Oral inoculation of sheep with the agent of bovine spongiform encephalopathy (BSE). 1. Onset and distribution of disease-specific PrP accumulation in brain and viscera.

Sixty-three Romney sheep aged 6 months, consisting of three groups (PrP(ARQ/ARQ), PrP(ARQ/ARR), and PrP(ARR/ARR)genotypes) of 21 animals, were infected orally with brain tissue from BSE-infected cattle. Sub-groups of the 21 PrP(ARQ/ARQ) animals were killed, together with uninfected controls 4, 10, 16, 22 or 24-28 (after the development of full clinical disease) months post-inoculation (mpi). One sheep from each of the two groups of four killed at 4 or 10 mpi were shown by immunohistochemical examination to possess disease-specific PrP accumulations in single lymph nodes. At 16 mpi, such accumulations were detected in two of four infected sheep in some viscera and in the spinal cord and brain. At 22 mpi, three of five infected sheep had widespread disease-specific PrP accumulations in all tissues examined, but the remaining two animals gave positive results only in the central nervous system. Clinical disease appeared at 20-28 mpi. Three sheep killed with advanced clinical signs showed widespread PrP accumulation in brain, spinal cord and peripheral tissues. These results confirmed that PrP(ARQ/ARQ) Romney sheep are susceptible to experimental infection with the BSE agent. The different sites at which initial PrP accumulations were detected suggested that the point of entry of infection varied. Once established, however, infection appeared to spread rapidly throughout the lymphoreticular system. The results suggested that in some BSE-infected sheep neuroinvasion occurred in the absence of detectable PrP accumulations in the viscera or peripheral nervous system. In contrast to cattle with BSE, however, most sheep showed disease-specific PrP accumulations in the lymphoreticular system. In this respect, BSE-infected resembled scrapie-infected sheep; it is possible, however, that future research will reveal differences in respect of targeting of cell types within the lymphoreticular and peripheral nervous systems. The PrP(ARQ/ARR)and PrP(ARR/ARR)sheep were also killed in sub-groups at intervals after inoculation. Up to 24 mpi, however, none of these animals showed disease-specific PrP accumulations. Further results will be reported later.     

Differential diagnosis of infections with the bovine spongiform encephalopathy (BSE) and scrapie agents in sheep.

Scrapie, bovine spongiform encephalopathy (BSE), and variant Creutzfeldt-Jakob disease belong to the group of disorders called transmissible spongiform encephalopathies or prion diseases. The possibility that some sheep may be infected with the BSE agent is of human and animal health concern. Immunohistochemical methods were used to identify specific prion protein (PrP) peptide sequences in specific cell types of the brain and lymphoreticular system (LRS) of sheep with natural scrapie and Suffolk and Romney sheep infected experimentally with the BSE agent. Clinically affected and some pre-clinical cases of BSE infection could be distinguished from scrapie cases by the lesser amount of labelling of PrP containing the 84-102 amino-acid peptide sequences in phagocytic cells of the LRS and brain. Additionally, BSE-infected sheep had higher degrees of intra-neuronal PrP accumulation in the brain, as detected by labelling for a range of PrP peptide sequences. These results suggest that there is strain-dependent processing of PrP in specific cell types within the nervous system and LRS which can be used to distinguish BSE- and scrapie-infected sheep.     

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.     

Transportation of prion protein across the intestinal mucosa of scrapie-susceptible and scrapie-resistant sheep

To determine the mechanisms of intestinal transport of infection, and early pathogenesis, of sheep scrapie, isolated gut-loops were inoculated to ensure that significant concentrations of scrapie agent would come into direct contact with the relevant ileal structures (epithelial, lymphoreticular, and nervous). Gut loops were inoculated with a scrapie brain pool homogenate or normal brain or sucrose solution. After surgery, animals were necropsied at time points ranging from 15 min to 1 month and at clinical end point. Inoculum-associated prion protein (PrP) was detected by immunohistochemistry in villous lacteals and in sub-mucosal lymphatics from 15 min to 3.5 h post-challenge. It was also detected in association with dendritic-like cells in the draining lymph nodes at up to 24 h post-challenge. Replication of infection, as demonstrated by the accumulation of disease-associated forms of PrP in Peyer's patches, was detected at 30 days and sheep developed clinical signs of scrapie at 18-22 months post-challenge. These results indicate discrepancies between the routes of transportation of PrP from the inoculum and sites of de novo-generated disease-associated PrP subsequent to scrapie agent replication. When samples of homogenized inoculum were incubated with alimentary tract fluids in vitro, only trace amounts of protease-resistant PrP could be detected by western blotting, suggesting that the majority of both normal and abnormal PrP within the inoculum is readily digested by alimentary fluids.     

Specificity of lymphoreticular accumulation of prion protein for variant Creutzfeldt-Jakob disease

BACKGROUND: Immunocytochemical accumulation of prion protein (PrP) in lymphoid tissues is a feature of variant Creutzfeldt-Jakob disease (vCJD) that has been used both to aid in the diagnosis of patients and as a basis of large scale screening studies to assess the prevalence of preclinical disease in the UK. However, the specificity of this approach is unknown. AIM: To assess the specificity of lymphoreticular accumulation of PrP for vCJD by examining a range of human diseases. METHODS: Paraffin wax embedded lymphoreticular tissues from patients with several reactive conditions (58 cases), tumours (27 cases), vCJD (54 cases), and other human prion diseases (56 cases) were assessed. PrP accumulation was assessed by immunocytochemistry using two different monoclonal anti-PrP antibodies and a sensitive detection system. RESULTS: All cases of vCJD showed widespread lymphoreticular accumulation of PrP; however, this was not seen in the other conditions examined. CONCLUSION: Lymphoreticular accumulation of PrP, as assessed by immunocytochemistry, appears to be a highly specific feature of vCJD.     

Histopathological and immunohistochemical features of natural goat scrapie

Histopathological and immunohistochemical examinations were performed on the brain and spinal cord of 37 goats from two Greek herds in which scrapie had been reported. Of the 37 animals, 18 were from a herd consisting only of goats and 19 were from a herd of goats mixed with sheep. The goats studied were grouped on the basis of the presence or absence of clinical signs. Distinctive lesions and PrP(sc) (PrP, prion protein) deposition were found in the central nervous system (CNS) of eight clinically affected animals and six symptomless animals. The lesion profile and PrP(sc) distribution varied both between and within groups, variation being particularly pronounced in the symptomless goats. The results concerning the latter group suggested a poor correlation between the intensity of lesions, the amount of PrP(sc) in the CNS, and the manifestation of clinical signs. Immunohistochemical examination revealed 10 different PrP(sc) types, four of which are reported for the first time in goats. All scrapie-affected animals carried the VV(21)II(142)HH(143)RR(154) genotype, with the exception of two goats that carried the HR(143) dimorphism and had detectable PrP(sc) deposits. The results suggest that the histopathological and immunohistochemical profile of the natural disease in goats is influenced by the PrP genotype and age of the animals but may not be directly associated with the presence or otherwise of clinical signs.     

Post-mortem immunodiagnosis of scrapie and bovine spongiform encephalopathy

Two polyclonal antisera were raised in rabbits against the scrapie-associated fibril protein (PrP) prepared from sheep and mice which were terminally infected with experimental scrapie. The anti-mouse PrP serum identifies the proteins of scrapie-associated fibrils (SAF) from all the host species studied (mouse, hamster, sheep and goat) and bovine spongiform encephalopathy (BSE) fibrils from cow. The anti-sheep PrP serum displays species restricted immunoreactivity. While it identifies several PrP polypeptides from terminally affected sheep, goat and cow material, only the highest molecular weight band is recognised from hamster and there is no detection of mouse PrP. The use of these antisera in routine laboratory testing at post mortem provides a highly sensitive test for scrapie and BSE and may allow the identification of infected animals prior to the onset of clinical signs.     

Detection of PrPSc in lymphoid tissues of lambs experimentally exposed to the scrapie agent

Histoblotting and immunohistochemistry were used to detect disease-associated prion protein (PrP(Sc)) in lymphoid tissues of lambs of known PrP genotype infected with the scrapie agent by stomach tube at the age of 2 months. The ileal and jejunal Peyer's patches and retropharyngeal and distal jejunal lymph nodes were studied 1 week, 5 weeks, 5 months and 11 months after inoculation. Other lymphoid tissues examined included superficial cervical lymph node, tonsil and spleen. PrP(Sc) was not detected in any tissue of any lamb at 1 week post-inoculation. At 5 weeks, PrP(Sc) was detected in tissues of lambs of susceptible PrP genotypes (AV(136)QQ(171) and VV(136)QQ(171)), but not lambs of other PrP genotypes (AA(136)QQ(171), AA(136)QR(171) and AV(136)QR(171)). PrP(Sc) was present in the germinal centres of tonsils, distal jejunal and retropharyngeal lymph nodes, and spleen. In the nodules of ileal and jejunal Peyer's patches, only occasional solitary cells showed the presence of PrP(Sc). At 5 months post-inoculation, increased accumulations of PrP(Sc) were detected in ileal and jejunal Peyer's patches, as well as in the retropharyngeal and distal jejunal lymph nodes of a single lamb inoculated with the agent from a sheep of the same susceptible PrP genotype. Eleven months after exposure to the scrapie agent, PrP(Sc) was detected in all lymphoid tissues examined from sheep of susceptible PrP genotypes. These studies show that PrP(Sc) was detectable in lymphoid tissues 5 weeks after exposure to the scrapie agent by stomach tube in lambs as young as 3 months of age and indicate that the PrP genotype is a significant factor for the rapid uptake and spread of the agent through lymphoid tissues.     

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.     

Murine scrapie infection causes an abnormal germinal centre reaction in the spleen

Follicular dendritic cells (FDCs) of the lymphoreticular system play a role in the peripheral replication of prion proteins in some transmissible spongiform encephalopathies (TSEs), including experimental murine scrapie models. Disease-specific PrP (PrPd) accumulation occurs in association with the plasmalemma and extracellular space around FDC dendrites, but no specific immunological response has yet been reported in animals affected by TSEs. In the present study, morphology (light microscopical and ultrastructural) of secondary lymphoid follicles of the spleen were examined in mice infected with the ME7 strain of scrapie and in uninfected control mice, with or without immunological stimulation with sheep red blood cells (SRBCs), at 70 days post-inoculation or at the terminal stage of disease (268 days). Scrapie infection was associated with hypertrophy of FDC dendrites, increased retention of electron-dense material at the FDC plasma membrane, and increased maturation and numbers of B lymphocytes within secondary follicles. FDC hypertrophy was particularly conspicuous in immune-stimulated ME7-infected mice. The electron-dense material was associated with PrP Napoli accumulation, as determined by immunogold labelling. We hypothesize that immune system changes are associated with increased immune complex trapping by hypertrophic FDCs expressing PrP Napoli molecules at the plasmalemma of dendrites, and that this process is exaggerated by immune system stimulation. Contrary to previous dogma, these results show that a pathological response within the immune system follows scrapie infection.     

PrP gene polymorphism in sheep breeds in Slovakia and susceptibility to scrapie

We analyzed the prion protein (PrP) genotype based on the codons 136, 154 and 171 and assigned to five risk groups (R1-R5) in healthy and scrapie-affected sheep in Slovakia. In healthy (asymptomatic) population, 119 Merino, 106 Improved Valachian, 117 Tsigai, and 48 Suffolk breeds were tested. Among the asymptomatic sheep, the low-risk genotypes R1 and R2 were most abundant in Suffolk (94%) and Merino (84%) breeds, followed by Tsigai (58%) and Improved Valachian (40%) breeds. The medium-risk group R3 was most frequent in Improved Valachian (31%) breed, followed by Tsigai (21%), Merino (10%), and Suffolk (6%) breeds. The occurrence of high-risk groups R4 and R5 was none in Suffolk breed, followed by Merino (6%), Tsigai (21%), and Improved Valachian (30%) breeds. Since 2003, altogether 48 cases of scrapie have been confirmed in Tsigai (38), Merino (4), Improved Valachian (2), Improved Valachian x Tsigai (3), and Suffolk (1) breeds. Among sheep with scrapie, Merino breed belonged to the medium-risk group R3. The majority of scrapie-affected Tsigai sheep were classified into high-risk R5 (50%) and medium-risk R3 (42%) groups. We showed an association of scrapie with medium- and high-risk groups of PrP genotype in Slovakia. In particular, the glutamine at position 171 appears to be of major importance for the susceptibility to scrapie.     

Immunohistochemical distinction between preclinical bovine spongiform encephalopathy and scrapie infection in sheep

Sheep are susceptible experimentally to bovine spongiform encephalopathy (BSE), the clinical signs being indistinguishable from those of scrapie. Because of the possibility of natural ovine BSE infection, laboratory tests are needed to distinguish between scrapie and BSE infection. The objectives of this study were to determine whether (1) PrPSc accumulates in biopsy samples of the tonsil or third eyelid, or both, of BSE-infected sheep before the appearance of clinical disease, and (2) such samples from BSE- and scrapie-infected sheep differ in respect of PrPSc accumulations. Homozygous ARQ sheep (n = 10) were dosed orally at 4-5 months of age with a brain homogenate from BSE-infected cattle. Third eyelid and tonsillar biopsy samples were taken at < or = 6 monthly intervals post-infection and examined immunohistochemically for PrPSc. Third eyelid protuberances were difficult to identify, resulting in many unsuitable samples; however, third eyelid samples shown to contain lymphoid follicles were invariably negative for PrPSc. In contrast, tonsillar biopsy samples became positive for PrPSc from 11 to 20 months post-infection. Consistent differences in the morphology of PrPSc granules in tingible body macrophages (TBMs) between BSE- and scrapie-infected sheep were detected with anti-peptide antibodies directed towards amino acids 93-106 of the ovine prion protein: thus, PrPSc appeared as single granules in TBMs of tonsillar sections from BSE-infected sheep, whereas clusters of PrPSc granules were observed within TBMs in the tonsils of scrapie-infected sheep. In contrast, antibodies against epitopes situated N- and C-terminally from the 93-106 region of the ovine prion protein revealed no differences between BSE- and scrapie-infected sheep in terms of PrPSc granules in TBMs.     

Active surveillance for scrapie by third eyelid biopsy and genetic susceptibility testing of flocks of sheep in Wyoming

Control of scrapie, an ovine transmissible spongiform encephalopathy or prion disorder, has been hampered by the lack of conventional antemortem diagnostic tests. Currently, scrapie is diagnosed by postmortem examination of the brain and lymphoid tissues for PrP(Sc), the protein marker for this group of disorders. For live, asymptomatic sheep, diagnosis using tonsil or third-eyelid lymphoid tissue biopsy and PrP(Sc) assay has been described. To evaluate the feasibility and efficacy of third-eyelid testing for identification of infected flocks and individual infected sheep, 690 sheep from 22 flocks were sampled by third-eyelid lymphoid tissue biopsy and immunohistochemistry. Sheep were further evaluated for relative genetic susceptibility and potential contact exposure to scrapie. Third-eyelid testing yielded suitable samples for 80% of the sheep tested, with a mean of 18.1 lymphoid follicles (germinal centers) per histologic section. Three hundred eleven of the sheep were sampled through passive surveillance programs, in which only sheep with potential contact with an infected sheep at a lambing event were tested, regardless of their scrapie susceptibility genotype. In addition, 141 genetically susceptible sheep with no record of contact with an infected animal at a lambing event were sampled through a targeted active surveillance program. Ten PrP(Sc)-positive sheep were identified through the passive surveillance program, and an additional three PrP(Sc)-positive sheep, including two from flocks with no history of scrapie, were identified through the active surveillance program. All PrP(Sc)-positive sheep had the highly susceptible PrP genotype. Third-eyelid testing is a useful adjunct to flock monitoring programs, slaughter surveillance, and mandatory disease reporting in a comprehensive scrapie eradication and research program.     

Changes in tonsillar tissue in early HIV-1 infection and during 3 years of antiretroviral therapy

Tonsillar tissue from individuals in the early stages of HIV-1 infection was studied during the natural course of infection and during antiretroviral therapy with and without a protease inhibitor in order to investigate markers of clinical progression and evaluate the effects of therapy. Tonsillar biopsies and blood samples were collected at regular intervals during 3 years and clinical observations were noted. Tonsillar morphology was evaluated and the fragmentation of the follicular dendritic cell network was quantified by standardised follicular fragmentation rate (FR) analysis. Lymphocyte subsets were phenotyped by flow cytometry, and viral load was calculated by limiting dilution assay. The FRs were higher in the HIV-1-infected individuals than in the uninfected controls, although tonsillar tissue from both groups contained follicular fragmentation. During HIV-1 infection, the FR increased and the tonsillar CD4/CD8 ratio declined. During maximum viral suppression, FR approached that of controls while tonsillar T cell subsets and blood CD4 cell counts normalised. Even when virus suppression was incomplete, tonsillar improvements were observed in parallel with a resolution of the HIV-1-related dermatological disorders. However, persistent viral replication paralleled distortion of the tonsillar architecture. We suggest that a normalisation of the lymphoid tissue may have important functional and clinical implications in HIV-1 infection.     

Protease-resistant prion protein in brain and lymphoid organs of sheep within a naturally scrapie-infected flock

The hallmark of transmissible spongiform encephalopathies (TSE), such as scrapie in sheep, is the accumulation in tissues of an insoluble and protease resistant form (PrPres) of the cellular prion protein. In this study, we evaluated whether the diversity in both the clinical pattern and the PrP genotypes of scrapied sheep from the same flock was connected with different levels and/or glycoform patterns of the PrPres in the brain and lymphoid organs of the animals. Whereas the PrPres levels in spleen, lymph nodes and tonsils from sheep of different PrP genotypes and clinical status appeared comparable, they were highly variable in brain, particularly in the brain stem and the cerebellum. PrPres was only detected in sheep bearing at least one VRQ allele, including three asymptomatic sheep and the highest PrPres load was found in the cerebellum of VRQ/VRQ animals. All together, levels of PrPres in brain did not necessarily correlate with the severity of the clinical disease but might depend on the PrP genotype of the animals. Different brain regions from a given sheep displayed a similar glycopattern of PrPres, whereas the apparent molecular sizes of the unglycosylated and diglycosylated forms of the protein differed between brain and lymphoid tissues. We did not find any notifiable differences in the glycopattern of PrPres in brain from sheep of different PrP genotypes or different clinical status and this PrPres glycotype was also similar to that found in brain from four cattle BSE.     

Detection of PrP(Sc) in rectal biopsy and necropsy samples from sheep with experimental scrapie

Scrapie diagnosis is based on the demonstration of disease-associated prion protein (PrP(Sc)) in brain or, in the live animal, in readily accessible peripheral lymphoid tissue. Lymphatic tissues present at the rectoanal line were readily obtained from sheep without the need for anaesthesia. The presence of PrP(Sc) in such tissue was investigated in sheep infected orally with scrapie-infected brain material. The methods used consisted of immunohistochemistry and histoblotting on biopsy and post-mortem material. PrP(Sc) was detected in animals with PrP genotypes associated with high susceptibility to scrapie from 10 months after infection, i.e., from about the time of appearance of early clinical signs. In the rectal mucosa, PrP(Sc) was found in lymphoid follicles and in cells scattered in the lamina propria, often near and sometimes in the crypt epithelium. By Western blotting, PrP(Sc) was detected in rectal biopsy samples of sheep with the PrP genotype VRQ/VRQ, after electrophoresis of material equivalent to 8 mg of tissue. This study indicated that rectal biopsy samples should prove useful for the diagnosis of scrapie in sheep.     

Neuropathological changes in auditory brainstem nuclei in cattle with experimentally induced bovine spongiform encephalopathy

Bovine spongiform encephalopathy (BSE) is characterized by the appearance of spongy lesions in the brain, particularly in the brainstem nuclei. This study evaluated the degenerative changes observed in the central auditory brainstem of BSE-challenged cattle. The neuropathological changes in the auditory brainstem nuclei were assessed by determining the severity of vacuolation and the presence of disease-associated prion protein (PrP(Sc)). Sixteen female Holstein-Friesian calves, 2-4 months of age, were inoculated intracerebrally with BSE agent. BSE-challenged animals developed the characteristic clinical signs of BSE approximately 18 months post inoculation (mpi) and advanced neurological signs after 22 mpi. Before the appearance of clinical signs (i.e. at 3, 10, 12 and 16 mpi), vacuolar change was absent or mild and PrP(Sc) deposition was minimal in the auditory brainstem nuclei. The two cattle sacrificed at 18 and 19 mpi had no clinical signs and showed mild vacuolar degeneration and moderate amounts of PrP(Sc) accumulation in the auditory brainstem pathway. In the animals challenged with BSE agent that developed clinical sings (i.e. after 20 mpi), spongy changes were more prominent in the nucleus of the inferior colliculus compared with the other nuclei of the auditory brainstem and the medial geniculate body. Neuropathological changes characterized by spongy lesions accompanied by PrP(Sc) accumulation in the auditory brainstem nuclei of BSE-infected cattle may be associated with hyperacusia.