet matter) Narthecium ossifragum per kg live weight for two consecutive days. Their serum creatinine, urea and magnesium concentrations increased, whereas the serum calcium concentration decreased. Histopathological examination of the kidneys of the 5 calves that were killed revealed tubular epithelial cell degeneration and necrosis. There were signs of liver dysfunction in all the calves including increased aspartate aminotransferase (ASAT), glutamate dehydrogenase (GLDH) and gamma-glutamyltransferase activities. All the calves refused to ingest N. ossifragum after 2 days feeding, and their appetite for hay and concentrate was also reduced. It can be concluded that N. ossifragum is nephrotoxic and hepatotoxic to calves.

Nephrotoxicity in lambs apparently caused by experimental feeding with Narthecium ossifragum.

Seven lambs were fed a mixture of bog plants containing 15 g (wet matter) Narthecium ossifragum per kg live weight for 10 consecutive days. Their serum creatinine concentration increased from day 1 to 4 but then fell to normal by day 6 after feeding started. In the same animals, the serum magnesium concentration was increased on days 3, 4 and 5. Histopathological examination of the kidneys of 2 lambs killed after being fed 25 g N. ossifragum per kg live weight for one day revealed tubular epithelial cell degeneration and necrosis. There were no signs of disease or distress in the experimental animals and their appetite remained normal throughout the experimental period. It was concluded that N. ossifragum is probably nephrotoxic to lambs.

An ultrastructural and cytochemical study of the pulmonary lesions and multinucleate giant cells in porcine dermatosis vegetans.

The pulmonary lesions and multinucleate giant cell formations in porcine dermatosis vegetans were studied ultrastructurally and cytochemically in seven affected pigs that ranged from one to 120 days of age. At birth, no pulmonary lesions were observed. By seven days of age, there was a moderate infiltration of monocytes/macrophages in the alveoli and interstitium. These changes progressed, and by two weeks of age there was pronounced infiltration of macrophages and lymphocytes in the alveoli. Close apposition of macrophages and lymphocytes was observed, and cellular contact was demonstrated. By three to four weeks of age, small aggregates with epithelioid cells, macrophages, and lymphocytes resembling granulomas were present. In an older pig, aged four months, the inflammatory changes waned. Large macrophages, and large and small multinucleate cells shared common morphological characteristics of cytoplasm endowed with organelles, primary and secondary lysosomes, Golgi profiles, and granular endoplasmic reticulum. Cytochemically, macrophages and MGCs were positive for acid phosphatase. The present study showed that the initial pulmonary changes share morphological characteristics with a granulomatous inflammatory response, and evolve into small granulomas with macrophages, epithelioid cells and lymphocytes. Morphologically, these changes share characteristics with epithelioid cell granulomas. Macrophages and MGCs share common ultrastructural traits, and are positive for acid phosphatase. MGCs seem to evolve in the course of the granulomatous inflammatory response and are probably of monocyte/macrophage origin.

A sequential light microscopic study of the pulmonary lesions in porcine dermatosis vegetans.

The sequential pulmonary changes in dermatosis vegetans were studied in pigs aged one, seven, 14, 17, 21 and 28 days, and two and a half, four and 12 months. At one day old the lung tissues appeared normal. At seven days old there was a moderate infiltration of mononuclear inflammatory cells in the alveolar structures, and proliferation of type II pneumocytes was also observed. At 14 and 17 days old the alveolar structures were intensely infiltrated with mononuclear inflammatory cells. At 21 days old, loosely arranged epithelioid and multinucleate giant cell granulomas appeared, and there was a proliferation of alveolar epithelial cells. By four and 12 months old the inflammatory changes diminished, and small granulomas and moderate alveolar fibrosis were present. The initial lesions were characterised by an alveolitis, developing into loosely arranged granulomas by four months old, and persisting to the age of 12 months. These observations suggest that the interpretation of the lung changes and the disease in general should be revised.

Morphology and immunoperoxidase studies of intestinal adenomatosis in the blue fox, Alopex lagopus.

In a blue fox farm about 300 of 400 weanling pups were affected with diarrhoea and prolapse of the rectum. About 40 pups died in an emaciated state after a few days of disease. Eight pups were submitted for examination. Pathological findings were restricted to the caecum, proximal colon and rectum, with occasional extension to the ileum. Affected areas revealed a thick and rigid intestinal wall, a narrow lumen and a thickened, wrinkled and sometimes ulcerated mucosa. Histologically, the mucosa showed long tortuous crypts outlined by a high pseudostratified epithelium which lacked goblet cells. Epithelial cells revealed increased basophilia, and mitoses were seen along the entire crypt length and in the surface epithelium. Silver impregnation showed curved organisms in the apical cytoplasm of the altered epithelial cells. Penetration of the muscularis mucosa by the altered epithelial cells was frequent. The avidin-biotin-immunoperoxidase complex (ABC) technique revealed positive organisms in the apical cytoplasm of altered epithelial cells, when rabbit antisera against different biotypes and serotypes of Campylobacter jejuni and Campylobacter coli were used. Antisera against Campylobacter sputorum subsp. muscosalis and Campylobacter hyointestinalis were negative.

Myofibre changes and capsule formation in mice infected with different strains of Trichinella.

Mice were infected with 4 strains of Trichinella with the purpose of describing muscle changes (M. Gastrocnemius, diaphragma) by light and electron microscopy, paying particular attention to capsule formation in a polar bear isolate (T. no. 13). No differences in myofibre changes or capsule formation were observed between the different strains by light microscopy at 21 and 60 days post infection. Ultrastructural studies of meat samples 60 day post infection with T. no. 13 revealed active capsule formation in muscle fibres, characterized by protrusions and invaginations of the sarcolemma. Basal lamina-like material was located close to the sarcolemma and in a lattice-like pattern in the capsule matrix. T. no. 13 seems to have a migratory pattern similar to the other strains examined and induces muscle fibre changes undiscernible from those of the 3 other strains. Infected muscle cells are considered to be involved in capsule formation.

Non-neoplastic pulmonary multinucleate giant cells. Comparative study of multinucleate giant cells in porcine dermatosis vegetans and other polykaryocytic conditions.

Pulmonary multinucleate giant cells (MGCs) in porcine dermatosis vegetans (DV) were compared with virus-induced and inflammatory-associated MGCs, with particular attention to mechanisms involved in the formation of such cells. Ultrastructurally, pigs with DV showed numerous pulmonary MGCs with transversely-sectioned interdigitating microvillous structures. Microvilli were observed closely apposed to type-II pneumocytes and with morphological characteristics of membrane bridge formation. Rabbits infected with Mycobacterium bovis exhibited multiple pulmonary granulomas with prominent MGC formation 14 days after infection. Close apposition between MGCs and small macrophages and membrane bridge formation were demonstrated ultrastructurally. Pulmonary changes in calves infected with bovine respiratory syncytial virus (BRSV) included lobular consolidation and intense infiltration of inflammatory cells into the bronchial, bronchiolar and alveolar walls. Proliferation of bronchiolo-alveolar epithelial cells was prominent, but syncytiae were only occasionally demonstrated. This study of the morphological characteristics of MGCs is related to the present knowledge of mechanisms involved in membrane fusion and allows for an interpretation of an active fusion process involved in the formation of MGCs in porcine DV.