Experimental transmission of ovine atypical scrapie to cattle.

Classical bovine spongiform encephalopathy (BSE) in cattle was caused by the recycling and feeding of meat and bone meal contaminated with a transmissible spongiform encephalopathy (TSE) agent but its origin remains unknown. This study aimed to determine whether atypical scrapie could cause disease in cattle and to compare it with other known TSEs in cattle. Two groups of calves (five and two) were intracerebrally inoculated with atypical scrapie brain homogenate from two sheep with atypical scrapie. Controls were five calves intracerebrally inoculated with saline solution and one non-inoculated animal. Cattle were clinically monitored until clinical end-stage or at least 96 months post-inoculation (mpi). After euthanasia, tissues were collected for TSE diagnosis and potential transgenic mouse bioassay. One animal was culled with BSE-like clinical signs at 48 mpi. The other cattle either developed intercurrent diseases leading to cull or remained clinical unremarkable at study endpoint, including control cattle. None of the animals tested positive for TSEs by Western immunoblot and immunohistochemistry. Bioassay of brain samples from the clinical suspect in Ov-Tg338 and Bov-Tg110 mice was also negative. By contrast, protein misfolding cyclic amplification detected prions in the examined brains from atypical scrapie-challenged cattle, which had a classical BSE-like phenotype. This study demonstrates for the first time that a TSE agent with BSE-like properties can be amplified in cattle inoculated with atypical scrapie brain homogenate.

Test performance data demonstrates utility of a cattle DIVA skin test reagent (DST-F) compatible with BCG vaccination.

Bacillus Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis (M. bovis), is the lead candidate vaccine for control of bovine tuberculosis (TB) in cattle. However, BCG vaccination sensitises cattle to bovine tuberculin, thus compromising the use of the current bovine TB surveillance tests. To address this, we have developed a diagnostic skin test that is not compromised by BCG vaccination and is able to detect BCG vaccinated animals that subsequently develop bovine TB following exposure to M. bovis. Building on previous work using 'in house' formulated protein cocktail reagents, we herein present test performance data for a single fusion protein (DST-F) containing the mycobacterial antigens ESAT-6, CFP-10 and Rv3615c formulated as a 'ready to use' reagent by a commercial manufacturer. Our results demonstrate that, unlike tuberculin reagents, a diagnostic skin test using DST-F maintained high specificity in BCG vaccinated animals. Furthermore, the DST-F skin test demonstrated a high relative sensitivity in identifying M. bovis infected animals, including those where BCG vaccination failed to prevent bovine TB pathology following experimental exposure to M. bovis. The DST-F is currently undergoing field trials in Great Britain to support its licensure and commercialisation.

A review of cleaning and disinfection guidelines and recommendations following an outbreak of classical scrapie.

Classical scrapie is a prion disease of small ruminants, the infectious agent of which has been shown to be extremely persistent in the environment. Cleaning and disinfection (C&D) after a scrapie outbreak is currently recommended by many governments' veterinary advisors and implemented in most farms affected. Yet, the effectiveness of these procedures remains unclear. The aim of this study was to review existing literature and guidelines regarding farm C&D protocols following classical scrapie outbreaks and assess their effectiveness and the challenges that translation of policy and legislative requirements present at a practical level. A review of the literature was conducted to identify the on-farm C&D protocols used following outbreaks of scrapie, assess those materials with high risk for persistence of the scrapie agent on farms, and review the existing evidence of the effectiveness of recommended C&D protocols. An expert workshop was also organised in Great Britain (GB) to assess: the decision-making process used when implementing C&D protocols on GB farms, the experts' perceptions on the effectiveness of these protocols and changes needed, and their views on potential recommendations for policy and research. Outputs of the literature review revealed that the current recommended protocol for C&D [1 h treatment with sodium hypochlorite containing 20,000 ppm free chlorine or 2 M sodium hydroxide (NaOH)] is based on laboratory experiments. Only four field farm experiments have been conducted, indicating a lack of data on effectiveness of C&D protocols on farms by the re-occurrence of scrapie infection post re-stocking. Recommendations related to the control of outdoor environment, which are difficult and expensive to implement, vary between countries. The expert workshop concluded that there are no practical, cost-effective C&D alternatives to be considered at this time, with control therefore based on C&D only in combination with additional time restrictions on re-stocking and replacement with non-susceptible livestock or more genetically resistant types, where available. Participants agreed that C&D should still be completed on scrapie affected farms, as it is considered to be "good disease practice" and likely to reduce the levels of the prion protein. Participants felt that any additional protocols developed should not be "too prescriptive" (should not be written down in specific policies) because of significant variation in farm types, farm equipment and installations. Under this scenario, control of classical scrapie on farms should be designed with a level of C&D in combination with re-stocking temporal ban and replacement with livestock of limited susceptibility.

Temporal dynamics of intradermal cytokine response to tuberculin in Mycobacterium bovis BCG-vaccinated cattle using sampling microneedles.

Bovine tuberculosis (bTB) is a disease of livestock with severe and worldwide economic, animal welfare and zoonotic consequences. Application of test-and-slaughter-based control polices reliant on tuberculin skin testing has been the mainstay of bTB control in cattle. However, little is known about the temporal development of the bovine tuberculin skin test response at the dermal sites of antigen injection. To fill this knowledge gap, we applied minimally-invasive sampling microneedles (SMNs) for intradermal sampling of interstitial fluid at the tuberculin skin test sites in Mycobacterium bovis BCG-vaccinated calves and determined the temporal dynamics of a panel of 15 cytokines and chemokines in situ and in the peripheral blood. The results reveal an orchestrated and coordinated cytokine and local chemokine response, identified IL-1RA as a potential soluble biomarker of a positive tuberculin skin response, and confirmed the utility of IFN-γ and IP-10 for bTB detection in blood-based assays. Together, the results highlight the utility of SMNs to identify novel biomarkers and provide mechanistic insights on the intradermal cytokine and chemokine responses associated with the tuberculin skin test in BCG-sensitized cattle.

Classical Scrapie Did Not Re-occur in Goats After Cleaning and Disinfection of the Farm Premises.

After an outbreak of classical scrapie in a dairy goat herd with over 1,800 goats, all goats in the herd were culled in 2008, cleaning and disinfection of the premises was implemented, and restocking with goats took place ~4 months after depopulation. Ten years later the new herd population is over 3,000 goats. This study was carried out to determine whether the measures were effective to prevent re-occurrence of scrapie to the 1% prevalence level seen when scrapie was first detected on this farm. A total of 280 goats with a minimum age of 18 months, which were predominantly at the end of their productive life, were euthanized, and brain and retropharyngeal lymph node examined by immunohistochemistry for disease-associated prion protein. Genotyping was done in all euthanized goats and live male goats used or intended for breeding to determine prion protein gene polymorphisms associated with resistance to classical scrapie. None of the goats presented with disease-associated prion protein in the examined tissues, and 34 (12.2%) carried the K222 allele associated with resistance. This allele was also found in four breeding male goats. The study results suggested that classical scrapie was not re-introduced on this goat farm through mass restocking or inadequate cleaning and disinfection procedures. Further scrapie surveillance of goats on this farm is desirable to confirm absence of disease. Breeding with male goats carrying the K222 allele should be encouraged to increase the scrapie-resistant population.

Assessing the aggregated probability of entry of a novel prion disease agent into the United Kingdom.

In 2018 prion disease was detected in camels at an abattoir in Algeria for the first time. The emergence of prion disease in this species made it prudent to assess the probability of entry of the pathogen into the United Kingdom (UK) from this region. Potentially contaminated products were identified as evidenced by other prion diseases. The aggregated probability of entry of the pathogen was estimated as very high and high for legal milk and cheese imports respectively and very high, high and high for illegal meat, milk and cheese products respectively. This aggregated probability represents a qualitative assessment of the probability of one or more entry events per year into the UK; it gives no indication of the number of entry events per year. The uncertainty associated with these estimates was high due to the unknown variation in prevalence of infection in camels and an uncertain number and type of illegal products entering the UK. Potential public health implications of this pathogen are unknown although there is currently no evidence of zoonotic transmission of prion diseases other than bovine spongiform encephalopathy to humans.

The Scrapie Prevalence in a Goat Herd Is Underestimated by Using a Rapid Diagnostic Test.

Current European surveillance regulations for scrapie, a naturally occurring transmissible spongiform encephalopathy (TSE) or prion disease in sheep and goats, require testing of fallen stock or healthy slaughter animals, and outline measures in the case of confirmation of disease. An outbreak of classical scrapie in a herd with 2500 goats led to the culling of the whole herd, providing the opportunity to examine a subset of goats, take samples, and examine them for the presence of disease-associated prion protein (PrPSc) to provide further information on scrapie test sensitivity, pathology, and association with prion protein genotype. Goats were examined clinically prior to cull, and the brains examined post mortem by Bio-Rad ELISA, a rapid screening test used for active surveillance in sheep and goats, and two confirmatory tests, Western blot and immunohistochemistry. Furthermore, up to 10 lymphoid tissues were examined by immunohistochemistry. Of 151 goats examined, three (2.0%) tested positive for scrapie by ELISA on brain, confirmed by confirmatory tests, and a further five (3.3%) were negative by ELISA but positive by at least one of the confirmatory tests. Only two of these, both positive by ELISA, displayed evident signs of scrapie. In addition, 10 (6.6%) goats, which also included two clinical suspects, were negative on brain examination but had detectable PrPSc in lymphoid tissue. PrPSc was detected most frequently in the medial retropharyngeal lymph node (LN; 94.4% of all 18 cases) and palatine tonsil (88.9%). Abnormal behavior and circling or loss of balance when blindfolded were the best clinical discriminators for scrapie status. None of the goats that carried a single allele in the prion protein gene associated with increased resistance to scrapie (Q211, K222, S146) were scrapie-positive, and the percentage of goats with these alleles was greater than expected from previous surveys. Significantly more goats that were scrapie-positive were isoleucine homozygous at codon 142 (II142). The results indicate that the sensitivity of the applied screening test is poor in goats compared to the confirmatory tests as gold standard, particularly for asymptomatic animals. Sensitivity of surveillance could be improved by testing retropharyngeal LN or palatine tonsil in addition to brain.

Rapid recontamination of a farm building occurs after attempted prion removal.

The transmissible spongiform encephalopathy scrapie of sheep/goats and chronic wasting disease of cervids are associated with environmental reservoirs of infectivity. Preventing environmental prions acting as a source of infectivity to healthy animals is of major concern to farms that have had outbreaks of scrapie and also to the health management of wild and farmed cervids. Here, an efficient scrapie decontamination protocol was applied to a farm with high levels of environmental contamination with the scrapie agent. Post-decontamination, no prion material was detected within samples taken from the farm buildings as determined using a sensitive in vitro replication assay (sPMCA). A bioassay consisting of 25 newborn lambs of highly susceptible prion protein genotype VRQ/VRQ introduced into this decontaminated barn was carried out in addition to sampling and analysis of dust samples that were collected during the bioassay. Twenty-four of the animals examined by immunohistochemical analysis of lymphatic tissues were scrapie-positive during the bioassay, samples of dust collected within the barn were positive by month 3. The data illustrates the difficulty in decontaminating farm buildings from scrapie, and demonstrates the likely contribution of farm dust to the recontamination of these environments to levels that are capable of causing disease.

Estimating the impact on food and edible materials of changing scrapie control measures: The scrapie control model.

Multiple controls established during the bovine spongiform encephalopathy (BSE) epidemic were not solely applied to BSE in cattle, but were implemented for scrapie in sheep and goats due to concerns over the occurrence of BSE in sheep. In the absence of BSE in sheep being observed, control measures for prion diseases are now being evaluated to ensure they remain proportionate to risk. This risk assessment, aims to estimate, by use of stochastic simulation, the impact of reducing controls for Specified Risk Materials (SRM) from sheep at abattoir. Three scenarios have been included: 1) current list of SRM; 2) brain and spinal cord of adult sheep; and 3) the brain of adult sheep. Results indicate the total amount of infectivity passing through British abattoirs is highest for atypical scrapie with nearly 3,500,000 Ovine Oral (OO) ID50 per year. The majority of this infectivity enters Category 1 waste for incineration, with only 13,000 OO ID50 per year within edible products. Under Scenario 2, an additional 4000 OO ID50 per year would be classified as edible products from the lifting of restrictions on the distal ileum of adult sheep. However, if SRM removal was limited to brain, an additional 110,000 OO ID50 per year would be permitted into edible products with the lifting of restrictions on the spinal cord of adult sheep. For classical scrapie, there is a mean estimate of infectivity of 30,000 OO ID50 per year at abattoir. This is lower than for atypical scrapie due to the lower occurrence of this disease in Great Britain. However, more infectivity is destined to reach the food chain as the disease is peripherally distributed in the carcase. The highest contributor to the total amount of infectivity consumed per year is the intestines (duodenum and jejunum). If SRM removal is limited to the brain and spinal cord of sheep over 12 months of age, there is an approximate mean increase from 19,000 to 21,000 OO ID50 per year diverted to edible products. If the SRM list is restricted to brain only, this increases to over 23,000 OO ID50 per year. For the potential of sheep-BSE, there is a very low estimate of 29 OO ID50 per year in total from carcases entering abattoir, due to the potential very rare occurrence of this disease. Given changes in SRM regulations there is a change of an additional 4 OO ID50 per year being diverted to edible products.

Codon 141 polymorphisms of the ovine prion protein gene affect the phenotype of classical scrapie transmitted from goats to sheep.

BACKGROUND: A study to investigate transmission of classical scrapie via goat milk was carried out in sheep: firstly, lambs were challenged orally with goat scrapie brain homogenate to confirm transmission of scrapie from goats to sheep. In the second study phase, milk from scrapie-infected goats was fed to lambs. Lambs were selected according to their prion protein gene (PRNP) genotype, which was either VRQ/VRQ or ARQ/ARQ, with or without additional polymorphisms at codon 141 (FF141, LF141 or LL141) of the ovine PRNP. This report describes the clinical, pathological and molecular phenotype of goat scrapie in those sheep that progressed to clinical end-stage. RESULTS: Ten sheep (six VRQ/VRQ and four ARQ/ARQ, of which three FF141 and one LL141) challenged with one of two scrapie brain homogenates, and six pairs of sheep (ARQ, of which five LL141 and seven LF141) fed milk from six different goats, developed clinical disease, which was characterised by a pruritic (all VRQ/VRQ and LL141 sheep) or a non-pruritic form (all LF141 and FF141 sheep). Immunohistochemical (IHC) examination revealed that the pattern of intra- and extracellular accumulation of disease-associated prion protein in the brain was also dependent on PRNP polymorphisms at codon 141, which was similar in VRQ and LL141 sheep but different from LF141 and FF141 sheep. The influence of codon 141 was also seen in discriminatory Western blot (WB), with LF141 and FF141 sheep showing a bovine spongiform encephalopathy-like profile (diminished reactivity with P4 antibody) on brain tissue. However, discriminatory WB in lymphoid tissues, and IHC pattern and profile both in lymphoid and brain tissue was consistent with classical scrapie in all sheep. CONCLUSIONS: This study provided further evidence that the clinical presentation and the pathological and molecular phenotypes of scrapie in sheep are influenced by PRNP polymorphisms, particularly at codon 141. Differences in the truncation of disease-associated prion protein between LL141 sheep and those carrying the F141 allele may be responsible for these observations.

L-BSE experimentally transmitted to sheep presents as a unique disease phenotype.

Apart from prion protein genotype, the factors determining the host range and susceptiblity for specific transmissible spongiform encephalopathy agents remain unclear. It is known that bovine atypical L-BSE can transmit to a range of species including primates and humanised transgenic mice. It is important, therefore, that there is as broad an understanding as possible of how such isolates might present in food animal species and how robust they are on inter- and intra-species transmission to inform surveillance sytems and risk assessments. This paper demonstrates that L-BSE can be intracerebrally transmitted to sheep of several genotypes, with the exception of ARR/ARR animals. Positive animals mostly present with a cataplectic form of disease characterized by collapsing episodes and reduced muscle tone. PrP accumulation is confined to the nervous system, with the exception of one animal with lymphoreticular involvement. In Western blot there was maintenance of the low molecular mass and glycoform profile associated with L-BSE, irrespective of ovine host genotype, but there was a substantially higher N-terminal antibody signal relative to the core-specific antibody, which is similar to the ratio associated with classical scrapie. The disease phenotype was maintained on experimental subpassage, but with a shortened survival time indicative of an original species barrier and subsequent adaptation. Passive surveillance approaches would be unlikely to identify such cases as TSE suspects, but current statutory active screening methods would be capable of detecting such cases and classifying them as unusual and requiring further investigation if they were to occur in the field.

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.

Abnormalities in Brainstem Auditory Evoked Potentials in Sheep with Transmissible Spongiform Encephalopathies and Lack of a Clear Pathological Relationship.

Scrapie is transmissible spongiform encephalopathy (TSE), which causes neurological signs in sheep, but confirmatory diagnosis is usually made postmortem on examination of the brain for TSE-associated markers like vacuolar changes and disease-associated prion protein (PrP(Sc)). The objective of this study was to evaluate whether testing of brainstem auditory evoked potentials (BAEPs) at two different sound levels could aid in the clinical diagnosis of TSEs in sheep naturally or experimentally infected with different TSE strains [classical and atypical scrapie and bovine spongiform encephalopathy (BSE)] and whether any BAEP abnormalities were associated with TSE-associated markers in the auditory pathways. BAEPs were recorded from 141 clinically healthy sheep of different breeds and ages that tested negative for TSEs on postmortem tests to establish a reference range and to allow comparison with 30 sheep clinically affected or exposed to classical scrapie (CS) without disease confirmation (test group 1) and 182 clinically affected sheep with disease confirmation (test group 2). Abnormal BAEPs were found in 7 sheep (23%) of group 1 and 42 sheep (23%) of group 2. The proportion of sheep with abnormalities did not appear to be influenced by TSE strain or PrP(Sc) gene polymorphisms. When the magnitude of TSE-associated markers in the auditory pathways was compared between a subset of 12 sheep with and 12 sheep without BAEP abnormalities in group 2, no significant differences in the total PrP(Sc) or vacuolation scores in the auditory pathways could be found. However, the data suggested that there was a difference in the PrP(Sc) scores depending on the TSE strain because PrP(Sc) scores were significantly higher in sheep with BAEP abnormalities infected with classical and L-type BSE, but not with CS. The results indicated that BAEPs may be abnormal in sheep infected with TSEs but the test is not specific for TSEs and that neither vacuolation nor PrP(Sc) accumulation appears to be responsible for the clinical abnormalities.

Developing a Collaborative Agenda for Humanities and Social Scientific Research on Laboratory Animal Science and Welfare.

Improving laboratory animal science and welfare requires both new scientific research and insights from research in the humanities and social sciences. Whilst scientific research provides evidence to replace, reduce and refine procedures involving laboratory animals (the '3Rs'), work in the humanities and social sciences can help understand the social, economic and cultural processes that enhance or impede humane ways of knowing and working with laboratory animals. However, communication across these disciplinary perspectives is currently limited, and they design research programmes, generate results, engage users, and seek to influence policy in different ways. To facilitate dialogue and future research at this interface, we convened an interdisciplinary group of 45 life scientists, social scientists, humanities scholars, non-governmental organisations and policy-makers to generate a collaborative research agenda. This drew on methods employed by other agenda-setting exercises in science policy, using a collaborative and deliberative approach for the identification of research priorities. Participants were recruited from across the community, invited to submit research questions and vote on their priorities. They then met at an interactive workshop in the UK, discussed all 136 questions submitted, and collectively defined the 30 most important issues for the group. The output is a collaborative future agenda for research in the humanities and social sciences on laboratory animal science and welfare. The questions indicate a demand for new research in the humanities and social sciences to inform emerging discussions and priorities on the governance and practice of laboratory animal research, including on issues around: international harmonisation, openness and public engagement, 'cultures of care', harm-benefit analysis and the future of the 3Rs. The process outlined below underlines the value of interdisciplinary exchange for improving communication across different research cultures and identifies ways of enhancing the effectiveness of future research at the interface between the humanities, social sciences, science and science policy.

Objects in Contact with Classical Scrapie Sheep Act as a Reservoir for Scrapie Transmission.

Classical scrapie is an environmentally transmissible prion disease of sheep and goats. Prions can persist and remain potentially infectious in the environment for many years and thus pose a risk of infecting animals after re-stocking. In vitro studies using serial protein misfolding cyclic amplification (sPMCA) have suggested that objects on a scrapie-affected sheep farm could contribute to disease transmission. This in vivo study aimed to determine the role of field furniture (water troughs, feeding troughs, fencing, and other objects that sheep may rub against) used by a scrapie-infected sheep flock as a vector for disease transmission to scrapie-free lambs with the prion protein genotype VRQ/VRQ, which is associated with high susceptibility to classical scrapie. When the field furniture was placed in clean accommodation, sheep became infected when exposed to either a water trough (four out of five) or to objects used for rubbing (four out of seven). This field furniture had been used by the scrapie-infected flock 8 weeks earlier and had previously been shown to harbor scrapie prions by sPMCA. Sheep also became infected (20 out of 23) through exposure to contaminated field furniture placed within pasture not used by scrapie-infected sheep for 40 months, even though swabs from this furniture tested negative by PMCA. This infection rate decreased (1 out of 12) on the same paddock after replacement with clean field furniture. Twelve grazing sheep exposed to field furniture not in contact with scrapie-infected sheep for 18 months remained scrapie free. The findings of this study highlight the role of field furniture used by scrapie-infected sheep to act as a reservoir for disease re-introduction although infectivity declines considerably if the field furniture has not been in contact with scrapie-infected sheep for several months. PMCA may not be as sensitive as VRQ/VRQ sheep to test for environmental contamination.

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.

Phenotype shift from atypical scrapie to CH1641 following experimental transmission in sheep.

The interactions of host and infecting strain in ovine transmissible spongiform encephalopathies are known to be complex, and have a profound effect on the resulting phenotype of disease. In contrast to classical scrapie, the pathology in naturally-occurring cases of atypical scrapie appears more consistent, regardless of genotype, and is preserved on transmission within sheep homologous for the prion protein (PRNP) gene. However, the stability of transmissible spongiform encephalopathy phenotypes on passage across and within species is not absolute, and there are reports in the literature where experimental transmissions of particular isolates have resulted in a phenotype consistent with a different strain. In this study, intracerebral inoculation of atypical scrapie between two genotypes both associated with susceptibility to atypical forms of disease resulted in one sheep displaying an altered phenotype with clinical, pathological, biochemical and murine bioassay characteristics all consistent with the classical scrapie strain CH1641, and distinct from the atypical scrapie donor, while the second sheep did not succumb to challenge. One of two sheep orally challenged with the same inoculum developed atypical scrapie indistinguishable from the donor. This study adds to the range of transmissible spongiform encephalopathy phenotype changes that have been reported following various different experimental donor-recipient combinations. While these circumstances may not arise through natural exposure to disease in the field, there is the potential for iatrogenic exposure should current disease surveillance and feed controls be relaxed. Future sheep to sheep transmission of atypical scrapie might lead to instances of disease with an alternative phenotype and onward transmission potential which may have adverse implications for both public health and animal disease control policies.

The pathological and molecular but not clinical phenotypes are maintained after second passage of experimental atypical bovine spongiform encephalopathy in cattle.

BACKGROUND: Atypical bovine spongiform encephalopathies (BSEs), classified as H-type and L-type BSE based on the Western immunoblot profiles, are naturally occurring diseases in cattle, which are phenotypically different to classical BSE. Transmission studies in cattle using the intracerebral route resulted in disease where the phenotypes were maintained irrespective of BSE type but clinically affected cattle with a shorter survival time displayed a nervous form whereas cattle with a longer survival time displayed a dull form. A second transmission study is reported here where four cattle were intracerebrally inoculated with brain tissue from experimentally infected cattle presenting with either the nervous or dull form of H- or L-type BSE to determine whether the phenotype is maintained. RESULTS: The four inoculated cattle were culled at 16.5-19.5 months post inoculation after presenting with difficulty getting up, a positive scratch response (all) and dullness (three cattle), which was not observed in two non-inoculated control cattle, each housed with either group of inoculated cattle. Only the inoculated cattle had detectable prion protein in the brain based on immunohistochemical examination, and the Western immunoblot profile was consistent with the H-type or L-type BSE of the respective donor cattle. CONCLUSIONS: Second passage of H-type and L-type BSE in cattle produced a TSE where the majority of cattle displayed the dull form regardless of clinical disease form of the donor cattle. The pathological and molecular phenotypes of H- and L-type BSE were maintained.

Clinical examination protocol to detect atypical and classical scrapie in sheep.

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated prion protein by post mortem tests. Unless a random sample of the sheep or goat population is actively monitored for scrapie, identification of scrapie cases relies on the reporting of clinical suspects, which is dependent on the individual's familiarization with the disease and ability to recognize clinical signs associated with scrapie. Scrapie may not be considered in the differential diagnosis of neurological diseases in small ruminants, particularly in countries with low scrapie prevalence, or not recognized if it presents as nonpruritic form like atypical scrapie. To aid in the identification of clinical suspects, a short examination protocol is presented to assess the display of specific clinical signs associated with pruritic and nonpruritic forms of TSEs in sheep, which could also be applied to goats. This includes assessment of behavior, vision (by testing of the menace response), pruritus (by testing the response to scratching), and movement (with and without blindfolding). This may lead to a more detailed neurologic examination of reporting animals as scrapie suspects. It could also be used in experimental TSE studies of sheep or goats to evaluate disease progression or to identify clinical end-point.