L.J.M. van Keulen

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.

Tonsillar biopsy and PrPSc detection in the preclinical diagnosis of scrapie

Preliminary findings have indicated that in naturally infected sheep, fully susceptible to scrapie (vRQ-homozygous), PrPSc can be detected in the tonsils approximately one year before the expected onset of clinical disease, whereas no immunostaining can be detected in animals with a semi-resistant genotype. This paper describes the technique for taking tonsillar biopsies from sheep and gives the results of the completed experiment. In another experiment PrPSc was detected even earlier in comparable vRQ-homozygous sheep born and raised in different surroundings. At three-and-a-half months of age no PrPSc could be detected in three homozygous susceptible sheep (VRQ/VRQ), but PrpSc was detected at four months in one similar sheep. At eight months of age all seven sampled VRQ/VRQ sheep showed positive immunostaining in the biopsies, but none of the biopsies from three VRQ/ARQ heterozygotes showed any immunostaining; they were positive when sampled at 14 to 15 months of age. Biopsies from VRQ/ARR sheep were negative throughout this period. On the basis of the established or expected incubation period, PrPSc could thus be detected in the tonsils of live susceptible animals at between one-third and a half of the incubation period, more than oneand- a-half years before clinical signs normally appear in both these genotypes.

Pathogenesis of natural scrapie in sheep.

Although scrapie has been known for a long time as a natural disease of sheep and goats, the pathogenesis in its natural host still remains unclear. To study the pathogenesis of natural scrapie, we used immunohistochemistry to monitor the deposition of PrP(Sc) in various tissues, collected during a natural scrapie infection from sheep with the PrP(VRQ)/PrP(VRQ) genotype which were purposely bred for their short incubation period for natural scrapie. PrP(Sc) was present in the lymphoid tissues of all animals from the age of 5 months onwards. At this age, PrP(Sc) was detected in the neural tissues only in the enteric nervous system (ENS) at the level of the duodenum and ileum. At the age of 10 months, PrP(Sc) was not only found in the ENS but also in the ganglion mesentericum cranialis/coeliacum, the dorsal motor nucleus of the vagus, and the intermediolateral column of the thoracic segments T8-T10. PrP(Sc) was detected for the first time in the nucleus tractus solitarius and ganglion nodosus at 17 months of age and in the ganglion trigeminale and several spinal ganglia at 21 months of age. Since the scrapie agent consists largely, if not entirely of PrP(Sc), these results indicate that the ENS acts as a portal of entry to the neural tissues for the scrapie agent followed by centripetal and retrograde spread through sympathetic and parasympathetic efferent fibers of the autonomic nervous system to the spinal cord and medulla oblongata respectively. PrP(Sc) accumulation in sensory ganglia occurs after infection of the CNS and is therefore probably due to centrifugal and anterograde spread of the scrapie agent from the CNS through afferent nerve fibers.

Early and late pathogenesis of natural scrapie infection in sheep.

The pathogenesis of scrapie infection was studied in sheep carrying the PrPVRQ/PrPVRQ genotype, which is associated with a high susceptibility for natural scrapie. The sheep were killed at sequential time points during a scrapie infection covering both the early and late stages of scrapie pathogenesis. Various lymphoid and neural tissues were collected and immunohistochemically examined for the presence of the scrapie‐associated prion protein PrPSc, a marker for scrapie infectivity. The first stage of scrapie infection consisted of invasion of the palatine tonsil and Peyers patches of the caudal jejunum and ileum, the so‐called gut‐associated lymphoid tissues (GALT). At the same time, PrPSc was detected in the medial retropharyngeal lymph nodes draining the palatine tonsil and the mesenteric lymph nodes draining the jejunal and ileal Peyers patches. From these initial sites of scrapie replication, the scrapie agent disseminated to other non‐GALT‐related lymphoid tissues. Neuroinvasion started in the enteric nervous system followed by retrograde spread of the scrapie agent via efferent parasympathetic and sympathetic nerve fibres innervating the gut, to the dorsal motor nucleus of the vagus in the medulla oblongata and the intermediolateral column of the thoracic spinal cord segments T8–T10, respectively.

Discrimination between Scrapie and Bovine Spongiform Encephalopathy in Sheep by Molecular Size, Immunoreactivity, and Glycoprofile of Prion Protein

ABSTRACT A procedure for discrimination between scrapie and bovine spongiform encephalopathy (BSE) in sheep is of importance for establishing whether BSE has entered the sheep population. Since BSE has not yet been found in sheep at the farm level, such discrimination procedures can be developed only with experimental sheep BSE. Two distinctive molecular features of the prion protein (PrP)—molecular size and glycosylation profile—in proteinase K digests of brain stem tissue from sheep were used here; upon Western blotting, these features led to an unequivocal discrimination among natural scrapie, experimental scrapie, and experimental BSE. The higher electrophoretic mobility of PrP in sheep BSE could be best observed after deglycosylation treatment with N-glycosidase F. A simpler method for confirmation of this size difference involved comparison of the ratios for the binding of two monoclonal antibodies: P4 and 66.94b4. Based on epitope mapping studies with P4 and peptides, it appeared that N-terminal amino acid sequence WGQGGSH was intact only in sheep scrapie digests. Another feature typical for PrP in sheep BSE was the large fraction of diglycosylated PrP (70% or more). These data were obtained for a large group of positive sheep, consisting of 7 sheep with experimental BSE infection (genotypes: six ARQ/ARQ and one AHQ/AHQ), 48 sheep naturally infected with scrapie (six different genotypes), and 3 sheep with primary experimental scrapie infection. Routine tests of slaughter material serve well for the initial detection of both BSE and scrapie. With Western blotting as a rapid follow-up test, a 66.94b4/P4 antibody binding ratio above 1.5 is a practical indicator for serious suspicion of BSE infection in sheep.

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.

Paratuberculosis in sheep: its possible role in the epidemiology of paratuberculosis in cattle

A total of 50 sheep originating from 15 Dutch farms with a known paratuberculosis infection in their cattle herd, but with no history of paratuberculosis infection in their sheep flock, were examined for infection with Mycobacterium avium subsp. paratuberculosis (Map). The sheep had been grazing on the same pastures as the cattle or on pastures fertilised with manure from these cows. The sheep were screened for paratuberculosis by serum biochemistry, serology and intradermal skin tests. At necropsy they were examined macroscopically, microscopically and bacteriologically for paratuberculosis. From 10 sheep, originating from eight flocks, Map could be isolated from various tissues but not from the intestinal contents, after an incubation period of 2.5-4 months. Six of these culture-positive sheep had no macroscopic signs of paratuberculosis at necropsy. Seven sheep were Map culture negative but showed macroscopic and microscopic lesions consistent with a paratuberculosis infection. Results of serology and skin tests did not correlate with the results of bacteriological culture. Serum concentrations of calcium, albumin and total protein of the infected, suspected and negative sheep were not different. These results indicate that a substantial number of the sheep examined were infected with Map. Even though this bacterium was not isolated from their faeces, the possibility that these sheep could have been shedding Map with their faeces below detection level or at a later stage of the disease cannot be eliminated. Map infected sheep should, therefore, be considered as a possible factor in the epidemiology of with Map infected cattle herds in The Netherlands. At necropsy bacteriological culture of Map should be performed on a routine basis to improve the diagnosis of paratuberculosis in sheep.

Studies on the efficacy of hyperbaric rendering procedures in inactivating bovine spongiform encephalopathy (BSE) and scrapie agents

The efficacy of the procedures in use at the two rendering plants in the Netherlands was assessed on a laboratory.scale using procedures that simulated the pressure cooking part of the rendering process. A pool of bovine spongiform encephalopathy (BsE).infected brainstem from the United Kingdom and a pool of scrapie.infected brainstem from Dutch sheep were used to spike the rendering materials. The mixtures were subjected to various time.temperature combinations of hyperbaric heat treatment related to the conditions used in Dutch rendering plants in the early 1990s, and to the combination of 20 minutes at 133°C required by the EU Directive on rendering of 1996. The efficacy of the procedures in inactivating BSE or scrapie infectivity was measured by titrating the materials before and after heat treatment in inbred mice, by combined intracerebral and intraperitoneal inoculations at limiting dilutions. Two independent series of experiments were carried out. The design of the study allowed for minimum inactivations of up to 2.2 log (2.0 in the second series) to be measured in the diluted infective material and 3.1 log in the undiluted material. After 20 minutes at 133°C there was a reduction of BSE infectivity of about 2.2 log in the first series (with some residual infectivity detected), and in the second series more than 2.0 log (with no residual infectivity detected). With undiluted brain material there was an inactivation of about 3.0 log (with some residual infectivity detected). With the same procedure, scrapie infectivity was reduced by more than 1.7 log in the first series and by more than 2.2 log in the second series. With undiluted brain material there was an inactivation of more than 3.1 log. In each case no residual scrapie infectivity was detected. The BSE agent consistently appeared to be more resistant to heat inactivation procedures than the scrapie agent, particularly at lower temperatures and shorter times.

Proteinase K-resistant material in ARR/VRQ sheep brain affected with classical scrapie is composed mainly of VRQ prion protein.

ABSTRACT Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so-called ARR allele, in the sheep population have been demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrPC) to the scrapie-associated form (PrPSc) could play a key role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrPres, the proteinase K-resistant PrPSc core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrPres was mostly composed of the 171Q allelotype. Furthermore, using a novel tool for prion research, endoproteinase Lys-C-digested PrPres yielded substantial amounts of a nonglycosylated and a monoglycosylated PrP fragment comprising codons 114 to 188. Following two-dimensional gel electrophoresis, only marginal amounts (<9%) of 171R PrPres were detected. Enhanced 171Rres proteolytic susceptibility could be excluded. Thus, these data support a nearly zero contribution of 171R PrP in PrPres of 171R/Q field scrapie-infected animals. This is suggestive of a poor adaptation of classical scrapie to this resistance allele under these natural conditions.

Characterisation of a recently isolated lyssavirus in frugivorous zoo bats

Summary. In July 1997 a lyssavirus was isolated in Denmark from a colony of Egyptian flying foxes (Rousettus aegyptiacus) originating from a Dutch zoo. Sequencing of a 400 nucleotides coding region of the nucleoprotein and of a major part of the G-protein ectodomain encoding region of the newly isolated virus, revealed a very high similarity with European Bat Lyssavirus subtype 1a (EBL-1a). For characterisation of the recently isolated lyssavirus in frugivorous zoo bats, 16 frugivorous bats (Rousettus aegyptiacus) of the same colony and 80 mice were experimentally infected with the Rousettus isolate or with a well defined EBL-1a strain isolated from a Dutch insectivorous bat (Eptesicus serotinus). Inoculation viruses were titrated in mice to determine LD50‘s of both isolates. Clinical signs of inoculated bats were recorded during 6 weeks. After showing neurological signs or at the end of the experimental infection all animals were euthanized. During the experimental infection sera and various tissues of inoculated bats were collected. Immunoassays, mouse inoculation tests (MIT) and polymerase chain reaction (PCR) were employed for detection of lyssavirus specific antibodies, antigen or RNA. Five bats inoculated with the Rousettus isolate and 2 bats inoculated with the Eptesicus isolate showed neurological signs. The remaining 9 bats survived and cleared the virus; at least under the detection limit of the used assays. Despite a much higher pathogenicity of the Rousettus isolate observed in mice, LD25’s in bats were quite the same for the 2 isolates. The pathogenicity of both isolates suggested that like many other mammals, Rousettus aegyptiacus bats could be victims of lyssavirus infection besides reservoir hosts of infectious EBL1a. There was no significant difference in detecting the different lyssavirus isolates in Rousettus aegyptiacus bats. An employed immunoperoxidase staining (IP) method was very useful for sensitive detection and localization of lyssavirus antigen in histologic preparates.