Olivier Andreoletti

Early accumulation of PrPSc in gut-associated lymphoid and nervous tissues of susceptible sheep from a Romanov flock with natural scrapie

The immune system is known to be involved in the early phase of scrapie pathogenesis. However, the infection route of naturally occurring scrapie and its spread within the host are not entirely known. In this study, the pathogenesis of scrapie was investigated in sheep of three PrP genotypes, from 2 to 9 months of age, which were born and raised together in a naturally scrapie-affected Romanov flock. The kinetics of PrP(Sc) accumulation in sheep organs were determined by immunohistochemistry. PrP(Sc) was detected only in susceptible VRQ/VRQ sheep, from 2 months of age, with an apparent entry site at the ileal Peyers patch as well as its draining mesenteric lymph node. At the cellular level, PrP(Sc) deposits were associated with CD68-positive cells of the dome area and B follicles before being detected in follicular dendritic cells. In 3- to 6-month-old sheep, PrP(Sc) was detected in most of the gut-associated lymphoid tissues (GALT) and to a lesser extent in more systemic lymphoid formations such as the spleen or the mediastinal lymph node. All secondary lymphoid organs showed a similar intensity of PrP(Sc)-immunolabelling at 9 months of age. At this time-point, PrP(Sc) was also detected in the autonomic myenteric nervous plexus and in the nucleus parasympathicus nervi X of the brain stem. These data suggest that natural scrapie infection occurs by the oral route via infection of the Peyers patches followed by replication in the GALT. It may then spread to the central nervous system through the autonomic nervous fibres innervating the digestive tract.

BSE agent signatures in a goat

SIR, – One of the concerns about BSE is the potential presence of the agent in small ruminants, sheep and goats, as well as cattle. With the objective of documenting this, seven French laboratories have analysed 438 brain samples from confirmed cases of TSE in sheep and goats. These comprised

Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein

Transmissible spongiform encephalopathies, or prion diseases, are fatal degenerative disorders of the central nervous system that affect humans and animals. Prions are nonconventional infectious agents whose replication depends on the host prion protein (PrP). Transmission of prions to cultured cells has proved to be a particularly difficult task, and with a few exceptions, their experimental propagation relies on inoculation to laboratory animals. Here, we report on the development of a permanent cell line supporting propagation of natural sheep scrapie. This model was obtained by stable expression of a tetracycline-regulatable ovine PrP gene in a rabbit epithelial cell line. After exposure to scrapie agent, cultures were repeatedly found to accumulate high levels of abnormal PrP (PrPres). Cell extracts induced a scrapie-like disease in transgenic mice overexpressing ovine PrP. These cultures remained healthy and stably infected upon subpassaging. Such data show that (i) cultivated cells from a nonneuronal origin can efficiently replicate prions; and (ii) species barrier can be crossed ex vivo through the expression of a relevant PrP gene. This approach led to the ex vivo propagation of a natural transmissible spongiform encephalopathy agent (i.e., without previous experimental adaptation to rodents) and might be applied to human or bovine prions.

PrPSc accumulation in placentas of ewes exposed to natural scrapie: influence of foetal PrP genotype and effect on ewe-to-lamb transmission

Placentas from scrapie-affected ewes are known to be infectious. Nevertheless, placenta infectivity in such ewes is not systematic. Maternal transmission to lambs is highly suspected but contamination of the foetus in utero has not been demonstrated. Using ewes from a naturally scrapie-infected flock, it was demonstrated that abnormal prion protein (PrP(Sc)) accumulation in the placenta (i) is controlled by polymorphisms at codons 136, 154 and 171 of the foetal PrP gene and (ii) is restricted mainly to placentome foetal trophoblastic cells. In order to go deeper into the role of the placenta in scrapie transmission, the pattern of PrP(Sc) dissemination was established in susceptible lambs (genotype VRQ/VRQ) sampled from 140 days post-insemination to the age of 4 months from either VRQ/VRQ ewes with PrP(Sc)-positive placentas or ARR/VRQ ewes with PrP(Sc)-negative placentas. In both VRQ/VRQ lamb groups, PrP(Sc) spatial and temporal accumulation patterns were similar, suggesting post-natal rather than in utero contamination.

PrPSc accumulation in myocytes from sheep incubating natural scrapie.

Because variant Creutzfeldt–Jakob disease (vCJD) in humans probably results from consumption of products contaminated with tissue from animals with bovine spongiform encephalopathy, whether infectious prion protein is present in ruminant muscles is a crucial question. Here we show that experimentally and naturally scrapie-affected sheep accumulate the prion protein PrPSc in a myocyte subset. In naturally infected sheep, PrPSc is detectable in muscle several months before clinical disease onset. The relative amounts of PrPSc suggest a 5,000-fold lower infectivity for muscle as compared to brain.

A Bovine Prion Acquires an Epidemic Bovine Spongiform Encephalopathy Strain-Like Phenotype on Interspecies Transmission

Implementation in Europe of large-scale testing to detect bovine spongiform encephalopathy (BSE)-infected cattle and prevent the transmission of this prion disease to humans has recently led to the discovery of novel types of bovine prions. We characterized atypical isolates called BSE L-type by analyzing their molecular and neuropathological properties during transmission to several mouse lines transgenic for the prion protein (PrP). Unexpectedly, such isolates acquired strain features closely similar to those of BSE-type agents when propagated in mice expressing ovine PrP, although they retained phenotypic traits distinct from BSE in other lines, including bovine PrP mice. These findings further underline the relationship between the crossing of species barrier and prion strain diversification, and, although the origin of the epidemic BSE agent has only been speculative until now, they provide new insight into the nature of the events that could have led to the appearance of this agent.

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.

State-of-the-art review of goat TSE in the European Union, with special emphasis on PRNP genetics and epidemiology

Scrapie is a fatal, neurodegenerative disease of sheep and goats. It is also the earliest known member in the family of diseases classified as transmissible spongiform encephalopathies (TSE) or prion diseases, which includes Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE), and chronic wasting disease in cervids. The recent revelation of naturally occurring BSE in a goat has brought the issue of TSE in goats to the attention of the public. In contrast to scrapie, BSE presents a proven risk to humans. The risk of goat BSE, however, is difficult to evaluate, as our knowledge of TSE in goats is limited. Natural caprine scrapie has been discovered throughout Europe, with reported cases generally being greatest in countries with the highest goat populations. As with sheep scrapie, susceptibility and incubation period duration of goat scrapie are most likely controlled by the prion protein (PrP) gene (PRNP). Like the PRNP of sheep, the caprine PRNP shows significantly greater variability than that of cattle and humans. Although PRNP variability in goats differs from that observed in sheep, the two species share several identical alleles. Moreover, while the ARR allele associated with enhancing resistance in sheep is not present in the goat PRNP, there is evidence for the existence of other PrP variants related to resistance. This review presents the current knowledge of the epidemiology of caprine scrapie within the major European goat populations, and compiles the current data on genetic variability of PRNP.

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.

Sheep and goat BSE propagate more efficiently than cattle BSE in human PrP transgenic mice.

A new variant of Creutzfeldt Jacob Disease (vCJD) was identified in humans and linked to the consumption of Bovine Spongiform Encephalopathy (BSE)-infected meat products. Recycling of ruminant tissue in meat and bone meal (MBM) has been proposed as origin of the BSE epidemic. During this epidemic, sheep and goats have been exposed to BSE-contaminated MBM. It is well known that sheep can be experimentally infected with BSE and two field BSE-like cases have been reported in goats. In this work we evaluated the human susceptibility to small ruminants-passaged BSE prions by inoculating two different transgenic mouse lines expressing the methionine (Met) allele of human PrP at codon 129 (tg650 and tg340) with several sheep and goat BSE isolates and compared their transmission characteristics with those of cattle BSE. While the molecular and neuropathological transmission features were undistinguishable and similar to those obtained after transmission of vCJD in both transgenic mouse lines, sheep and goat BSE isolates showed higher transmission efficiency on serial passaging compared to cattle BSE. We found that this higher transmission efficiency was strongly influenced by the ovine PrP sequence, rather than by other host species-specific factors. Although extrapolation of results from prion transmission studies by using transgenic mice has to be done very carefully, especially when human susceptibility to prions is analyzed, our results clearly indicate that Met129 homozygous individuals might be susceptible to a sheep or goat BSE agent at a higher degree than to cattle BSE, and that these agents might transmit with molecular and neuropathological properties indistinguishable from those of vCJD. Our results suggest that the possibility of a small ruminant BSE prion as vCJD causal agent could not be ruled out, and that the risk for humans of a potential goat and/or sheep BSE agent should not be underestimated.