PrPd Accumulation Research Articles

CATTLE ( BOS TAURUS) RESIST CHRONIC WASTING DISEASE FOLLOWING ORAL INOCULATION CHALLENGE OR TEN YEARS' NATURAL EXPOSURE IN CONTAMINATED ENVIRONMENTS.

We conducted a 10-yr study to establish whether chronic wasting disease (CWD) was readily transmissible to domestic cattle ( Bos taurus) following oral inoculation or by cohousing cattle with captive cervids in outdoor research facilities where CWD was enzootic. Calves ( n=12) were challenged orally on one occasion using brain homogenate derived from CWD-infected mule deer ( Odocoileus hemionus). Five uninoculated cattle served as unchallenged controls. Two other groups of cattle ( n=10-11/group) were housed outdoors for 10 yr in captive cervid research facilities. The environmentally challenged cattle were exposed to CWD-associated prions through common paddocks, feed, and water and via direct daily contact with known and potentially infected mule deer or wapiti ( Cervus canadensis) throughout the decade-long study period. None of the exposed cattle developed neurologic disease during the study. We euthanized cattle surviving to 10 yr postchallenge and examined all for lesions or disease-associated prion protein (PrPd) by histopathology, immunohistochemistry, and western immunoblot analysis of central nervous system and lymphoid tissue. None had evidence of PrPd accumulation. We conclude that the risks of CWD transmission to cattle following oral inoculation or after prolonged exposure to contaminated environments are low.

Altered trafficking of abnormal prion protein in atypical scrapie: prion protein accumulation in oligodendroglial inner mesaxons.

Prion diseases exist in classical and atypical disease forms. Both forms are characterized by disease-associated accumulation of a host membrane sialoglycoprotein known as prion protein (PrPd ). In classical forms of prion diseases, PrPd can accumulate in the extracellular space as fibrillar amyloid, intracellularly within lysosomes, but mainly on membranes in association with unique and characteristic membrane pathology. These membrane changes are found in all species and strains of classical prion diseases and consist of spiral, branched and clathrin-coated membrane invaginations on dendrites. Atypical prion diseases have been described in ruminants and man and have distinct biological, biochemical and pathological properties when compared to classical disease. The purpose of this study was to determine whether the subcellular pattern of PrPd accumulation and membrane changes in atypical scrapie were the same as those found in classical prion diseases. Immunogold electron microscopy was used to examine brains of atypical scrapie-affected sheep and Tg338 mice. Classical prion disease-associated membrane lesions were not found in atypical scrapie-affected sheep, however, white matter PrPd accumulation was localized mainly to the inner mesaxon and paranodal cytoplasm of oligodendroglia. Similar lesions were found in myelinated axons of atypical scrapie Tg338-infected mice. However, Tg338 mice also showed the unique grey matter membrane changes seen in classical forms of disease. These data show that atypical scrapie infection directs a change in trafficking of abnormal PrP to axons and oligodendroglia and that the resulting pathology is an interaction between the agent strain and host genotype.

Minimum Effective Dose of Cattle and Sheep BSE for Oral Sheep Infection.

The minimum dose required to cause infection of Romney and Suffolk sheep of the ARQ/ARQ or ARQ/ARR prion protein gene genotypes following oral inoculation with Romney or Suffolk a sheep Bovine spongiform encephalopathy (BSE)-derived or cattle BSE-derived agent was investigated using doses ranging from 0.0005g to 5g. ARQ/ARQ sheep which were methionine (M) / threonine (T) heterozygous or T/T homozygous at codon 112 of the Prnp gene, dosed ARQ/ARR sheep and undosed controls did not show any evidence of infection. Within groups of susceptible sheep, the minimum effective oral dose of BSE was found to be 0.05g, with higher attack rates following inoculation with the 5g dose. Surprisingly, this study found no effect of dose on survival time suggesting a possible lack of homogeneity within the inoculum. All clinical BSE cases showed PrPd accumulation in brain; however, following cattle BSE inoculation, LRS involvement within Romney recipients was found to be significantly lower than within the Suffolk sheep inoculated group which is in agreement with previous reports.

Pathogenesis of scrapie in ARQ/ARQ sheep after subcutaneous infection: Effect of lymphadenectomy and immune cell subset changes in relation to prion protein accumulation

It is well established that the infectious agent of scrapie can replicate in the lymphoreticular system (LRS). However, the effects of removal of LRS target tissues on the pathogenesis of the infection and the accumulation of disease-associated prion protein (PrPd) in LRS tissues on specific immune cell subsets are poorly understood aspects. To address these questions 16 ARQ/ARQ sheep were subcutaneously inoculated in the drainage area of the prefemoral lymph node with brain homogenate derived from Suffolk sheep naturally infected with scrapie. Fourteen sheep were then subjected to either early (14–17 days post-inoculation [dpi]) or late (175–201 dpi) lymphadenectomy and culled at preclinical or clinical stages of infection. Neither late nor even early lymphadenectomy prevented infection or had any effect on the accumulation of PrPd in the LRS or CNS suggesting a rapid organic dissemination of the infectious agent after inoculation. Lymph nodes from eight scrapie inoculated sheep selected on the basis of the amount of PrPd in their LRS tissues (negative, low or high) were examined for six different immune cell markers. The PrPd negative lymph nodes from two sheep with no evidence of scrapie infection showed lower numbers of cluster of determination (CD) 21 positive cells than PrPd positive nodes, irrespective of their location (hind leg or head). However, quantitative differences in the expression of this marker were not detected when comparing lymph nodes with low and high levels of PrPd accumulation, suggesting that proliferation of CD21 positive cells is related to scrapie infection, but not directly linked to the magnitude of PrPd accumulation. An additional observation of the study was that sheep that were methionin–threonine at codon 112 of the prion protein gene showed lower attack rates than methionine homozygotes (67% and 100%, respectively) and also generally lower levels of PrPd accumulation in the LRS and brain and increased survival times, suggesting an influence of such polymorphism in the susceptibility to scrapie.

Time and frequency domain analysis of heart rate variability in cattle affected by bovine spongiform encephalopathy

BackgroundHeart rate variability (HRV) analysis is a method to assess the function of the autonomic nervous system. Brainstem nuclei that influence HRV are affected by vacuolar changes and accumulation of disease-associated prion protein (PrPd) in bovine spongiform encephalopathy (BSE) resulting in clinical signs suggestive of an increased parasympathetic tone. It was hypothesised that BSE in cattle causes changes in the autonomic nervous system; this was tested by comparing HRV indices derived from 1048 electrocardiograms, which were recorded from 51 naturally or experimentally infected cattle with BSE confirmed by postmortem tests, 321 clinical suspect cases or cattle inoculated with potentially infectious tissue without disease confirmation and 78 BSE-free control cattle.FindingsStatistically significant differences were found for low or high frequency power, their normalised values and ratio when the last recording prior to cull or repeated recordings were compared but only between male and female cattle of the three groups and not between groups of the same gender, even though BSE cases of each gender appeared to be more nervous during the recording. The same findings were made for heart rate, deviation from the mean RR interval and vasovagal tonus index when repeated recordings were compared. BSE cases with severe vacuolar changes in the parasympathetic nucleus of the vagus nerve had a significantly lower low:high frequency power ratio but not a lower heart rate than BSE cases with mild vacuolation, whereas severity of vacuolar changes in the solitary tract nucleus or intensity of PrPd accumulation in both nuclei did not appear to have any affect on either index. Abnormalities in the electrocardiogram were detected in 3% of the recordings irrespective of the BSE status; sinus arrhythmia was present in 93% of the remaining recordings.ConclusionsHRV analysis was not useful to distinguish BSE-positive from BSE-negative cattle grouped by gender, and HRV indices appeared to be mainly influenced by gender. There is agreement with earlier studies that vacuolar changes in the brainstem may be associated with an increased parasympathetic tone in BSE and that abnormalities in an electrocardiogram can be detected in cattle without evidence of heart disease.

Relationship between clinical signs and postmortem test status in cattle experimentally infected with the bovine spongiform encephalopathy agent

BackgroundVarious clinical protocols have been developed to aid in the clinical diagnosis of classical bovine spongiform encephalopathy (BSE), which is confirmed by postmortem examinations based on vacuolation and accumulation of disease-associated prion protein (PrPd) in the brain. The present study investigated the occurrence and progression of sixty selected clinical signs and behaviour combinations in 513 experimentally exposed cattle subsequently categorised postmortem as confirmed or unconfirmed BSE cases. Appropriate undosed or saline inoculated controls were examined similarly and the data analysed to explore the possible occurrence of BSE-specific clinical expression in animals unconfirmed by postmortem examinations.ResultsBased on the display of selected behavioural, sensory and locomotor changes, 20 (67%) orally dosed and 17 (77%) intracerebrally inoculated pathologically confirmed BSE cases and 21 (13%) orally dosed and 18 (6%) intracerebrally inoculated but unconfirmed cases were considered clinical BSE suspects. None of 103 controls showed significant signs and were all negative on diagnostic postmortem examinations. Signs indicative of BSE suspects, particularly over-reactivity and ataxia, were more frequently displayed in confirmed cases with vacuolar changes in the brain. The display of several BSE-associated signs over time, including repeated startle responses and nervousness, was significantly more frequent in confirmed BSE cases compared to controls, but these two signs were also significantly more frequent in orally dosed cattle unconfirmed by postmortem examinations.ConclusionsThe findings confirm that in experimentally infected cattle clinical abnormalities indicative of BSE are accompanied by vacuolar changes and PrPd accumulation in the brainstem. The presence of more frequently expressed signs in cases with vacuolar changes is consistent with this pathology representing a more advanced stage of disease. That BSE-like signs or sign combinations occur in inoculated animals that were not confirmed as BSE cases by postmortem examinations requires further study to investigate the potential causal relationship with prion disease.

Monitoring of clinical signs in goats with transmissible spongiform encephalopathies

BackgroundAs there is limited information about the clinical signs of BSE and scrapie in goats, studies were conducted to describe the clinical progression of scrapie and BSE in goats and to evaluate a short clinical protocol for its use in detecting scrapie-affected goats in two herds with previously confirmed scrapie cases. Clinical assessments were carried out in five goats intracerebrally infected with the BSE agent as well as five reported scrapie suspects and 346 goats subject to cull from the two herds, 24 of which were retained for further monitoring. The brain and selected lymphoid tissue were examined by postmortem tests for disease confirmation.ResultsThe sensitivity and specificity of the short clinical protocol in detecting a scrapie case in the scrapie-affected herds was 3.9% and 99.6%, respectively, based on the presence of tremor, positive scratch test, extensive hair loss, ataxia and absent menace response. All BSE- and scrapie-affected goats displayed abnormalities in sensation (over-reactivity to external stimuli, startle responses, pruritus, absent menace response) and movement (ataxia, tremor, postural deficits) at an advanced clinical stage but the first detectable sign associated with scrapie or BSE could vary between animals. Signs of pruritus were not always present despite similar prion protein genotypes. Clinical signs of scrapie were also displayed by two scrapie cases that presented with detectable disease-associated prion protein only in lymphoid tissues.ConclusionsBSE and scrapie may present as pruritic and non-pruritic forms in goats. Signs assessed for the clinical diagnosis of scrapie or BSE in goats should include postural and gait abnormalities, pruritus and visual impairment. However, many scrapie cases will be missed if detection is solely based on the display of clinical signs. PrPd accumulation in the brain appeared to be related to the severity of clinical disease but not to the display of individual neurological signs.

Neuroinvasion in sheep transmissible spongiform encephalopathies: the role of the haematogenous route

It is generally believed that after oral exposure to transmissible spongiform encephalopathy (TSE) agents, neuroinvasion occurs via the enteric nervous system (ENS) and the autonomic nervous system. As a result, the dorsal motor nucleus of the vagus nerve is the initial point of disease-associated prion protein (PrP(d)) accumulation in the brain. HYPOTHESIS AND AIM: If direct ENS invasion following oral infection results in an early and specific brain targeting for PrP(d) accumulation, such topographical distribution could be different when other routes of infection were used, highlighting distinct routes for neuroinvasion. An immunohistochemical study has been conducted on the brain of 67 preclinically infected sheep exposed to natural scrapie or to experimental TSE infection by various routes. Initial PrP(d) accumulation consistently occurred in the dorsal motor nucleus of the vagus nerve followed by the hypothalamus, regardless of the breed of sheep, PrP genotype, TSE source and, notably, route of infection; these factors did not appear to affect the topographical progression of PrP(d) deposition in the brain either. Moreover, the early and consistent appearance of PrP(d) aggregates in the circumventricular organs, where the blood-brain barrier is absent, suggests that these organs can provide a portal for entry of prions when infectivity is present in blood. The haematogenous route, therefore, can represent a parallel or alternative pathway of neuroinvasion to ascending infection via the ENS/autonomic nervous system.

Frequency and Distribution of Nerves in Scrapie-affected and Unaffected Peyer's Patches and Lymph Nodes

Transmission of sheep scrapie and some other prion diseases, including variant Creutzfeldt-Jakob disease of man, probably occurs via the oral route. A disease-associated variant of the host-coded prion protein (PrP(d)) accumulates in germinal center follicles of lymphoid tissues, including Peyer's patches of the gut, where it can be detected before its accumulation in the central nervous system. To investigate the potential role of lymphoid tissue nerves in neuroinvasion, we used immunohistochemical methods to study the frequency and distribution of nerves and PrP(d) accumulation in Peyer's patches and other lymphoid tissues from scrapie-affected and unaffected sheep. Nerves were infrequently found in secondary follicles of Peyer's patches, but never in germinal centers of the other lymphoid tissues tested. No differences in the frequency or distribution of nerves were found in relation to the presence or absence of PrP(d) accumulation. PrP(d) accumulation and nerves were only infrequently present together in Peyer's patches. These results suggest that, even if amplification of infectivity in lymphoid tissues facilitates neuroinvasion, nerves within lymph nodes and germinal centers of Peyer's patches do not play a primary role in transport of infectivity to the central nervous system. However, sheep between 2 and 4 months of age had significantly more nerve fibers within follicles than older groups. It is therefore possible that a general increase in nerve density of the intestine during early phases of life may contribute to an increased susceptibility of young animals to oral prion infection.

Frequency and Distribution of Nerves in Scrapie-affected and Unaffected Peyer's Patches and Lymph Nodes

Transmission of sheep scrapie and some other prion diseases, including variant Creutzfeldt-Jakob disease of man, probably occurs via the oral route. A disease-associated variant of the host-coded prion protein (PrPd) accumulates in germinal center follicles of lymphoid tissues, including Peyer's patches of the gut, where it can be detected before its accumulation in the central nervous system. To investigate the potential role of lymphoid tissue nerves in neuroinvasion, we used immunohistochemical methods to study the frequency and distribution of nerves and PrPd accumulation in Peyer's patches and other lymphoid tissues from scrapie-affected and unaffected sheep. Nerves were infrequently found in secondary follicles of Peyer's patches, but never in germinal centers of the other lymphoid tissues tested. No differences in the frequency or distribution of nerves were found in relation to the presence or absence of PrPd accumulation. PrPd accumulation and nerves were only infrequently present together in Peyer's patches. These results suggest that, even if amplification of infectivity in lymphoid tissues facilitates neuroinvasion, nerves within lymph nodes and germinal centers of Peyer's patches do not play a primary role in transport of infectivity to the central nervous system. However, sheep between 2 and 4 months of age had significantly more nerve fibers within follicles than older groups. It is therefore possible that a general increase in nerve density of the intestine during early phases of life may contribute to an increased susceptibility of young animals to oral prion infection.

Occurrence and cellular localization of PrPd in kidneys of scrapie‐affected sheep in the absence of inflammation

Following a preliminary description of disease-associated prion protein (PrPd) deposition in the kidneys of scrapie-affected sheep, detailed studies have been undertaken in order to evaluate the factors that could account for such PrPd accumulation and to determine the precise location of PrPd in the renal papillae. Immunohistochemical (IHC) examinations for PrPd were conducted in kidneys collected at post-mortem from 30 naturally and 37 experimentally infected sheep. In addition, PrPd detection by western blot analysis (WB) and ultrastructural examination was carried out in a selection of kidneys. PrPd-specific, multifocal IHC labelling with antibody R145 was achieved in the kidneys of 44% and 51% of the naturally and experimentally infected sheep, respectively. The specificity of these results was confirmed by further IHC and WB using several PrP antibodies raised to different amino acid sequences, and by examination of control tissues. PrPd was shown to accumulate in the interstitium of the renal papillae, in association with the cell membrane and lysosomes of fibroblast-like cells, or extracellularly, in close contact with collagen and basal membranes. These deposits were unrelated to inflammatory changes in the kidney as shown by routine histology and by IHC for different immune cell markers. PrPd accumulated in the kidney of sheep that showed widespread PrPd deposition in the lymphoreticular system and had long incubation periods; these findings argue for a haematogenous origin of renal PrPd, although the precise site and mechanism-glomerular filtration and reabsorption at Henle's loop, or extravasation from vasa recta capillaries, or both-by which PrPd leaves the blood to accumulate in the interstitium of renal papillae remain to be determined. Either of these pathogenetic mechanisms could lead to environmental contamination via urine.

Diagnosis of preclinical scrapie in live sheep by the immunohistochemical examination of rectal biopsies

In most sheep infected with a transmissible spongiform encephalopathy (tse) the disease-associated prion protein (PrPd) accumulates in tissues of the lymphoreticular system, suggesting that it might be detected in biopsy...

Classical sheep transmissible spongiform encephalopathies: pathogenesis, pathological phenotypes and clinical disease

Scrapie is a prion disease or transmissible spongiform encephalopathy (TSE) of sheep, goats and moufflon. As with its human counterparts, pathology consists of vacuolation, gliosis and accumulations of abnormal forms of a host prion protein (PrPd) in the brain of affected individuals. Immunohistochemical methods can be used to identify both the intracellular truncation sites of PrPd in different cell types (PrPd epitope mapping) and the different morphological patterns of accumulation (PrPd profiling). Differences in the inferred truncation sites of PrPd are found for different strains of sheep TSEs and for different infected cell types within individual strains. Immunochemical methods of characterizing strains broadly correspond to PrPd mapping discriminatory results, but distinct PrPd profiles, which provide strain- and source-specific information on both the cell types which sustain infection (cellular tropisms) and the cellular processing of PrPd, have no immunoblotting counterparts. The cause of neurological dysfunction in human is commonly considered to be neuronal loss secondary to a direct or indirect effect of the accumulation of PrPd. However, in sheep scrapie there is no significant neuronal loss, and relationships between different magnitudes, topographical and cytological forms of PrPd accumulation and clinical signs are not evident. PrPd accumulation also occurs in lymphoid tissues, for which there is indirect evidence of a pathological effect, in the peripheral nervous system and in other tissues. It is generally assumed that neuroinvasion results from infection of the enteric nervous system neurones subsequent to amplification of infectivity in lymphoid tissues and later spread via sympathetic and parasympathetic pathways. The evidence for this is, however, circumstantial. Accumulation of PrPd and presence of infectivity in tissues other than the nervous and lymphoreticular systems gives insights on the ways of transmission of infection and on food safety.

The neuropathologic phenotype of experimental ovine BSE is maintained after blood transfusion

Iatrogenic transmission by blood transfusion has been described in cases of human variant Creutzfeldt-Jakob disease (vCJD), experimental ovine bovine spongiform encephalopathy (BSE), and natural sheep scrapie, demonstrating that blood in these prion diseases is infectious. However, the possible effect of the transfusion, derived from differences in the inoculum (blood) and the route of infection (intravenous), on the pathologic phenotype of the disease in the recipients is not known. This study describes the neuropathologic phenotype of PrP(d) accumulation in sheep succumbing to neurologic disease after blood transfusion from donors experimentally infected with BSE; these were either clinically or subclinically affected at the time of donation. We demonstrate that blood can become infectious at early stages of ovine BSE infection and that the PrP(d) immunohistochemical phenotype is maintained after transfusion. This suggests that a change in the pathologic phenotype of vCJD would not be expected as a result of exposure to infected blood.

Phenotype of disease-associated PrP accumulation in the brain of bovine spongiform encephalopathy experimentally infected sheep

In view of the established link between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease and of the susceptibility of sheep to experimental BSE, the detection of potential cases of naturally occurring BSE in sheep has become of great importance. In this study, the immunohistochemical (IHC) phenotype of disease-associated prion protein (PrP(d)) accumulation has been determined in the brain of 64 sheep, of various breeds and PrP genotypes, that had developed neurological disease after experimental BSE challenge with different inocula by a range of routes. Sheep BSE was characterized by neuron-associated intra- and extracellular PrP(d) aggregates and by conspicuous and consistent deposits in the cytoplasm of microglia-like cells. The stellate PrP(d) type was also prominent in most brain areas and marked linear deposits in the striatum and midbrain were distinctive. Sheep of the ARR/ARR and ARQ/AHQ genotypes displayed lower levels of PrP(d) than other sheep, and intracerebral BSE challenge resulted in higher levels of PrP(d) accumulating in the brain compared with other routes. The PrP genotype and the route of challenge also appeared to affect the incubation period of the disease, giving rise to complex combinations of magnitude of PrP(d) accumulation and incubation period. Despite these differences, the phenotype of PrP(d) accumulation was found to be very consistent across the different factors tested (notably after subpassage of BSE in sheep), thus highlighting the importance of detailed IHC examination of the brain of clinically affected sheep for the identification of potential naturally occurring ovine BSE.

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 (PrPd) 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 PrPdtypes, which were subjectively quantified in eight different brain regions. The PrPdtypes were grouped into four PrPdpatterns, the relative magnitude of which provided the PrPdprofile of each sheep examined. The analysis of the differences in magnitude and relative proportion of each of these PrPdtypes and patterns indicated (1) an effect of the scrapie strain on the PrPdprofile, and (2) a possible effect of the host genotype on the magnitude of PrPdaccumulation in the brain, apparently related to the incubation period. Furthermore, intraneuronal deposition of PrPdwas 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.