Susan J Bellworthy

Evidence of scrapie transmission via milk

BackgroundThe risk of scrapie infection increases with increased duration and proximity of contact between sheep at lambing. Scrapie infectivity has not been detected in milk but cellular prion protein, the precursor of disease-associated prion protein PrPd, has been found in milk from ruminants. To determine whether milk is able to transmit scrapie, 18 lambs with a prion protein genotype associated with high susceptibility to scrapie (VRQ/VRQ) were fed milk from twelve scrapie-affected ewes of the same genotype, and 15 VRQ/VRQ sheep reared on scrapie-free dams served as controls.ResultsThree lambs fed milk from scrapie-affected ewes were culled due to intercurrent diseases at 43, 44 and 105 days of age respectively, and PrPd was detected in the distal ileum of the first two lambs, whilst PrPd was not found in lymphoreticular tissues in the third lamb. A control lamb, housed in a separate pen and culled at 38 days of age, was also negative for PrPd in a range of tissues. Samples of recto-anal mucosa associated lymphoid tissue collected from the remaining 15 live lambs at seven months of age (between five to seven months after mixing) were positive for PrPd in the scrapie milk recipients, whereas PrPd was not detected in the remaining 14 controls at that time. A subsequent sample collected from control lambs revealed PrPd accumulation in two of five lambs eight months after mixing with scrapie milk recipients suggestive of an early stage of infection via lateral transmission. By contrast, the control sheep housed in the same building but not mixed with the scrapie milk recipients were still negative for PrPd.ConclusionThe presence of PrPd in distal ileum and rectal mucosa indicates transmission of scrapie from ewe to lamb via milk (or colostrum) although it is not yet clear if such cases would go on to develop clinical disease. The high level of infection in scrapie-milk recipients revealed by rectal mucosal testing at approximately seven months of age may be enhanced or supplemented by intra-recipient infection as these lambs were mixed together after feeding with milk from scrapie-affected ewes and we also observed lateral transmission from these animals to lambs weaned from scrapie-free ewes.

Tissue distribution of bovine spongiform encephalopathy infectivity in Romney sheep up to the onset of clinical disease after oral challenge

Sixty Romney sheep of three prion protein genotypes were dosed orally at six months of age with an inoculum prepared from the brains of cattle clinically affected with BSE, and 15 sheep were left undosed as controls. They were randomly assigned within genotype to groups and were sequentially euthanased and examined postmortem at intervals of six or 12 months, depending on their predicted susceptibility. Tissue pools prepared from the three, four or five dosed animals in each group were inoculated into groups of 20 RIII mice as a bioassay for infectivity. Separate inocula were prepared from the matched control sheep killed at each time. In the ARQ/ARQ sheep killed four months after inoculation, infectivity was detected in the Peyers patch tissue pool, and at 10 months it was detected in the spleen pool; from 16 months, infectivity was detected in a range of nervous and lymphoreticular tissues, including the spinal cord pool, distal ileum excluding Peyers patches, liver, Peyers patches, mesenteric and prescapular lymph nodes, spleen, tonsil and cervical thymus. No infectivity was detected in the tissue pools from the ARQ/ARR and ARR/ARR sheep killed 10 months or 22 months after infection.

Postmortem diagnosis of preclinical and clinical scrapie in sheep by the detection of disease-associated PrP in their rectal mucosa

Samples of tissue from the central nervous system (CNS), the lymphoreticular system (LRS) and the rectal mucosa of a large number of scrapie-exposed sheep, with and without signs of clinical disease, were examined immunohistochemically for evidence of disease-associated prion protein (PrPd). The rectal mucosa has received almost no attention so far in scrapie diagnosis, despite its abundant rectoanal mucosa-associated lymphoid tissue, and its accessibility. The scrapie-confirmed cases included 244 with clinical disease, of which 237 (97·1 per cent) were positive in the rectal mucosa, and 121 apparently healthy sheep, of which 104 (86 per cent) were positive in the rectal mucosa. PrPd was detected in 86·4 to 91·5 per cent of the other LRS tissues of the healthy sheep examined and in 77·7 per cent of their CNS tissues. The stage of infection, therefore, affected the probability of a positive result in the rectal mucosa, whereas the breed, PrP genotype, age and sex had little or no independent effect. Accumulations of PrPd were observed in the rectal mucosa and other LRS tissues of VRQ/ARR sheep with preclinical and clinical scrapie, albeit with a lower frequency and magnitude than in sheep of other PrP genotypes. Western immunoblotting analyses of samples of rectal mucosa gave the characteristic PrP glycoprofile, with a sensitivity similar to that of immunohistochemistry.

Prions Are Secreted into the Oral Cavity in Sheep with Preclinical Scrapie

A major concern in prion disease transmission is the spread of the disease agent by means of secretions and excretions. We analyzed buccal swab samples obtained from preclinical scrapie-infected sheep by concentrating the collected prions on silicon dioxide, followed by amplification by serial protein misfolding cyclic amplification. Data clearly demonstrate that prions are present in buccal swab samples from sheep with a VRQ/VRQ PRNP genotype during preclinical scrapie infection. These data describe for the first time to our knowledge the secretion of prions into the oral cavity of sheep, a finding with implications for the transmission of ovine scrapie and very likely other prion diseases.

Natural transmission of BSE between sheep within an experimental flock.

SIR, – The recognition of bovine spongiform encephalopathy (BSE) in a French goat ([Eloit and others 2005][1]) has heightened the debate in Europe as to whether BSE has been maintained in small ruminants following historical exposure via feed. Key to the debate and associated risk assessments,

Environmental Sources of Scrapie Prions

ABSTRACT Ovine scrapie and cervine chronic wasting disease show considerable horizontal transmission. Here we report that a scrapie-affected sheep farm has a widespread environmental contamination with prions. Prions were amplified by protein-misfolding cyclic amplification (sPMCA) from seven of nine environmental swab samples taken, including those from metal, plastic, and wooden surfaces. Sheep had been removed from the areas from which the swabs were taken up to 20 days prior to sampling, indicating that prions persist for at least that long. These data implicate inanimate objects as environmental reservoirs for prion infectivity that are likely to contribute to facile disease transmission.

Prions Are Secreted in Milk from Clinically Normal Scrapie-Exposed Sheep

ABSTRACT The potential spread of prion infectivity in secreta is a crucial concern for prion disease transmission. Here, serial protein misfolding cyclic amplification (sPMCA) allowed the detection of prions in milk from clinically affected animals as well as scrapie-exposed sheep at least 20 months before clinical onset of disease, irrespective of the immunohistochemical detection of protease-resistant PrPSc within lymphoreticular and central nervous system tissues. These data indicate the secretion of prions within milk during the early stages of disease progression and a role for milk in prion transmission. Furthermore, the application of sPMCA to milk samples offers a noninvasive methodology to detect scrapie during preclinical/subclinical disease.

Detection of PrPsc in Blood from Sheep Infected with the Scrapie and Bovine Spongiform Encephalopathy Agents

ABSTRACT The role of blood in the iatrogenic transmission of transmissible spongiform encephalopathy (TSE) or prion disease has become an increasing concern since the reports of variant Creutzfeldt-Jakob disease (vCJD) transmission through blood transfusion from humans with subclinical infection. The development of highly sensitive rapid assays to screen for prion infection in blood is of high priority in order to facilitate the prevention of transmission via blood and blood products. In the present study we show that PrPsc, a surrogate marker for TSE infection, can be detected in cells isolated from the blood from naturally and experimentally infected sheep by using a rapid ligand-based immunoassay. In sheep with clinical disease, PrPsc was detected in the blood of 55% of scrapie agent-infected animals (n = 80) and 71% of animals with bovine spongiform encephalopathy (n = 7). PrPsc was also detected several months before the onset of clinical signs in a subset of scrapie agent-infected sheep, followed from 3 months of age to clinical disease. This study confirms that PrPsc is associated with the cellular component of blood and can be detected in preclinical sheep by an immunoassay in the absence of in vitro or in vivo amplification.

Pruritus is a common feature in sheep infected with the BSE agent

BackgroundThe variability in the clinical or pathological presentation of transmissible spongiform encephalopathies (TSEs) in sheep, such as scrapie and bovine spongiform encephalopathy (BSE), has been attributed to prion protein genotype, strain, breed, clinical duration, dose, route and type of inoculum and the age at infection. The study aimed to describe the clinical signs in sheep infected with the BSE agent throughout its clinical course to determine whether the clinical signs were as variable as described for classical scrapie in sheep. The clinical signs were compared to BSE-negative sheep to assess if disease-specific clinical markers exist.ResultsForty-seven (34%) of 139 sheep, which comprised 123 challenged sheep and 16 undosed controls, were positive for BSE. Affected sheep belonged to five different breeds and three different genotypes (ARQ/ARQ, VRQ/VRQ and AHQ/AHQ). None of the controls or BSE exposed sheep with ARR alleles were positive. Pruritus was present in 41 (87%) BSE positive sheep; the remaining six were judged to be pre-clinically infected. Testing of the response to scratching along the dorsum of a sheep proved to be a good indicator of clinical disease with a test sensitivity of 85% and specificity of 98% and usually coincided with weight loss. Clinical signs that were displayed significantly earlier in BSE positive cases compared to negative cases were behavioural changes, pruritic behaviour, a positive scratch test, alopecia, skin lesions, teeth grinding, tremor, ataxia, loss of weight and loss of body condition. The frequency and severity of each specific clinical sign usually increased with the progression of disease over a period of 16–20 weeks.ConclusionOur results suggest that BSE in sheep presents with relatively uniform clinical signs, with pruritus of increased severity and abnormalities in behaviour or movement as the disease progressed. Based on the studied sheep, these clinical features appear to be independent of breed, affected genotype, dose, route of inoculation and whether BSE was passed into sheep from cattle or from other sheep, suggesting that the clinical phenotype of BSE is influenced by the TSE strain more than by other factors. The clinical phenotype of BSE in the genotypes and breed studied was indistinguishable from that described for classical scrapie cases.

Oral transmission of bse to vrq/vrq sheep in an experimental flock

SIR, — We report here the first confirmed case of oral transmission of bovine spongi-form encephalopathy (bse) to sheep homozygous for valine at codon 136 of the prion protein (PrP) gene. Although intracerebral transmission has been previously reported ([Martin and others 2005][1]), our finding is