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1.
Sci Rep ; 11(1): 11931, 2021 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-34099797

RESUMEN

To understand the possible role of mixed-prion infections in disease presentation, the current study reports the co-infection of sheep with bovine spongiform encephalopathy (BSE) and scrapie. The bovine BSE agent was inoculated subcutaneously into sheep with ARQ/ARQ or VRQ/ARQ PRNP genotypes either at the same time as subcutaneous challenge with scrapie, or three months later. In addition, VRQ/VRQ sheep naturally infected with scrapie after being born into a scrapie-affected flock were challenged subcutaneously with BSE at eight or twenty one months-of-age. Sheep were analysed by incubation period/attack rate, and western blot of brain tissue determined the presence of BSE or scrapie-like PrPSc. Serial protein misfolding cyclic amplification (sPMCA) that can detect very low levels of BSE in the presence of an excess of scrapie agent was also applied to brain and lymphoreticular tissue. For VRQ/ARQ sheep challenged with mixed infections, scrapie-like incubation periods were produced, and no BSE agent was detected. However, whilst ARQ/ARQ sheep developed disease with BSE-like incubation periods, some animals had a dominant scrapie western blot phenotype in brain, but BSE was detected in these sheep by sPMCA. In addition, VRQ/VRQ animals challenged with BSE after natural exposure to scrapie had scrapie-like incubation periods and dominant scrapie PrPSc in brain, but one sheep had BSE detectable by sPMCA in the brain. Overall, the study demonstrates for the first time that for scrapie/BSE mixed infections, VRQ/ARQ sheep with experimental scrapie did not propagate BSE but VRQ/VRQ sheep with natural scrapie could propagate low levels of BSE, and whilst BSE readily propagated in ARQ/ARQ sheep it was not always the dominant PrPSc strain in brain tissue. Indeed, for several animals, a dominant scrapie biochemical phenotype in brain did not preclude the presence of BSE prion.


Asunto(s)
Enfermedades de los Bovinos/diagnóstico , Coinfección/diagnóstico , Encefalopatía Espongiforme Bovina/diagnóstico , Scrapie/diagnóstico , Enfermedades de las Ovejas/diagnóstico , Animales , Encéfalo/metabolismo , Bovinos , Enfermedades de los Bovinos/metabolismo , Coinfección/genética , Coinfección/metabolismo , Encefalopatía Espongiforme Bovina/complicaciones , Encefalopatía Espongiforme Bovina/metabolismo , Genotipo , Fenotipo , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Scrapie/complicaciones , Scrapie/metabolismo , Ovinos , Enfermedades de las Ovejas/genética , Enfermedades de las Ovejas/metabolismo
3.
J Neurol Neurosurg Psychiatry ; 90(4): 424-427, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30355606

RESUMEN

OBJECTIVE: To assess whether the involvement of the peripheral nervous system (PNS) belongs to the phenotypic spectrum of sporadic Creutzfeldt-Jakob disease (sCJD). METHODS: We examined medical records of 117 sCJDVV2 (ataxic type), 65 sCJDMV2K (kuru-plaque type) and 121 sCJDMM(V)1 (myoclonic type) subjects for clinical symptoms, objective signs and neurophysiological data. We reviewed two diagnostic nerve biopsies and looked for abnormal prion protein (PrPSc) by western blotting and real-time quaking-induced conversion (RT-QuIC) in postmortem PNS samples from 14 subjects. RESULTS: Seventy-five (41.2%) VV2-MV2K patients, but only 11 (9.1%) MM(V)1, had symptoms or signs suggestive of PNS involvement occurring at onset in 18 cases (17 VV2-MV2K, 9.3%; and 1 MM(V)1, 0.8%) and isolated in 6. Nerve biopsy showed a mixed predominantly axonal and demyelinating neuropathy in two sCJDMV2K. Electromyography showed signs of neuropathy in half of the examined VV2-MV2K patients. Prion RT-QuIC was positive in all CJD PNS samples, whereas western blotting detected PrPSc in the sciatic nerve in one VV2 and one MV2K. CONCLUSIONS: Peripheral neuropathy, likely related to PrPSc deposition, belongs to the phenotypic spectrum of sCJDMV2K and VV2 and may mark the clinical onset. The significantly lower prevalence of PNS involvement in typical sCJDMM(V)1 suggests that the PNS tropism of sCJD prions is strain dependent.


Asunto(s)
Síndrome de Creutzfeldt-Jakob/epidemiología , Encefalopatía Espongiforme Bovina/epidemiología , Enfermedades del Sistema Nervioso Periférico/epidemiología , Nervio Ciático/patología , Nervio Sural/patología , Ataxia , Síndrome de Creutzfeldt-Jakob/complicaciones , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/fisiopatología , Enfermedades Desmielinizantes , Electromiografía , Encefalopatía Espongiforme Bovina/complicaciones , Encefalopatía Espongiforme Bovina/metabolismo , Encefalopatía Espongiforme Bovina/fisiopatología , Humanos , Mioclonía , Nervios Periféricos/patología , Nervios Periféricos/fisiopatología , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/patología , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Proteínas Priónicas/metabolismo
6.
J Alzheimers Dis ; 59(1): 329-337, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28598840

RESUMEN

BACKGROUND: The polymorphism at codon 129 of the prion protein gene (PRNP) and the PrPSc types 1 and 2 belong to a molecular classification of sporadic Creutzfeldt-Jakob disease (sCJD) that correlates well with the clinical and neuropathological phenotype of sCJD. OBJECTIVE: The aim of the study was to perform the first detailed evaluation of neuropsychological deficits in a large group of definite sCJD patients with known molecular subtype. METHODS: We analyzed neuropsychological symptoms in a cohort of 248 sCJD patients with known M129 V polymorphism of PRNP and prion protein type. RESULTS: Neuropsychological symptoms were very frequent in our patients (96%) and occurred as early as in the first third of the disease course. Besides amnesia and impaired attention (89% each), frontal lobe syndrome (75%), aphasia (63%), and apraxia (57%) were the most common neuropsychological deficits. There was no statistically significant difference with regard to frequency of neuropsychological symptoms between the subtypes. In MV2 and VV2 patients, the onset of neuropsychological symptoms was significantly later than in all other subtypes. CONCLUSION: We provide the first detailed analysis of neuropsychological symptoms in a large group of sCJD patients with known M129 V genotype and prion protein type. We suggest that the rate of progression of neuropsychological symptoms is subtype-specific. These data may improve the diagnosis in atypical sCJD subtypes.


Asunto(s)
Trastorno por Déficit de Atención con Hiperactividad/etiología , Trastornos del Conocimiento/etiología , Síndrome de Creutzfeldt-Jakob/complicaciones , Encefalopatía Espongiforme Bovina/complicaciones , Trastornos del Humor/etiología , Trastornos Psicomotores/etiología , Proteínas 14-3-3/líquido cefalorraquídeo , Adulto , Anciano , Anciano de 80 o más Años , Afasia/etiología , Apraxias/etiología , Trastorno por Déficit de Atención con Hiperactividad/diagnóstico , Trastornos del Conocimiento/diagnóstico , Trastornos del Conocimiento/diagnóstico por imagen , Estudios de Cohortes , Síndrome de Creutzfeldt-Jakob/diagnóstico por imagen , Síndrome de Creutzfeldt-Jakob/epidemiología , Síndrome de Creutzfeldt-Jakob/genética , Encefalopatía Espongiforme Bovina/diagnóstico por imagen , Encefalopatía Espongiforme Bovina/epidemiología , Encefalopatía Espongiforme Bovina/genética , Femenino , Alemania/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Pruebas Neuropsicológicas , Proteínas Priónicas/genética , Adulto Joven , Proteínas tau/líquido cefalorraquídeo
8.
Neuropathology ; 35(4): 336-42, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25783686

RESUMEN

We describe an atypical neuropathological phenotype of sporadic Creutzfeldt-Jakob disease in a 76-year-old man. The clinical symptoms were characterized by progressive dementia, gait ataxia, rigidity and urinary incontinence. The disease duration was 6 weeks. MRI did not show prominent atrophy or hyperintensities in cortical areas, striatum or thalamus. Biomarker examination of the cerebrospinal fluid deviated from that seen in pure Alzheimer's disease. Triphasic waves in the EEG were detected only later in the disease course, while 14-3-3 assay was positive. PRNP genotyping revealed methionine homozygosity (MM) at codon 129. Neuropathology showed classical CJD changes corresponding to the MM type 1 cases. However, a striking feature was the presence of abundant kuru-type plaques in the white matter. This rare morphology was associated with neuropathological signs of intranuclear inclusion body disease and advanced stage of argyrophilic grain disease. These alterations did not show correlation with each other, thus seemed to develop independently. This case further highlights the complexity of neuropathological alterations in the ageing brain.


Asunto(s)
Encéfalo/patología , Síndrome de Creutzfeldt-Jakob/patología , Encefalopatía Espongiforme Bovina/patología , Kuru/patología , Tauopatías/patología , Sustancia Blanca/patología , Anciano , Síndrome de Creutzfeldt-Jakob/complicaciones , Síndrome de Creutzfeldt-Jakob/genética , Encefalopatía Espongiforme Bovina/complicaciones , Encefalopatía Espongiforme Bovina/genética , Humanos , Cuerpos de Inclusión Intranucleares/patología , Kuru/complicaciones , Masculino , Metionina/genética , Fenotipo , Tauopatías/complicaciones
9.
PLoS One ; 9(8): e104343, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25090610

RESUMEN

Obesity has become one of the largest public health challenges worldwide. Recently, certain bacterial and viral pathogens have been implicated in the pathogenesis of obesity. In the present study, we retrospectively analyzed clinical data, plasma samples and post-mortem tissue specimens derived from a risk assessment study in bovine spongiform encephalopathy (BSE)-infected female cynomolgus monkeys (Macaca fascicularis). The original study design aimed to determine minimal infectious doses after oral or intracerebral (i.c.) infection of macaques to assess the risk for humans. High-dose exposures resulted in 100% attack rates and a median incubation time of 4.7 years as described previously. Retrospective analyses of clinical data from high-dosed macaques revealed that foodborne BSE transmission caused rapid weight gain within 1.5 years post infection (ß = 0.915; P<0.0001) which was not seen in age- and sex-matched control animals or i.c. infected animals. The rapid-onset obesity was not associated with impaired pancreatic islet function or glucose metabolism. In the early preclinical phase of oral transmission associated with body weight gain, prion accumulation was confined to the gastrointestinal tract. Intriguingly, immunohistochemical findings suggest that foodborne BSE transmission has a pathophysiological impact on gut endocrine cells which may explain rapid weight gain. To our knowledge, this is the first experimental model which clearly demonstrates that foodborne pathogens can induce obesity.


Asunto(s)
Encefalopatía Espongiforme Bovina/complicaciones , Enfermedades Transmitidas por los Alimentos/fisiopatología , Obesidad/etiología , Priones/patogenicidad , Animales , Bovinos , Encefalopatía Espongiforme Bovina/metabolismo , Encefalopatía Espongiforme Bovina/fisiopatología , Encefalopatía Espongiforme Bovina/transmisión , Femenino , Humanos , Macaca fascicularis , Obesidad/metabolismo , Obesidad/fisiopatología , Primates , Priones/genética , Aumento de Peso/fisiología
10.
J Med Case Rep ; 8: 269, 2014 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-25098795

RESUMEN

INTRODUCTION: Slowly progressive cognitive decline is the most frequent initial manifestation in MM2-cortical-type sporadic Creutzfeldt-Jakob disease. Agraphia has never been noted in patients with this type of sporadic Creutzfeldt-Jakob disease, however, we report the case of a Japanese patient with sporadic Creutzfeldt-Jakob disease in whom agraphia of Kanji was an initial cardinal symptom. CASE PRESENTATION: A 59-year-old right-handed Japanese woman complained of agraphia of Kanji (Chinese characters) as an initial symptom. A neurological examination revealed mild word-finding difficulty, constructive disturbance, hyperreflexia in her jaw and lower limbs, and bilateral extensor plantar reflexes. An examination of her cerebrospinal fluid revealed increased levels of 14-3-3 and total tau proteins, and abnormal conformation of the proteinase K-resistant prion protein. Diffusion-weighted magnetic resonance imaging showed diffuse hyperintensity in bilateral cerebral cortices. Single-photon emission computed tomography scans revealed hypoperfusion in the left temporal lobe, bilateral parietal and occipital lobes. An analysis of the prion protein gene demonstrated no mutation with homozygous for methionine at the codon 129. We diagnosed our patient with sporadic Creutzfeldt-Jakob disease. Although a histological examination was not performed, it was assumed that our patient could be the MM2-cortical type according to the clinical findings and the elevated levels of 14-3-3 protein in her cerebrospinal fluid. The left posterior inferior temporal area, which was affected in our patient as a hypoperfusion area, is associated with selecting and recalling Kanji characters. CONCLUSIONS: Focal signs as an early symptom and hypoperfusion areas in sporadic Creutzfeldt-Jakob disease are critical to recognize initial brain lesions damaged by the proteinase K-resistant prion protein accumulation.


Asunto(s)
Agrafia/diagnóstico , Corteza Cerebral/patología , Síndrome de Creutzfeldt-Jakob/diagnóstico , Encefalopatía Espongiforme Bovina/diagnóstico , Agrafia/etiología , Corteza Cerebral/diagnóstico por imagen , Síndrome de Creutzfeldt-Jakob/complicaciones , Imagen de Difusión por Resonancia Magnética , Encefalopatía Espongiforme Bovina/complicaciones , Femenino , Humanos , Persona de Mediana Edad , Tomografía Computarizada de Emisión de Fotón Único
12.
Vet Pathol ; 50(5): 909-13, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23381923

RESUMEN

A 9-year-old cynomolgus monkey (Macaca fascicularis) infected orally with bovine spongiform encephalopathy (BSE) was presented for necropsy following euthanasia 4 years post infection (p.i.). This macaque R984 was exposed to a BSE dose that causes a simian form of variant Creutzfeldt-Jakob disease (vCJD) within 5 years p.i. in other macaques. All orally BSE-infected macaques developed a significant weight gain within the first 2 years p.i. compared with non-BSE-infected age- and sex-matched control animals, suggesting increased risk of type 2 diabetes (T2D). In contrast, macaque R984 developed rapid weight loss, hyperglycemia, and glucosuria and had to be euthanatized 4 years p.i. before clinical signs of vCJD. Pancreas histopathological evaluation revealed severe islet degeneration but, remarkably, no islet amyloid deposits were present. Immunostaining of pancreas sections for insulin and glucagon confirmed the loss of endocrine cells. In addition, prions were present in the adenohypophysis but not in other areas of the brain, indicating centripetal prion spread from the gut during the preclinical phase of BSE infection. Plasma glucose and insulin concentrations of macaque R984 became abnormal with age and resembled T2D. This unusual case of spontaneous T2D in the absence of islet amyloid deposits could have been due to early prion spread from the periphery to the endocrine system or could have occurred spontaneously.


Asunto(s)
Diabetes Mellitus Tipo 2/patología , Encefalopatía Espongiforme Bovina/complicaciones , Macaca fascicularis , Enfermedades de los Monos/patología , Obesidad/complicaciones , Animales , Glucemia/metabolismo , Peso Corporal , Bovinos , Diabetes Mellitus Tipo 2/etiología , Femenino , Técnicas Histológicas/veterinaria , Insulina/sangre , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Páncreas/patología , Factores Sexuales
13.
J Comp Pathol ; 147(1): 84-93, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22018806

RESUMEN

Squirrel monkeys (Saimiri sciureus) were infected experimentally with the agent of classical bovine spongiform encephalopathy (BSE). Two to four years later, six of the monkeys developed alterations in interactive behaviour and cognition and other neurological signs typical of transmissible spongiform encephalopathy (TSE). At necropsy examination, the brains from all of the monkeys showed pathological changes similar to those described in variant Creutzfeldt-Jakob disease (vCJD) of man, except that the squirrel monkey brains contained no PrP-amyloid plaques typical of that disease. Constant neuropathological features included spongiform degeneration, gliosis, deposition of abnormal prion protein (PrP(TSE)) and many deposits of abnormally phosphorylated tau protein (p-Tau) in several areas of the cerebrum and cerebellum. Western blots showed large amounts of proteinase K-resistant prion protein in the central nervous system. The striking absence of PrP plaques (prominent in brains of cynomolgus macaques [Macaca fascicularis] with experimentally-induced BSE and vCJD and in human patients with vCJD) reinforces the conclusion that the host plays a major role in determining the neuropathology of TSEs. Results of this study suggest that p-Tau, found in the brains of all BSE-infected monkeys, might play a role in the pathogenesis of TSEs. Whether p-Tau contributes to development of disease or appears as a secondary change late in the course of illness remains to be determined.


Asunto(s)
Encefalopatía Espongiforme Bovina/patología , Enfermedades de los Monos/patología , Saimiri , Tauopatías/patología , Proteínas tau/metabolismo , Animales , Bovinos , Síndrome de Creutzfeldt-Jakob/complicaciones , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patología , Modelos Animales de Enfermedad , Encefalopatía Espongiforme Bovina/complicaciones , Encefalopatía Espongiforme Bovina/metabolismo , Masculino , Tauopatías/complicaciones , Tauopatías/metabolismo
14.
Vet Rec ; 155(21): 659-66, 2004 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-15581140

RESUMEN

The clinical findings in 59 cows with bovine spongiform encephalopathy (BSE) were compared with those in 19 cattle that were submitted as BSE suspects but not confirmed by immunohistochemistry. Both groups were also compared with a control group of 20 healthy cows. Abnormalities in behaviour, temperament, mental status and activity, neurogenic disorders of gait and hyperreactivity to touch were frequently observed in the cattle with BSE. Not every animal with BSE displayed clinical signs in all these categories, and the severity of the signs was not always useful for differentiating them from the BSE suspects that were not confirmed by pathology. The neurological examination was better than passive observations for the clinical diagnosis of BSE. Tests of the animals' responses to sudden auditory, visual and tactile stimuli were very useful for distinguishing cases of BSE from unconfirmed BSE suspects if the cases did not display signs in all the categories.


Asunto(s)
Encefalopatía Espongiforme Bovina/diagnóstico , Animales , Conducta Animal , Peso Corporal , Encéfalo/patología , Bovinos , Encefalopatía Espongiforme Bovina/complicaciones , Encefalopatía Espongiforme Bovina/fisiopatología , Oftalmopatías/etiología , Oftalmopatías/veterinaria , Femenino , Locomoción , Masculino , Reino Unido
16.
Med Hypotheses ; 57(5): 555-60, 2001 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11735310

RESUMEN

The new prion diseases that have emerged in the last 15 years are bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (variant CJD). Although initially confined to the UK, these diseases have recently emerged in other European countries. The accepted cause of the human disease is that BSE spread from cattle to humans by the consumption of infected beef. However, the evidence that supports this is very thin. This article describes this evidence and lists a series of hypotheses concerning the cause of both BSE and variant CJD. The final hypothesis is based on recent evidence linking prion diseases to environmental factors including manganese. High environmental availability of manganese is associated with the prevalence of those prion diseases not linked to BSE. Therefore it is quite possible that BSE and variant CJD have emerged as a result of manganese-rich industrial pollution that has only occurred in the last century.


Asunto(s)
Síndrome de Creutzfeldt-Jakob/etiología , Encefalopatía Espongiforme Bovina/complicaciones , Contaminantes Ambientales/efectos adversos , Animales , Bovinos , Síndrome de Creutzfeldt-Jakob/inducido químicamente , Humanos , Productos de la Carne
18.
Infect Immun ; 67(12): 6591-5, 1999 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-10569779

RESUMEN

Bovine spongiform encephalopathy (BSE) is a neurological disorder, predominantly of British cattle, which belongs to the group of transmissible spongiform encephalopathies together with Creutzfeldt-Jakob disease (CJD), kuru, and scrapie. Autoantibodies to brain neurofilaments have been previously described in patients with CJD and kuru and in sheep affected by scrapie. Spongiform-like changes have also been observed in chronic experimental allergic encephalomyelitis, at least in rabbits and guinea pigs, and in these conditions autoantibodies to myelin occur. We report here that animals with BSE have elevated levels of immunoglobulin A autoantibodies to brain components, i.e., neurofilaments (P < 0.001) and myelin (P < 0.001), as well as to Acinetobacter calcoaceticus (P < 0.001), saprophytic microbes found in soil which have sequences cross-reacting with bovine neurofilaments and myelin, but there were no antibody elevations against Agrobacterium tumefaciens or Escherichia coli. The relevance of such mucosal autoantibodies or antibacterial antibodies to the pathology of BSE and its possible link to prions requires further evaluation.


Asunto(s)
Acinetobacter calcoaceticus/inmunología , Anticuerpos Antibacterianos/sangre , Autoanticuerpos/sangre , Encéfalo/inmunología , Encefalopatía Espongiforme Bovina/inmunología , Acinetobacter calcoaceticus/química , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Bovinos , Encefalopatía Espongiforme Bovina/complicaciones , Vaina de Mielina/inmunología , Proteínas de Neurofilamentos/química , Proteínas de Neurofilamentos/inmunología , Conejos
19.
Vet Rec ; 144(26): 715-7, 1999 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-10423814

RESUMEN

Fifteen cows with bovine spongiform encephalopathy (BSE) and 90 healthy cows were given xylazine intramuscularly at a dosage of 0.15 mg/kg bodyweight. The onset of sedation and of drooling was recorded, and the heart and respiratory rates and the systolic and diastolic blood pressure were measured every five minutes for 40 minutes. All the healthy cows but only five of the 15 cows with BSE became sedated, and the period between the administration of xylazine and the onset of sedation was twice as long in the cows with BSE than in the healthy cows (15.0 [7.5] and 7.6 [2.6] minutes). Throughout the observation period, the blood pressure of the cows with BSE was significantly higher than that of the healthy cows, and the blood pressure of the healthy cows, but not of the cows with BSE, decreased significantly towards the end of the observation period.


Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Presión Sanguínea/fisiología , Sedación Consciente/veterinaria , Encefalopatía Espongiforme Bovina/complicaciones , Xilazina/farmacología , Animales , Bovinos , Femenino , Frecuencia Cardíaca , Respiración
20.
Vox Sang ; 76(4): 203-8, 1999.
Artículo en Inglés | MEDLINE | ID: mdl-10394138

RESUMEN

Creutzfeldt-Jakob disease (CJD) has been transmitted in the laboratory and also by iatrogenic accident. However, research has failed to find evidence that its most common form (sporadic CJD) is a natural infection and, in particular, that there is a causal link with scrapie. Bovine spongiform encephalopathy (BSE) probably resulted from scrapie infection in cattle food. In the wake of the BSE epidemic, a novel clinico-pathological form of CJD has been recognized: new variant CJD (nvCJD). This paper reviews the relationship between nvCJD and BSE and presents the accumulated evidence supporting the view that nvCJD resulted from BSE contamination of human food.


Asunto(s)
Síndrome de Creutzfeldt-Jakob/etiología , Síndrome de Creutzfeldt-Jakob/genética , Encefalopatía Espongiforme Bovina/complicaciones , Encefalopatía Espongiforme Bovina/transmisión , Animales , Bovinos , Variación Genética , Humanos
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