Occurrence and distribution of infection-specific PrP in tissues of clinical scrapie cases and cull sheep from scrapie-affected farms in Shetland.

The prion protein (PrP) genotypes of all cull sheep originating from four scrapie-affected farms in Shetland in 1998-1999 were determined and a representative sample of the different genotypes was selected for necropsy. Samples of brain and selected viscera were removed from 159 such sheep aged 2-11 years. These samples were examined immunohistochemically and by Western blotting for infection-specific forms of PrP. None of the sheep bearing the following genotypes showed any evidence of PrP accumulation in brain, intestine, selected lymph nodes or the cranial mesenteric ganglia: ARQ/ARQ (n = 41), ARQ/ARH (n = 12), ARH/ARH (n = 2), ARQ/ARR (n = 24), ARR/ARR (n= 2). In five of 71 sheep bearing a single VRQ allele, PrP accumulation was detected immunohistochemically in viscera or brain, or both. These results suggested that only a small proportion of susceptible sheep showed evidence of infection (accumulation of PrP) on the farms studied, and that even sheep of the most susceptible genotype (VRQ/VRQ) did not invariably develop disease in an infected environment. Furthermore, there was no evidence that, in sheep of semi-resistant or fully resistant genotypes, infection could be sequestered within the lymphoreticular system or peripheral nervous system and thereby provide a possible "carrier" source of infection. Rather, the data suggested that some sheep, possibly because they had been exposed to a relatively low infective dose, became infected and accumulated the infective agent over a protracted pre-clinical phase of the disease. Such sheep might be potentially infective for many years. In two VRQ/ARR genotype sheep, PrP was confined to the brain. Infection-specific PrP was also confined to the brain in two of 24 clinical cases of VRQ/ARQ scrapie. Thus, direct neuroinvasion, apparently without a prior phase of replication in the lymphoreticular system, occurred in a proportion of VRQ/ARQ sheep. Possibly it may occur in all sheep of the VRQ/ARR genotype. The factors responsible for direct neuroinvasion are not understood. However, it cannot be attributed to genotype alone.

Vacuolar lesion profile in sheep scrapie: factors influencing its variation and relationship to disease-specific PrP accumulation.

Detailed neuropathological examination for vacuolar lesions was performed on the brains of 42 sheep with clinical signs compatible with scrapie. The sheep were grouped according to their breed (Poll-Dorset, Cheviot, Welsh Mountain, Shetland and Suffolk), their PrP genotype at codons 136, 154 and 171 (VRQ/VRQ, VRQ/ARQ, VRQ/ARR and ARQ/ARQ) and the type of infection (experimental infection with SSBP/1, or natural disease). Twenty-two neuroanatomical sites from seven brain regions were examined for vacuolation in the neuropil and five sites at the level of the obex were examined for intraneuronal vacuolation. In 36 sheep, immunohistochemical examination for disease-specific PrP (PrP(d)) accumulation had also been performed in the same brain regions in an earlier study. The magnitude of total neuropil vacuolation was highest in the naturally affected ARQ/ARQ Suffolk sheep and lowest in the experimentally infected VRQ/VRQ Cheviot sheep and VRQ/ARR Poll-Dorset sheep. The severity of neuropil vacuolation at nine of the 22 neuroanatomical sites examined was used to generate a vacuolar lesion profile, which showed variations between the different sheep groups. These variations could be attributed to both PrP genotype and sheep breed and also possibly to scrapie agent; there was, however, considerable individual variation in lesion profile within sheep groups. All groups showed a similar ratio of neuropil vacuolation to neuronal vacuolation at the level of the obex. Although a positive correlation between neuropil vacuolation and PrP(d) deposition was generally observed, it was low except for the astrocyte-associated pattern of PrP(d) accumulation. The study suggests that vacuolar lesion profiles in sheep are affected by several factors and, by comparison with lesion profiles in mice, are of no more than limited value for discriminating between scrapie strains.

Effects of agent strain and host genotype on PrP accumulation in the brain of sheep naturally and experimentally affected with scrapie.

Different cellular and neuroanatomical types of disease-specific prion protein (PrP(d)) accumulation in the brain were identified in sheep of different breeds and PrP genotypes exposed to experimental or natural scrapie infection. Immunohistochemical examination of the brains of 43 sheep with clinical signs compatible with scrapie revealed 12 different PrP(d)types, which were subjectively quantified in eight different brain regions. The PrP(d)types were grouped into four PrP(d)patterns, the relative magnitude of which provided the PrP(d)profile of each sheep examined. The analysis of the differences in magnitude and relative proportion of each of these PrP(d)types and patterns indicated (1) an effect of the scrapie strain on the PrP(d)profile, and (2) a possible effect of the host genotype on the magnitude of PrP(d)accumulation in the brain, apparently related to the incubation period. Furthermore, intraneuronal deposition of PrP(d)was the type most closely associated with the development of clinical disease. We conclude that different scrapie strains can be distinguished by PrP immunohistochemical examination of brains of affected animals.

Onset and distribution of tissue prp accumulation in scrapie-affected suffolk sheep as demonstrated by sequential necropsies and tonsillar biopsies.

Tonsillar biopsies (single or multiple) or necropsies, or both, were performed on sheep taken from a Suffolk flock in which frequent cases of scrapie had occurred over a period of several years. Clinically affected sheep of the susceptible PrP(AQ/AQ)genotype had widespread disease-specific PrP accumulation in the central nervous system (CNS), lymphoreticular system and peripheral ganglia. In nine healthy PrP(AQ/AQ)Suffolk sheep between 4 and 7 years of age, PrP could not be demonstrated post mortem in any of the lymphoreticular tissues, or in the peripheral ganglia or CNS. Tonsillar biopsies taken from animals of the resistant PrP(AR/AR)and PrP(AR/AQ)genotypes at age 3, 8, 14, 20 or 26 months did not show PrP accumulation. Disease- specific PrP accumulation in tonsillar biopsies from PrP(AQ/AQ)sheep was not seen in 20 animals aged 3 months, but was found in two of 10 animals at age 8 months and in eight of 10 animals at age 20 months. The numbers of PrP-positive tonsillar biopsies obtained from sheep previously biopsied on more than one occasion was greater than the number of positive tonsils obtained from other susceptible sheep of comparable ages. The earliest disease-specific PrP accumulation seen was in tingible body macrophages within germinal centres and only later was it detected in cells resembling follicular dendritic cells. Fourteen PrP(AQ/AQ)sheep examined post mortem at up to 17 months of age and which had not previously been biopsied or were biopsied only once had no CNS or tonsillar PrP accumulations. Two of these sheep subjected to necropsy at 14 months had PrP accumulation in lymphoreticular tissue, where it was confined to the mesenteric lymph nodes. In susceptible sheep, only low levels of immunohistochemically detectable PrP were present in a minority of follicles from tonsillar biopsies of young lambs, but by 14 months of age widespread PrP accumulation, affecting many or even all follicles, was present. Although clinical cases had widespread PrP accumulations in viscera, susceptible survivors had no such accumulations in tissues of the lymphoreticular system, peripheral nervous system or CNS, suggesting that some animals were not exposed to infection or were exposed to a non-infectious dose.

Prevalence of vacuolar lesions consistent with scrapie in the brains of healthy cull sheep of the Shetland Islands.

To determine the levels of background scrapie-like pathology in the brains of clinically normal adult sheep, the brains of 1106 sheep from 28 known scrapie-infected flocks and nine apparently uninfected flocks were examined during 1998 and 1999. One per cent of the brains had vacuolar pathology and disease-specific accumulations of prion protein consistent with a diagnosis of scrapie. All the positive animals had at least one allele of the prion protein gene encoding valine at codon 136, and originated from flocks in which cases of clinical scrapie had been confirmed within the last four years. The parasympathetic nucleus of the vagal nerve was the most consistently and severely affected nucleus in the medulla oblongata, suggesting that the infection enters the brain via ascending fibres of the vagus nerve.

Early immunopathological events in experimental ovine paratuberculosis.

An experimental oral infection of neonatal (< 2 weeks old) lambs with a cervine isolate of Mycobacterium avium subspecies paratuberculosis (M.a. paratuberculosis), the causal agent of ruminant paratuberculosis (Johne's disease) was used to investigate bacteriological, histopathological and immunological changes during the early (up to 8 weeks) post-infection phase. In vitro culture for mycobacteria was positive in one faecal and three mesenteric lymph node (MLN) samples from the eight infected lambs. All mycobacterial isolates from MLN were identified as M.a. paratuberculosis by polymerase chain reaction (PCR). Small-to-medium sized focal granulomata were observed in jejunal (JPP) and ileal Peyer's patches (IPP) from four of the eight infected lambs. Compared with controls, JPP from all infected lambs had significantly (p < 0.05) higher proportions of CD8+ and CD2+ lymphocytes, and there were significantly (p < 0.05) fewer cells expressing B lymphocyte-associated markers in IPP and MLN. The T/B cell ratio was significantly (p < 0.05) increased in both JPP and MLN from infected lambs. The expression of a range of genes for cytokines was examined using specific reverse transcriptase PCR (RT-PCR) amplification of messenger RNA (mRNA) template isolated from MLN, JPP and IPP from both groups of animals. Densitometric analyses indicated that, in infected animals, MLN expressed significantly (p < 0.05) more mRNA for TNF-alpha: JPP had significantly increased (p < 0.05) mRNA for GM-CSF and significantly decreased (p < 0.05) mRNA for IL-4 and IFN-gamma. Infected lambs had significantly (p < 0.05) decreased titres of both circulating IgG and gut mycobacteria-associated IgG antibody. Infection was not associated with any consistent changes in lymphocyte reactivity to specific mycobacterial antigens, IFN-gamma release into supernatants from in vitro intestinal lymphocyte cultures or gut IgA antibody levels.