RESUMEN
Ovine scrapie can be transmitted via environmental reservoirs. A pool of ovine scrapie isolates were incubated on soil for one day or thirteen months and eluted prion was used to challenge tg338 mice transgenic for ovine PrP. After one-day incubation on soil, two PrP(Sc) phenotypes were present: G338 or Apl338ii. Thirteen months later some divergent PrP(Sc) phenotypes were seen: a mixture of Apl338ii with either G338 or P338, and a completely novel PrP(Sc) deposition, designated Cag338. The data show that prolonged ageing of scrapie prions within an environmental matrix may result in changes in the dominant PrP(Sc) biological/biochemical properties.
Asunto(s)
Reservorios de Enfermedades/veterinaria , Proteínas PrPSc/metabolismo , Scrapie/metabolismo , Animales , Animales Modificados Genéticamente , Ratones , Ovinos , SueloRESUMEN
INTRODUCTION: Scrapie and bovine spongiform encephalopathy (BSE) are transmissible spongiform encephalopathies (TSEs) which naturally affect small and large ruminants respectively. However, small ruminants, which are susceptible to BSE under experimental conditions, have been exposed to the same or similar contaminated food additives as cattle. To date two natural cases of BSE in small ruminants have been reported. As a result surveillance projects, combined with appropriate control measures, have been established throughout the European Union (EU) to minimize the overall incidence of small ruminant TSEs. Although BSE can be differentiated from classical scrapie (subsequently referred to as scrapie) if appropriate discriminatory tests are applied, the value of these tests in BSE/scrapie co-infection scenarios has not been evaluated fully. Mouse bioassay is regarded as the gold standard regarding differentiation of distinct TSE strains and has been used as to resolve TSE cases were laboratory tests produced equivocal results. However, the ability of this method to discriminate TSE strains when they co-exist has not been examined systematically. To address this issue we prepared in vitro mixtures of ovine BSE and scrapie and used them to challenge RIII, C57BL/6 and VM mice. RESULTS: Disease phenotype analysis in all three mouse lines indicated that most phenotypic parameters (attack rates, incubation periods, lesion profiles and Western blots) were compatible with scrapie phenotypes as were immunohistochemistry (IHC) data from RIII and C57BL/6 mice. However, in VM mice that were challenged with BSE/scrapie mixtures a single BSE-associated IHC feature was identified, indicating the existence of BSE in animals where the scrapie phenotype was dominant. CONCLUSIONS: We conclude that wild type mouse bioassay is of limited value in detecting BSE in the presence of scrapie particularly if the latter is in relative excess.
Asunto(s)
Bioensayo/métodos , Ratones Endogámicos , Fenotipo , Enfermedades por Prión/fisiopatología , Scrapie/fisiopatología , Especificidad de la Especie , Animales , Western Blotting , Bovinos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Enfermedades por Prión/metabolismo , Scrapie/metabolismoRESUMEN
Several transgenic mouse models have been developed which facilitate the transmission of chronic wasting disease (CWD) of cervids and allow prion strain discrimination. The present study was designed to assess the susceptibility of the prototypic mouse line, Tg(CerPrP)1536(+/-), to bovine spongiform encephalopathy (BSE) prions, which have the ability to overcome species barriers. Tg(CerPrP)1536(+/-) mice challenged with red deer-adapted BSE resulted in 90% to 100% attack rates, and BSE from cattle failed to transmit, indicating agent adaptation in the deer.
Asunto(s)
Ciervos/metabolismo , Modelos Animales de Enfermedad , Encefalopatía Espongiforme Bovina/metabolismo , Ratones , Priones/metabolismo , Enfermedad Debilitante Crónica/metabolismo , Animales , Bovinos , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Susceptibilidad a Enfermedades , Encefalopatía Espongiforme Bovina/patología , Encefalopatía Espongiforme Bovina/transmisión , Femenino , Masculino , Ratones Transgénicos , Especificidad de la Especie , Enfermedad Debilitante Crónica/patología , Enfermedad Debilitante Crónica/transmisiónRESUMEN
Mouse-adapted transmissible spongiform encephalopathy (TSE) strains are routinely distinguished based on reproducible disease characteristics in a given mouse line following inoculation via a consistent route. We investigated whether different administration routes (oral, intragastric (i.g.) and intracerebral (i.c.)) can alter the disease characteristics in IM mice after serial dilution of a stabilized mouse-adapted bovine spongiform encephalopathy (BSE) strain (301V). In addition, the infectivity of distal ileum and mesenteric lymph nodes (ln) sampled at three time points (35 days postinoculation (dpi), 70 dpi and terminal disease) after i.g. inoculation of 301V strain was assessed in mice by i.c. challenge. Strain characteristics were assessed according to standard methodology and PrP(Sc) immunohistochemistry deposition patterns. Mean incubation periods were prolonged following oral or i.g. inoculations compared to the i.c. route. Lesion profiles following i.c. challenges were elevated compared to i.g. and oral routes although vacuolation in the dorsal medulla was consistently high irrespective of the route of administration. Nevertheless, the same PrP(Sc) deposition pattern was associated with each route of administration. Distal and mesenteric ln infectivity was detected as early as 35 dpi and displayed consistent lesion profiles and PrP(Sc) deposition patterns. Our data suggest that although 301V retained its properties, some phenotypic parameters were affected by the route of inoculation. We conclude that bioassay data should be interpreted carefully and should be standardized for route of inoculation.
Asunto(s)
Encefalopatía Espongiforme Bovina/patología , Encefalopatía Espongiforme Bovina/transmisión , Priones/administración & dosificación , Priones/patogenicidad , Animales , Encéfalo/metabolismo , Encéfalo/patología , Bovinos , Modelos Animales de Enfermedad , Encefalopatía Espongiforme Bovina/metabolismo , Íleon/metabolismo , Íleon/patología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Ratones , Ratones Endogámicos , Proteínas PrPSc/metabolismo , Factores de TiempoRESUMEN
Development of transgenic mouse models expressing heterologous prion protein (PrP) has facilitated and advanced in vivo studies of prion diseases affecting humans and animals. Here, novel transgenic mouse lines expressing a chimaeric murine/ovine (Mu/Ov) PrP transgene, including amino acid residues alanine, histidine and glutamine at ovine polymorphic codons 136, 154 and 171 (A136H154Q171), were generated to provide a means of assessing the susceptibility of the ovine AHQ allele to ruminant prion diseases in an in vivo model. Transmission studies showed that the highest level of transgene overexpression, in Tg(Mu/OvPrP(AHQ))EM16 (EM16) mice, conferred high susceptibility to ruminant prions. Highly efficient primary transmission of atypical scrapie from sheep was shown, irrespective of donor sheep PrP genotype, with mean incubation periods (IPs) of 154178 days post-inoculation (p.i.), 100% disease penetrance and early Western blot detection of protease-resistant fragments (PrP(res)) of the disease-associated isoform, PrP(Sc), in EM16 brain from 110 days p.i. onwards. EM16 mice were also highly susceptible to classical scrapie and bovine spongiform encephalopathy (BSE), with mean IPs 320 and 246 days faster, respectively, than WT mice. Primary passage of atypical scrapie, classical scrapie and BSE showed that the PrP(res) profiles associated with disease in the natural host were faithfully maintained in EM16 mice, and were distinguishable based on molecular masses, antibody reactivities and glycoform percentages. Immunohistochemistry was used to confirm PrP(Sc) deposition in brain sections from terminal phase transmissible spongiform encephalopathy-challenged EM16 mice. The findings indicate that EM16 mice represent a suitable bioassay model for detection of atypical scrapie infectivity and offer the prospect of differentiation of ruminant prions.
Asunto(s)
Ratones Transgénicos/metabolismo , Enfermedades por Prión/metabolismo , Enfermedades por Prión/transmisión , Priones/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Rumiantes/metabolismo , Regulación hacia Arriba , Animales , Encéfalo/metabolismo , Encéfalo/patología , Bovinos , Encefalopatía Espongiforme Bovina/metabolismo , Encefalopatía Espongiforme Bovina/transmisión , Humanos , Ratones , Priones/genética , Proteínas Recombinantes de Fusión/genética , Rumiantes/genética , Scrapie/metabolismo , Scrapie/transmisión , Ovinos , TransgenesRESUMEN
According to traditional murine bioassay methodology, prions must be serially passaged within a new host before a stable phenotype, and therefore a strain, can be assigned. Prions often transmit with difficulty from one species to another; a property termed the transmission barrier. Transgenic mouse lines that over express prion protein (PrP) genes of different species can circumvent the transmission barrier but serial passages may still be required, particularly if unknown strains are encountered. Here we sought to investigate whether protein misfolding cyclic amplification (PMCA), an in-vitro method of PrP(Sc) replication, could be used to replace serial passage of VRQ/VRQ classical scrapie isolates undergoing strain typing in ovine transgenic tg338 mice. Two classical scrapie field isolates that do not readily transmit to wild-type mice underwent bioassay in tg338 mice pre- and post- PMCA and the phenotype of disease in inoculated mice was compared. For one of the sources investigated, the PMCA product gave rise to the same disease phenotypes in tg338 mice as traditional bioassay, as indicated by lesion profile, IHC analysis and Western blot, whilst the second source produced phenotypic characteristics which were not identical with those that arose through traditional bioassay. These data show that differences in the efficiency of PMCA as a strain-typing tool may vary between ovine classical scrapie isolates and therefore suggest that the ability of PMCA to replace serial passage of classical scrapie in tg338 mice may depend on the strain present in the initial source.
Asunto(s)
Bioensayo/métodos , Proteínas PrPSc/clasificación , Scrapie/metabolismo , Animales , Genotipo , Técnicas para Inmunoenzimas , Inmunohistoquímica , Ratones , Ratones Transgénicos , Tipificación Molecular , Fenotipo , Proteínas PrPSc/genética , Pliegue de Proteína , Isoformas de Proteínas , Scrapie/genética , Ovinos , TransgenesRESUMEN
In individual animals affected by transmissible spongiform encephalopathies, different disease phenotypes can be identified which are attributed to different strains of the agent. In the absence of reliable technology to fully characterise the agent, classification of disease phenotype has been used as a strain typing tool which can be applied in any host. This approach uses standardised data on biological parameters, established for a single host, to allow comparison of different prion sources. Traditionally prion strain characterisation in wild type mice is based on incubation periods and lesion profiles after the stabilisation of the agent into the new host which requires serial passages. Such analysis can take many years, due to prolonged incubation periods. The current study demonstrates that the PrPSc patterns produced by one serial passage in wild type mice of bovine or ovine BSE were consistent, stable and showed minimal and predictable differences from mouse-stabilised reference strains. This biological property makes PrPSc deposition pattern mapping a powerful tool in the identification and definition of TSE strains on primary isolation, making the process of characterisation faster and cheaper than a serial passage protocol. It can be applied to individual mice and therefore it is better suited to identify strain diversity within single inocula in case of co-infections or identify strains in cases where insufficient mice succumb to disease for robust lesion profiles to be constructed. The detailed description presented in this study provides a reference document for identifying BSE in wild type mice.
Asunto(s)
Encefalopatía Espongiforme Bovina/genética , Proteínas PrPSc/genética , Enfermedades por Prión/veterinaria , Animales , Encéfalo/patología , Bovinos , Encefalopatía Espongiforme Bovina/etiología , Encefalopatía Espongiforme Bovina/patología , Inmunohistoquímica/veterinaria , Ratones , Adhesión en Parafina/veterinaria , Proteínas PrPSc/metabolismo , Enfermedades por Prión/etiología , Enfermedades por Prión/genética , Enfermedades por Prión/patología , Estudios Retrospectivos , OvinosRESUMEN
Mouse bioassay can be readily employed for strain typing of naturally occurring transmissible spongiform encephalopathy cases. Classical scrapie strains have been characterised historically based on the established methodology of assessing incubation period of disease and the distribution of disease-specific vacuolation across the brain following strain stabilisation in a given mouse line. More recent research has shown that additional methods could be used to characterise strains and thereby expand the definition of strain "phenotype". Here we present the phenotypic characteristics of classical scrapie strains isolated from 24 UK ovine field cases through the wild-type mouse bioassay. PrPSc immunohistochemistry (IHC), paraffin embedded tissue blots (PET-blot) and Western blotting approaches were used to determine the neuroanatomical distribution and molecular profile of PrPSc associated with each strain, in conjunction with traditional methodologies. Results revealed three strains isolated through each mouse line, including a previously unidentified strain. Moreover IHC and PET-blot methodologies were effective in characterising the strain-associated types and neuroanatomical locations of PrPSc. The use of Western blotting as a parameter to define classical scrapie strains was limited. These data provide a comprehensive description of classical scrapie strain phenotypes on isolation through the mouse bioassay that can provide a reference for further scrapie strain identification.
Asunto(s)
Bioensayo/métodos , Proteínas PrPSc/clasificación , Scrapie/metabolismo , Animales , Western Blotting/métodos , Encéfalo/metabolismo , Inmunohistoquímica/métodos , Ratones , Ratones Endogámicos C57BL , Tipificación Molecular/métodos , Adhesión en Parafina/métodos , Proteínas PrPSc/genética , Scrapie/genética , OvinosRESUMEN
It is widely accepted that abnormal forms of the prion protein (PrP) are the best surrogate marker for the infectious agent of prion diseases and, in practice, the detection of such disease-associated (PrP(d)) and/or protease-resistant (PrP(res)) forms of PrP is the cornerstone of diagnosis and surveillance of the transmissible spongiform encephalopathies (TSEs). Nevertheless, some studies question the consistent association between infectivity and abnormal PrP detection. To address this discrepancy, 11 brain samples of sheep affected with natural scrapie or experimental bovine spongiform encephalopathy were selected on the basis of the magnitude and predominant types of PrP(d) accumulation, as shown by immunohistochemical (IHC) examination; contra-lateral hemi-brain samples were inoculated at three different dilutions into transgenic mice overexpressing ovine PrP and were also subjected to quantitative analysis by three biochemical tests (BCTs). Six samples gave 'low' infectious titres (106·5 to 106·7 LD50 g⻹) and five gave 'high titres' (108·¹ to ≥ 108·7 LD50 g⻹) and, with the exception of the Western blot analysis, those two groups tended to correspond with samples with lower PrP(d)/PrP(res) results by IHC/BCTs. However, no statistical association could be confirmed due to high individual sample variability. It is concluded that although detection of abnormal forms of PrP by laboratory methods remains useful to confirm TSE infection, infectivity titres cannot be predicted from quantitative test results, at least for the TSE sources and host PRNP genotypes used in this study. Furthermore, the near inverse correlation between infectious titres and Western blot results (high protease pre-treatment) argues for a dissociation between infectivity and PrP(res).
Asunto(s)
Encefalopatía Espongiforme Bovina , Priones/genética , Priones/patogenicidad , Scrapie , Animales , Bioensayo/métodos , Encéfalo , Bovinos , Femenino , Inmunohistoquímica , Masculino , Ratones , OvinosRESUMEN
Two cases of unusual transmissible spongiform encephalopathy (TSE) were diagnosed on the same farm in ARQ/ARQ PrP sheep showing attributes of both bovine spongiform encephalopathy (BSE) and scrapie. These cases, UK-1 and UK-2, were investigated further by transmissions to wild-type and ovine transgenic mice. Lesion profiles (LP) on primary isolation and subpassage, incubation period (IP) of disease, PrP(Sc) immunohistochemical (IHC) deposition pattern and Western blot profiles were used to characterize the prions causing disease in these sheep. Results showed that both cases were compatible with scrapie. The presence of BSE was contraindicated by the following: LP on primary isolation in RIII and/or MR (modified RIII) mice; IP and LP after serial passage in wild-type mice; PrP(Sc) deposition pattern in wild-type mice; and IP and Western blot data in transgenic mice. Furthermore, immunohistochemistry (IHC) revealed that each case generated two distinct PrP(Sc) deposition patterns in both wild-type and transgenic mice, suggesting that two scrapie strains coexisted in the ovine hosts. Critically, these data confirmed the original differential IHC categorization that these UK-1 and UK-2 cases were not compatible with BSE.