Evidence of scrapie transmission to sheep via goat milk.

BACKGROUND: Previous studies confirmed that classical scrapie can be transmitted via milk in sheep. The current study aimed to investigate whether scrapie can also be transmitted via goat milk using in vivo (new-born lambs fed milk from scrapie-affected goats due to the unavailability of goat kids from guaranteed scrapie-free herds) and in vitro methods (serial protein misfolding cyclic amplification [sPMCA] on milk samples). RESULTS: In an initial pilot study, new-born lambs of two different prion protein gene (PRNP) genotypes (six VRQ/VRQ and five ARQ/ARQ) were orally challenged with 5 g brain homogenate from two scrapie-affected goats to determine susceptibility of sheep to goat scrapie. All sheep challenged with goat scrapie brain became infected based on the immunohistochemical detection of disease-associated PrP (PrP(sc)) in lymphoid tissue, with an ARQ/ARQ sheep being the first to succumb. Subsequent feeding of milk to eight pairs of new-born ARQ/ARQ lambs, with each pair receiving milk from a different scrapie-affected goat, resulted in scrapie in the six pairs that received the largest volume of milk (38-87 litres per lamb), whereas two pairs fed 8-9 litres per lamb, and an environmental control group raised on sheep milk from healthy ewes, did not show evidence of infection when culled at up to 1882 days of age. Infection in those 12 milk recipients occurred regardless of the clinical status, PrP(sc) distribution, caprine arthritis-encephalitis virus infection status and PRNP polymorphisms at codon 142 (II or IM) of the donor goats, but survival time was influenced by PRNP polymorphisms at codon 141. Serial PMCA applied to a total of 32 milk samples (four each from the eight donor goats collected throughout lactation) detected PrP(sc) in one sample each from two goats. CONCLUSIONS: The scrapie agent was present in the milk from infected goats and was able to transmit to susceptible species even at early preclinical stage of infection, when PrP(sc) was undetectable in the brain of the donor goats. Serial PMCA as a PrP(sc) detection method to assess the risk of scrapie transmission via milk in goats proved inefficient compared to the bioassay.

Dynamics of the natural transmission of bovine spongiform encephalopathy within an intensively managed sheep flock.

Sheep are susceptible to the bovine spongiform encephalopathy (BSE) agent and in the UK they may have been exposed to BSE via contaminated meat and bone meal. An experimental sheep flock was established to determine whether ovine BSE could be naturally transmitted under conditions of intensive husbandry. The flock consisted of 113 sheep of different breeds and susceptible PRNP genotypes orally dosed with BSE, 159 sheep subsequently born to them and 125 unchallenged sentinel controls. BSE was confirmed in 104 (92%) orally dosed sheep and natural transmission was recorded for 14 of 79 (18%) lambs born to BSE infected dams, with rates varying according to PRNP genotype. The likelihood of natural BSE transmission was linked to stage of incubation period of the dam: the attack rate for lambs born within 100 days of the death of BSE infected dams was significantly higher (9/22, 41%) than for the rest (5/57, 9%). Within the group of ewes lambing close to death, those rearing infected progeny (n = 8, for 9/12 infected lambs) showed a significantly greater involvement of lymphoid tissues than those rearing non-infected offspring (n = 8, for 0/10 infected lambs). Horizontal transmission to the progeny of non-infected mothers was recorded only once (1/205, 0.5%). This low rate of lateral transmission was attributed, at least partly, to an almost complete absence of infected placentas. We conclude that, although BSE can be naturally transmitted through dam-lamb close contact, the infection in this study flock would not have persisted due to low-efficiency maternal and lateral transmissions.

Transcriptional changes in the brains of cattle orally infected with the bovine spongiform encephalopathy agent precede detection of infectivity.

Bovine spongiform encephalopathy (BSE) is a fatal, transmissible, neurodegenerative disease of cattle. BSE can be transmitted experimentally between cattle through the oral route, and in this study, brain tissue samples from animals at different time points postinoculation were analyzed for changes in gene expression. The aims of this study were to identify differentially regulated genes during the progression of BSE using microarray-based gene expression profiling and to understand the effect of prion pathogenesis on gene expression. A total of 114 genes were found to be differentially regulated over the time course of the infection, and many of these 114 genes encode proteins involved in immune response, apoptosis, cell adhesion, stress response, and transcription. This study also revealed a broad correlation between gene expression profiles and the progression of BSE in cattle. At 21 months postinoculation, the largest number of differentially regulated genes was detected, suggesting that there are many pathogenic processes in the animal brain even prior to the detection of infectivity in the central nervous systems of these orally infected cattle. Moreover, evidence is presented to suggest that it is possible to predict the infectious status of animals using the expression profiles from this study.

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

BACKGROUND: Various 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. RESULTS: Based 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. CONCLUSIONS: The 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.

BACKGROUND: As 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. RESULTS: The 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. CONCLUSIONS: BSE 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.

Transmissibility studies of vacuolar changes in the rostral colliculus of pigs.

BACKGROUND: Histopathological examinations of brains from healthy pigs have revealed localised vacuolar changes, predominantly in the rostral colliculus, that are similar to the neuropil vacuolation featured in the transmissible spongiform encephalopathies and have been described in pigs challenged parenterally with the agent causing bovine spongiform encephalopathy (BSE). Feedstuff containing BSE-contaminated meat and bone meal (MBM) may have been fed to pigs prior to the ban of mammalian MBM in feed of farmed livestock in the United Kingdom in 1996, but there is no evidence of the natural occurrence of a transmissible spongiform encephalopathy (TSE) in the domestic pig. Furthermore, experimental transmission of BSE to pigs by the oral route has been unsuccessful. A study was conducted to investigate whether the localised vacuolar changes in the porcine brain were associated with a transmissible aetiology and therefore biologically significant. Two groups of ten pigs were inoculated parenterally with vacuolated rostral colliculus from healthy pigs either born before 1996 or born after 1996. Controls included ten pigs similarly inoculated with rostral colliculus from New Zealand-derived pigs and nine pigs inoculated with a bovine BSE brain homogenate. RESULTS: None of the pigs inoculated with rostral colliculus developed a TSE-like neurological disease up to five years post inoculation when the study was terminated, and disease-associated prion protein, PrPd, was not detected in the brains of these pigs. By contrast, eight of nine BSE-inoculated pigs developed neurological signs, two of which had detectable PrPd by postmortem tests. No significant histopathological changes were detected to account for the clinical signs in the PrPd-negative, BSE-inoculated pigs. CONCLUSION: The findings in this study suggest that vacuolation in the porcine rostral colliculus is not caused by a transmissible agent and is probably a clinically insignificant change. The presence of neurological signs in pigs inoculated with BSE without detectable PrPd raises the possibility that the BSE agent may produce a prion disease in pigs that remains undetected by the current postmortem tests.

Different prion disease phenotypes result from inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain.

BACKGROUND: Given the theoretical proposal that bovine spongiform encephalopathy (BSE) could have originated from sheep scrapie, this study investigated the pathogenicity for cattle, by intracerebral (i.c.) inoculation, of two pools of scrapie agents sourced in Great Britain before and during the BSE epidemic. Two groups of ten cattle were each inoculated with pools of brain material from sheep scrapie cases collected prior to 1975 and after 1990. Control groups comprised five cattle inoculated with sheep brain free from scrapie, five cattle inoculated with saline, and for comparison with BSE, naturally infected cattle and cattle i.c. inoculated with BSE brainstem homogenate from a parallel study. Phenotypic characterisation of the disease forms transmitted to cattle was conducted by morphological, immunohistochemical, biochemical and biological methods. RESULTS: Disease occurred in 16 cattle, nine inoculated with the pre-1975 inoculum and seven inoculated with the post-1990 inoculum, with four cattle still alive at 83 months post challenge (as at June 2006). The different inocula produced predominantly two different disease phenotypes as determined by histopathological, immunohistochemical and Western immunoblotting methods and biological characterisation on transmission to mice, neither of which was identical to BSE. Whilst the disease presentation was uniform in all scrapie-affected cattle of the pre-1975 group, the post-1990 inoculum produced a more variable disease, with two animals sharing immunohistochemical and molecular profile characteristics with animals in the pre-1975 group. CONCLUSION: The study has demonstrated that cattle inoculated with different pooled scrapie sources can develop different prion disease phenotypes, which were not consistent with the phenotype of BSE of cattle and whose isolates did not have the strain typing characteristics of the BSE agent on transmission to mice.

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.

Persistence of ovine scrapie infectivity in a farm environment following cleaning and decontamination.

Scrapie of sheep/goats and chronic wasting disease of deer/elk are contagious prion diseases where environmental reservoirs are directly implicated in the transmission of disease. In this study, the effectiveness of recommended scrapie farm decontamination regimens was evaluated by a sheep bioassay using buildings naturally contaminated with scrapie. Pens within a farm building were treated with either 20,000 parts per million free chorine solution for one hour or were treated with the same but were followed by painting and full re-galvanisation or replacement of metalwork within the pen. Scrapie susceptible lambs of the PRNP genotype VRQ/VRQ were reared within these pens and their scrapie status was monitored by recto-anal mucosa-associated lymphoid tissue. All animals became infected over an 18-month period, even in the pen that had been subject to the most stringent decontamination process. These data suggest that recommended current guidelines for the decontamination of farm buildings following outbreaks of scrapie do little to reduce the titre of infectious scrapie material and that environmental recontamination could also be an issue associated with these premises.

Further characterisation of transmissible spongiform encephalopathy phenotypes after inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain.

BACKGROUND: The infectious agent responsible for the bovine spongiform encephalopathy (BSE) epidemic in Great Britain is a transmissible spongiform encephalopathy (TSE) strain with uniform properties but the origin of this strain remains unknown. Based on the hypothesis that classical BSE may have been caused by a TSE strain present in sheep, cattle were inoculated intracerebrally with two different pools of brains from scrapie-affected sheep sourced prior to and during the BSE epidemic to investigate resulting disease phenotypes and characterise their causal agents by transmission to rodents. RESULTS: As reported in 2006, intracerebral inoculation of cattle with pre-1975 and post-1990 scrapie brain pools produced two distinct disease phenotypes, which were unlike classical BSE. Subsequent to that report none of the remaining cattle, culled at 10 years post inoculation, developed a TSE. Retrospective Western immunoblot examination of the brains from TSE cases inoculated with the pre-1975 scrapie pool revealed a molecular profile similar to L-type BSE. The inoculation of transgenic mice expressing the bovine, ovine, porcine, murine or human prion protein gene and bank voles with brains from scrapie-affected cattle did not detect classical or atypical BSE strains but identified two previously characterised scrapie strains of sheep. CONCLUSIONS: Characterisation of the causal agents of disease resulting from exposure of cattle to naturally occurring scrapie agents sourced in Great Britain did not reveal evidence of classical or atypical BSE, but did identify two distinct previously recognised strains of scrapie. Although scrapie was still recognizable upon cattle passage there were irreconcilable discrepancies between the results of biological strain typing approaches and molecular profiling methods, suggesting that the latter may not be appropriate for the identification and differentiation of atypical, particularly L-type, BSE agents from cattle experimentally infected with a potential mixture of classical scrapie strains from sheep sources.

Elements modulating the prion species barrier and its passage consequences.

The specific characteristics of Transmissible Spongiform Encephalopathy (TSE) strains may be altered during passage across a species barrier. In this study we investigated the biochemical and biological characteristics of Bovine Spongiform Encephalopathy (BSE) after transmission in both natural host species (cattle, sheep, pigs and mice) and in transgenic mice overexpressing the corresponding cellular prion protein (PrPC) in comparison with other non-BSE related prions from the same species. After these passages, most features of the BSE agent remained unchanged. BSE-derived agents only showed slight modifications in the biochemical properties of the accumulated PrPSc, which were demonstrated to be reversible upon re-inoculation into transgenic mice expressing bovine-PrPC. Transmission experiments in transgenic mice expressing bovine, porcine or human-PrP revealed that all BSE-derived agents were transmitted with no or a weak transmission barrier. In contrast, a high species barrier was observed for the non-BSE related prions that harboured an identical PrP amino acid sequence, supporting the theory that the prion transmission barrier is modulated by strain properties (presumably conformation-dependent) rather than by PrP amino acid sequence differences between host and donor. As identical results were observed with prions propagated either in natural hosts or in transgenic mouse models, we postulate that the species barrier and its passage consequences are uniquely governed by the host PrPC sequence and not influenced by other host genetic factors. The results presented herein reinforce the idea that the BSE agent is highly promiscuous, infecting other species, maintaining its properties in the new species, and even increasing its capabilities to jump to other species including humans. These data are essential for the development of an accurate risk assessment for BSE.

Ultrastructural findings in pigs experimentally infected with bovine spongiform encephalopathy agent.

We report here an electron microscopic study of selected nervous system tissues from pigs infected experimentally with the agent of bovine spongiform encephalopathy (BSE). Generally, the ultrastructural neuropathology of BSE-affected pig brain resembled that of BSE-affected cattle brain. Spongiform change, in the form of membrane-bound vacuoles separated by septae into secondary chambers, dominated the pathology. Numerous astrocytic processes were visible in close conjunction with elongated microglial cells. Neuronal degeneration presented as either dystrophic neurites or by the formation of autophagic vacuoles. Altered subcellular organelles: mitochondria, electron-dense bodies, autophagic vacuoles, neurofilaments and "branching-cisterns" accumulated in abnormal neurites. Autophagic vacuoles appeared as neuronal cytoplasm of increased electron-density sequestrated by intracytoplasmic membranes. Tubulovesicular structures were numerous, particularly in the cerebellum. Unusual crystalloids were observed in the white matter. In conclusion, experimental BSE in pigs demonstrated ultrastructural pathology in keeping with that observed in other spongiform encephalopathies.

Bovine spongiform encephalopathy: the effect of oral exposure dose on attack rate and incubation period in cattle - an update.

BACKGROUND: To provide information on dose-response and aid in modelling the exposure dynamics of the BSE epidemic in the United Kingdom groups of cattle were exposed orally to a range of different doses of brainstem homogenate of known infectious titre from clinical cases of classical bovine spongiform encephalopathy (BSE). Interim data from this study was published in 2007. This communication documents additional BSE cases, which occurred subsequently, examines possible influence of the bovine prion protein gene on disease incidence and revises estimates of effective oral exposure. FINDINGS: Following interim published results, two further cattle, one dosed with 100 mg and culled at 127 months post exposure and the other dosed with 10 mg and culled at 110 months post exposure, developed BSE. Both had a similar pathological phenotype to previous cases. Based on attack rate and incubation period distribution according to dose, the dose estimate at which 50% of confirmed cases would be clinically affected was revised to 0.15 g of the brain homogenate used in the experiment, with a 95% confidence interval of 0.03-0.79 g. Neither the full open reading frame nor the promoter region of the prion protein gene of dosed cattle appeared to influence susceptibility to BSE, but this may be due to the sample size. CONCLUSIONS: Oral exposure of cattle to a large range of doses of a BSE brainstem homogenate produced disease in all dose groups. The pathological presentation resembled natural disease. The attack rate and incubation period were dependent on the dose.