Classical scrapie appears to consist of several strains which have most likely evolved gradually under the influence of different ovine PRNP genotypes and other yet unknown factors . It is important that distinct classical scrapie strains can be properly identified, both as a means of monitoring their presence in the national flock and in identifying emerging strains with possible zoonotic potential. As there is evidence of an association between ovine genotype and predilection for classical scrapie strains [28–30], it is also important to determine whether schemes that alter the genetic background of the flock change the repertoire of strains.
Ideally strain typing would be possible in the natural host however attempts to link phenotype with different strains have been met with limited success because it is difficult to isolate single entities of the agent from the original host. Whilst it is not currently possible to correlate with certainty different disease phenotypes with a given classical scrapie agent in the natural host, previous studies have suggested that TSE phenotype diversity in mice may reflect strain diversity in sheep . However, other parameters such as PrP genotype may also play a significant role .
Applying the traditional methodology of IP and LP analysis, three strains were identified following bioassay of natural scrapie cases through each of the C57BL/6 and VM mouse lines including ME7, 87A and 221C through the C57BL/6 line [8, 9] and ME7 and 87V through the VM line . Notably the IP characteristics of 87V in two out of three isolates in the current study differed to a previous study  where clinical disease in C57BL/6 or C57BL/6 × VM lines was either longer than 700 days or exceeded the lifespan of the mice. A third previously unidentified strain, termed VLA-V1 was also observed in VM mice from over half of the scrapie sources with a prolonged IP of over 400 days in both C57BL/6 and C57BL/6 × VM mice. ME7 was observed in both mouse lines, where both the LP and the absolute and relative IP of each mouse line infected with this strain were remarkably consistent, irrespective of the mouse line through which the strain was stabilised. In accordance with the protein only hypothesis  strains are said to manifest in the tertiary conformation of the prion protein and the ability for a strain to replicate in a given host depends on the conformational flexibility of the host PrPC molecule to adopt the conformation of a specific strain . The ME7 strain may therefore represent a more stable conformation  and may not be influenced by host factors to the extent that other strains may be. Indeed ME7 is reportedly the most prevalent strain to have been isolated from natural scrapie sources both pre- and post- the BSE epidemic [9, 33].
It has been previously reported that the deposition pattern of PrPSc across the brain of infected mice varies according to the agent strain [10–12], although the molecular basis of this is not yet fully known. Strain specific differences in PrPSc conformation may reasonably evoke differences in neuronal targeting, trafficking and processing/degradation of the protein [12, 34–36]. In the current study IHC was used to characterise the identified classical scrapie strains according to the specific types and neuroanatomical location of PrPSc. In order to demonstrate the predominant topographical distribution and PrPSc type associated with each strain a comparative whole brain mapping method was employed. This approach has been used previously to describe the PrPSc distribution in wild-type mice following primary passage of classical scrapie sources  and second passage of BSE , although in both studies only general neuropil deposition and aggregates and plaques were recorded. In a previous study the predominant PrPSc deposition types associated with ME7 and 87V were described . Here we have expanded the description of each strain to account for 8 specific forms of PrPSc accumulation (granular, aggregates, plaques, intraglial, punctate, intraneuronal, perineuronal and linear) in conjunction with the whole brain mapping approach, thus increasing the discriminatory power of this method to distinguish different strains. A distinct advantage of this method is that it is less affected by titre and patterns can be identified on an individual mouse basis, in contrast with traditional IP and LP approaches which are based on mean data of all mice inoculated with a given source. Thus with the IHC method it is possible to compare the PrPSc pattern in mice associated with a given TSE strain both within and between inocula derived from different sources.
Each of the strains identified in the current study gave distinct PrPSc deposition patterns: ME7 through both C57BL/6 and VM mouse lines gave a very similar pattern of predominantly widespread granular deposition within the neuropil and the presence of many small aggregates. The characteristic perpendicular linear streaks observed were in agreement with those reported in previous IHC  and PET-blot  studies with this strain and are proposed to be associated with purkinje cells dendrites . 87A through the C57BL/6 line and 87V through the VM line were the only strains to present with many large plaques and aggregates. 221C was associated with a widespread, diffuse granular deposition. The PrPSc pattern for VLA-V1 was very similar but had not been previously observed in association with any other strain in this mouse line.
In an earlier study the PrPSc patterns following transmission of classical field scrapie sources to wild-type mice were reported . Notably patterns were detected on primary isolation that share great similarity with those reported in the current study. For example an ME7 PrPSc-like pattern was reported in RIII and C57BL/6 mice, which suggests that this parameter stabilises before IP and LP. If used on primary passage IHC may therefore better reflect the repertoire of strains in the host and reduce the number of murine passages required for strain typing. IHC can also help to resolve strain identification when IP and LP are uninformative, even after full characterisation as was shown for VLA-C1 in the current study. Conversely this method may help to establish identification of novel strains, i.e. VLA-V1.
The PET-blot method has previously been proven to be a highly sensitive method for PrPSc detection. It has been reported that in C57BL/6 mice inoculated with ME7, PrPSc was detected in the brain by PET-blot 30 days after inoculation and 145 days before clinical signs , with greater sensitivity than both IHC and Western blotting. In sheep, PET-blot has been recently used to demonstrate the differential routes of spreading of PrPSc across the brain associated with different prion types  and as a method of discriminating classical scrapie and experimental BSE . For strain typing, the PET-blot method was used previously in C57BL/6 mice to compare the distribution of BSE from different species with ovine and mouse adapted scrapie . In the current study PET-blots proved a useful tool to clearly demonstrate the differences in neuroanatomical location of PrPSc associated with different classical scrapie strains and in this respect correlated well with IHC staining. In agreement with previous studies, IHC showed comparably better microscopic resolution at the cellular and subcellular levels, enabling different PrPSc types to be more clearly defined, although certain strain specific markers, i.e. perpendicular streaks of PrPSc through the molecular layer of the cerebellum, associated with ME7, were also identifiable by PET-blot as were denser structures including aggregates and plaques.
According to our data, Western blotting cannot be utilised to strain type classical scrapie following passage in wild-type mice: it was not possible to distinguish the scrapie strains identified in C57BL/6 mice by Western blotting whilst in VM mice only 87V could be distinguished from ME7 and VLA-V1. This was in agreement with a previous study  where 87V gave a highly glycosylated profile in VM mice compared to ME7. Indeed with the exception of 87V, the electrophoretic mobility and glycoform ratio of each strain identified was consistent, irrespective of the mouse line. However, Western blot may be a very useful phenotypic characteristic in helping to discriminate BSE from classical scrapie in wild-type mice or distinguishing TSE strains in transgenic mice, usually in conjunction with IHC .
In conclusion we have characterised 29 transmissions of classical scrapie field sources through the wild-type mouse bioassay and attained a comprehensive phenotypic description of each identified strain based on standard and alternative methodologies. Numerous scrapie strains have been reported using the wild-type mouse bioassay  however, only a very small sample of sheep scrapie cases have been thoroughly tested (20 pre-1985, 10 post-1985) . The largest published study of natural classical scrapie isolates characterised through the mouse bioassay detailed transmissions of ten individual cases , although some of these previously reported cases originated from closed experimental flocks . Additionally many of the mouse-passaged strains that have been described were derived from experimentally infected animals and may not reflect, either in terms of identity or relative prevalence, the classical scrapie strains which are endemic in the UK national flock [5, 8].
The current study will serve as a standard reference which will be used to further analyse the dynamics of strain evolution and adaptation of classical scrapie through serial passage in wild-type mice, including the identification of strain specific parameters which can be used to identify strains during primary passage. Upon identification of the strains from at least 100 field cases of classical scrapie using the methodology presented here, we will attempt to analyse the strain demographics of classical scrapie in the UK and identify host parameters that may influence them. These data suggest that analysis of these sources should include whole brain mapping based on the distribution of different PrPSc types as revealed by IHC on individual mice which may negate the requirement for serial passages currently required for strain typing in mice and in addition, may reveal more than one phenotype emerging from a single source.