Correctional psychopharmacology: pitfalls, challenges, and victories of prescribing in a correctional setting.

Mentally ill patients are entering the criminal justice system at alarming rates, representing a significant percentage of those incarcerated. Correctional facilities are mandated to provide mental health treatment to inmates. The increasing number of inmate patients has made psychiatry an important part of institutional operations. Psychiatrists are called to provide psychopharmacological interventions to aid in the safe operation of institutions and provide effective treatment to those with mental illness. This article discusses the uniqueness of prescribing within correctional settings and delineates important aspects of correctional psychiatry. It includes information on navigating the rigid structure of correctional environments, providing effective interventions for behaviourally disruptive and manipulative inmates, prescribing within the limits of formulary restrictions, considerations for dispensing medication, preventing medication diversion, involuntarily medication practices, and treatment of aggressive inmates. The article concludes with case illustrations that depict the pitfalls, challenges, and victories of working with inmate patients.

The oral secretion of infectious scrapie prions occurs in preclinical sheep with a range of PRNP genotypes.

Preclinical sheep with the highly scrapie-susceptible VRQ/VRQ PRNP genotype secrete prions from the oral cavity. In order to further understand the significance of orally available prions, buccal swabs were taken from sheep with a range of PRNP genotypes and analyzed by serial protein misfolding cyclic amplification (sPMCA). Prions were detected in buccal swabs from scrapie-exposed sheep of genotypes linked to high (VRQ/VRQ and ARQ/VRQ) and low (ARR/VRQ and AHQ/VRQ) lymphoreticular system involvement in scrapie pathogenesis. For both groups, the level of prion detection was significantly higher than that for scrapie-resistant ARR/ARR sheep which were kept in the same farm environment and acted as sentinel controls for prions derived from the environment which might contaminate the oral cavity. In addition, sheep with no exposure to the scrapie agent did not contain any measurable prions within the oral cavity. Furthermore, prions were detected in sheep over a wide age range representing various stages of preclinical disease. These data demonstrate that orally available scrapie prions may be a common feature in sheep incubating scrapie, regardless of the PRNP genotype and any associated high-level accumulation of PrP(Sc) within lymphoreticular tissues. PrP(Sc) was present in buccal swabs from a large proportion of sheep with PRNP genotypes associated with relatively low disease penetrance, indicating that subclinical scrapie infection is likely to be a common occurrence. The significance of positive sPMCA reactions was confirmed by the transmission of infectivity in buccal swab extracts to Tg338 mice, illustrating the likely importance of orally available prions in the horizontal transmission of scrapie.

Evidence of effective scrapie transmission via colostrum and milk in sheep.

BACKGROUND: Evidence for scrapie transmission from VRQ/VRQ ewes to lambs via milk was first reported in 2008 but in that study there were concerns that lateral transmission may have contributed to the high transmission rate observed since five control lambs housed with the milk recipients also became infected. This report provides further information obtained from two follow-up studies, one where milk recipients were housed separately after milk consumption to confirm the validity of the high scrapie transmission rate via milk and the second to assess any difference in infectivity from colostrum and subsequent milk. Protein misfolding cyclic amplification (PMCA) was also used to detect prion protein in milk samples as a comparison with the infectivity data and extended to milk samples from ewes without a VRQ allele. RESULTS: Seven pairs of lambs fed colostrum and milk individually from seven scrapie-affected sheep (pre-clinical or clinical) presented with disease-associated prion protein, PrPd, in rectal lymphoid tissue at 4-5 months of age. Five further pairs of lambs fed either colostrum or subsequent milk from five pre-clinical scrapie-affected sheep equally presented with PrPd in lymphoid tissue by 9 months of age. Nine sheep were lost due to intercurrent diseases but all remaining milk or colostrum recipients, including those in the original study with the lateral transmission controls, developed clinical signs of scrapie from 19 months of age and scrapie was confirmed by brain examination. Unexposed control sheep totalling 19 across all three studies showed no evidence of infection.Scrapie PrP was amplified repeatedly by PMCA in all tested milk samples from scrapie-affected VRQ/VRQ sheep, and in one scrapie-affected ARQ/ARQ sheep. By contrast, milk samples from five VRQ/VRQ and 11 ARQ/ARQ scrapie-free sheep did not have detectable scrapie PrP on repeated tests. CONCLUSIONS: Feeding of milk from scrapie-affected sheep results in a high transmission rate in VRQ/VRQ sheep and both colostrum and milk transmit scrapie. Detection of scrapie prion protein in individual milk samples from scrapie-affected ewes confirms PMCA as a valuable in vitro test.

Infectious titres of sheep scrapie and bovine spongiform encephalopathy agents cannot be accurately predicted from quantitative laboratory test results.

It is widely accepted that abnormal forms of the prion protein (PrP) are the best surrogate marker for the infectious agent of prion diseases and, in practice, the detection of such disease-associated (PrP(d)) and/or protease-resistant (PrP(res)) forms of PrP is the cornerstone of diagnosis and surveillance of the transmissible spongiform encephalopathies (TSEs). Nevertheless, some studies question the consistent association between infectivity and abnormal PrP detection. To address this discrepancy, 11 brain samples of sheep affected with natural scrapie or experimental bovine spongiform encephalopathy were selected on the basis of the magnitude and predominant types of PrP(d) accumulation, as shown by immunohistochemical (IHC) examination; contra-lateral hemi-brain samples were inoculated at three different dilutions into transgenic mice overexpressing ovine PrP and were also subjected to quantitative analysis by three biochemical tests (BCTs). Six samples gave 'low' infectious titres (106·5 to 106·7 LD50 g⁻¹) and five gave 'high titres' (108·¹ to ≥ 108·7 LD50 g⁻¹) and, with the exception of the Western blot analysis, those two groups tended to correspond with samples with lower PrP(d)/PrP(res) results by IHC/BCTs. However, no statistical association could be confirmed due to high individual sample variability. It is concluded that although detection of abnormal forms of PrP by laboratory methods remains useful to confirm TSE infection, infectivity titres cannot be predicted from quantitative test results, at least for the TSE sources and host PRNP genotypes used in this study. Furthermore, the near inverse correlation between infectious titres and Western blot results (high protease pre-treatment) argues for a dissociation between infectivity and PrP(res).

The interpretation of disease phenotypes to identify TSE strains following murine bioassay: characterisation of classical scrapie.

Mouse bioassay can be readily employed for strain typing of naturally occurring transmissible spongiform encephalopathy cases. Classical scrapie strains have been characterised historically based on the established methodology of assessing incubation period of disease and the distribution of disease-specific vacuolation across the brain following strain stabilisation in a given mouse line. More recent research has shown that additional methods could be used to characterise strains and thereby expand the definition of strain "phenotype". Here we present the phenotypic characteristics of classical scrapie strains isolated from 24 UK ovine field cases through the wild-type mouse bioassay. PrPSc immunohistochemistry (IHC), paraffin embedded tissue blots (PET-blot) and Western blotting approaches were used to determine the neuroanatomical distribution and molecular profile of PrPSc associated with each strain, in conjunction with traditional methodologies. Results revealed three strains isolated through each mouse line, including a previously unidentified strain. Moreover IHC and PET-blot methodologies were effective in characterising the strain-associated types and neuroanatomical locations of PrPSc. The use of Western blotting as a parameter to define classical scrapie strains was limited. These data provide a comprehensive description of classical scrapie strain phenotypes on isolation through the mouse bioassay that can provide a reference for further scrapie strain identification.

In vitro amplification of ovine prions from scrapie-infected sheep from Great Britain reveals distinct patterns of propagation.

BACKGROUND: Protein misfolding cyclic amplification (PMCA) is a method that facilitates the detection of prions from many sources of transmissible spongiform encephalopathy (TSE). Sheep scrapie represents a unique diversity of prion disease agents in a range of susceptible PRNP genotypes. In this study PMCA was assessed on a range of Great Britain (GB) sheep scrapie isolates to determine the applicability to veterinary diagnosis of ovine TSE. RESULTS: PrPSc amplification by protein misfolding cyclic amplification (PMCA) was assessed as a diagnostic tool for field cases of scrapie. The technique was initially applied to thirty-seven isolates of scrapie from diverse geographical locations around GB, and involved sheep of various breeds and PRNP genotypes. All samples were amplified in either VRQ and/or ARQ PrPC substrate. For PrPSc from sheep with at least one VRQ allele, all samples amplified efficiently in VRQ PrPC but only PrPSc from ARH/VRQ sheep amplified in both substrates. PrPSc from ARQ/ARQ sheep displayed two amplification patterns, one that amplified in both substrates and one that only amplified in ARQ PrPC. These amplification patterns were consistent for a further 14/15 flock/farm mates of these sheep. Furthermore experimental scrapie strains SSBP1, Dawson, CH1641 and MRI were analysed. SSBP1 and Dawson (from VRQ/VRQ sheep) amplified in VRQ but not ARQ substrate. MRI scrapie (from ARQ/ARQ sheep) nor CH1641 did not amplify in ARQ or VRQ substrate; these strains required an enhanced PMCA method incorporating polyadenylic acid (poly(A)) to achieve amplification. CONCLUSIONS: PrPsc from 52 classical scrapie GB field isolates amplified in VRQ or ARQ or both substrates and supports the use of PMCA as a rapid assay for the detection of a wide range of ovine classical scrapie infections involving multiple PRNP genotypes and scrapie strains.

Isolation of prion with BSE properties from farmed goat.

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that include variant Creutzfeldt-Jakob disease in humans, scrapie in small ruminants, and bovine spongiform encephalopathy (BSE) in cattle. Scrapie is not considered a public health risk, but BSE has been linked to variant Creutzfeldt-Jakob disease. Small ruminants are susceptible to BSE, and in 2005 BSE was identified in a farmed goat in France. We confirm another BSE case in a goat in which scrapie was originally diagnosed and retrospectively identified as suspected BSE. The prion strain in this case was further characterized by mouse bioassay after extraction from formaldehyde-fixed brain tissue embedded in paraffin blocks. Our data show that BSE can infect small ruminants under natural conditions and could be misdiagnosed as scrapie. Surveillance should continue so that another outbreak of this zoonotic transmissible spongiform encephalopathy can be prevented and public health safeguarded.

Experimental oral transmission of atypical scrapie to sheep.

To investigate the possibility of oral transmission of atypical scrapie in sheep and determine the distribution of infectivity in the animals' peripheral tissues, we challenged neonatal lambs orally with atypical scrapie; they were then killed at 12 or 24 months. Screening test results were negative for disease-specific prion protein in all but 2 recipients; they had positive results for examination of brain, but negative for peripheral tissues. Infectivity of brain, distal ileum, and spleen from all animals was assessed in mouse bioassays; positive results were obtained from tissues that had negative results on screening. These findings demonstrate that atypical scrapie can be transmitted orally and indicate that it has the potential for natural transmission and iatrogenic spread through animal feed. Detection of infectivity in tissues negative by current surveillance methods indicates that diagnostic sensitivity is suboptimal for atypical scrapie, and potentially infectious material may be able to pass into the human food chain.

Environmental sources of scrapie prions.

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.

Detection of prions in the faeces of sheep naturally infected with classical scrapie.

Classical scrapie is a naturally transmitted prion disease of sheep and goats. Contaminated environments may contribute to the spread of disease and evidence from animal models has implicated urine, blood, saliva, placenta and faeces as possible sources of the infection. Here we sought to determine whether sheep naturally infected with classical scrapie shed prions in their faeces. We used serial protein misfolding cyclic amplification (sPMCA) along with two extraction methods to examine faeces from sheep during both the clinical and preclinical phases of the disease and showed amplification of PrP(Sc) in 7 of 15 and 14 of 14 sheep respectively. However PrP(Sc) was not amplified from the faeces of 25 sheep not exposed to scrapie. These data represent the first demonstration of prion shedding in faeces from a naturally infected host and thus a likely source of prion contamination in the environment.

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.

2020 White Paper on Recent Issues in Bioanalysis: BAV Guidance, CLSI H62, Biotherapeutics Stability, Parallelism Testing, CyTOF and Regulatory Feedback (Part 2A - Recommendations on Biotherapeutics Stability, PK LBA Regulated Bioanalysis, Biomarkers Assays, Cytometry Validation & Innovation Part 2B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 2A) BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation and (Part 2B) Regulatory Input. Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 4, and 6 (2021), respectively.

Update to the European Bioanalysis Forum recommendation on biomarkers assays; bringing context of use into practice.

In 2012, the European Bioanalysis Forum published a recommendation on biomarker method development and the bioanalysis of biomarkers in support of drug development. Since then, there has been significant discussion on how to bring the topic of context of use of biomarker assays to the forefront so that the purpose of the assay, the use of the data and the decisions being made with the data are well defined and clearly understood, not just by the bioanalytical scientist, but across all stakeholders. Therefore, it is imperative that discussions between the bioanalytical laboratory and the end users of the data happen early (and regularly) in the drug development process to enable the right assays to be developed and appropriately validated to generate the correct data and allow suitable decisions to be made. This updated refinement to the previous European Bioanalysis Forum recommendation will highlight the items to consider when discussing context of use for biomarker assay development and validation, thus enabling the correct conversations to occur and the move away from the misapplication of PK assay validation criteria to biomarker assays.

Incomplete inactivation of atypical scrapie following recommended autoclave decontamination procedures.

Prions are highly resistant to the decontamination procedures normally used to inactivate conventional pathogens. This is a challenging problem not only in the medical and veterinary fields for minimizing the risk of transmission from potentially infective sources but also for ensuring the safe disposal or subsequent use of animal by-products. Specific pressure autoclaving protocols were developed for this purpose, but different strains of prions have been reported to have differing resistance patterns to established prion decontamination procedures, and as additional TSE strains are identified it is necessary to determine the effectiveness of such procedures. In this study we assessed the efficacy of sterilization using the EU recommended autoclave procedure for prions (133°C, 3 Bar for 20 min) on the atypical or Nor98 (AS/Nor98) scrapie strain of sheep and goats. Using a highly sensitive murine mouse model (tg338) that overexpresses ovine PrPC , we determined that this method of decontamination reduced the infectivity titre by 1010 . Infectivity was nonetheless still detected after applying the recommended autoclaving protocol. This shows that AS/Nor98 can survive the designated legislative decontamination conditions, albeit with a significant decrease in titre. The infectivity of a classical scrapie isolate subjected to the same decontamination conditions was reduced by 106 suggesting that the AS/Nor98 isolate is less sensitive to decontamination than the classical scrapie source.

Idiopathic Brainstem Neuronal Chromatolysis (IBNC): a novel prion protein related disorder of cattle?

BACKGROUND: The epidemic form of Bovine Spongiform Encephalopathy (BSE) is generally considered to have been caused by a single prion strain but at least two strain variants of cattle prion disorders have recently been recognized. An additional neurodegenerative condition, idiopathic brainstem neuronal chromatolysis and hippocampal sclerosis (IBNC), a rare neurological disease of adult cattle, was also recognised in a sub-set of cattle submitted under the BSE Orders in which lesions of BSE were absent. Between the years of 1988 and 1991 IBNC occurred in Scotland with an incidence of 7 cases per 100,000 beef suckler cows over the age of 6 years. RESULTS: When the brains of 15 IBNC cases were each tested by immunohistochemistry, all showed abnormal labelling for prion protein (PrP). Immunohistological labelling for PrP was also present in the retina of a single case available for examination. The pattern of PrP labelling in brain is distinct from that seen in other ruminant prion diseases and is absent from brains with other inflammatory conditions and from normal control brains. Brains of IBNC cattle do not reveal abnormal PrP isoforms when tested by the commercial BioRad or Idexx test kits and do not reveal PrPres when tested by Western blotting using stringent proteinase digestion methods. However, some weakly protease resistant isoforms of PrP may be detected when tissues are examined using mild proteinase digestion techniques. CONCLUSION: The study shows that a distinctive neurological disorder of cattle, which has some clinical similarities to BSE, is associated with abnormal PrP labelling in brain but the pathology and biochemistry of IBNC are distinct from BSE. The study is important either because it raises the possibility of a significant increase in the scope of prion disease or because it demonstrates that widespread and consistent PrP alterations may not be confined to prion diseases. Further studies, including transmission experiments, are needed to establish whether IBNC is a condition in which prion protein is abnormally regulated or it is yet a further example of an infectious cattle prion disease.

Adaptation and evaluation of a rapid test for the diagnosis of sheep scrapie in samples of rectal mucosa.

In recent publications, it was shown that disease-associated prion protein (PrP(d)) accumulates in the lymphoid tissue of the rectal mucosa of a high proportion of scrapie-infected sheep at clinical and preclinical stages, regardless of several host factors; PrP(d) can also be detected in biopsy specimens of rectal mucosa, with an increased probability proportional to age or incubation period and with an efficiency almost identical to that of tonsil biopsies. Rectal biopsies have the advantages of providing higher numbers of lymphoid follicles and of being simpler to perform, which makes them suitable for scrapie screening in the field. In biopsy samples, PrP(d) could be demonstrated by immunohistochemical (IHC) and Western immunoblotting methods, and the purpose of the present study was to optimize and evaluate a "rapid test" for the diagnosis of scrapie in rectal biopsy samples. The HerdChek CWD (chronic wasting disease) antigen EIA (enzyme immunoassay) test was chosen and, once optimized, provided specificity and sensitivity figures of 99.2% and 93.5%, respectively, compared with IHC results in the same samples obtained at a postmortem. The sensitivity of the assay increased from 82.1%, when a single rectal mucosa sample was tested to 99.4% for those sheep in which 3 or more samples were analyzed. Similarly, sensitivity values of the HerdChek CWD antigen EIA test on biopsy samples increased from 95% to 100% for sheep subjected to 1 or 2 sequential biopsies 4 months apart, respectively. Thus, a preclinical diagnosis of scrapie in live sheep can be achieved by a combination of a simple sampling procedure, which can be repeated several times with no detrimental effect for the animals, and a rapid and efficient laboratory method.

Decreased cell surface prion protein in mouse models of prion disease.

Transmissible spongiform encephalopathies are infectious neurodegenerative diseases caused by prions, composed of ordered aggregates of misfolded cellular prion protein. Neural antigen density of prion protein, Thy-1 and glial fibrillary acidic protein was analyzed using flow cytometry of dissociated mouse brain cells after inoculation with mouse-adapted transmissible spongiform encephalopathy agents. Transmissible spongiform encephalopathy gliosis was demonstrated by increased intracellular immunoreactivity for glial fibrillary acidic protein compared with controls. Immunoreactivity for cell surface prion protein was reduced 2.8-3.8-fold compared with control brain cells, whereas surface Thy-1 protein was reduced 1.5-4-fold. Double-staining protocols revealed loss of brain cells highly immunoreactive for prion protein and Thy-1, with a preferential reduction of prion protein, suggesting that prion protein expression, trafficking or consumption may be affected early in disease.

Ability of wild type mouse bioassay to detect bovine spongiform encephalopathy (BSE) in the presence of excess scrapie.

INTRODUCTION: Scrapie and bovine spongiform encephalopathy (BSE) are transmissible spongiform encephalopathies (TSEs) which naturally affect small and large ruminants respectively. However, small ruminants, which are susceptible to BSE under experimental conditions, have been exposed to the same or similar contaminated food additives as cattle. To date two natural cases of BSE in small ruminants have been reported. As a result surveillance projects, combined with appropriate control measures, have been established throughout the European Union (EU) to minimize the overall incidence of small ruminant TSEs. Although BSE can be differentiated from classical scrapie (subsequently referred to as scrapie) if appropriate discriminatory tests are applied, the value of these tests in BSE/scrapie co-infection scenarios has not been evaluated fully. Mouse bioassay is regarded as the gold standard regarding differentiation of distinct TSE strains and has been used as to resolve TSE cases were laboratory tests produced equivocal results. However, the ability of this method to discriminate TSE strains when they co-exist has not been examined systematically. To address this issue we prepared in vitro mixtures of ovine BSE and scrapie and used them to challenge RIII, C57BL/6 and VM mice. RESULTS: Disease phenotype analysis in all three mouse lines indicated that most phenotypic parameters (attack rates, incubation periods, lesion profiles and Western blots) were compatible with scrapie phenotypes as were immunohistochemistry (IHC) data from RIII and C57BL/6 mice. However, in VM mice that were challenged with BSE/scrapie mixtures a single BSE-associated IHC feature was identified, indicating the existence of BSE in animals where the scrapie phenotype was dominant. CONCLUSIONS: We conclude that wild type mouse bioassay is of limited value in detecting BSE in the presence of scrapie particularly if the latter is in relative excess.

Correlation between infectivity and disease associated prion protein in the nervous system and selected edible tissues of naturally affected scrapie sheep.

The transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative disorders characterised by the accumulation of a pathological form of a host protein known as prion protein (PrP). The validation of abnormal PrP detection techniques is fundamental to allow the use of high-throughput laboratory based tests, avoiding the limitations of bioassays. We used scrapie, a prototype TSE, to examine the relationship between infectivity and laboratory based diagnostic tools. The data may help to optimise strategies to prevent exposure of humans to small ruminant TSE material via the food chain. Abnormal PrP distribution/accumulation was assessed by immunohistochemistry (IHC), Western blot (WB) and ELISA in samples from four animals. In addition, infectivity was detected using a sensitive bank vole bioassay with selected samples from two of the four sheep and protein misfolding cyclic amplification using bank vole brain as substrate (vPMCA) was also carried out in selected samples from one animal. Lymph nodes, oculomotor muscles, sciatic nerve and kidney were positive by IHC, WB and ELISA, although at levels 100-1000 fold lower than the brain, and contained detectable infectivity by bioassay. Tissues not infectious by bioassay were also negative by all laboratory tests including PMCA. Although discrepancies were observed in tissues with very low levels of abnormal PrP, there was an overall good correlation between IHC, WB, ELISA and bioassay results. Most importantly, there was a good correlation between the detection of abnormal PrP in tissues using laboratory tests and the levels of infectivity even when the titre was low. These findings provide useful information for risk modellers and represent a first step toward the validation of laboratory tests used to quantify prion infectivity, which would greatly aid TSE risk assessment policies.

Strain typing of classical scrapie by transgenic mouse bioassay using protein misfolding cyclic amplification to replace primary passage.

According to traditional murine bioassay methodology, prions must be serially passaged within a new host before a stable phenotype, and therefore a strain, can be assigned. Prions often transmit with difficulty from one species to another; a property termed the transmission barrier. Transgenic mouse lines that over express prion protein (PrP) genes of different species can circumvent the transmission barrier but serial passages may still be required, particularly if unknown strains are encountered. Here we sought to investigate whether protein misfolding cyclic amplification (PMCA), an in-vitro method of PrP(Sc) replication, could be used to replace serial passage of VRQ/VRQ classical scrapie isolates undergoing strain typing in ovine transgenic tg338 mice. Two classical scrapie field isolates that do not readily transmit to wild-type mice underwent bioassay in tg338 mice pre- and post- PMCA and the phenotype of disease in inoculated mice was compared. For one of the sources investigated, the PMCA product gave rise to the same disease phenotypes in tg338 mice as traditional bioassay, as indicated by lesion profile, IHC analysis and Western blot, whilst the second source produced phenotypic characteristics which were not identical with those that arose through traditional bioassay. These data show that differences in the efficiency of PMCA as a strain-typing tool may vary between ovine classical scrapie isolates and therefore suggest that the ability of PMCA to replace serial passage of classical scrapie in tg338 mice may depend on the strain present in the initial source.