La fantástica historia de la increíble prion / The fantastic story of the incredible prion

Resumen Desde el scrapie de la oveja a la encefalopatía espongiforme bovina y desde el kuru a la enfermedad de Creutzfeldt-Jakob, tenaces investigadores buscaron los misteriosos agentes de estos desórdenes neurológicos, hasta que Stanley Prusiner descubriera y describiera las priones en los ochenta, obteniendo el Premio Nobel en 1997. Pero, este no fue el final de esta fantástica historia de la increible proteina designada prion por Prusiner, porque ahora, la investigación en neurociencia ha encontrado proteínas prion-like jugando un importante papel en la génesis de la memoria a largo plazo.

Abstract From the scrapie of the sheep to the bovine spongiform encephalitis, and from the kuru to the Creutzfeldt-Jakob disease, tenacious investigators searched for the mysterious agent of these neurological disorders, till Stanley Prusiner discovered and described the prion in the eighties, wining the Nobel Prize in 1997. But this was not the end of the fantastic history of the incredible protein designed prion by Prusiner, because now the investigation on neuroscience has founded prion-like proteins playing an important role in the genesis of the long-term memory.

Re-transmissibility of mouse-adapted ME7 scrapie strain to ovine PrP transgenic mice

Scrapie is a mammalian transmissible spongiform encephalopathy or prion disease that predominantly affects sheep and goats. Scrapie has been shown to overcome the species barrier via experimental infection of other rodents. To confirm the re-transmissibility of the mouse-adapted ME7 scrapie strain to ovine prion protein (PrP) transgenic mice, mice of an ovinized transgenic mouse line carrying the Suffolk sheep PrP gene that contained the A₁₃₆ R₁₅₄ Q₁₇₁/ARQ allele were intracerebrally inoculated with brain homogenates obtained from terminally ill ME7-infected C57BL/6J mice. Herein, we report that the mouse-adapted ME7 scrapie strain was successfully re-transmitted to the transgenic mice expressing ovine PrP. In addition, we observed changes in the incubation period, glycoform profile, and pattern of scrapie PrP (PrP(Sc)) deposition in the affected brains. PrP(Sc) deposition in the hippocampal region of the brain of 2nd-passaged ovine PrP transgenic mice was accompanied by plaque formation. These results reveal that the mouse-adapted ME7 scrapie strain has the capacity to act as a template for the conversion of ovine normal monomeric precursors into a pathogenic form in ovine PrP transgenic mice. The change in glycoform pattern and the deposition of plaques in the hippocampal region of the brain of the 2nd-passaged PrP transgenic mice are most likely cellular PrP species dependent rather than being ME7 scrapie strain encoded.

Identification of single nucleotide polymorphisms in the prion protein gene in Santa Ines and Dorset sheep / Identificação de polimorfismos de nucleotídeos únicos em ovinos Santa Inês e Dorset através do gene da proteína priônica

Scrapie is a transmissible spongiform encephalopathy (TSE) that affects sheep and goats and results from accumulation of the abnormal isoform of a prion protein in the central nervous system. Resistance or susceptibility to the disease is dependent on several factors, including the strain of infecting agent, the degree of exposure, and the presence of single nucleotide polymorphisms (SNPs) in the prion protein gene. The most important polymorphisms are present in codons 136, 154, and 171. SNPs have also been identified in other codons, such as 118, 127, 141, 142, and 143. The objective of this study was to investigate the genotypic profile of Santa Ines (n=94) and Dorset (n=69) sheep and identify polymorphisms in the prion protein gene using real-time PCR techniques and sequencing. We analyzed SNPs in 10 different codons (127, 136, 138, 140, 141, 142, 143, 154, 171, and 172) in Santa Ines sheep. Classification of the flock into risk groups associated with scrapie revealed that approximately 68% of the Santa Ines herd was considered at moderate risk (group 3), and the most frequent haplotype was ARQ/ARQ (47.8%). For Dorset sheep, 42% of the herd was considered at moderate risk (group 3), 40% at low risk (group 2), and 12% at very low risk (group 1). These findings improve our understanding of the genotype breed and further highlight the importance of genotyping and identification of polymorphisms in Brazilian herds to assess their effects on potential infections upon exposure to the sheep prion.(AU)

Scrapie é uma encefalopatia espongiforme transmissível que afeta ovinos e caprinos, resultante do acúmulo de uma isoforma anormal da proteína priônica no sistema nervoso central. A resistência ou susceptibilidade está relacionada a diversos fatores, tais como, a cepa do agente infectante, o grau de exposição e o polimorfismo de nucleotídeo único (SNPs) do gene da proteína priônica. Os principais polimorfismos estão presentes nos códons 136, 154 e 171. SNPs também são identificadas em outros códons, tais como, 118, 127, 141, 142, e 143. O objetivo do trabalho foi descrever o perfil genotípico de um rebanho da raça Santa Inês (n=94) e um rebanho da raça Dorset (n=89) para identificar potenciais polimorfismos através da técnica de PCR em tempo real e sequenciamento. Os achados no rebanho Santa Inês indicaram a presença de polimorfismos de nucleotídeos únicos em 10 códons diferentes (127, 136, 138, 140, 141, 142, 143, 154, 171 e 172). A classificação do rebanho, quanto aos grupos de risco associados ao scrapie, relevaram que aproximadamente 68% dos ovinos foram considerados do grupo de risco moderado (grupo 3), onde o haplótipo mais frequente foi ARQ/ARQ (47,8%). Para os ovinos da raça Dorset, 42% do rebanho foi considerado do grupo de risco moderado (grupo 3), 40% do grupo de risco baixo (grupo 2) e 12% do grupo de risco muito baixo. Os dados encontrados contribuem para o conhecimento do genótipo das raças, destacando a importância de trabalhos que relatam os polimorfismos genéticos para a identificação de rebanhos brasileiros, bem como o seu impacto a infecções com exposição ao príon ovino.(AU)

Scrapie diagnosis in a goat and four Santa Inês sheep from the same herd in Brazil / Diagnóstico de scrapie em um caprino e quatro ovinos Santa Inês de um mesmo rebanho no Brasil

Scrapie is a fatal and progressive transmissible spongiform encephalopathy (TSE) of natural occurrence in sheep and goats. The suspicion of scrapie may be based on clinical signs; however, the detection of pathological features of the prionic protein (PrP) in target tissues is necessary to diagnose the disease. The presence of an abnormal protein form (PrPSc) in lymphoreticular and nervous tissues is an important characteristic in diagnosis. This paper reports a case of scrapie in a flock of 55 Suffolk crossbred sheep, 19 Santa Inês sheep and 21 goats in the Mato Grosso state, midwestern Brazil. The animals were euthanized after the confirmation of a scrapie case with clinical signs in a Suffolk sheep in the same farm...

Scrapie é uma encefalopatia espongiforme transmissível (EET) progressiva e fatal de ocorrência natural em ovinos e caprinos. A suspeita de scrapie é baseada nos sinais clínicos, porém a manifestação patológica da proteína priônica (PrP) nos tecidos-alvo é necessária para a confirmação da doença. A presença de uma forma anormal da proteína (PrPSc) em tecido linforreticular e tecido nervoso constitui uma característica importante para o diagnóstico. Este trabalho é o relato de um foco de scrapie ocorrido em rebanho com 55 ovinos mistos Suffolk, 21 caprinos e 19 ovinos Santa Inês, na região Centro-Oeste do Brasil. Os animais foram eutanasiados após a confirmação de um caso de scrapie com sinais clínicos em um ovino Suffolk nessa propriedade...

Prion protein gene polymorphisms and accumulation of pathogenic prion protein (PrPSc) in a herd with previously confirmed scrapie cases / Polimorfismos do gene da proteína priônica celular (PRNP) e distribuição da proteína priônica alterada (PrPSc) em rebanho com surto anterior de scrapie

Scrapie in sheep is associated with at least three polymorphisms in the prion protein gene (PRNP) on codons 136, 154, and 171. Countries where scrapie is endemic have been using breeding programs based on selection for the most resistant alleles. There are some PRNP genotyping data on sheep in Brazil, and scrapie has sporadically been observed since 1978. Paraná is the Brazilian state where most of the cases of scrapie have been diagnosed. A flock that had three clinical scrapie cases in 2003 and 2004 was genotyped (128 sheep: 53 pure Hampshire Down and 75 crossbred) and slaughtered (111 sheep: 47 pure Hampshire Down and 64 crossbred) in 2006. Samples of lymphoid and central nervous tissues were examined by immunohistochemistry (IHC) for altered prion protein (PrPSc). Six genotypes were detected in the 128 genotyped animals: ARR/ARQ was the most frequent (45.3%), followed by ARQ/ARQ (28.1%), ARR/ARR (14.1%), and ARQ/VRQ (8.6%). ARR/VRQ and ARQ/AHQ showed less than 2.5% genotype frequency. IHC identified 16 positive sheep. Palatine tonsil tissue had the highest percentage of reactive samples: 81.25% of the total positive samples. Of these 16 positive animals, nine (56.25%) had genotype ARR/ARQ, five (31.25%) had genotype ARQ/ARQ, and the remaining two (12.5%) had genotype ARQ/VRQ. All the positive animals were clinically healthy, and therefore represented 14.14% of pre-clinical cases of scrapie in this flock.

Scrapie nos ovinos está associada a pelo menos três polimorfismos do gene da proteína priônica celular (PRNP) nos códons 136, 154 e 171. Países onde o scrapie é endêmico têm utilizado programas de melhoramento, com a seleção para os alelos mais resistentes. Há alguns dados disponíveis de genotipagem do PRNP em ovinos no Brasil, e o scrapie tem sido observado esporadicamente desde 1978. O Paraná é o Estado brasileiro onde a maioria dos casos de scrapie foi diagnosticada. Um rebanho, que teve três casos clínicos de scrapie em 2003 e 2004, foi genotipado (128 ovinos - 53 Hampshire Down e 75 mestiços) e abatido (111 ovinos - 47 Hampshire Down e 64 mestiços) em 2006. Amostras de tecido linfóide e sistema nervoso central foram examinadas por imunohistoquímica (IHQ) para presença de proteína priônica alterada (PrPSc). Seis genótipos foram encontrados nos 128 animais genotipados: ARR/ARQ foi o mais frequente (45,3%), seguido por ARQ/ARQ (28,1%), ARR/ARR (14,1%) e ARQ/VRQ (8,6%). ARR/VRQ e ARQ/AHQ apresentaram menos de 2,5% de freqüência do genótipo. Na IHC, 16 animais com exame positivo para a presença da proteína priônica celular alterada (PrPSc) foram detectados. As tonsilas foram o tecido com a mais alta porcentagem de amostras reativas: 81,25% do total das amostras positivas. Considerando os 16 animais positivos, nove (56,25%) tinham o genótipo ARR/ARQ, seguido pelo genótipo ARQ/ARQ com 31,25% (n = 5) e ARQ/VRQ com 12,5% (n = 2). Todos os animais positivos estavam clinicamente saudáveis, representando, portanto, 14,14% de casos pré-clínicos de scrapie neste rebanho.

Classical natural ovine scrapie prions detected in practical volumes of blood by lamb and transgenic mouse bioassays

Scrapie is diagnosed antemortem in sheep by detecting misfolded isoforms of prion protein (PrP(Sc)) in lymphoid follicles of the rectal mucosa and nictitating membranes. Assay sensitivity is limited if (a) the biopsy is collected early during disease development, (b) an insufficient number of follicles is collected, or (c) peripheral accumulation of PrP(Sc) is reduced or delayed. A blood test would be convenient for mass live animal scrapie testing. Currently approved techniques, however, have their own detection limits. Novel detection methods may soon offer a non-animal-based, rapid platform with detection sensitivities that rival the prion bioassay. In anticipation, we sought to determine if diseased animals could be routinely identified with a bioassay using B lymphocytes isolated from blood sample volumes commonly collected for diagnostic purposes in small ruminants. Scrapie transmission was detected in five of six recipient lambs intravenously transfused with B lymphocytes isolated from 5~10 mL of blood from a naturally scrapie-infected sheep. Additionally, scrapie transmission was observed in 18 ovinized transgenic Tg338 mice intracerebrally inoculated with B lymphocytes isolated from 5~10 mL of blood from two naturally scrapie-infected sheep. Based on our findings, we anticipate that these blood sample volumes should be of diagnostic value.

Breast cancer in Latin America: global burden, patterns, and risk factors / Cáncer de mama en América Latina: carga, patrones y factores de riesgo

Breast cancer is a major public health problem in Latin America (LA) and the most common form of cancer among women. An important variability according to ethnicity/race with respect to incidence/mortality, clinical characteristics, and prognosis is observed throughout LA. In addition, women are more likely to develop breast cancer (BC) at younger age and to be diagnosed at an advanced stage compared to western women. While little is known about specific risk factors, changes in reproductive pattern (parity, breastfeeding) and lifestyle factors including sedentary behaviours, unhealthy diet, and alcohol intake may contribute to the increase of BC incidence. In this paper we give an overview of the burden and patterns of BC, review the leading causes of BC and discuss the possible ways to improve BC prevention and control in LA.

El cáncer de mama (CaMa) es uno de los mayores problemas de salud pública en América Latina (AL) y el cáncer más frecuente en mujeres. Se observa una importante variabilidad en la incidencia/mortalidad, las características clínicas y el pronóstico según la etnia/raza a lo largo de AL. Además, las mujeres latinoamericanas son más propensas a desarrollar CaMa en edades más tempranas y a ser diagnosticadas en una etapa más avanzada, comparando con mujeres occidentales. Aunque poco se sabe sobre sus factores de riesgo específicos, cambios en los patrones reproductivos (paridad y lactancia) y estilos de vida, incluyendo los hábitos sedentarios, las dietas poco saludables y el consumo de alcohol, podrían contribuir al incremento de la incidencia del CaMa. En este artículo se da una visión general de la carga y los patrones del CaMa, se revisan las causas principales del CaMa y se discuten posibles vías para mejorar la prevención y el control del CaMa en AL.

Polimorfismo do gene da proteína prion celular (PrPC) e imunohistoquímica de tecido linfóide em ovinos / Polymorphism of cellular prion protein (PrPC) and immunohistochemistry of lymphoid tissue of sheep

O objetivo deste trabalho foi avaliar o polimorfismo do gene da proteína prion celular (PRPN) de ovinos introduzidos numa propriedade onde ocorreu um surto de scrapie, e relacionar com a suscetibilidade à doença por meio da análise da presença da proteína prion celular alterada (PrPSc), utilizando imunohistoquímica (IHQ) de tecido linfóide associado à mucosa reto-anal. Foram avaliados 42 ovinos, mestiços Texel. Eram fêmeas entre um e oito anos de idade, sendo que sete (16,67%) ovelhas foram introduzidas adultas na propriedade em 2006. As demais, 83,33%, eram nascidas na fazenda. A genotipagem do PRPN foi feita pela análise do polimorfismo de comprimento de fragmento de restrição - RFLP ("Restriction Fragment Lenght Polimorphism"). O genótipo ARQ/ARQ foi o mais freqüente, encontrado em 73,81% dos animais, seguido do genótipo ARR/ARQ, com 16,67% e do ARQ/VRQ, com 9,52%. Os alelos ARH e AHQ não foram encontrados nestes animais. O resultado da IHQ foi negativo em todas as amostras. Não foi possível, portanto, estabelecer uma relação entre genótipo e maior susceptibilidade ao scrapie, devido à ausência de PrPSc na amostras examinadas. No Brasil, há poucos dados de genotipagem do gene da proteína prion celular (PRNP) em ovinos e, até o momento, nenhum tipo de controle baseado em cruzamentos direcionados foi implementado.

The aim of this work was to study the polymorphism of the prion protein gene (PRNP) of a sheep flock raised in a farm where a scrapie outbreak had occurred, and to relate to disease susceptibility of possible animals infected with altered prion protein (PrPSc), by immunohistochemical analysis of recto-anal mucosa-associated lymphoid tissue (RAMALT). Forty two sheep, crossbred with Texel, Ile de France, Dorper and Suffolk were used. Females were between one and eight years old, and seven (16.67%) were adult ewes when they entered the flock in 2006. The rest, 83.33% were born in the farm. The PRNP genotyping was performed by RFLP ("restriction fragment length polymorphism") analysis. The most frequent genotype was ARQ/ARQ, found in 73.81% of the animals, followed by ARR/ARQ, with 16.67% and ARQ/VRQ, with 9.52%. The ARH and AHQ alleles were not found. All RAMALT samples were negative in immunohistochemical analysis. It was not possible to establish a relation between PRNP polymorphisms and susceptibility to scrapie, due to the lack of positive samples to PrPSc. In Brazil, there is little available PRNP genotyping data of sheep and, so far, no type of controlled breeding scheme for scrapie has been implemented.

Doenças de ovinos diagnosticadas no Laboratório de Anatomia Patológica Animal da Universidade Federal de Mato Grosso do Sul (1996-2010) / Sheep diseases diagnosed at the Laboratory of Animal Pathology, Federal University of Mato Grosso do Sul, Brazil (1996-2010)

Nas últimas décadas a pecuária ovina cresceu significativamente no Brasil. Concomitantemente, grupos de pesquisas e laboratórios de diagnósticos realizam estudos retrospectivos com a finalidade de fornecer subsídios técnico-científicos para os médicos veterinários. Desta forma, realizou-se um estudo de prevalência nos arquivos do Laboratório de Anatomia Patológica Animal (LAP) da Universidade Federal de Mato Grosso do Sul (UFMS) no período de Janeiro de 1996 a Dezembro de 2010. O Laboratório de Bacteriologia da UFMS e o Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul forneceram apoio diagnóstico nos casos de mannheimiose pulmonar e scrapie, respectivamente. Os laudos da espécie ovina foram revisados e agrupados em conclusivos e inconclusivos, dos quais foram excluídos os casos experimentais e de outros estados e países. Os casos conclusivos foram classificados de acordo com a etiologia da doença. Os exames da espécie ovina somaram 331 laudos (3,97 %) de um total de 8.333 casos diagnosticados no período. Destes, foram excluídos sessenta e quatro (19,3%) casos experimentais e materiais oriundos de outros estados ou países. Dos 267 casos remanescentes, 87 (32,6%) foram inconclusivos e 180 (67,4%) considerados conclusivos, sendo 60 (33,3%) doenças infecciosas e parasitárias; 45 (25%) intoxicações e toxi-infecções; 41 (22,8%) "lesões sem causa definida"; 22 (12,2%) doenças metabólicas e nutricionais; 10 (5,6%) foram classificadas como "outros distúrbios" e 2 (1,1%) neoplasmas. A hemoncose, intoxicação por Brachiaria spp., pleuropneumonias, broncopneumonias, pneumonias fibrinonecrosante ou fibrinossupurativa sem causa definida e a intoxicação por cobre foram as doenças mais prevalentes no período estudado. Dois casos de scrapie foram diagnosticados no período.

Sheep farming has increased significantly in Brazil during the last decades. Concurrently, research groups and diagnostic laboratories compile data and perform retrospective studies to provide important insight for professionals. A prevalence study from January 1996 to December 2010 was carried out in the archives of Laboratório de Anatomia Patológica Animal (LAP), Universidade Federal de Mato Grosso do Sul (UFMS). Laboratório de Bacteriologia, UFMS, and Setor de Patologia Veterinária at Universidade Federal do Rio Grande do Sul helped on the diagnostic of pulmonary mannheimiosis and scrapie respectively. The reports for sheep were reviewed and grouped into conclusive and inconclusive ones. The conclusive cases were classified according to the etiology of the disease. In the period, 331 exams (3.97%) were done. Sixty-four experimental cases and materials from other states or countries (19.3%) were excluded. Remaining cases (267), eighty-seven (32.6%) were inconclusive and 180 (67.4%) were considered conclusive reports, were classified according to the etiology: 60 (33.3%) infectious and parasitary diseases; 45 (25%) were poisonings and toxi-infections; 41 (22.8%) were summarized as "injuries without apparent cause"; 22 (12.2%) cases of metabolic and nutritional diseases; 10 (5.6%) were classified as "other disorders" and 2 (1.1%) case of neoplasms. Haemonchosis, fibrinonecrotic or fibrinopurulent pleuropneumonia, bronchopneumonia and pneumonia, poisonings by Brachiaria spp. and copper poisoning were the most prevalent diseases in sheep. Two cases of scrapie have been diagnosed in this period.

Trends in genotype frequency resulting from breeding for resistance to classical scrapie in Belgium (2006~2011)

In sheep, susceptibility to scrapie is mainly determined by codons 136, 154, and 171 of the PRNP gene. Five haplotypes are usually present (ARR, ARQ, ARH, AHQ, and VRQ). The ARR haplotype confers the greatest resistance to classical scrapie while VRQ renders animals most susceptible. In 2004, the European Union implemented a breeding program that promotes selection of the ARR haplotype while reducing the incidence of VRQ. From 2006 to 2011 in Belgium, frequency for the ARR/ARR genotypes increased from 38.3% to 63.8% (n = 6,437), the ARQ haplotype diminished from 21.1% to 12.9%, and the VRQ haplotype decreased from 2.0% to 1.7%. The status of codon 141, a determinant for atypical scrapie, was also evaluated. Out of 27 different breeds (n = 5,163), nine were abundant. The ARR/ARR frequency increased in eight of these nine major breeds. The selection program has had a major impact on the ARR haplotype frequency in Belgium. However, the occurrence of atypical scrapie represents a critical point for this program that warrants the continuous monitoring of scrapie. Additionally, genotype frequencies among the breeds varied greatly. Texel, a breed that is common in Belgium, can still be selected for due to its average ARR frequency.

Scrapie e seu diagnóstico diferencial em ovinos no Mato Grosso do Sul / Scrapie and differential diagnosis in sheep in Mato Grosso do Sul, Brazil

Scrapie é uma doença infecciosa, neurodegenerativa fatal, causada pelo príon scrapie (PrPsc). Apresenta-se tanto na forma clássica em ovinos e caprinos geneticamente susceptíveis quanto na forma atípica em ovinos. A primeira notificação oficial do Brasil à Organização Mundial de Saúde Animal (OIE), um caso da forma clássica diagnosticado no Rio Grande do Sul ocorreu em 1985, mas a doença já havia sido diagnosticada no mesmo Estado em 1978. Este trabalho objetivou descrever dois surtos de Scrapie em ovinos em Mato Grosso do Sul (MS), Brasil e investigar, por meio de imuno-histoquímica (IHQ) a presença de PrPsc no Sistema Nervoso Central (SNC) de ovinos examinados entre 2003 e 2010. Na primeira parte observaram-se dois ovinos com sinais clínicos típicos de scrapie, detalhando-se os sinais neurológicos, dados epidemiológicos, histopatológicos e amostras teciduais em duplicata desses ovinos foram encaminhadas para realização de diagnóstico de Raiva e para diagnóstico IHQ para príon. Na segunda parte realizou-se levantamento de laudos de necropsia e diagnósticos histopatológicos de ovinos, no período de maio de 2003 a março de 2010. Amostras de sistema nervoso central de 51 casos foram selecionados, incluindo os dois já com diagnóstico de Scrapie mencionados acima; os tecido de todos esses ovinos foram submetidos à IHQ para detecção de proteína priônica. Os 49 ovinos avaliados apresentaram resultado negativo na IHQ para príon.

Scrapie is a fatal neurodegenerative infectious disease, caused by the scrapie prion (PrPsc), that can both in the as the classic form in genetically susceptible sheep and goats and in the atypical form in sheep. The first official notification of scrapie from Brazil was made to the World Organization for Animal Health (OIE) in 1985, in the state of Rio Grande do Sul, although the disease was first documented in this Brazilian state in 1978. The objective this paper was to describe two outbreaks of scrapie in sheep from Mato Grosso do Sul (MS), Brazil, and to investigate by immunohistochemistry (IHC) the presence of PrPsc in samples from the CNS of sheep examined during 2003 and 2010. The study was conducted in two stages; the first was the observation of two sheep with typical clinical signs of scrapie that underwent clinical examination with emphasis on neurological parameters, epidemiological data collection, necropsy and collection of samples in duplicate forwarded to the diagnosis of rabies, and for the IHC diagnosis of Transmissible Spongiform Encephalopathies. In the second part of the study, a survey was made the necropsy reviewing gross findings and histopathological diagnoses in sheep from May 2003 to March 2010. Samples of the central nervous system of fifty-one cases, including the two sheep mentioned above were subjected to IHC for detection of prion protein. The other 49 sheep, although displaying neurological-disease which should be included as scrapie differential diagnosis, had their tissues submitted to IHC resulting negative.

Genotipagem de polimorfismos no gene prnp em ovinos da raça Santa Inês no Estado de São Paulo / Genotyping of polymorphisms in the prnp gene in Santa Ines sheep in the State of São Paulo, Brazil

Scrapie ou paraplexia enzoótica dos ovinos é uma doença neurodegenerativa fatal que acomete ovinos e raramente caprinos. A doença é influenciada por polimorfismos nos códons 136, 154 e 171 do gene prnp que codifica a proteína priônica. Os animais podem ser susceptíveis ou resistentes, de acordo com as sequências alélicas observadas nos referidos códons. No Brasil ocorreram apenas casos de animais que foram importados, sendo o país considerado livre da doença. Neste trabalho foi realizada a genotipagem dos diferentes polimorfismos associados ao desenvolvimento do scrapie e a categorização em animais susceptíveis e resistentes. Foram sequenciadas 118 amostras provenientes de ovinos da raça Santa Inês criados em propriedades localizadas no Estado de São Paulo. Destas amostras foram identificados 6 alelos e 11 genótipos (ARQ/ARQ, ARR/ARQ, ARQ/AHQ, ARQ/VRQ, AHQ/AHQ, ARR/ARR, ARR/AHQ, VRQ/VRQ, ARQ/TRQ, TRR/TRR, TRQ/TRQ), dentre os quais o genótipo ARQ/ARQ teve ocorrência de 56,7%. Em nosso estudo foi detectada a presença da tirosina no códon 136, observação rara na medida em que não existem relatos nacionais e internacionais envolvendo a raça Santa Inês descrevendo este polimorfismo. Com os resultados obtidos, foi possível determinar a existência de grande variabilidade genética relacionada à raça Santa Inês no Estado de São Paulo. Apesar da variabilidade, apenas 1,69% dos genótipos observados mostraram-se extremamente resistentes ao scrapie. Estes dados demonstram que a raça nativa Santa Inês pode ser considerada potencialmente susceptível ao scrapie.

Enzootic paraplexia or scrapie is a fatal neurodegenerative disease affecting mainly sheep and rarely goats. The disease is influenced by polymorphisms at codons 136, 154 and 171 of prnp gene that encodes the prion protein. The animals may be susceptible or resistant to the development of the disease according to the allelic sequences observed in these codons. In Brazil there were only cases of scrapie in imported animals, therefore the country is considered free of the disease. This study performed the genotyping of different polymorphisms associated to the development of scrapie. Then, based on these findings the animals were categorized in resistant and susceptible. A total of 118 samples were sequenced from the Santa Ines sheep raised on properties located in the State of Sao Paulo. From these samples, 6 alleles and 11 genotypes were identified (ARQ / ARQ, ARR / ARQ, ARQ / AHQ, ARQ / VRQ, AHQ / AHQ, ARR / ARR, ARR / AHQ, VRQ / VRQ, ARQ / TRQ, TRR / TRR, TRQ / TRQ), the genotype ARQ / ARQ presented a frequency of 56.7%. It was also detected the presence of tyrosine at codon 136, which may be considered a rare observation, since there is no report regarding Santa Ines breeding presenting this polymorphism. These results showed the great genetic variability in Santa Ines in Sao Paulo and only 1,69% of the genotypes observed are extremely resistant to scrapie. These data demonstrate that the Santa Ines sheep can be considered potentially susceptible to scrapie.

Polimorfismos de nucleotídeos únicos em 15 códons do gene da proteína priônica em um rebanho Suffolk afetado com scrapie no Brasil / Single nucleotide polymorphisms at 15 codons of the prion protein gene from a scrapie-affected herd of Suffolk sheep in Brazil

Scrapie is a transmissible spongiform encephalopathy of sheeps and goats, associated with the deposition of a isoform of the prion protein (PrPsc). This isoform presents an altered conformation that leads to aggregation in the host's central nervous and lymphoreticular systems. Predisposition to the prion agent infection can be influenced by specific genotypes related to mutations in amino acids of the PrPsc gene. The most characterized mutations occur at codons 136, 154 and 171, with genotypes VRQ being the most susceptible and ARR the most resistant. In this study we have analyzed polymorphisms in 15 different codons of the PrPsc gene in sheeps from a Suffolk herd from Brazil affected by an outbreak of classical scrapie. Amplicons from the PrPsc gene, encompassing the most relevant altered codons in the protein, were sequenced in order to determine each animal's genotype. We have found polymorphisms at 3 of the 15 analyzed codons (136, 143 and 171). The most variable codon was 171, where all described alleles were identified. A rare polymorphism was found at the 143 codon in 4 percent of the samples analyzed, which has been described as increasing scrapie resistance in otherwise susceptible animals. No other polymorphisms were detected in the remaining 12 analyzed codons, all of them corresponding to the wild-type prion protein. Regarding the risk degree of developing scrapie, most of the animals (96 percent) had genotypes corresponding to risk groups 1 to 3 (very low to moderate), with only 4 percent in the higher risks group. Our data is discussed in relation to preventive measures involving genotyping and positive selection to control the disease.

Scrapie é uma encefalopatia espongiforme transmissível de ovinos e caprinos, associado a deposição da isoforma da proteína priônica (PrPsc). Essa isoforma apresenta uma alteração conformacional que leva ao acúmulo da proteína no sistema nervoso central e linforeticular do hospedeiro. A predisposição a infecção pelo agente priônico pode ser influenciado por genótipos específicos relacionados a mutações na sequência de aminoácidos do gene PrPsc. As principais mutações caracterizadas ocorrem nos códons 136, 154 e 171, sendo o genótipo VRQ o mais suscetível e o genótipo ARR o mais resistente. Nesse estudo nós analisamos os polimorfismos de 15 códons diferentes da gene PrPsc em ovinos de um rebanho da raça Suffolk no Brasil afetado com scrapie clássico. Os amplicons do gene da PrPsc, que contem os códons mais frequentemente encontrados foram sequenciados para determinar o genótipo de cada animal. Nós encontramos 3 polimorfismos do 15 códons analisados (136, 143 e 171). O códon que mais teve variações foi o códon 171, onde todos os alelos foram identificados. Um polimorfismo raro foi encontrado no códon 143, em 4 por cento das amostras analisadas, o qual tem sido descrito por aumentar a resistência a scrapie em animais suscetíveis. Nenhum outro polimorfismo foi detectado nos 12 códons restantes, todos então, correspondendo à proteína priônica selvagem. De acordo com a grau de risco a desenvolver scrapie, a maioria dos animais (96 por cento) tiveram genótipo correspondentes aos grupos de risco 1 a 3 (muito baixo a moderado), e somente 4 por cento no grupo de risco alto. Nossos dados discutem a relação das medidas de prevenção envolvendo a genotipagem e a seleção positiva para o controle da doença.

Interaction between various 14-3-3beta segments and PrP in vitro / 中华实验和临床病毒学杂志

<p><b>UNLABELLED</b>OBJECTIVE To study the potential interaction between PrP protein.</p><p><b>METHODS</b>The supernatant of health and scrapie-infected hamsters' brain homogenate was prepared, while various recombinant 14-3-3beta or PrP proteins were purified. The possible molecular interaction between 14-3-3beta proteins and PrP was tested by pull-down and immunoprecipitation assays.</p><p><b>RESULTS</b>Both native PrP(c) and its protease-resistant isoform (PrP(Sc)) formed complexes with 14-3-3beta. The full-length recombinant 14-3-3beta proteins interacted with PrP. The domain responsible for interacting 14-3-3beta was located at N-terminal of 14-3-3beta (residues 1 to 38).</p><p><b>CONCLUSION</b>The studies of the association of PrP with 14-3-3beta may further provide insight into a potential role of 14-3-3beta in the biological function of PrP and the pathogenesis of prion disease.</p>

Analysis of monoclonal antibody binding sites in ovine prion protein / 生物工程学报

Binding sites of five monoclonal antibodies were obtained by reinforceable method of overlapping recombinant prion protein and synthetic peptide. Overlapping peptides of PrP core were expressed in Escherichia coli by insertion of serial PCR amplicons of ovine PrP gene fragments into pET32a. The expressed fusion peptides were then tested for the binding activity to PrP monoclonal antibodies in Western blotting. The binding sites of 5 monoclonal antibodies of ovine PrP were located respectively as follows: 2H3 in 199 aa-213 aa, 4C6, 5F11 and 7F11 in 139 aa-168 aa and 7F1 in 214 aa-227 aa. There oligo peptides were synthesized and used in ELISA test for more accurate localization of the binding sites. The binding sites of 4C6, 5F11 and 7F11 were further confirmed to be in 149 aa-158 aa. This conclusion may contribute to the research for pathogenesis and diagnostic method of scrapie and bovine transmissible spongiform encephalopathy.

Establishment of a stable PrP(Sc) panel from brain tissues of experimental hamsters with scrapie strain 263K / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To establish a stable PrP(Sc) panel from brain tissues of experimental hamsters infected with scrapie agent 263K for evaluating diagnostic techniques of human and animals' prion diseases.</p><p><b>METHODS</b>Thirty brain tissue samples from hamsters intracerebrally infected with scrapie strain 263K and another 30 samples from normal hamsters were selected to prepare 10%, 1%, and 0.5% brain homogenates, which were aliquoted into stocks. PrP(Sc) in each brain homogenate was determined by proteinase K digestions followed by Western blot assay and partially by immunohistochemistry. Stability and glycoforms of PrP(Sc) were repeatedly detected by PrP(Sc)-specific Western blots in half a year and 3 years later.</p><p><b>RESULTS</b>PrP(Sc) signals were observed in all 10% brain homogenates of infected hamsters. Twenty out of 30 stocks and 19 out of 30 stocks were PrP(Sc) positive in 1% and 0.5% brain homogenatesof infected hamsters, respectively. Twenty-seven out of 30 stocks presented three positive bands in 10% brain homogenates, whereas none of 1% and 0.5% homogenates contained 3 bands. The detection of PrP(Sc)-specific signals stored in half a year and 3 years later demonstrated that the ratio of PrP(Sc) positive samples and glycoforms was almost unchanged. All normal hamsters' brain homogenates were PrP(Sc) negative.</p><p><b>CONCLUSION</b>A PrP(Sc) panel of prion disease can be established, which displays reliably stable PrP(Sc)-specific signals and glycoforms.</p>

Establishment of a prion disease PrP(Sc) panel from the brain tissues of experimental hamsters infected with scrapie agent 263K / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a prion disease PrP(Sc) panel from the brain tissues of experimental hamsters and to address the stability of the panel conserved under the specific condition, for evaluating the diagnostic techniques of human and animal's prion diseases.</p><p><b>METHODS</b>30 brain tissues of hamsters infected with scrapie strain 263K intracerebrally and 30 ones of normal hamsters were enrolled in this panel. Each brain sample was prepared to 10%, 1% and 0.5% homogenates and aliquoted into stocks. The presences of PrP(Sc) in each brain sample were evaluated with PrP-specific Western Blots and partially with immunohistochemistry, and the stability of PrP(Sc) signals in each sample were repeatedly assessed half a year later and 3 years later.</p><p><b>RESULTS</b>PrP(Sc) signals were detected in all stocks of 10% brain homogenates from infected hamsters, 26 out of 30 stocks of 1% homogenates and 19 out of 30 stocks of 0.5% homogenates. The assessments of PrP(Sc) signals in all samples half-year and three years later demonstrated almost unchanged. All homogenates of brain tissues of normal hamsters were PrP(Sc) negative.</p><p><b>CONCLUSION</b>A prion disease PrP(Sc) panel of the brain tissues, which includes 90 PrP(Sc) positive stocks and PrP(Sc) negative ones, was successfully established, with a reliable stability of PrP(Sc) signals.</p>

Treatment of scrapie pathogen 263K with tetracycline partially abolishes protease-resistant activity in vitro and reduces infectivity in vivo / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To study the possible effect of tetracycline on protease-resistant activity in vitro and infectivity in vivo of a scrapie strain 263K.</p><p><b>METHODS</b>Scrapie pathogens were incubated with tetracycline at different concentrations for various periods of time and protease-resistant PrP signals were evaluated with proteinase K-treatment and Western blots. The preparations treated with tetracycline were intracerebrally inoculated into golden hamsters and typical TSE manifestations were noted. PrPSc in brain tissues of the infected animals was detected by PrP specific Western blot assays.</p><p><b>RESULTS</b>Protease-resistant PrP was significantly reduced in or removed from the preparations treated with tetracycline in a dose-dependant manner. Compared with the control group after incubated for 53.75 +/- 0.50 days, the preparations treated with 5 mmol/L and 20 mmol/L tetracycline prolonged the incubation time of 61.5 +/- 1.73 and 59.5 +/- 0.58 days (P < 0.05).</p><p><b>CONCLUSION</b>Treatment of scrapie pathogen 263K with tetracycline reduces or removes its protease-resistant activity in vitro.</p>

Dynamic analyses of PrP and PrP(Sc) in brain tissues of golden hamsters infected with scrapie strain 263K revealed various PrP forms / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To expatiate dynamic changes in hamsters infected with scrapie strain 263K, to observe the presence and aggravation of various forms of PrP and PrP(Sc) during incubation period, and to probe primarily the relationship between the onset of clinic manifestations and the presence of different PrP(Sc) forms.</p><p><b>METHODS</b>Hamster-adapted scrapie strain 263K was intracerebrally inoculated into hamsters. Different forms of PrP and PrP(Sc) were monitored dynamically by Western blot and immuno-histochemical assays. The presence of scrapie-associated fibril (SAF) was assayed by electron microscopy analysis (EM) and immuno-golden EM.</p><p><b>RESULTS</b>PrP(Sc) was initially detected in the brain tissues of the animals in 20 days post-inoculation by immunohistochemistry and 40 days with Western blot. Quantitative evaluations revealed that the amounts of PrP and PrP(Sc) in brain tissues increased along with the incubation. Several high and low molecular masses of PrP were seen in the brains of the long-life span infected animals. Deglycosylation assays identified that the truncated PrP in the infected brains showed similar glycosylation patterns as the full-length PrP. The presence of short fragments was seemed to relate with the onset of clinical conditions.</p><p><b>CONCLUSION</b>These results indicate that infectious agents exist and accumulate in central nerve system prior to the onset of the illness. Various molecular patterns of PrP(Sc) may indwell in brain tissues during the infection.</p>

Comparison study on clinical and neuropathological characteristics of hamsters inoculated with scrapie strain 263K in different challenging pathways / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To understand the infectious characteristics of a hamster-adapted scrapie strain 263K with five different routes of infection including intracerebral (i.c.), intraperitoneal (i.p.), intragastrical (i.g.), intracardiac and intramuscular (i.m.) approaches.</p><p><b>METHODS</b>Hamsters were infected with crude- or fine-prepared brain extracts. The neuropathological changes, PrP(Sc) deposits, and patterns of PK-resistant PrP were analyzed by HE stain, immnunohistochemistry (IHC) assay and Western blot. Reactive gliosis and neuron loss were evaluated by glial fibrillary acidic protein (GFAP) and neuron specific enolase (NSE) specific IHC.</p><p><b>RESULTS</b>The animals inoculated in i.m. and i.p. ways with crude PrP(Sc) extracts showed clinical signs at the average incubation of 69.2 +/- 2.8 and 65.5 +/- 3.9 days. Inoculation in i.c. and intracardiac ways with fine PrP(Sc) extracts (0.00035 g) caused similar, but relative long incubation of around 90 days. Only one out of eight hamsters challenged in i.g. way with low dosage (0.01 g) became ill after a much longer incubation (185 d), while all animals (4/4) with high dosage (0.04 g) developed clinical signs 105 days postinfection. The most remarkable spongiform degeneration and PrP(Sc) deposits were found in brain stem among the five challenge groups generally. The number of GFAP-positive astrocytes increased distinctly in brain stems in all infection groups, while the number of NSE-positive cells decreased significantly in cerebrum, except i.c. group. The patterns of PK-resistant PrP in brains were basically identical among the five infection routes.</p><p><b>CONCLUSION</b>Typical TSE could be induced in hamsters by inoculating strain 263K in the five infection ways. The incubation periods in bioassays depend on infective dosage, administrating pathway and preparation of PrP(Sc). The neuropathological changes and PrP(Sc) deposits seem to be related with regions and inoculating pathways.</p>