Public and animal health controls to limit human exposure to animal prions are focused on bovine spongiform encephalopathy (BSE), but other prion strains in ruminants may also have zoonotic potential. One example is atypical/Nor98 scrapie, which evaded statutory diagnostic methods worldwide until the early 2000s. To investigate whether sheep infected with scrapie prions could be another source of infection, we inoculated transgenic mice that overexpressed human prion protein with brain tissue from sheep with natural field cases of classical and atypical scrapie, sheep with experimental BSE, and cattle with BSE. We found that these mice were susceptible to BSE prions, but disease did not develop after prolonged postinoculation periods when mice were inoculated with classical or atypical scrapie prions. These data are consistent with the conclusion that prion disease is less likely to develop in humans after exposure to naturally occurring prions of sheep than after exposure to epizootic BSE prions of ruminants. Bovine spongiform encephalopathy (BSE) is the transmissible spongiform encephalopathy (TSE) or prion disease of domestic cattle. The BSE prion is an epizootic agent and causes variant Creutzfeldt-Jakob disease (vCJD) in humans after dietary exposure  in number of confirmed cases of classical scrapie in the national flock. However, atypical scrapie continues to affect sheep bred for their relative resistance to the classical form of this prion disease, and the proportion of sheep with resistant genotypes in the national flock is likely to have increased over the past decade because of the National Scrapie Plan for Great Britain. This increase has rekindled speculation that atypical scrapie in small ruminants might be a source of human prion disease  on a mouse PrP null background (2+ and Mg2+ ions (D-PBS) by extrusion through syringe needles of decreasing diameter. Brains from cattle with neuropathologically confirmed cases of BSE  were provided by the UK Central Veterinary Laboratory (now AHVLA). We used 10% (w/v) homogenates prepared from the brainstems of 5 cattle with natural BSE to generate pooled inocula, designated I038, which was previously shown to transmit prion disease to wild-type FVB/N and C57Bl/6 mice, and to transgenic mice overexpressing human PrP , we obtained 10% (w/v) brain homogenates prepared in sterile saline from sheep with neuropathologically confirmed prion disease and demonstrated ability to transmit prion disease to transgenic mice expressing ovine PrP or to wild-type mice . We obtao/oPrnp), designated Tg(HuPrP129V+/+o/oPrnp)-152 mice (129VV Tg152 mice), or homozygous for a human PrP 129M transgene array and murine PrP null alleles (o/oPrnp), designated Tg(HuPrP129M+/+o/oPrnp)-35 mice (129MM Tg35 mice), have been described  by using 84 FVB-specific PCR microsatellite markers covering 19 chromosomes at ≈20-cM intervals, to select breeding pairs positive for 100% of the FVB-specific markers. Selected congenic pairs were interbred to remove the endogenous murine PrP gene and to establish homozygosity of the human PrP transgene array. The resulting congenic lines, designated 129MM Tg35c and 129VV Tg152c, overexpress human PrP in brain at levels of 2× and 6× that of pooled human brain, respectively.Transgenic mice homozygous for a human PrP 129V transgene array and murine PrP null alleles . Brain homogenates (10% w/v) were diluted to 1% (w/v) in sterile D-PBS and passed through a 25-gauge needle. Each mouse was inoculated with 30-μL of 1% (w/v) brain homogenate because this avoids excessive animal losses within the first 48 hours postinoculation  were pretreated by boiling for 10 min in a low ionic strength buffer  before exposure to 98% formic acid for 5 min. Abnormal PrP accumulation was examined by using monoclonal antibody ICSM 35 against PrP  on an automated IHC staining machine  by using proprietary secondary detection reagents  before development with 3′3-diaminobenzedine tetrachloride as the chromogen (Sc) after analysis of 10 μL 10% (w/v) brain homogenate were reanalyzed by sodium phosphotungstic acid (NaPTA) precipitation of PrPSc (Proteinase K (PK) digestion , electrophoresis, and immunoblotting of 10% (w/v) transgenic mouse brain homogenates or 10% (w/v) brain homogenates from sheep with classical scrapie (prepared in D-PBS) were performed as described  sodium lauroylsarcosine (sarkosyl) in D-PBS. After incubation at 37°C for 30 min with constant agitation and centrifugation at 500 × g for 5 min, 150 μL of the supernatant was transferred to a new tube. The supernatant fraction was treated with 2 μL of Benzonase  for 30 min at 37°C with agitation and adjusted to a final concentration of 50 μg/mL PK (by adding 8 μL of a 1 mg/mL PK stock solution) and incubated at 37°C for 60 min with agitation. Samples were treated with 4 μL 100 mmol/L 4-(2-aminoethyl)-benzene sulfonyl fluoride, heated at 100°C for 5 min, adjusted with an equal volume of 2% (w/v) sarkosyl in D-PBS and 3 μL of Benzonase; they were then incubated for 30 min at 37°C with agitation before addition of 4% (w/v) NaPTA containing 170 mmol/L MgCl2, pH 7.4, to give a final concentration in the sample of 0.3% (w/v) NaPTA. After incubation for 60 min at 37°C, with constant agitation, samples were centrifuged at 16,100 × g for 30 min, and the supernatant fraction was discarded. The pellet fraction was resuspended to a final volume of 10 μL in D-PBS containing 0.1% (w/v) sarkosyl and analyzed by electrophoresis, immunoblotting, and high sensitivity chemiluminescence  that contain PK-resistant ovine PrPvine PrP , togethevine PrP . All natvine PrP . Consistent with the inability of IHC or high sensitivity immunoblotting to detect pathologic PrP in the brains of inoculated mice, neuropathologic examination of the brain showed no difference in spongiform change or gliosis from that observed in the brains of age-matched control mice (data not shown). From these findings, we conclude that both methionine and valine residue 129 variants of human PrP are refractory to pathologic conversion by these ovine prion strains in transgenic mice.Brain isolates from sheep with classical and atypical scrapie (including those with demonstrated prion infectivity in transgenic mice expressing ovine PrP) did not transmit prion disease to transgenic mice that were overexpressing human PrP. This fact contrasts markedly with the known susceptibility of these mice to transmission of multiple cattle BSE isolates  to type 4 PrPSc seen in vCJD brain (In the transgenic mice expressing human PrP, clinical prion disease was not produced by either of the 2 experimental sheep BSE isolates after postinoculation intervals >600 days . ExaminaJD brain , panel BJD brain , panel CWhy the efficiency of transmission of experimental sheep BSE prions to 129MM Tg35c mice is low compared with that reported in different lines of human PrP 129 methionine–expressing mice . TherefoIn this study, we have shown that disease does not develop in transgenic mice overexpressing human PrP when mice are inoculated with ovine prions from sheep with natural cases of classical scrapie and atypical scrapie from Great Britain and Germany. These transgenic mice are susceptible to infection, and clinical disease develops when mice are challenged with brain tissue from cattle affected by classical BSE (Our findings complement those of other recent studies that have investigated the zoonotic potential of ruminant prion strains using other lines of human PrP–expressing mice. Gene-targeted human PrP–expressing mice have been shown to be resistant to infection with classical and atypical scrapie prions from sheep (Although we found evidence for transmission of experimental ovine BSE to transgenic mice expressing human PrP 129 methionine, the relative attack rate was lower than observed in the other lines of mice (No strain variation has been found so far in the transmission, biochemical, or histopathologic characteristics of atypical scrapie prions (


Spinal cord homogenates prepared from BSE-infected cows were treated with SCW at 230–280°C for 5–7.5 min and used to intracerebrally inoculate transgenic mice overexpressing bovine prion protein. Serial protein misfolding cyclic amplification (sPMCA) analysis detected no PrPSc in the SCW-treated homogenates, and the mice treated with these samples survived for more than 700 days without any signs of disease. However, sPMCA analyses detected PrPSc accumulation in the brains of all inoculated mice. Furthermore, secondary passage mice, which inoculated with brain homogenates derived from a western blotting (WB)-positive primary passage mouse, died after an average of 240 days, similar to mice inoculated with untreated BSE-infected spinal cord homogenates. The PrPSc accumulation and vacuolation typically observed in the brains of BSE-infected mice were confirmed in these secondary passage mice, suggesting that the BSE prions maintained their infectivity after SCW treatment. One late-onset case, as well as asymptomatic but sPMCA-positive cases, were also recognized in secondary passage mice inoculated with brain homogenates from WB-negative but sPMCA-positive primary passage mice. These results indicated that SCW-mediated hydrolysis was insufficient to eliminate the infectivity of BSE prions under the conditions tested.The global outbreak of bovine spongiform encephalopathy (BSE) has been attributed to the recycling of contaminated meat and bone meals (MBMs) as feed supplements. The use of MBMs has been prohibited in many countries; however, the development of a method for inactivating BSE prions could enable the efficient and safe use of these products as an organic resource. Subcritical water (SCW), which is water heated under pressure to maintain a liquid state at temperatures below the critical temperature (374°C), exhibits strong hydrolytic activity against organic compounds. In this study, we examined the residual  Sc) comprise the infectious agents that cause prion diseases such as Creutzfeldt-Jacob disease (CJD) in humans, scrapie in sheep and goats, chronic wasting disease (CWD) in deer and elk, and bovine spongiform encephalopathy (BSE) in cattle  was performed in quadruplicate. For serial PMCA analysis, 1:5 dilution of the amplified product and subsequent amplification was repeated 3 times.In a previous study, we developed an ultrasensitive method for BSE PrPte (DSP) . The PMCc levels , 17. Brievels [C , and PrPPMCA samples (10 μl) were incubated with 10 μl proteinase K solution (100 μg/ml) at 37°C for 1 h, and then mixed with 20 μl of 2× sodium dodecyl sulfate (SDS) sample buffer and incubated at 100°C for 5 min. The samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes . After blocking, the membranes were incubated for 30 min with a horseradish peroxidase (HRP)-conjugated T2 monoclonal antibody , which rSc present in the primary passage mice, 10% BH of several western blot (WB)-positive or sPMCA-positive mice were subsequently injected intracerebrally into four to seven TgBoPrP mice (20 μl per mouse). After inoculation, the mice were evaluated daily for signs of disease.The SCW-treated bovine spinal cord samples were injected intracerebrally into five or six TgBoPrP mice (20 μl per mouse). To examine the infectivity of the PrPThe left hemispheres of mouse brains were fixed in 10% buffered formalin for subsequent neuropathological analyses. Coronal slices of the brain were immersed in 98% formic acid to reduce the infectivity, and embedded in paraffin wax. Sections (4 μm thickness) were cut and stained with hematoxylin and eosin, and subjected to immunohistochemical analysis using with SAF84 monoclonal antibody , as described previously .Sc present in the SCH samples subjected to the SCW treatments listed in Sc signal was detected by conventional WB analysis in any of the SCW-treated samples (Sc derived from BSE-infected cattle could be efficiently amplified by sPMCA in the presence of DSP [Sc in the SCW-treated SCH samples. PrPSc were amplified from the untreated BSE-infected SCH (10%) samples diluted up to 10−6 and10-9 after one and two rounds of amplification, respectively  after two or three rounds of sPMCA, the ratio of positive tests in the quadruplicate samples varied among these mice (50–100%) after four rounds of sPMCA. Additionally, PrPSc were detected in each of the mice inoculated with SCH treated at 250°C for 5 min  used for inactivation of BSE-SCH. While relatively higher levels of PrPSc accumulation were detected in three primary passage mice . Such undecomposed BSE PrPIn conclusion, we demonstrated that SCW treatments suitable for recycle use of MBM failed to completely inactivate BSE prions. Although prion inactivation may proceed more effectively at higher temperature regions around supercritical water or in the presence of longer heating times, such harsh treatments would cause severe damage to the valuable organic materials present in the MBM, resulting in reduced economic merits.S1 Fig(SCH) were treated with SCW at 250°C for 7.5 min, and serially amplified by PMCA. After each round (R1–R4) of amplification, 80 samples (lane 1–80) were digested with proteinase K and subjected to western blot analysis. The lanes labeled “PC” indicate the positive control samples, which contained untreated BSE-SCH diluted from 10−6 to 10−9. The lanes labeled “Ns” indicate samples in which only the PrPC substrate was treated in the same manner.Bovine spongiform encephalopathy (BSE)-infected spinal cord homogenates (TIF)Click here for additional data file.S2 Fig(SCH) (10%) were diluted to 10−4, and amplified in the presence of 1–8 μl of BSE-SCH treated with SCW at 250°C for 7.5 min or PBS. After amplification, the samples were digested with proteinase K and analyzed by western blot. Densitometric analysis indicated a reduction in the PrPSc signal intensity to 84% (1 μl), 72% (2 μl), 68% (4 μl), and 62% (8 μl) of that of the respective PBS-added samples after the addition of the SCW-treated product.Bovine spongiform encephalopathy (BSE)-infected spinal cord homogenates (TIF)Click here for additional data file.


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.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.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.The online version of this article (doi:10.1186/s13104-015-1260-3) contains supplementary material, which is available to authorized users. Epidemiological studies indicated that the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom (UK) was caused by food-borne exposure of cattle to a transmissible spongiform encephalopathy (TSE) agent , but thePrP) gene  and five cattle inoculated intracerebrally with saline solution. The study was terminated at 120 months post inoculation (mpi), and all remaining cattle  were euthanased with pentobarbitone. Pathological examinations were carried out as described previously [PrP sequence. In addition, mAb SAF84  was used because of its particular usefulness in identifying H-type BSE by specific downward molecular mass shift [Briefly, two groups of cattle were inoculated intracerebrally with brain homogenate from pathologically confirmed scrapie cases sourced prior to 1975 (ten cattle) and after 1990 (ten cattle). Both inocula were characterised by biochemical (Western immunoblot (WB) hybrid technique ) and bioeviously . These mres) is detected as three protein bands that relate to diglycosylated, monoglycosylated and unglycosylated forms of the abnormal protein, and the migration as well as the relative intensity (expressed as glycoform ratio) of the protein bands of PrPres enables differentiation of scrapie from BSE. Discrimination is also possible by parallel testing with the two specific mAbs: mAb Sha31 detects PrPres in both cattle and sheep, while mAb P4 is more selective for scrapie PrPres under the test conditions, as reported previously [res bands detected using mAbs Sha31, 6H4 and P4. Stringent digestion was undertaken with 500 μg/ml PK at pH 8.0, and mild digestion with 50 μg/ml PK at pH 6.5. The PK susceptibility ratio was obtained by comparing the optical density of the signal strengths of the PrPres bands produced by mild and stringent digestion, which is >0.7 for C-type BSE and <0.6 for L-type BSE cases [With this WB technique proteinase-resistant prion protein  from either blood  or brain  according to methods described previously [Determination of the bovine eviously .The original study design included strain characterisation only in wild-type mice but brain tissue was subsequently distributed to other research institutes to further characterise bovine passaged scrapie in additional rodent lines. This was carried out independent of the original study and selection of material was restricted by availability.Myodes glareolus) were inoculated with brain tissue  from steers P75-7 (inoculated with the pre-1975 pool) and P90-4 (inoculated with the post-1990 pool), both positive for PrPSc in brain. Both inocula were further tested by WB for presence of PrPres as described previously using mAb SAF84  and mAb P4  [Bank voles  .PrP gene) were inoculated with 20 μl of brain homogenate (as 10% w/v in PBS) derived from each of the two steers. Inoculation procedure, clinical monitoring and euthanasia at terminal stage of disease were as described previously [res characterisation and lesion profiling. Brains were examined by WB with mAb SAF84 targeting PrP aa residues 163–173 of the bank vole PrP sequence and 12B2  [Each of two groups of 15 bank voles (homozygous for methionine at codon 109 of the eviously . Brains equence) , and lesThe disease phenotypes observed in voles after transmission of P75-7 and P90-4 brain samples were compared with those previously derived from different scrapie sources, including natural ovine scrapie isolates SS-UK6 (10 brains) and SCR6 (single brain)  and the Sc) from the clinically affected steers P75-7 (as above) and P90-1 (inoculated with the post-1990 pool). Both of the original inocula and the brain of steer P90-1 had previously been inoculated into conventional mice , resulting in successful transmission with lesion profile features uncharacteristic of BSE (pre-1975 pool), or transmissions with low attack rates, insufficient to establish a lesion profile in RIII mice (post-1990 pool and P90-1) [Inocula comprised the original two scrapie brain pools (pre-1975 and post-1990) and brain tissue  ; see alsPrP gene of various species as follows: tg338 mice , tg110 PrP gene ), tg001 PrP gene ), tga20 PrP gene ) and tg3PrP gene ).Groups of 6–12 mice were inoculated intracerebrally with 20 μl of either 2% homogenate of the original ovine brain pools or 10% homogenate of the bovine brain tissue (prepared in sterile 5% glucose). The former inoculum dilution was determined by restricted availability of source tissue. The procedures for inoculation, clinical monitoring and cull of affected mice were as described previously .res by WB with mAb Sha31 (BioRad Laboratories) [res positive mice, where available, were used for further passages. When, on primary passage, all mice of an inoculum group were negative for PrPres a second passage of the pooled brain homogenates was carried out. For second passages, mice were inoculated intracerebrally with 20 μl of a 10% w/v brain homogenates (prepared in sterile 5% glucose).Disease in mice was confirmed according to previously published protocols for detection of PrPatories)  or parafatories) . Brain hData were compared to those derived from mice inoculated with classical BSE bovine and ovine brain homogenates and with L-type and H-type BSE bovine brain homogenates, some of which were obtained from separate studies , 28–31.Sc or PrPres could not be detected on examination of the brain by postmortem tests [From the time of publication of interim results  to termiem tests –34 and cThe brainstem samples of the nine positive cases inoculated with the pre-1975 pool, where the initial WB results resembled classical BSE, but with some differences regarding lower molecular mass migration and glycoform ratio, produced a WB profile with similarities to the L-type BSE control sample see Fig. .Fig. 1DiThe brain samples of the seven positive cattle inoculated with the post-1990 scrapie pool, the molecular profile of which previously resembled classical scrapie, maintained the classical scrapie profile with the WB protocol adapted for the detection of atypical BSE cases. They also exhibited variation in the molecular mass migration as reported previously . After aNone of the samples from the pre-1975 or the post-1990 scrapie pools resembled an H-type BSE-like profile using mAbs Sha31 or P4 (no higher unglycosylated band) or showed the distinctive molecular mass downward shift and sharp band at 14 kDa, as illustrated by the H-type BSE control when SAF 84 was applied  of animal P90-5 was not found in any other steer, the genotype of P90-4 was identical to that of P90-2 despite having a different molecular mass profile. The inoculation of cattle with a pool of scrapie brain material, containing possibly multiple strains, remains the most likely reason for the observed diversity in the molecular profiles as hypothesised previously [PrP gene polymorphism did not appear to be responsible for the lack of transmission in some animals since the same polymorphism was found in inoculated cattle with or without PrPSc accumulation in the brain (see Table Sc in the brain were heterozygous carriers of the 12 base pair (bp) deletion allele, which is associated with a higher risk of having BSE [We previously reported phenotype diversity in cattle inoculated with the post-1990 scrapie pool: two cattle [P90-4 (inoculated into bank voles) and P90-5, see Table ving BSE .res were detectable by WB with mAb SAF84 in the inoculum from P75-7, whilst PrPres was not detected in the inoculum from P90-4 , were detected in the inoculum from P90-4. These findings were consistent with those made by separate WB analysis of the bovine brainstems (see above), which indicated that the molecular profile was maintained within different brain areas regardless of the choice of antibodies Sha31 or SAF84.Prior to inoculation the bovine donor brain samples were subject to molecular analyses. Low levels of PrPres pattern in infected vole brains analysed by WB was not uniform  or low molecular mass unglycosylated PrPres fragment . This partial similarity with classical BSE was confirmed by discriminatory WB, which showed that the 17 kDa PrPres fragment in voles infected with P75-7 was poorly detected by mAb 12B2, the epitope of which (aa 93WGQGG97) is near the N-terminus of the PrPres fragment  and a low attack rate (7/12). The PrPres fragment. The survival times and attack rates are displayed in Table Second and third passages were made using donor voles displaying either the 18 or 17 kDa PrPres fragments after primary transmission . Also, the vole-adapted “sub-strains” derived from CH1641 gave survival times of ~110 dpi for the 17K sub-strain and of ~140 dpi for the 18K sub-strain  and an attack rate of 11/15 although, based on the low level of PrP9 dpi andSecond and third passages gave short and consistent survival times .Lesion profiles of vole-adapted P90-4 were different from those observed in P75-7 Fig. . The surIn summary the findings in bank voles suggest that the prion strains isolated from the cattle inoculated with the pre-1975 and the post-1990 scrapie brain pools were different and distinct from classical BSE and L-type BSE, but similar, or identical, to scrapie strains previously isolated from European natural sheep scrapie cases.Table Neither of the ovine scrapie pools or inocula from P75-7 or P90-1 transmitted to tg001 mice on first or second passage Table . Lack ofVRQ PrP transgenic mouse line) inoculated with VRQ/VRQ scrapie sheep isolates, whereas longer survival times have been observed in tg338 mice inoculated with ARQ/ARQ scrapie sheep isolates [VRQ/VRQ, ARQ/VRQ and ARQ/ARQ sheep in similar proportions . It is more likely that the post-1990 brain pool contained isolates which have been shown not to propagate in wild-type mice but transmit to tg338 mice with similar long incubation periods regardless of genotype of the sheep (VRQ/VRQ or ARQ/ARQ) source [Although both original scrapie pools transmitted to tg338 mice, survival times were almost seven times shorter in mice inoculated with the pre-1975 scrapie pool. As both inocula produced disease in cattle with similar survival time ranges, it seems unlikely that this finding is due to a lower infectious titre in the post-1990 scrapie pool. Short survival periods are observed in tg338 mice (a isolates . Both po) source . In fact) source , and his) source ; J FosteThe survival times in tg338 mice inoculated with the inoculum from P75-7 were more than three times shorter than with inocula from ovine or bovine BSE sources. By contrast, the inoculum from P90-1 failed to transmit at primary passage but transmitted weakly on second passage; a phenomenon, which has not previously been documented for any isolate from naturally infected cattle with TSEs in this mouse line.Sc distribution in the brain of tg110 mice was different to L-type BSE and as reported for CH1641 [PrP transgenic mice (tg340) (Table PrP gene (Tg540 mouse line) [None of the WB profiles obtained after passage in tg110 mice resembled classical BSE. The profile in mice inoculated with the pre-1975 pool and P75-7 inoculum showed an unglycosylated band of 19 kDa, which was maintained after passage in all tg110 inoculated with this inoculum  . Indeed,se line) .Fig. 7WeThe WB profile obtained after inoculation of tg110 mice with the inoculum from P90-1 gave an unglycosylated 21 kDa band that has also been observed in tg110 mice inoculated with some sources of classical scrapie . A WB profile with an unglycosylated band of 19 kDa (L-type BSE-like) was also obtained after inoculation of tg110 mice with the post-1990 scrapie brain pool but the profiles obtained from the brain of steer P90-1 and tg110 mice inoculated with the inoculum from P90-1 were different (unglycosylated band of 21 kDa, in Fig. Two different disease phenotypes were produced after intracerebral inoculation of cattle with scrapie brain pools sourced pre-1975 and post-1990 in GB, which were not readily explained by any differences in PrP genotype of the cattle. Based on pathological and molecular characteristics and biological characterisation in bank voles and transgenic mice there was no clear evidence of an agent derived from the cattle resembling classical or atypical forms of BSE. Transmissions in bank voles identified previously isolated scrapie strains and some similarities to the experimental isolate CH1641. Contrary to the transmission results in rodents, the results for the molecular techniques, which have been adopted for the detection of atypical BSE cases, suggest that they may not be appropriate for differentiating WB profiles in cattle following infection from an ovine scrapie source.


Sc). H-BSE is transmissible to wild-type mice—with infected mice showing a long survival period that is close to their normal lifespan—but not to hamsters. Therefore, rodent-adapted H-BSE with a short survival period would be useful for analyzing H-BSE characteristics. In this study, we investigated the transmissibility of H-BSE to hamster prion protein transgenic (TgHaNSE) mice with long survival periods. Although none of the TgHaNSE mice manifested the disease during their lifespan, PrPSc accumulation was observed in some areas of the brain after the first passage. With subsequent passages, TgHaNSE mice developed the disease with a mean survival period of 220 days. The molecular characteristics of proteinase K-resistant PrPSc (PrPres) in the brain were identical to those observed in first-passage mice. The distribution of immunolabeled PrPSc in the brains of TgHaNSE mice differed between those infected with H-BSE as compared to C-BSE or L-BSE, and the molecular properties of PrPres in TgHaNSE mice infected with H-BSE differed from those of the original isolate. The strain-specific electromobility, glycoform profiles, and proteolytic cleavage sites of H-BSE in TgHaNSE mice were indistinguishable from those of C-BSE, in which the diglycosylated form was predominant. These findings indicate that strain-specific pathogenic characteristics and molecular features of PrPres in the brain are altered during cross-species transmission. Typical H-BSE features were restored after back passage from TgHaNSE to bovinized transgenic mice, indicating that the H-BSE strain was propagated in TgHaNSE mice. This could result from the overexpression of the hamster prion protein.Two distinct forms of atypical bovine spongiform encephalopathies (H-BSE and L-BSE) can be distinguished from classical (C-) BSE found in cattle based on biochemical signatures of disease-associated prion protein (PrP Sc) that is thought to harbor a post-translational conformational change of the normal, host-encoded cellular prion protein (PrPC) in the CNS of affected hosts [Sc (PrPres) identified by western blotting (WB) [Bovine spongiform encephalopathy (BSE), a type of prion disease or transmissible spongiform encephalopathy, is a fatal and progressive degenerative central nervous system (CNS) disorder in cattle. BSE was first identified in the United Kingdom in 1986 [ed hosts . BSE in ing (WB) –6. The oing (WB) , 8. Arou136R154Q171 allele of ovine PrP [136R154Q171 allele [BSE is transmissible in a wide range of host species. The transmission of H-BSE to cattle –15, tranvine PrP , and wilvine PrP –20 has b1 allele  and to S1 allele , suggest1 allele –20. Ther1 allele , 24. On 1 allele , 23, 25.Animal experiments were carried out in strict accordance with the regulations outlined in the Guide for the Care and Use of Laboratory Animals of the National Institute of Animal Health and the Guidelines for Proper Conduct of Animal Experiments, 2006 by the Science Council of Japan. Procedures involving animals were approved by the Institutional Animal Care and Use Committee at the National Institute of Animal Health , with all possible effort made to minimize the pain and discomfort of each animal in accordance with the Guidelines for Animal Transmissible Spongiform Encephalopathy Experiments of the Ministry of Agriculture, Forestry, and Fisheries of Japan. All intracerebral inoculations were performed under sevoflurane anesthesia.n = 5; 3 weeks old) were intracerebrally inoculated with 20 μl of 10% brainstem homogenate (weight/volume) from cattle experimentally infected with H-BSE [Sc-positive brains of first-passage TgHaNSE mice were used for subsequent passages to obtain the TgHaNSE mouse-adapted H-BSE prion. The expression level of hamster PrP in the brain of TgHaNSE mice (provided by Dr. B. Chesebro) was approximately 4–5 times higher than that of Syrian hamsters [Female TgHaNSE mice  from National Institute of Infectious Diseases   were injCharacteristics of the five primary antibodies used in this study are summarized in Sc immunohistochemistry (IHC).At necropsy, the left hemisphere and selected tissues—including lymphoid organs—were fixed in 10% buffered formalin containing 10% methanol. Formalin-fixed tissue specimens were immersed in 98% formic acid for 60 min to reduce the infectivity, then embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) for histological evaluation or processed for PrPAfter appropriate epitope retrieval by hydrate autoclaving in 10 mM citrate buffer (pH 6.0) at 121°C for 3 min or with a combination of enzymatic and chemical treatments , sectionres was detected with mAbs 3F4, 4E10, T2, 6H4, or SAF84 and a chemiluminescent substrate  as previously described [The right half of each brain was digested with 40 μg/ml PK . Tissue samples were dissolved in sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer and assayed using standard WB procedures. PrPescribed . After PTo compare disease phenotypes and molecular characteristics of the H-BSE inoculum before and after passage in TgHaNSE mice, brain homogenates from diseased first-passage TgHaNSE mice were intracerebrally injected back into TgBoPrP mice. The susceptibility of these mice (kindly provided by Dr. S.B. Prusiner ) to H-BSNone of the TgHaNSE mice injected with the H-BSE prion exhibited typical signs of the disease at first passage . Four ofAfter one passage of H-BSE from cattle in TgHaNSE mice, subsequent transmission to the mice resulted in shortened mean survival periods, with mice showing signs of prion disease. A third passage did not further reduce the survival period .Lesion profiles based on average scores for histological vacuolation in nine neuroanatomical regions of coronal HE-stained brain sections  from TgHSc immunoreactivity at first and subsequent passages by IHC with the mAb 3F4, which reacts with hamster PrP , and muscle bundles or skeletal muscle fibers  was similar to that of H-BSE from cattle   [Sc distribution patterns, and molecular features of PrPres in the brains of TgBoPrP mice inoculated with H-BSE from TgHaNSE mice were identical to those in TgBoPrP mice inoculated with H-BSE from cattle  . Patholom cattle . The mosAb SAF84 .Sc of H-BSE from cattle was propagated with hamster PrPC substrate in TgHaNSE mouse brains, as determined by WB and IHC analyses. These results demonstrate the transmissibility of H-BSE across a species barrier. Adaptation of prions to a new host requires several sequential passages. Third-passage mice showed the same shortened survival periods, characteristics of prion disease, and pathological and molecular features of PrPSc in the brain as second-passage mice. These data indicate that the infectivity and virulence of H-BSE increased in the new species during adaptation [PrPaptation . This waaptation . After aaptation .C and PrPSc may create a species barrier that influences interspecies transmission [Sc is converted by hamster PrPC and may explain the species barrier with respect to C-BSE prion transmission [C expression level, host genetic factors, or the host microenvironment, which may contribute to the adaptation of heterologous prions and their propagation [Sc aggregation before primary passage TgHaNSE mice reach the end of their lifespan [Differences in amino acid sequence between host PrPsmission . Only eismission . Plausibpagation , 39, 40.lifespan .res from different species cannot be directly compared for strain characterization, the WB analysis revealed different molecular features for PrPres in TgHaNSE mice as compared to the original H-BSE isolate from cattle, including differences in PK cleavage sites, glycoform patterns, and molecular weight. Interestingly, H-BSE-infected TgHaNSE mice did not show an additional 10–12-kDa band when analyzed using C-terminal-specific antibodies such as SAF84, which is typical in cattle and bovine transgenic or wild-type mice infected with H-BSE [res glycoform in TgHaNSE mice were altered relative to those in the original cattle inoculum. Although the glycoprofiles of TgHaNSE mice infected with H-BSE as compared to C- or L-BSE were similar, the unglycosylated molecular mass of PrPres from L-BSE was less than that of PrPres from H- and C-BSE. In addition, immunolabeled PrPSc distribution patterns in the brain differed for the three BSE agents in TgHaNSE mice. However, alterations in neuropathological phenotype and biochemical characteristics may be a general phenomenon in interspecies transmission [res in TgHaNSE mice may be influenced by characteristics of the host species rather than those of the prion strain [res has been reported in the transmission from sporadic Creutzfeldt-Jakob disease (CJD) to humanized transgenic mice [C and/or selection of a PrPSc subpopulation from heterogeneous PrPSc, resulting in the emergence of a new prion strain [Although PrPth H-BSE , 20, 35.smission , 43. Then strain , 44, 45.nic mice , variantnic mice , and hamnic mice . These cn strain .The origin of prions that emerged in TgHaNSE mice inoculated with H-BSE was investigated in reverse transmission studies of H-BSE inoculum after passage from TgHaNSE to TgBoPrP mice, which revealed H-BSE-like phenotypes, a phenomenon known as traceback . This inSc under artificial conditions, that is, by overexpressing transgenes in a heterogeneous environment. Nonetheless, our results provide a better understanding of how interspecies transmission of prion agents contributes to PrPSc diversity in prion pathogenesis. Additional studies are needed to clarify the transmissibility of H-BSE from TgHaNSE mice to hamsters and to examine PrPSc of H-BSE in TgHaNSE mice, as well as the influence of environment on its propagation in hamster brains.In this study, we investigated PrPSc in the brain as well as several peripheral tissues. Furthermore, H-BSE adapted and was stabilized after the third passage. The pathogenic features of H-BSE in TgHaNSE mice differed from those of C- and L-BSE. This mouse model is a useful tool for further investigations of the mechanisms underlying the species barrier of BSE prions.In conclusion, H-BSE from cattle was efficiently transmitted to TgHaNSE mice, as determined by the accumulation of PrPS1 FigSc mainly accumulated in the gray matter of the spinal cord. (B) Fine punctate to coarse particulate PrPSc deposits were present in the ganglion cell layer, inner nucleus layer, and inner and outer plexiform layers of the retina. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nucleus layer; OPL, outer plexiform layer; ONL, outer nucleus layer; PL, photoreceptor layer; RPE, retinal pigment epithelium.  Granular PrPSc immunoreactivity was observed in ganglionic cells (arrows) of the trigeminal ganglion (C) and dorsal root ganglion (D). (E) Granular PrPSc immunoreactivity was detected in the intrafusal myofibers of muscle spindles of skeletal muscle. (F) Serial section of (E) with HE staining.(A) Coarse particulate PrP(PDF)Click here for additional data file.


Successful transmission of Transmissible Mink Encephalopathy (TME) to cattle supports the bovine hypothesis for the still controversial origin of TME outbreaks. Human and primate susceptibility to classical Bovine Spongiform Encephalopathy (c-BSE) and the transmissibility of L-type BSE to macaques indicate a low cattle-to-primate species barrier. We therefore evaluated the zoonotic potential of cattle-adapted TME. In less than two years, this strain induced in cynomolgus macaques a neurological disease similar to L-BSE but distinct from c-BSE. TME derived from another donor species (raccoon) induced a similar disease with even shorter incubation periods. L-BSE and cattle-adapted TME were also transmissible to transgenic mice expressing human prion protein (PrP). Secondary transmissions to transgenic mice expressing bovine PrP maintained the features of the three tested bovine strains  regardless of intermediate host. Thus, TME is the third animal prion strain transmissible to both macaques and humanized transgenic mice, suggesting zoonotic potentials that should be considered in the risk analysis of animal prion diseases for human health. Moreover, the similarities between TME and L-BSE are highly suggestive of a link between these strains, and therefore the possible presence of L-BSE for many decades prior to its identification in USA and Europe. Transmissible Mink Encephalopathy (TME) is a rare prion disease affecting ranch-reared mink that was reported in four isolated outbreaks in the USA in 1947, 1961, 1963 and 1985 . EpidemiSeveral experimental exposures of mink to ruminant prions were performed to identify the exact origin of TME. Low efficiency and rate of transmission were observed after inoculation of mink with sheep scrapie  and elk-Currently, classical BSE is the only animal transmissible spongiform encephalopathy (TSE) considered as a zoonotic disease, since it induces a variant of Creutzfeldt-Jakob disease (CJD) in humans ,14,15. WWe chose to assess the risk for human health linked to TME-related prion strains by evaluating the transmissibility of cattle-adapted TME in this cynomolgus macaque model, in comparison to raccoon TME as a non-ruminant source of the same prion strain. In parallel, we used transgenic mice overexpressing human or bovine prion protein (PrP) to assess the relevance of our results for human situation.A primate intracerebrally inoculated with the equivalent of 40 mg of a TME-infected cattle brain (second passage) developed the first neurological signs of disease after less than twenty months of incubation . It firsIn parallel, several but not all the transgenic mice overexpressing human (Met/Met) PrP (tg650 mice) intracerebrally inoculated with cattle-adapted TME inoculum exhibited cerebral PrPres: partial transmission (75 %) occurred in humanized mice that died after about 18 months of incubation .From these results, cattle-adapted TME represents the third cattle prion strain (together with c-BSE and L-BSE) experimentally demonstrated to be transmissible to non-human primates. We confirmed in this study the previously described transmissibility of both L-BSE and c-BSE in both experimental primates ,21 and tIn the primate model, exposure to L-BSE-infected cattle brain induced a clinical picture with incubation time and duration of illness that are similar to those observed after exposure to cattle-adapted TME, even after exposure to as little as 2.5 mg of brain tissue . ConversA comparison of incubation periods confirmed and magnified the higher virulence of L-BSE for macaque compared to c-BSE, which we had previously observed . Moreoveet al., in this transgenic model [In the model of transgenic mice overexpressing human (Met/Met) PrP (tg650 mice), an incomplete transmission rate of 25% of L-BSE was observed after an incubation period of similar duration (18 months) to those from cattle TME-exposed animals, while a 100% transmission rate was observed with c-BSE, but with longer incubations . These observations are consistent with the results obtained with L-BSE strain by Beringue ic model  and by KThe overexpression of PrP in transgenic mice is often criticized as an element helping to force the way through the species barrier and extrapolation of our results in this model to the human situation should be taken with caution, since transgenic mice expressing physiological levels of human PrP are resistant to L-BSE . NeverthThe macaque inoculated with cattle-adapted TME showed widespread cortical spongiosis similar to that in both primates exposed to L-BSE . The spoPrimates inoculated with L-BSE or cattle TME exhibited a similar diffuse laminar synaptic pattern of PrPres depositions (either fine and sandy or roughly granular) but no evidence of plaques, even when stained with thioflavine T (data not shown), whereas c-BSE-infected animals had weak diffuse synaptic labeling but multiple intensely-stained PrPres aggregates and characteristic plaques . We previously demonstrated that the technique that we developed for typing and classifying prion strains in small ruminants might also be used to discriminate L-BSE from c-BSE in experimentally infected macaques . BrieflyUnder these experimental conditions, PrPres in both primates and Tg650 exposed to cattle-adapted TME behaved like PrPres derived from corresponding animals infected with L-BSE  Figure . A 19 kDStrain signatures were also assessed in bioassays in transgenic mice overexpressing bovine PrP (tg110). Those mice were intracerebrally inoculated with cattle-adapted TME, L-BSE or c-BSE isolates issued from cattle, macaque or tg650 recipients  . All theThis biochemical strain typing protocol was adapted  and applied to Tg110 mice . The resPrimates and mice were housed and handled in accordance with the European Directive 2010/63 related to animal protection and welfare in research, under the constant internal surveillance of veterinarians. Animals were handled under anesthesia to limit stress, and euthanasia was performed for ethical reasons when animals lost autonomy. Macaca fascicularis) were provided by Noveprim (Mauritius), checked for the absence of common primate pathogens before importation, and handled in accordance to national guidelines. Transgenic mice overexpressing human  or bovin (tg650 ) PrP werThe TME inocula were derived from a second passage in cattle  or the Tissues were fixed in formalin 4% for histological examination. Neuropathology and immunohistochemical detection of protease-resistant prion protein (PrPres) were performed on brain sections as previously described . PrP was purified according to the TeSeE purification protocol (Bio-Rad), in adapted conditions of proteolysis for strain discrimination as previously described , using BWe have shown that cattle-adapted TME is the third cattle prion strain  to be transmissible both to non-human primates and transgenic mice overexpressing human PrP. However, the successful transmission of raccoon TME to primate, inducing a disease with similar features as cattle TME, extends this notion to TME-related strains independent of host origin. Pathological, biochemical and bioassay investigations converged to demonstrate the similarity between cattle-adapted TME and L-BSE. Together with previous experiments performed in ovinized and bovinized transgenic mice and hamsters ,9 indica


The H-type of atypical bovine spongiform encephalopathy (H-BSE) was serially passaged in bovinized transgenic (TgBoPrP) mice. At the fourth passage, most challenged mice showed a typical H-BSE phenotype with incubation periods of 223 ± 7.8 days. However, a different phenotype of BSE prion with shorter incubation periods of 109 ± 4 days emerged in a minor subset of the inoculated mice. The latter showed distinct clinical signs, brain pathology, and abnormal prion protein profiles as compared to H-BSE and other known BSE strains in mice. This novel prion was transmitted intracerebrally to cattle, with incubation periods of 14.8 ± 1.5 months, with phenotypes that differed from those of other bovine prion strains. These data suggest that intraspecies transmission of H-BSE in cattle allows the emergence of a novel BSE strain. Therefore, the continuation of feed ban programs may be necessary to exclude the recycling of H-BSE prions, which appear to arise spontaneously, in livestock. Such measures should help to reduce the risks from both novel and known strains of BSE. Sc). PrPSc is a disease-associated isoform of the host-encoded prion proteinC) to PrPSc is thought represent a central event in prion pathogenesis. PrPSc has been recognized as the major component of prionsSc are associated with different prion strains that, in turn, cause distinct disease phenotypesPrions cause transmissible spongiform encephalopathies (TSEs), which are characterized by a spongiform change in the central nervous system and the accumulation of an abnormal prion protein  is a TSE of cattle. Most BSE cases show a unique phenotype that is thought to be caused by a single prion strain55The worldwide occurrence of BSE is declining, primarily because of effective feed ban programsC, respectively1516H-BSE was first reported in France59101113Serial passages of H-BSE in wild type18Sc, and histopathological features from the primary to third passage mice. At the fourth passage, mice from a single experimental group (#3), out of eight experiments, showed shorter incubation periods (108.8 ± 4.0 days) than the other groups. This group was challenged with brain homogenates of a mouse with 221-day incubation period. Group #3 animals showed weight loss, but no constant chewing of the bedding. This short incubation-type of BSE was designed BSE-SW (short incubation with weight loss) strain. BSE-SW was transmitted to TgBoPrP mice with 97.3 ± 3.7 day incubation periods, and their clinical signs were identical to those of mice in the experimental group #3. The other mice in the fourth passage groups showed the symptomatic chewing of the bedding, and their incubation periods were 223.3 ± 7.8 days  of BSE-SW was lower than that of H-BSE, but similar to C-BSE  were observed for H-BSE and BSE-SW (14) was detected in C-BSE and BSE-SW, whereas a slightly higher molecular weight (approximately 19 kDa) of PrPcore #1 was detected in H-BSE. Unglycosylated PrPcore with a molecular weight of 12 kDa (PrPcore #214) was observed in H-BSE and BSE-SW  6H4 revealed that the molecular mass of proteinase K (PK) digested PrPto C-BSE . MAb P4,to C-BSE . This red BSE-SW . Deglycod BSE-SW . On the d BSE-SW . These bSc in TgBoPrP mice with BSE-SW were different from H-BSE and C-BSE  for PrPSc denaturation in the H-BSE and BSE-SW brains were 3.8 ± 0.4 M and 3.0 ± 0.2 M, respectively , which included disturbance, mild fear or anxiety, mild gait changes, and, occasionally, low head carriage. After one to two months of the initial clinical signs, the animals were leaning towards the floor and rested their heads against the wall, which was followed by ataxia of the hind limbs that progressed to difficulty in getting up without assistance at the clinically terminal stage of the disease. The bodily condition gradually worsened because of a loss of weight during the two to three month clinical duration. None of the animals exhibited anorexia, nervousness, or aggression, and responded to visual, acoustic, and tactile stimuli throughout the course of the disease. The cattle were eventually culled at 13.3 mpi, 15 mpi, and 16.2 mpi before astasia, in accordance with the welfare guidelines for animal experiments. The incubation periods of cattle infected with BSE-SW (14.8 ± 1.5 mpi) were shorter than those for H-BSE, C-BSE, and L-BSE . Obex saSc with mAb F99/97.6.1 resulted in intraneuronal and intraglial patterns throughout the obex , the solitary nucleus, the nucleus of trigeminal nerve spinal tract, and the olivary nucleus in all animals. No intraneuronal vacuolation was seen. Spongy changes were not prominent in the gray matter of medulla oblongata at the obex . Immunolthe obex . IntraneSc from the obex tissue of the challenged cattle. The molecular features of PrPcore of BSE-SW-affected cattle were distinctly different from C-BSE, L-BSE, and H-BSE . PrPSc of BSE-SW has some similarity to H-BSE on the account of the presence of truncated 12-kDa fragments (PrPcore #2). Sc from BSE-SW, as assessed by immunoreactivity with mAbs P4, 6H4, and SAF84. These results argue against the possibility that the BSE-SW prion resulted from a contamination of other laboratory prion strains.Our previous reports have revealed the usefulness of TgBoPrP mice for characterizing BSE prions16211621It is known that sheep scrapie comprises different prion strains241819Sc are involved in the prion strain diversitySc from a uniform conformation causes the emergence of new host-adapted PrPSc Sc of BSE-SW exhibited different conformational stability from H-BSE. It is also known that strain “mutation or transformation” may occur upon intraspecies transmission, where the PrP amino acid sequences of the host and the donor are identicalSc of BSE-SW has a different conformation than H-BSE.It has been suggested that different conformations of PrPSc accumulation in the obex, and the challenged cattle fulfilled BSE criteria . Animal experiments were performed in accordance with the Guidelines for Animal Transmissible Spongiform Encephalopathy Experiments of the Ministry of Agriculture, Forestry, and Fisheries of Japan.PrP gene (encoding BoPrP) in a mouse PrP deficient background were used. These mice harbored the cattle PrP gene containing six copies of the octarepeat sequence , and produced approximately eight times more BoPrP per gram of protein than found in the cattle brainTgBoPrP mice overexpressing the bovine Brain samples of Canadian H-BSE cattle, courteously provided by Dr. S. Czub, were used in this study211032g for 5 min at room temperature (RT). Three-week-old female TgBoPrP mice were inoculated intracerebrally with 20 μl supernatant. Following inoculation, clinical status of the mice was monitored daily to assess the onset of neurological signs. The brains of diseased mice were removed and stored at -80 °C for biochemical analysis or fixed for histopathology. For the fourth passage, eight diseased mice brains from the third passage were used to independently challenge to TgBoPrP mice.Brain tissues from BSE-affected animals were homogenized in nine volumes of phosphate buffered saline (PBS) using a multi-bead shocker (Yasui Kikai) and centrifuged at 1,000 × Three female 3-4-month-old Holstein calves were challenged intracerebrally with 1 ml of 10% brain homogenate of BSE-SW-affected TgBoPrP mice, as described previouslySc fragments (PrPcore). PK digestion was terminated with 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride . The samples were mixed with equal volumes of 2-butanol:methanol mixture (5:1) and centrifuged at 20,000 × g for 10 min. The pellets were resuspended in gel-loading buffer containing 2% (w/v) SDS, and were then boiled for 10 min before western blotting. After PK treatment, some samples were deglycosylated with N-glycosidase F , following the manufacturer’s instruction.Brain tissues were homogenized in PBS  using a multi-bead shocker. The brain homogenate (125 μl) was mixed with an equal volume of buffer containing 4% (w/v) Zwittergent 3–14 , 1% (w/v) Sarkosyl, 100 mM NaCl, and 50 mM Tris-HCl (pH 7.6), and incubated with 6.25 μl of 40 mg/ml collagenase solution. The samples  were then subjected to PK (Roche Diagnostic) digestion at 37 °C for 1 h to detect PK-resistant PrPThe following monoclonal antibodies (mAbs) against PrP were used in this study: P4 (R-Biopharm), 6H4 (Prionics), SAF84 (SPI-bio), and F99/97.6.1 (VMRD). MAb P4 recognizes amino acid residues 101–107 of bovine PrP sequenceSamples were separated by SDS-PAGE and blotted electrically onto a PVDF membrane (Millipore). The blotted membrane was incubated with mAbs P4, 6H4, and SAF84 at RT for 1 h. MAb binding was detected by horseradish peroxidase-conjugated anti-mouse IgG. Signals were developed with a chemiluminescent substrate .Sc immunohistochemistry (IHC), sections were incubated with mAbs SAF84 or F99/97.61, followed by incubation with anti-mouse universal immunoperoxidase polymer (Histofine Simple Stain MAX-PO (M), Nichirei) as the secondary antibody, and visualized using 3,3′-diaminobenzidine tetrachloride as the chromogen. Finally, the sections were counterstained with hematoxylin. Paraffin embedded tissue (PET) blot was performed as described previouslyHistopathological analysis of TgBoPrP mice and cattle was performed according to a previously described method1016Sc concentration and western blot analysis were carried out as described above. Conformational stability was examined using mAbs 6H4 and SAF84. Denaturation curves were obtained by densitometric analysis using Fluorochem software (Alpha Innotech Co.). GdnHCl concentration at half maximal denaturation ([GdnHCl]1/2) was used as a measure of the relative conformational stability of PrPSc. [GdnHCl]1/2 was calculated based on the denaturation curves.Conformation stability assay was performed according to a previously described method with minor modificationHow to cite this article: Masujin, K. et al. Emergence of a novel bovine spongiform encephalopathy (BSE) prion from an atypical H-type BSE. Sci. Rep.6, 22753; doi: 10.1038/srep22753 (2016).


Sc) was detected in the brain and/or lymphoid tissues during the lifespan of mice that were asymptomatic subclinical carriers, indicating that wild-type mice were susceptible to sheep-passaged L-BSE. The morphological characteristics of the PrPSc of sheep-passaged L-BSE included florid plaques that were distributed mainly in the cerebral cortex and hippocampus of subsequent passaged mice. The PrPSc glycoform profiles of wild-type mice infected with sheep-passaged L-BSE were similar to those of the original isolate. The data indicate that sheep-passaged L-BSE has an altered host range and acquired transmissibility to wild-type mice.L-type bovine spongiform encephalopathy (L-BSE) is an atypical form of BSE that is transmissible to cattle and several lines of prion protein (PrP) transgenic mice, but not to wild-type mice. In this study, we examined the transmissibility of sheep-passaged L-BSE prions to wild-type mice. Disease-associated prion protein (PrP Sc) has higher (H-BSE) or lower (L-BSE) molecular mass than that of classical (C-) BSE [Bovine spongiform encephalopathy (BSE) was originally thought to be caused by a single prion strain, based on analysis of its biological and biochemical characteristics. However, since 2003, different pathological and molecular phenotypes of BSE  have been reported in approximately 90 cases worldwide, mainly in aged cattle. Currently, atypical BSE is classified into two groups depending on whether the proteinase K (PK)-resistant abnormal, disease-associated form of the prion protein (PrP(C-) BSE . The oriExperimentally, L-BSE prions have shown transmissibility by intracerebral challenge to cattle -6; bovinAll the experiments involving animals were performed with the approval of the Animal Ethics Committee and the Animal Care and Use Committee of the National Institute of Animal Health . Fifteen 3-week-old outbred ICR (CD-1) mice  were inoculated intracerebrally with 20 μL of 10% brain homogenates of Japanese L-BSE  passagedSc immunohistochemistry (IHC). Selected sections were stained with phenol Congo red and examined under a polarizing microscope, and the presence of amyloid was confirmed by observation of its characteristic dichroism [At necropsy, the left hemisphere and selected tissues including the lymphoid organs were removed and fixed in 10% buffered formalin containing 10% methanol. Formalin-fixed tissues were immersed in 98% formic acid for 60 min to reduce the infectivity, embedded in paraffin, and sectioned for histological evaluation by staining with hematoxylin and eosin (HE), and using PrPichroism .After appropriate epitope retrieval with either hydrate autoclaving or a combination of enzymatic and chemical treatment, IHC was carried out using the monoclonal antibodies (mAbs) 2G11, 12F10, or SAF84  followed by an anti-mouse, universal horseradish peroxidase (HRP)-conjugated polymer (Nichirei Histofine Simple Stain MAX-PO (M); Nichirei Biosciences Inc., Tokyo, Japan) as the secondary antibody, and visualized with 3,3′-diaminobenzedine tetrachloride as the chromogen, as previously described . Finallyg for 10 min. The pellets were mixed with a gel-loading buffer containing 2% sodium dodecyl sulfate, boiled for 5 min before electrophoresis, and loaded onto a 12% polyacrylamide gel. The separated proteins were transferred onto an Immobilon-P polyvinylidene fluoride membrane (EMD Millipore). The blotted membranes were incubated with the mAbs SAF84 and T2 [The right hemisphere and spleen were removed and stored at −80 °C until use. The tissues (200 ± 10 mg) were homogenized at 20% concentration (w/v) in a buffer containing 100 mM NaCl and 50 mM Tris–HCl (pH 7.6). The homogenates (250 μL) were mixed with an equal volume of detergent buffer containing 4% (w/v) Zwittergent 3–14 , 1% (w/v) Sarkosyl, 100 mM NaCl, and 50 mM Tris–HCl (pH 7.6), and treated with 6.25 μL of 40 mg/mL collagenase. The sample was then digested with 40 μg/mL proteinase K  and the digestion was terminated using 2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride . After PK treatment, the samples were mixed with a 2-butanol: methanol mixture (5:1) and centrifuged at 20000 × 4 and T2  followedSc in wild-type mice inoculated with L-BSE/sheep, the brains of wild-type mice [To compare the molecular features of PrPype mice ,23, cattype mice ,9, or shype mice  inoculatThe mAbs 2G11, SAF84, and T2 were found to react with bovine, ovine, and mouse PrP; the mAb 12F10 reacts with both bovine and ovine, but not mouse PrP.To compare the phenotypic features of L-BSE inoculum before and after passage in sheep, reverse transmission to bovinized PrP expressing transgenic (TgBoPrP) mice was carried out to examine whether the L-BSE/sheep would maintain its specific strain properties in bovids. TgBoPrP mice were kindly provided by Dr Prusiner . The susSc bands were analyzed using Instat3 software  and ImageReader software  after background subtraction, respectively; p values <0.05 were considered statistically significant.Incubation periods expressed as mean ± standard deviation of the mean (SD) and signal intensities of PK-resistant PrPSc signal was detected by WB, IHC, or both techniques in the brain from 1 case at 710 dpi, and in lymphoid tissues  from 9 of 15 mice after 200 dpi after the first passage. Positive IHC results in a mouse were typically composed of sparse granular deposits in some areas of the brain such as the vestibular nucleus or dorsal motor nucleus of the vagal nerve, midbrain tegmentum, hypothalamus, medial preoptic nucleus, and habenular nucleus  (Table Sc-positive spleens of mice was used for subsequent second passages in ICR mice (Table n = 6) was statistically unchanged on second passage, without any clinical signs and a 100% transmission rate. Histopathological examination showed prominent florid and non-florid plaques with large confluent vacuoles in the cerebral cortex and hippocampus of all inoculated mice  and 152 ± 2 days (n = 24) at primary and secondary passage, respectively. In contrast, the mean incubation period of L-BSE/sheep to TgBoPrP mice was 249 ± 28 (n = 5) and 269 ± 17 days (n = 13) at primary and secondary passage, respectively, which indicated a significantly longer incubation period compared to L-BSE/cattle, the original isolate  showed a lack of any evidence of transmission during their lifespan by both WB and IHC tests  that are affected by L-BSE/sheep [Sc accumulation has not been detected in cattle [Sc in lymphoid tissues does not necessarily result in neuroinvasion [Sc deposits accumulated in the brain. This result indicates that L-BSE/sheep may not have pathogenicity and virulence towards wild-type mice. The discrimination and typing of prion strains are dependent on biological characteristics that include clinical signs, incubation times, histopathological vacuolar lesion profiles, PrPSc deposition patterns in the brain, and the biochemical features of PrPSc over several mouse passages [A transmission study performed on experimental animals is a useful approach for the isolation and characterization of prion strains. In the present study, we were able to demonstrate the transmissibility of L-BSE/sheep to wild-type mice across a species barrier during their lifespans, in the absence of clinical signs of the disease. According to the protein-only hypothesis, PrPhenotype . The PrPE prions . Since t passage . Interesn cattle ,28 or shn cattle  affectedn cattle . In addin cattle . Thus, sn cattle ,31. Conspassages ,33.Sc has been reported in cross-species transmission of sporadic Creutzfeldt-Jakob disease (CJD) to humanized transgenic mice [Sc, but gained the transmissibility to wild-type mice. Although the key event that determines the shift in the size of PK-resistant PrPSc remains unknown, it seems likely that the molecular characteristics may be influenced by the host-environment factors rather than the nature of the prion strain. The specific strain features of L-BSE observed in TgBoPrP mice affected with L-BSE/cattle [In the results of WB tests, a strain-specific molecular signature such as the glycoform pattern was conserved in the transmitted wild-type mice. The occurrence of size shifts in PK-digested PrPnic mice , variantnic mice , and hamnic mice . The traE/cattle ,37 or L-Sc in the brain. The first two possibilities were completely ruled out by the authors [Sc was undetectable in the brain of these mice, a faint positive signal was identified in one RIII mouse that showed biochemical characteristics of PrPSc identical to those of C-BSE-infected mice by WB analyses.To the best of our knowledge, the transmission of L-BSE/cattle to wild-type mice has only been reported in one study, and even in this case the L-BSE prions were converted to a C-BSE-like prion using serial passages, and had indistinguishable phenotypic traits compared with mouse-passaged C-BSE . A pheno authors . The las authors , should  authors . No tranFour L-BSE isolates from Japan , GermanyC and the PrPSc of inocula result in species barriers to the cross-species transmission of prions [Finally, the results indicate that L-BSE/sheep is transmissible to wild-type mice and it results in low virulence compared with C-BSE . In contf prions . In this


Bovine spongiform encephalopathy (BSE) in cattle and variant Creutzfeldt–Jakob disease in humans have previously been shown to be caused by the same strain of transmissible spongiform encephalopathy agent. It is hypothesized that the agent spread to humans following consumption of food products prepared from infected cattle. Despite evidence supporting zoonotic transmission, mouse models expressing human prion protein (HuTg) have consistently shown poor transmission rates when inoculated with cattle BSE. Higher rates of transmission have however been observed when these mice are exposed to BSE that has been experimentally transmitted through sheep or goats, indicating that humans may potentially be more susceptible to BSE from small ruminants. Here we demonstrate that increased transmissibility of small ruminant BSE to HuTg mice was not due to replication of higher levels of infectivity in sheep brain tissue, and is instead due to other specific changes in the infectious agent. C (Sc) is present in infected tissues of the central nervous system (CNS) and lymphoreticular system, and propagates via a templated seeding mechanism, where the abnormal protein binds to and converts normal PrPC into the abnormal conformation  diseases  are fatal, infectious neurodegenerative diseases of animals, and include bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and Creutzfeldt–Jakob disease (CJD) in humans. TSEs are unusual diseases, as the infectious agent is thought to be composed solely of a misfolded conformer of the normal host glycoprotein PrPC . The abnormation .Ruminant TSEs were for many years thought to be of low risk to humans due to the lack of association between incidence of natural scrapie in sheep and CJD in humans. However, in 1996, a new variant of CJD (vCJD) was identified in humans that presented with a different clinical and pathological phenotype (n = 177) compared with the size of the UK population that may have been exposed to the agent in the 1980s and 1990s, and the predicted number of possible silent carriers in the general population, evaluated from analysis of anonymized lymphoid tissue  in the presence of human PrP, and it was hoped these models would provide evidence on routes and risks of exposure of humans to caBSE. Considerable variation in susceptibility was observed between different transgenic lines with various constructs and protein expression levels. However, in general, transmission rates in mice overexpressing human 129M-PrP were found to be low (0–30 %) (Although sheep and goats were exposed to the same sources of contaminated feed (but in lower quantities) as cattle during the BSE epidemic  into groups of 12 Bov6 transgenic mice. The 10−1 homogenate was also inoculated into control 129/Ola mice and HuMM transgenic mice, to ensure the exp-shBSE inoculum used was able to cause the same TSE pathology in HuMM transgenic mice as observed previously. All animals were monitored daily and scored weekly for signs of clinical disease and culled either due to intercurrent illness or at a pre-defined clinical end point. Tissue from each mouse was analysed post-mortem for presence of TSE-associated vacuolation  for caBSE  and 105.0 ID50 g−1 tissue for exp-shBSE . The difference of less than half a log between titres was not statistically significant . Several survivors were found to show TSE pathology (vacuolation and/or PrP deposition) when culled due to intercurrent illness after the last clinical positive animal in each group. Titre calculations based on TSE pathology alone also showed no significant difference between caBSE and exp-shBSE titres . While no signs of disease were observed in HuMM mice inoculated with caBSE  and Romney sheep (105.4 ID50 g−1) in the study by Gonzalez et al. (2009) were equivalent to those measured here in Bov6 mice (105.0 ID50 g−1). Incubation times for 10−1 dilution in RIII mice (389 days)  more efficiently than sheep scrapie   were sim89 days) . More regh sheep , indicatTogether, these data show that the increased transmissibility of exp-shBSE and exp-gtBSE to mice expressing human 129M PrP is not due solely to PrP sequence, infectivity level or the convertibility of human 129M-PrP by ovine/caprine prions. The increased transmissibility of exp-shBSE in HuTg mice must therefore be due to other specific changes in the agent and its ability to interact with the host. These may include differences in cellular factors such as routing of the inoculum and trafficking of PrP-res, or the composition and interaction of specific conformers of abnormal PrP in the inoculum. The identification of such factors that are critical for zoonosis may aid in the assessment of future TSE agent outbreaks and the associated risk to humans from these isolates. As long as TSE agents remain in the environment, the opportunity for cross-species transmission to occur remains. Our data have shown that such cross-species transmission events can have major effects on the host range and transmissibility of different TSE agents. In particular we have established that the emergence or re-emergence of BSE in small ruminants could have serious public health implications. There is therefore a strong requirement to continue surveillance for new emerging TSE strains, or for the appearance of old strains in new hosts, and quickly assess the risks of zoonosis.


PRNP). A proline to leucine substitution at PrP residue 102 (P102L) is classically associated with Gerstmann-Sträussler-Scheinker (GSS) disease but shows marked clinical and neuropathological variability within kindreds that may be caused by variable propagation of distinct prion strains generated from either PrP 102L or wild type PrP. To-date the transmission properties of prions propagated in P102L patients remain ill-defined. Multiple mouse models of GSS have focused on mutating the corresponding residue of murine PrP (P101L), however murine PrP 101L, a novel PrP primary structure, may not have the repertoire of pathogenic prion conformations necessary to accurately model the human disease. Here we describe the transmission properties of prions generated in human PrP 102L expressing transgenic mice that were generated after primary challenge with ex vivo human GSS P102L or classical CJD prions. We show that distinct strains of prions were generated in these mice dependent upon source of the inoculum (either GSS P102L or CJD brain) and have designated these GSS-102L and CJD-102L prions, respectively. GSS-102L prions have transmission properties distinct from all prion strains seen in sporadic and acquired human prion disease. Significantly, GSS-102L prions appear incapable of transmitting disease to conventional mice expressing wild type mouse PrP, which contrasts strikingly with the reported transmission properties of prions generated in GSS P102L-challenged mice expressing mouse PrP 101L. We conclude that future transgenic modeling of IPDs should focus exclusively on expression of mutant human PrP, as other approaches may generate novel experimental prion strains that are unrelated to human disease.Inherited prion disease (IPD) is caused by autosomal-dominant pathogenic mutations in the human prion protein (PrP) gene ( Inherited prion disease (IPD) is caused by pathogenic mutations in the human prion protein (PrP) gene leading to the formation of lethal prions in the brain. To-date the properties of prions causing IPD and their similarities to prions causing other forms of human prion disease remain ill-defined. In the present study we have investigated the properties of prions seen in patients with Gerstmann-Sträussler-Scheinker (GSS) disease associated with the substitution of leucine for proline at amino acid position 102 (GSS P102L). We examined the ability of these prions to infect transgenic mice expressing human mutant 102L PrP, human wild-type PrP or wild-type mice. We found that GSS-102L prions have properties distinct from other types of human prions by showing that they can only infect transgenic mice expressing human PrP carrying the same mutation. Mice expressing wild-type human PrP or wild-type mouse PrP were entirely resistant to infection with GSS-102L prions. We conclude that accurate modeling of inherited prion disease requires the expression of authentic mutant human PrP in transgenic models, as other approaches may generate results that do not mirror the human disease. Prion diseases are a closely related group of neurodegenerative conditions which affect both humans and animals ,2. They C) to alternative isoforms designated PrPSc  sugtant PrP ,27. Herer clone) –50 minorutations –56.www.nc3rs.org.uk/ARRIVE/).Storage and biochemical analyses of post-mortem human brain samples and transmission studies to mice were performed with written informed consent from patients with capacity to give consent. Where patients were unable to give informed consent, assent was obtained from their relatives in accordance with UK legislation and Codes of Practice. Samples were stored and used in accordance with the Human Tissue Authority Codes of Practice and in line with the requirements of the Human Tissue Authority licence held by UCL Institute of Neurology. This study was performed with approval from the National Hospital for Neurology and Neurosurgery and the UCL Institute of Neurology Joint Research Ethics Committee —REC references: 03/N036, 03/N038 and 03/N133. Work with mice was performed under approval and licence granted by the UK Home Office  Act 1986; Project Licence number 70/6454) and conformed to University College London institutional and ARRIVE guidelines  designated Tg(HuPrP102L 129M+/+Prnpo/o)-27 mice (102LL Tg27) [129M transgene array and murine PrP null alleles (Prnpo/o) designated Tg(HuPrP129M+/+Prnpo/o)-35 congenic (129MM Tg35c) were derived by subjecting previously described 129MM Tg35 mice [129V transgene array and murine PrP null alleles (Prnpo/o) designated Tg(HuPrP129V+/+Prnpo/o)-152 congenic (129VV Tg152c) were derived by subjecting previously described 129VV Tg152 mice [Transgenic mice homozygous for a human PrPLL Tg27)  have beeg35 mice ,57 to co152 mice ,39,42 to2, and intra-cerebrally inoculated into the right parietal lobe with 30 μl of 1% (w/v) brain homogenate prepared in Dulbecco’s phosphate buffered saline lacking Ca2+ or Mg2+ ions (D-PBS). All mice were thereafter examined daily for clinical signs of prion disease. Mice were killed if they exhibited any signs of distress or once a diagnosis of prion disease was established. At post-mortem brains from inoculated mice were removed, divided sagittally with half frozen and half fixed in 10% buffered formol saline.Strict bio-safety protocols were followed. Inocula were prepared, using disposable equipment for each inoculum, in a microbiological containment level 3 laboratory and inoculations performed within a class 1 microbiological safety cabinet. Ten mice per group from three transgenic lines, 102LL Tg27, 129MM Tg35c, 129VV Tg152c and FVB/N wild type mice were inoculated with a panel of prion isolates, all previously passaged in 102LL Tg27 transgenic mice and therefore adapted to human 102L PrP. The primary inocula comprised human brain homogenates from three IPD P102L patients, one sporadic CJD patient and three iatrogenic CJD patients. Diagnosis of all cases had been neuropathologically confirmed. The genotype of each mouse was confirmed by PCR of DNA prior to inclusion and all mice were uniquely identified by sub-cutaneous transponders. Disposable cages were used and all cage lids and water bottles were also uniquely identified by transponder and remained with each cage of mice throughout the incubation period. Care of the mice was according to institutional and ARRIVE guidelines. Mice were anaesthetised with a mixture of halothane and OPrnpo/o mice against α or β PrP as described elsewhere [1 with an epitope spanning residues 142–153 of human PrP [2b with an epitope spanning residues 93–105 of human PrP [Anti-PrP monoclonal antibodies ICSM 18 and ICSM 35 were supplied by D-Gen Ltd, London, UK. ICSM antibodies were raised in lsewhere . ICSM 18uman PrP . ICSM 35uman PrP ,59. ICSMuman PrP .Brain homogenates (10% (w/v)) were prepared in D-PBS and aliquots analysed in duplicate with or without proteinase K digestion  by electrophoresis and immunoblotting as described previously ,61. Duplwww.ventana.com). Visualization was accomplished with diaminobenzidine staining. Bright field photographs were taken on an ImageView digital camera (www.soft-imaging.de) and composed with Adobe Photoshop.Fixed brain was immersed in 98% formic acid for 1 h and paraffin wax embedded. Serial sections of 4 μm nominal thickness were pre-treated with Tris-Citrate EDTA buffer for antigen retrieval . PrP dep


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 Disease phenotype analysis in all three mouse lines indicated that most phenotypic parameters  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.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.The online version of this article (doi:10.1186/s40478-015-0194-2) contains supplementary material, which is available to authorized users. Sc or prion) of a cellular protein (PrPC), spongiosis and gliosis. Even though scrapie has been endemic in sheep and goats for centuries it is believed not to have represented a risk for human health [Classical scrapie (subsequently referred to as scrapie) and Bovine Spongiform Encephalopathy (BSE) are transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative disorders of animals and man which are characterised by the deposition of a misfolded isoform  and Western blot.The AR and HR of all isolates are presented in Table The IP data are expressed as days post inoculation (dpi) and are based on TSE positive mice that exhibited clinical signs of neurological disease Table . GeneralBoth 100% scrapie and 100% BSE generated high AR. Compared to other inocula of the same TSEs prepared from terminally ill animals and bioassayed in our laboratory the IP values observed in this study were similar to the lower end of the IP data range indicating that that each source represented an origin of high infectivity ,31.Due to the high infectivity of all inocula there were at least five clinically and pathologically positive mice in each mouse group. Therefore it was feasible to construct LP from each mouse group Figure .Figure 2In the RIII mice the LP from mixtures appear to be more akin to the LP of the 100% scrapie source Figure a. HoweveThe LP differences between the 100% BSE and 100% scrapie sources were more profound in C57BL/6 and VM mice, where they resulted in different LPs. The LPs that were generated from the various mixtures in these two mouse lines aligned more closely to the LP from the 100% scrapie control than the 100% BSE isolate.In addition application of cluster analysis based on the lesion scores, showed that in all three mouse lines the BSE/scrapie mixtures grouped together with the 100% scrapie control which was distinct from the 100% BSE control  were indistinguishable from those that were challenged with the scrapie control, showing a high molecular weight unglycosylated band compared to the BSE control  in different neuroanatomical areas, located at four coronal levels  in all BSE/scrapie challenged mice. This assessment, which was performed blind, revealed that this pattern was more abundant in mice that were challenged with mixtures with a higher BSE concentration, both in terms of quantity in the different neuroanatomical areas . The presence of BSE-associated punctate deposits in specific neuroanatomical areas allocated in the four coronal levels was recorded. At each coronal level the number of neuroanatomical areas affected by BSE-associated punctate deposits was used to predict the presence and percentage of BSE in the original inoculum. The test predictions were compared to the actual values and the associations between test and true status were not random (p < 0.0001 by Fisher’s exact test). The sensitivity, specificity and accuracy of the approach were 78% (62–89), 91% (75–98) and 84% (73–91); values in brackets denote 95% confidence intervals. The predictions were more accurate in the high BSE content inocula (50% and 10%) whilst the low BSE content mixtures (2% and 1%) proved more difficult to predict accurately.Based on the previous finding that the BSE-associated punctate PrPin vitro and used them as a substitute to co-infected tissue. Both BSE and scrapie inocula were prepared from animals at the terminal stage of the disease to ensure maximum infectivity. However, measurement of PrPSc levels indicated that the concentration of PrPSc in the scrapie source was at least two logs higher compared to the BSE material. Conversely, it has been shown the TSE infectivity cannot be accurately predicted from quantitative laboratory test results [Despite the significant progress and development of tests that can discriminate BSE from scrapie in a single infection scenario, limited studies have been conducted that address the performance of the discriminatory tests in cases where BSE and scrapie exist in the same animal. Ideally, the best possible experimental materials should derive from sheep co-infected experimentally with natural scrapie and BSE sources via natural routes of inoculation i.e. under conditions that would reflect most closely a naturally occurring co-infection. To the best of our knowledge, however, materials from such experiments are not widely available. Therefore we used the best possible alternative i.e. we produced BSE and scrapie mixtures  results . A simil results . In thatBased on AR, IP and LP our data suggest that all three mouse lines that were challenged with BSE/scrapie mixtures exhibited a scrapie phenotype, and it was not possible to identify any BSE attributes in the mixtures. However, AR, IP and LP data are considered to be less reliable parameters with lower discriminatory power since they are based on average values derived from a group of animals ,38.Each mouse that was challenged with either a BSE/scrapie mixture or 100% scrapie control in the current study, exhibited a stable scrapie IHC pattern which is usually isolated from ARQ/ARQ scrapie cases ,31,38 alSc levels between the scrapie and BSE sources could be a possible explanation of this result.The Western blots from C57BL/6 and RIII mice that were challenged with BSE/scrapie mixtures showed that even with the highest BSE fraction (50%) the scrapie characteristics dominated and any BSE signal was undetectable within the resolution limits of this method. Using IHC it was not feasible to identify any BSE characteristics in either C57BL/6 or RIII mice that were challenged with BSE/scrapie mixtures so there is complete agreement between IHC and Western blot data regarding these two mouse lines. The difference in the PrPCollectively the above data indicate that in the C57BL/6 and RIII mice that were challenged with BSE/scrapie mixtures only scrapie phenotypic traits were identified, suggesting that either scrapie propagated preferentially at the expense of BSE or, that although BSE also propagated, it had a recessive phenotype that was not observed in the mice. The similarity of results obtained from C57BL/6 and RIII mice is likely explained by the fact that these two mouse lines share the same PrP sequence (Prnp-a), that influences the phenotypic features of TSE strains -40.Propagation of a mixture of strains is supported by the data from the VM mice where, while the overall IHC characteristics of scrapie prevailed, a subtle but distinct BSE feature was observed in mice challenged with BSE/scrapie mixtures, and that feature was more prominent in the mice that were exposed to high content of BSE (10% or 50%) compared to those that received a low content of BSE (1% or 2%). Although we were able to identify a BSE associated trait that could potentially be used to detect BSE in the presence of scrapie using VM mice, the current study shows that overall scrapie can dominate the phenotype of the disease even if BSE prions propagate in the background, while some phenotypic aspects of BSE may be evident. This disparity in the properties of different mouse lines could be attributed to the different PrP sequences between VM and C57BL/6 or RIII mice. However, the VM data show evidence that BSE and scrapie can co-exist in the same animal, although the presence of BSE may be masked by a dominant scrapie phenotype.Sc concentration. However, the content of PrPSc in an inoculum may not always correlate directly with infectivity [Sc levels in ovine BSE can be lower compared to cattle BSE infectivity titres between the two samples can be similar [Great care must be taken, however, when attempting to make any generalizations from this data. Firstly the infectious titre of each of the two sources that were used to produce the mixtures was not evaluated therefore the mixtures did not reflect titre ratios but simply volumetric fractions. Although, according to some researchers, it would have been preferable to use titrated material this would have prolonged the length of the study by at least another two years. In addition, titres are the function of the strain/ host combination used  and cross reading between different strain/host systems is not always informative or appropriate. Therefore it is questionable whether titre matching provides an optimal approach to co-infection experiments. It could also be argued that the mixtures could have been based on PrPectivity . Further similar -43. Neve similar ,32. The  similar ,38,44. Iin vitro [Sc within brain mixes with 100% specificity and 97% sensitivity when BSE agent was diluted into scrapie-infected brain homogenates at 1% (vol/vol) [Since the initiation of the current study transgenic mouse lines which are more sensitive to specific animal TSEs have been introduced and validated. Such lines are relatively new, but promising, as intermediate phenotypes have been identified after challenge with BSE/scrapie mixtures prepared in vitro ,46. Howevol/vol) . CompareThe fact that we were able to identify a marker to detect BSE against a specific scrapie strain background in VM mice does not mean that this is an acceptable test for BSE detection in co-infected material. It does, however, give further understanding of the potential and limitations of such an approach, which is relevant to the retrospective interpretation of historical data in this field, in particular to prevent the over-interpretation of ‘absence of BSE’ conclusions. It must be noted that the selected marker is subtle and only an experienced observer could interpret it correctly and consistently in the high content BSE inocula (10% or 50%), as in the low content BSE mixtures (1% and 2%) the levels of the marker dropped appreciably. Therefore, even using this IHC model, low BSE levels on a scrapie background may remain undetected. Theoretically similar caveats may apply to the natural host ie small ruminants, where there is a range of strain and host genotype combinations. Consequently, the diagnostic methodology applied in surveillance schemes may fail to identify co-infection cases where BSE is present. In the view of the authors, under these circumstances, the current policy of eradicating or minimizing scrapie rather than attempting to detect and manage BSE separately remains, scientifically and financially, the most feasible option to prevent BSE from entering the food chain via small ruminants.In conclusion, our data suggest that by applying immunohistochemistry to detect different PrPSc types in the brains of mice challenged with BSE/scrapie mixtures it is possible to detect BSE in a BSE/Scrapie co-infection scenario. We also provide evidence that in principle co-infection of BSE and scrapie cannot be excluded at least in an experimental model.All animal procedures were performed in compliance with the Animal  Act 1986 under license issued by the UK Home Office (license number PPL70/5155), and were approved by the local ethics committee.Initially a scrapie and an ovine BSE homogenate were prepared. The scrapie inoculum, hereafter referred to as 100% scrapie, was made using a pool of brains collected between 1996 and 1999 from confirmed cases of natural scrapie representing the most frequent PrP genotypes affected by scrapie Table . The oviEach inoculum was administered into three panels of wild type mice namely C57BL/6, RIII and VM. Each panel consisted of 20 mice and each mouse received 20 μl of homogenate intracerebrally and 100 μl intraperitonially. Intracerebral inoculations were performed under general anaesthesia using a 25 G hypodermic needle attached to a 0.5 ml insulin syringe. A plastic sheath was inserted along the needle allowing approximately 5 mm of free end of the needle to be exposed to ensure that the inoculum was deposited at similar depth in each animal and to minimize tissue injury. The point of entry was approximately 3 mm dorsal to a point lying halfway between the eye and the base of the ear. Insertion into the CNS was achieved by gently rotating the syringe along its axis at a right angle with respect to the skull surface.Mice were housed in standard mouse cages and were monitored for clinical signs of disease by experienced animal attendants. Mice were euthanized after exhibiting clinical signs of TSE for two consecutive weeks or having received scores of “definitely affected” in 2 out of 3 consecutive weeks unless the clinical progression of the disease was so rapid that animals had to be euthanized on welfare grounds. Mice that did not show clinical signs of TSE were allowed to live until they were euthanized on welfare grounds due to other conditions (intercurrent deaths).At postmortem the brain of each mouse was removed sectioned along a parasagittal plane into two parts. One third of the brain was stored at −80°C for further bioassay or biochemical studies and two thirds were immersed in buffered formalin for 72 hours at room temperature.After fixation was completed each brain was cut at 4 different coronal points at the level of medulla (including the cerebellum), midbrain, thalamus (including hippocampus and overlying cortex) and frontal cortex . Each coronal segment was embedded in paraffin wax and histological sections (3 μm thick) from each level were mounted on the same positively charged slide for interpretation.Slides were stained with haematoxylin and eosin according to standard methodology . TSE diaSc was achieved using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate for 20 minutes at RT and measuring colour development at 450 nm using a reference filter at 620 nm (Perkin Elmer Envision 2104 multi-label reader).Inocula samples were analysed using a modified version of the IDEXX Herdchek BSE – Scrapie EIA kit . Inocula samples were serially diluted in equal volumes of TSE negative ovine brain homogenate (10% (w/v)) prepared in IDEXX kit homogenisation buffer. Diluted samples were treated as if they were normal test homogenates ) and assayed following manufacturer’s instructions. Briefly, diluted samples were mixed 4:5 with kit plate diluent, distributed (100 μl) to the test plate and incubated for 180 minutes at ambient room temperature (RT). After washing, bound sample was incubated with conditioning buffer for 10 minutes at RT, washed a second time and incubated with horseradish peroxidase conjugated anti-PrP antibody (SRB-CC) for 90 minutes at RT. After final wash visualisation of bound PrPAll samples, solid tissue and inocula, were analysed using the Bio-Rad TeSeE™ Western blot .Sheep and mouse brain (solid) tissues were analysed according to manufacturer’s instructions. Briefly, 20% (w/v) tissue homogenates were treated with proteinase K before alcohol precipitation. After centrifugation, pellets were solubilised in Laemmli buffer. Following extraction proteins were separated on 12% Bis/Tris gels then electrotransferred to PVDF membrane. After blocking the membrane with 5% bovine serum albumin (BSA) proteins were labelled with anti-prion antibodies Sha31 (epitope: ovine amino acid sequence 145–152) and 12B2 (epitope: ovine amino acid sequence 97–115). Visualisation was achieved using enhanced chemiluminesence (ECL) reagents .Prior to biochemical analysis of inoculum samples by Bio-Rad TeSeE™ Western blot, aliquots of finalised inocula were centrifuged at 350000 g for 30 minutes. The resulting pellet was re-homogenised at 20% (w/v) in Bio-Rad kit homogenisation buffer and the resultant homogenate treated as described above.Prior to biochemical analysis of inoculum samples by Bio-Rad TeSeE™ Western blot, aliquots of finalised inocula were centrifuged at 350000 g for 30 minutes. The resulting pellet was re-homogenised at 20% (w/v) in Bio-Rad kit homogenisation buffer and the resultant homogenate treated as described above.Sc was detected using the rabbit polyclonal anti-PrP antibody Rb486 according to standard methodology [Samples from clinically and histopathologically positive mice were further analysed with IHC as described previously ,31. PrPSCluster analysis was performed using Statistica (Version 10). For all other statistical analyses the STATA (Version 10) or the Graph Pad Prism (Version 6.04) programmes were used.