RESUMEN
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
BACKGROUND: Histopathological examinations of brains from healthy pigs have revealed localised vacuolar changes, predominantly in the rostral colliculus, that are similar to the neuropil vacuolation featured in the transmissible spongiform encephalopathies and have been described in pigs challenged parenterally with the agent causing bovine spongiform encephalopathy (BSE). Feedstuff containing BSE-contaminated meat and bone meal (MBM) may have been fed to pigs prior to the ban of mammalian MBM in feed of farmed livestock in the United Kingdom in 1996, but there is no evidence of the natural occurrence of a transmissible spongiform encephalopathy (TSE) in the domestic pig. Furthermore, experimental transmission of BSE to pigs by the oral route has been unsuccessful. A study was conducted to investigate whether the localised vacuolar changes in the porcine brain were associated with a transmissible aetiology and therefore biologically significant. Two groups of ten pigs were inoculated parenterally with vacuolated rostral colliculus from healthy pigs either born before 1996 or born after 1996. Controls included ten pigs similarly inoculated with rostral colliculus from New Zealand-derived pigs and nine pigs inoculated with a bovine BSE brain homogenate. RESULTS: None of the pigs inoculated with rostral colliculus developed a TSE-like neurological disease up to five years post inoculation when the study was terminated, and disease-associated prion protein, PrPd, was not detected in the brains of these pigs. By contrast, eight of nine BSE-inoculated pigs developed neurological signs, two of which had detectable PrPd by postmortem tests. No significant histopathological changes were detected to account for the clinical signs in the PrPd-negative, BSE-inoculated pigs. CONCLUSION: The findings in this study suggest that vacuolation in the porcine rostral colliculus is not caused by a transmissible agent and is probably a clinically insignificant change. The presence of neurological signs in pigs inoculated with BSE without detectable PrPd raises the possibility that the BSE agent may produce a prion disease in pigs that remains undetected by the current postmortem tests.
Asunto(s)
Encéfalo/patología , Encefalopatía Espongiforme Bovina/transmisión , Enfermedades de los Porcinos/patología , Enfermedades de los Porcinos/transmisión , Animales , Western Blotting , Trasplante de Tejido Encefálico/veterinaria , Bovinos , Encefalopatía Espongiforme Bovina/patología , Femenino , Inmunohistoquímica , Masculino , Priones/aislamiento & purificación , Distribución Aleatoria , PorcinosRESUMEN
BACKGROUND: In the wake of the epidemic of bovine spongiform encephalopathy the British government established a flock of sheep from which scrapie-free animals are supplied to laboratories for research. Three breeds of sheep carrying a variety of different genotypes associated with scrapie susceptibility/resistance were imported in 1998 and 2001 from New Zealand, a country regarded as free from scrapie. They are kept in a purpose-built Sheep Unit under strict disease security and are monitored clinically and post mortem for evidence of scrapie. It is emphasised that atypical scrapie, as distinct from classical scrapie, has been recognised only relatively recently and differs from classical scrapie in its clinical, neuropathological and biochemical features. Most cases are detected in apparently healthy sheep by post mortem examination. RESULTS: The occurrence of atypical scrapie in three sheep in (or derived from) the Sheep Unit is reported. Significant features of the affected sheep included their relatively high ages (6 y 1 mo, 7 y 9 mo, 9 y 7 mo respectively), their breed (all Cheviots) and their similar PRNP genotypes (AFRQ/AFRQ, AFRQ/ALRQ, and AFRQ/AFRQ, respectively). Two of the three sheep showed no clinical signs prior to death but all were confirmed as having atypical scrapie by immunohistochemistry and Western immunoblotting. Results of epidemiological investigations are presented and possible aetiologies of the cases are discussed. CONCLUSION: By process of exclusion, a likely explanation for the three cases of atypical scrapie is that they arose spontaneously and were not infected from an exterior source. If correct, this raises challenging issues for countries which are currently regarded as free from scrapie. It would mean that atypical scrapie is liable to occur in flocks worldwide, especially in older sheep of susceptible genotypes. To state confidently that both the classical and atypical forms of scrapie are absent from a population it is necessary for active surveillance to have taken place.
Asunto(s)
Scrapie/patología , Animales , Western Blotting , Encéfalo/patología , Genotipo , Inmunohistoquímica/veterinaria , Priones/genética , Scrapie/genética , Ovinos , Reino UnidoRESUMEN
Atypical scrapie is a relatively recent discovery, and it was unknown whether it was a new phenomenon or whether it had existed undetected in the United Kingdom national flock. Before 1998, the routine statutory diagnosis of transmissible spongiform encephalopathy (TSE) in sheep relied on the presence of TSE vacuolation in the brainstem. This method would not have been effective for the detection of atypical scrapie. Currently, immunohistochemistry (IHC) and Western blot are commonly used for the differential diagnosis of classical and atypical scrapie. The IHC pattern of PrPd deposition in atypical scrapie is very different from that in classical scrapie using the same antibody. It is thus possible that because of a lack of suitable diagnostic techniques and awareness of this form of the disease, historic cases of atypical scrapie remain undiagnosed. Immunohistochemistry was performed on selected formalin-fixed, paraffin-embedded (FFPE) blocks of ovine brain from the Veterinary Laboratories Agency archives that were submitted for various reasons, including suspect neurological disorders, between 1980 and 1989. It was found that PrPd deposits in a single case were consistent with atypical scrapie. A method was developed to obtain a PrP genotype from FFPE tissues and was applied to material from this single case, which was shown to be AHQ/AHQ. This animal was a scrapie suspect from 1987, but diagnosis was not confirmed by the available techniques at that time.
Asunto(s)
Scrapie/epidemiología , Scrapie/patología , Animales , Ganglios Basales/patología , Cerebelo/patología , Cerebro/patología , Enfermedades de las Cabras/epidemiología , Enfermedades de las Cabras/patología , Cabras , Estudios Retrospectivos , Ovinos , Nervio Trigémino/patología , Reino Unido/epidemiologíaRESUMEN
Collaboration was established in 2001 to evaluate a commercially available immunohistochemistry assay kit for the detection of bovine spongiform encephalopathy (BSE) disease-associated prion protein in formic acid-treated formalin-fixed samples of bovine brain. The kit protocol was evaluated at the National Centre for Foreign Animal Diseases (Winnipeg, Canada) and the Veterinary Laboratories Agency (Weybridge, U.K.). The U.K. laboratory provided paraffin-embedded blocks of brainstem (medulla oblongata at the level of the obex) from 100 positive cases defined by clinical signs and histopathology, and 100 clinically suspect but BSE-negative samples defined by histopathology and immunohistochemistry with anti-PrP monoclonal antibody R145. The Canadian laboratory provided 400 blocks from surveillance cases defined as clinically suspect but negative by histopathology and immunohistochemistry with anti-PrP antibody 6H4. Consecutive sections from each block were cut and coded. Each set of 600 slides was immunolabeled and read in each laboratory. Evaluation parameters included estimates of diagnostic sensitivity and specificity and reproducibility of the results. The kit performed with 100% sensitivity, specificity, and reproducibility in spite of minor differences between the laboratories in brain sample areas, fixation and processing, and in the immunolabeling protocol. Although enzyme linked immunosorbent assays are widely used in high throughput surveillance programs, standardized protocols and reagents for manual immunohistochemistry provide a useful adjunct to surveillance efforts, particularly in laboratories testing small numbers of samples or using immunohistochemistry for confirmation and characterization of BSE cases.
Asunto(s)
Encefalopatía Espongiforme Bovina/diagnóstico , Inmunohistoquímica/veterinaria , Juego de Reactivos para Diagnóstico/veterinaria , Animales , Anticuerpos , Tronco Encefálico/inmunología , Tronco Encefálico/patología , Canadá , Bovinos , Inmunohistoquímica/métodos , Sensibilidad y Especificidad , Reino UnidoRESUMEN
BACKGROUND: Active surveillance for transmissible spongiform encephalopathies in small ruminants has been an EU regulatory requirement since 2002. A number of European countries have subsequently reported cases of atypical scrapie, similar to previously published cases from Norway, which have pathological and molecular features distinct from classical scrapie. Most cases have occurred singly in flocks, associated with genotypes considered to be more resistant to classical disease. Experimental transmissibility of such isolates has been reported in certain ovinised transgenic mice, but has not previously been reported in the natural host. Information on the transmissibility of this agent is vital to ensuring that disease control measures are effective and proportionate. RESULTS: This report presents the successful experimental transmission, in 378 days, of atypical scrapie to a recipient sheep of homologous genotype with preservation of the pathological and molecular characteristics of the donor. This isolate also transmitted to ovinised transgenic mice (Tg338) with a murine phenotype indistinguishable from that of Nor 98. CONCLUSION: This result strengthens the opinion that these cases result from a distinct strain of scrapie agent, which is potentially transmissible in the natural host under field conditions.
Asunto(s)
Encéfalo/metabolismo , Brotes de Enfermedades/veterinaria , Proteínas PrPSc/metabolismo , Scrapie/transmisión , Animales , Western Blotting/veterinaria , Encéfalo/patología , Europa (Continente)/epidemiología , Unión Europea , Predisposición Genética a la Enfermedad , Genotipo , Inmunohistoquímica/veterinaria , Proteínas PrPSc/administración & dosificación , Scrapie/epidemiología , Scrapie/genética , Scrapie/metabolismo , OvinosRESUMEN
To detect prion protein, brains from 5 cattle naturally affected with bovine spongiform encephalopathy (BSE) and 3 sheep naturally affected with scrapie were examined and compared with brains of normal cattle and sheep using a histoblot technique. The technique enabled the in situ distinctive detection of the cellular (PrP(C)) and abnormal (PrP(Sc)) isoforms of the prion protein. In BSE- or scrapie-affected brains, the Prp(C) signal decreased, especially in those areas where the PrP(Sc) signal was detected.
Asunto(s)
Encefalopatía Espongiforme Bovina/diagnóstico , Inmunohistoquímica/métodos , Priones/análisis , Scrapie/diagnóstico , Animales , Encéfalo/patología , Bovinos , Encefalopatía Espongiforme Bovina/patología , Isoformas de Proteínas/análisis , Scrapie/patología , Sensibilidad y Especificidad , OvinosRESUMEN
The paraffin-embedded tissue (PET) blot was modified for use as a tool to differentiate between classical scrapie and experimental bovine spongiform encephalopathy (BSE) in sheep. Medulla (obex) from 21 cases of classical scrapie and 6 cases of experimental ovine BSE were used to develop the method such that it can be used as a tool to differentiate between BSE and scrapie in the same way that differential immunohistochemistry (IHC) has been used previously. The differential PET blot successfully differentiated between all of the scrapie and ovine BSE cases. Differentiation was permitted more easily with PET blot than by differential IHC, with accurate observations possible at the macroscopic level. At the microscopic level, sensitivity was such that discrimination by the differential PET blot could be made with more confidence than with differential IHC in cases where the immunohistochemical differences were subtle. The differential PET blot makes use of harsh epitope demasking conditions, and, because of the differences in the way prion protein is processed in different prion diseases, it can serve as a new, highly sensitive method to discriminate between classical scrapie and experimental BSE in sheep.
Asunto(s)
Encefalopatía Espongiforme Bovina/diagnóstico , Adhesión en Parafina/veterinaria , Scrapie/diagnóstico , Enfermedades de las Ovejas/diagnóstico , Animales , Encéfalo/patología , Bovinos , Encefalopatía Espongiforme Bovina/patología , Immunoblotting/veterinaria , Sensibilidad y Especificidad , Ovinos , Enfermedades de las Ovejas/patologíaRESUMEN
BACKGROUND: Transmission of the prion disease bovine spongiform encephalopathy (BSE) occurred accidentally to cattle and several other mammalian species via feed supplemented with meat and bone meal contaminated with infected bovine tissue. Prior to United Kingdom controls in 1996 on the feeding of mammalian meat and bone meal to farmed animals, the domestic chicken was potentially exposed to feed contaminated with the causal agent of BSE. Although confirmed prion diseases are unrecorded in avian species a study was undertaken to transmit BSE to the domestic chicken by parenteral and oral inoculations. Transmissibility was assessed by clinical monitoring, histopathological examinations, detection of a putative disease form of an avian prion protein (PrP) in recipient tissues and by mouse bioassay of tissues. Occurrence of a progressive neurological syndrome in the primary transmission study was investigated by sub-passage experiments. RESULTS: No clinical, pathological or bioassay evidence of transmission of BSE to the chicken was obtained in the primary or sub-passage experiments. Survival data showed no significant differences between control and treatment groups. Neurological signs observed, not previously described in the domestic chicken, were not associated with significant pathology. The diagnostic techniques applied failed to detect a disease associated form of PrP. CONCLUSION: Important from a risk assessment perspective, the present study has established that the domestic chicken does not develop a prion disease after large parenteral exposures to the BSE agent or after oral exposures equivalent to previous exposures via commercial diets. Future investigations into the potential susceptibility of avian species to mammalian prion diseases require species-specific immunochemical techniques and more refined experimental models.
RESUMEN
Of all the species exposed naturally to the bovine spongiform encephalopathy (BSE) agent, the greater kudu (Tragelaphus strepsiceros), a nondomesticated bovine from Africa, appears to be the most susceptible to the disease. We present the results of mouse bioassay studies to show that, contrary to findings in cattle with BSE in which the tissue distribution of infectivity is the most limited recorded for any of the transmissible spongiform encephalopathies (TSE), infectivity in greater kudu with BSE is distributed in as wide a range of tissues as occurs in any TSE. BSE agent was also detected in skin, conjunctiva, and salivary gland, tissues in which infectivity has not previously been reported in any naturally occurring TSE. The distribution of infectivity in greater kudu with BSE suggests possible routes for transmission of the disease and highlights the need for further research into the distribution of TSE infectious agents in other host species.