Sylvie L. Benestad

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/ A136 H154 Q171 or heterozygous A136 HI54Q171/A136 R154 Q171, 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.

Atypical/Nor98 scrapie: properties of the agent, genetics, and epidemiology

Atypical/Nor98 scrapie cases in sheep were diagnosed for the first time in Norway in 1998. They are now identified in small ruminants in most European countries and represent an increasingly large proportion of the scrapie cases diagnosed in Europe. Atypical/Nor98 scrapie isolates have shown to be experimentally transmissible into transgenic mice and sheep but the properties of the TSE agent involved, like its biological and biochemical features, are so clearly distinct from the agent involved in classical scrapie that they have provided a challenging diagnostic for many years. No strain diversity has yet been identified among the atypical/Nor98 scrapie sample cases. The genetic predisposition of the sheep affected by atypical/Nor98 scrapie is almost inverted compared to classical scrapie, and the exact origin of this sporadic TSE strain is still speculative, but a spontaneous, non-contagious origin, like sporadic Creutzfeldt-Jakob disease in humans, can not be excluded. Further transmission and epidemiological studies are needed to better address this hypothesis.

Similar Biochemical Signatures and Prion Protein Genotypes in Atypical Scrapie and Nor98 Cases, France and Norway

Similarities raise questions regarding the origin of these recently described cases.

Prions in Milk from Ewes Incubating Natural Scrapie

Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrPSc accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 µg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species.

Two Irish cases of scrapie resembling Nor98

SINCE 1998, 38 cases of scrapie with unusual features, des-ignated Nor98 (Benestad and others 2003), have been diag-nosed in Norway (B. Bratberg, personal communication).Differences exist between the more usual scrapie phenotypesand Nor98 in vacuolation patterns (Wood and others 1997,Begara-McGorum and others 2002, Ligios and others 2002),disease-specific, protease-resistant form of the prion protein(PrP

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.

Atypical/Nor98 Scrapie Infectivity in Sheep Peripheral Tissues

Atypical/Nor98 scrapie was first identified in 1998 in Norway. It is now considered as a worldwide disease of small ruminants and currently represents a significant part of the detected transmissible spongiform encephalopathies (TSE) cases in Europe. Atypical/Nor98 scrapie cases were reported in ARR/ARR sheep, which are highly resistant to BSE and other small ruminants TSE agents. The biology and pathogenesis of the Atypical/Nor98 scrapie agent in its natural host is still poorly understood. However, based on the absence of detectable abnormal PrP in peripheral tissues of affected individuals, human and animal exposure risk to this specific TSE agent has been considered low. In this study we demonstrate that infectivity can accumulate, even if no abnormal PrP is detectable, in lymphoid tissues, nerves, and muscles from natural and/or experimental Atypical/Nor98 scrapie cases. Evidence is provided that, in comparison to other TSE agents, samples containing Atypical/Nor98 scrapie infectivity could remain PrPSc negative. This feature will impact detection of Atypical/Nor98 scrapie cases in the field, and highlights the need to review current evaluations of the disease prevalence and potential transmissibility. Finally, an estimate is made of the infectivity loads accumulating in peripheral tissues in both Atypical/Nor98 and classical scrapie cases that currently enter the food chain. The results obtained indicate that dietary exposure risk to small ruminants TSE agents may be higher than commonly believed.

Evidence for zoonotic potential of ovine scrapie prions

Although Bovine Spongiform Encephalopathy (BSE) is the cause of variant Creutzfeldt Jakob disease (vCJD) in humans, the zoonotic potential of scrapie prions remains unknown. Mice genetically engineered to overexpress the human prion protein (tgHu) have emerged as highly relevant models for gauging the capacity of prions to transmit to humans. These models can propagate human prions without any apparent transmission barrier and have been used used to confirm the zoonotic ability of BSE. Here we show that a panel of sheep scrapie prions transmit to several tgHu mice models with an efficiency comparable to that of cattle BSE. The serial transmission of different scrapie isolates in these mice led to the propagation of prions that are phenotypically identical to those causing sporadic CJD (sCJD) in humans. These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions.

Scrapie case similar to Nor98 diagnosed in Belgium via active surveillance

SCRAPIE is a fatal transmissible spongiform encephalopathy (TSE) caused by prions. Other diseases caused by prions include Creutzfeldt-Jakob disease in human beings and bovine spongiform encephalopathy (BSE) in cattle. Typical features of these diseases are a long incubation period and the gradual vacuolation of brain neurons and neuropil. The pathogenesis of the diseases is believed to be due to the conversion of the normal protease-sensitive prion protein, PrPc, into a partly protease-resistant isoform, PrPSc, which accumulates progressively in the central nervous system of affected animals (Prusiner 1982, Bueler and others 1993). Several (classical) scrapie strains have been described based on lesion profiling in mice (Wood and others 1997, Ligios and others 2002). Since April 2002, all sheep older than 18 months in Belgium have been tested with a rapid test (BSE Platelia test; Bio-Rad) through the active TSE surveillance programme (Pastoret and others 2001, Roels and others 2002) (EC regulation 999/2001). The rapid test is used for the diagnosis of BSE in bovine brain tissues (Moynagh and Schimmel 1999), but is also suitable for the diagnosis of scrapie in sheep, as the monoclonal antibodies used recognise the ovine PrP (Andreoletti and others 2000). This short communication describes a case of scrapie which was similar to Nor98, an unusual case of scrapie first detected in Norway (Benestad and others 2003). Up until the end of 2003, more than 5000 sheep had been tested for scrapie, and six primary outbreaks were detected. Five of the outbreaks showed a classical scrapie lesion profile, but one sheep showed unusual features. The ewe was apparently healthy and presented for slaughter. According to the active epidemiosurveillance protocol, only part of the medulla oblongata around the region of the obex was removed. The sample repeatedly tested positive with the rapid test (Debecker and others 2000). Histopathological investigation revealed no vacuolar lesions either in the neurons or in the neuropil in the region of the obex. There was no detectable PrPSc as revealed by immunohistochemistry of the obex region and tonsils using polyclonal R524-7. The detection of scrapie-associated fibrils (SAFs) was also negative. PrPSc analysis using Western blotting (Bio Rad protocol) with antibodies 12F10 and SAF60 (Fig 1) and BAR226 and SAF60, gave a clear positive result, showing a PrPSc glycoprofile with a strong lower band at approximately 12 kDa, compared with a classical scrapie glycoprofile (Fig 1). The glycoprofile of the present case was confirmed by the National Veterinary Institute, Oslo. Under the terms of the surveillance protocol, the whole flock was culled and the brains of all the animals older than 18 months were examined, but no other animal in the flock tested positive with the rapid test. The affected sheep’s PrP genotype was A136R154Q171 homozygous; this was determined by denaturing gradient gel electrophoresis (Bossers and others 1996). The present case has unusual characteristics: only one of the 55 animals in the flock was affected; no lesions were present in the brainstem (obex) compared with the lesion profiles of classical scrapie cases (Wood and others 1997, Ligios and others 2002); there was no PrPSc immunolabelling in the area of the obex; and the PrPSc glycoprofile differed clearly from the glycoprofiles found in isolates of classical scrapie strains and the BSE strain, and was not distinguishable from the Nor98 glycoprofile. All these features corresponded very well with those reported of the unusual Nor98 strain detected in Norway (Benestad and others 1999, 2002, 2003, Bratberg and others 2003). This raises questions concerning the scrapie active epidemiosurveillance protocol because, from a diagnostic point of view, the positive results obtained with a rapid test require confirmation by standard methods such as histopathological examination and the immunohistochemical detection of PrPSc at the level of the obex. In the present case, the confirmation tests (histology, immunohistochemistry and detections of SAFs), which are officially recognised in Belgium, were all negative. Only Western blotting was positive for scrapie; thus, the diagnosis of scrapie could have been overlooked. This may be of significance for future sampling in scrapie surveillance programmes and confirmation tests.

First case of chronic wasting disease in Europe in a Norwegian free-ranging reindeer

Chronic wasting disease (CWD) is a fatal contagious prion disease in cervids that is enzootic in some areas in North America. The disease has been found in deer, elk and moose in the USA and Canada, and in South Korea following the importation of infected animals. Here we report the first case of CWD in Europe, in a Norwegian free-ranging reindeer in Southern Norway. The origin of the disease is unknown. Until now a low number of cervids, and among them a few reindeer, have been tested for CWD in Norway. Therefore the prevalence of CWD is unknown.