M.A. Smits

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.

Prion protein and scrapie susceptibility.

This article presents briefly current views on the role of prion protein (PrP) in Transmissible Spongiform Encephalopathies or prion diseases and the effect of PrP polymoryhisms on the susceptibility to these diseases, with special emphasis on sheep scrapie. The PrP genotype of sheep appears to be a major risk factor for scrapie, and polymorphisms at codons 136, 154, and 171 modulate the susceptibility of sheep for scrapie. Nevertheless, scrapie is not a spontaneous genetic disease alone. We describe an in vitro system in which sheep PrP variants show characteristics which reflect their linkage with in vivo scrapie susceptibility. Studies with this in vitro system not only confirm that scrapie susceptibility is determined by the PrP genotype of the target animal, but also suggest that the PrP genotype of the animal that is the source of the infectious agent plays an important role in determining scrapie susceptibility. The behaviour of PrP variants in this in vitro system may be an indicator for the transmissibility of prion diseases.

CONTROL OF SCRAPIE EVENTUALLY POSSIBLE

After a brief description of the scrapie situation in the Netherlands, the technical progress made in aspects of scrapie diagnosis is reported. Emphasis is placed on the use of immuno-histochemistry (IHC) in the post-mortem histological diagnosis and on the recently published preclinical test for scrapie, in which IHC is applied to tonsillar biopsies. These two approaches use the same IHC technique and enable us to confirm suspected scrapie in individual animals, and for certain genotypes even in the preclinical phase. The tonsillar biopsy method could eventually lead to an infection- or PrPSc-detection method at flock level. Further work is required, including validation of the assay for various breeds, genotypes, and strains of the agent, and the conversion of the test into a more practical assay. The article continues with a discussion of several scrapie control strategies, describing briefly schemes tried in various countries, and elaborates on a proposed scrapie control scheme that could be suitable for the Netherlands. This scheme is essentially based on breeding for resistance, based on PrP genotyping.

Applicability of three anti-PrP peptide sera including staining of tonsils and brainstem of sheep with scrapie.

Three rabbit antibodies (R521, R505, R524) were produced, and raised to synthetic peptides corresponding to residues 94–105, 100–111, and 223–234, respectively, of the sheep prion protein (PrP). Epitope mapping analysis revealed the monospecific character of antisera R505 and R524. In addition to the amino acid sequence against which it was raised, R521 also recognized other small epitopes. ELISA and radio‐immunoprecipitation were used to assess the relative immunoreactivities of the antisera to the normal sheep prion protein (PrPc). Highest reactivity was found for R521, followed by R505 and R524. According to Western blot analysis, all three sera specifically reacted with the prion proteins PrPSc and PrP27‐30, extracted from the brain stem of a scrapie‐affected sheep. Yet, with R505 not all of the lower molecular weight deglycosylated forms could be detected. Contrary to the immunoreactivities found with the PrPSc and PrP27‐30 isoforms, only R521 recognised PrPc from a healthy sheep. The usefulness of all three anti‐peptide sera in the immunohistochemical detection of PrPSc in brain stem and tonsils of scrapie‐affected sheep was demonstrated and compared with an established rabbit anti‐PrP serum. Microsc. Res. Tech. 50:32–39, 2000.

Diagnosis of bovine spongiform encephalopathy: A review

Summary Cows affected with bovine spongiform encephalopathy (BSE) display chronic neurological signs consisting of behavioural changes, abnormalities of posture and movement, and/or hyperaesthesia. At present, there are no laboratory test available to diagnose BSE in the live animal. In this article, we describe the post‐mortem diagnostic examination of brains from BSE‐suspected cattle as currently performed at ID‐Lelystad. The routine laboratory diagnosis of BSE consists of histopathological examination of the brain and detection of the modified prion protein, PrPBSE, in brain tissue. These tests, however, have the disadvantage of being laborious and time consuming, so that results are available only after several days. Recently, at ID‐Lelystad a new post‐mortem test has been developed that enables screening of larger volumes of brain samples for PrPBSE within 1 day. This BSE test is especially suited for slaughterline monitoring. A preliminary validation study has shown that both sensitivity and specificity are 100% compared to the gold diagnostic standard of histopathology.