Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 3 uppsatser.

Publicación a texto completo no autorizada por el autor / Manifiesta que en los ovinos se ha establecido la influencia del gen Prion (PRNP) en la capacidad de resistir o padecer la enfermedad de scrapie. Desde hace varios años los Estados Unidos y la Unión Europea, entre otros países del mundo, promueven programas de erradicación de la enfermedad siendo un requisito realizar el genotipado de los animales para su posterior selección. En el Perú se tiene 9’523,200 (INEI) de ovinos entre carneros y corderos, y menos del 4.1% incluye a la raza Junín (INEI). Hasta la fecha se desconocía los diferentes genotipos del gen Prion en el ovino Junín, el estudio permitió validar la técnica y determinar los genotipos del gen Prión en una muestra de 56 ovinos de raza Junín provenientes de una Unidad de Producción Privada ubicada en la región central del departamento de Junín. Se obtuvieron 4 de los 5 genotipos reportados: el ARR (48,21%), ARQ (35,71%), AHQ (10,71%) y VRQ (5,36%). En relación a los genotipos, se encontraron 7 de los 15 reportados para el gen Prion: ARR/ARR (17.86%); ARR/AHQ (10.71%); ARR/ARQ (42.86%); ARQ/AHQ (10.71%); ARQ/ARQ (7.14%); ARR/VRQ (7.14%) y ARQ/VRQ (3.57%). De acuerdo al resultado obtenido se tiene un porcentaje de 82.14 % de animales con genotipos resistente y un 17.85 % de genotipos susceptibles. Aunque estos valores solo corresponde a un grupo de muestras (N=56), puede servir de punto de partida para futuras investigaciones. El conocer el genotipo de los animales permitiría establecer un programa de prevención seleccionando animales resistentes a la enfermedad. / Consejo Nacional de Ciencia y Tecnología (Perú) / Universidad Nacional Mayor de San Marcos (Lima). Vicerrectorado de Investigación y Posgrado / Universidad Nacional Mayor de San Marcos (Lima). Medicina Veterinaria / Tesis

The dorsal vagal nucleus (DMNX) and nucleus ambiguus (NA) are two anatomically distinct regions of the medulla oblongata of the brainstem involved with the control of the heart on a beat to beat basis. The vagus nerve has parasympathetic cell bodies located in the DMNX and NA. The presence of the disease associated prion (PrPD) in the DMNX and NA is used in the post mortem diagnosis of transmissible spongiform encephalopathies (TSEs) in animals. It has been shown that PrPD alters the neuronal discharge properties of infected tissue (Barrow, Holmgren et al.1999; Collinge, Whittington et al. 1994). I wished to investigate whether a change in heart rate variability (HRV) influenced by the presence of PrPD deposits in brainstem areas of animals and people incubating TSEs would be detectable. Recordings from control and infected sheep, cattle and humans, consisting of three hundred-second samples of electrocardiogram (ECG) were collected from species specific healthy controls and subjects incubating TSE disease. Data were digitised at a sampling frequency of 1kHz and were translated and analysed using standard software (CED Spike2 ; IBM SPSS). Artefacts and missed beats were corrected based upon screening by eye. ECG R-wave timings were obtained in order to determine variability in the R-R intervals. An instantaneous tachogram was constructed from which power spectra were calculated. Power spectral analysis along with simpler time domain estimates of HRV, such as RMSSD, were employed to investigate differences between control and infected animals. In addition R wave variability within each breath was utilized to examine the vagal control of the heart in relation to breathing and thus investigate a change in function of the specific neurological areas of the brainstem used as diagnostic criteria for such diseases. It was found there were significant differences (p<0.05) in the HRV of infected sheep, cattle and humans incubating TSE disease compared to control samples. Repeated non-invasive longitudinal tests may provide a means to screen animals and humans for the presence of disease associated prions and may give applications in the objective assessments of putative therapeutics in addition to identifying TSE disease at a preclinical stage.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

The Transmissible Spongiform Encephalopathies (TSEs) or “prion” diseases are a group of fatal neurodegenerative diseases the aetiology of which is not fully understood. These diseases are characterised by a number of pathological changes in the central nervous system (CNS) including; vacuolation of the neuropil, gliosis and deposition of PrPSc; the abnormal form of the host glycoprotein PrP. Although the major pathology in these diseases is associated with the CNS the immune system is central to the pathogenesis of many natural and experimental TSEs including natural scrapie in sheep, chronic wasting disease in free ranging and captive deer and variant CJD (vCJD) in humans. Unlike many infectious diseases where deficiencies in immune function are opportunistic for the invading pathogen a competent immune system is required for efficient TSE infection via peripheral routes. As infection of the lymphoid tissues in many TSEs can occur many months before the detection of infectivity in the CNS, the determination of those cells in the lymphoid system has been the focus of much research and a number of studies now point towards the importance of the follicular dendritic cell (FDC), a long-lived radio resistant cell, in TSE pathogenesis. The involvement of FDCs in peripheral TSE pathogenesis relates to the inability of ionising radiation to influence pathogenesis, the association of PrP protein with FDCs in both uninfected and infected lymphoid tissues, and the demonstration that TSE pathogenesis is severely impaired in mice devoid of these cells. The aims of this thesis were to further understand the role of FDCs in the pathogenesis of a range of mouse-adapted experimental TSE strains and to determine if peripherally acquired TSE infections are influenced by host age or by stimulation of the immune system. Using chimaeric mouse models where a mismatch in the expression of PrP protein between FDCs and lymphoid/myeloid cells was produced, further evidence for a critical role for in the pathogenesis of the ME7 TSE strain was produced. Although these findings produced strong evidence that FDCs were important for the ME7 strain the possibility that different TSE strains may target different cell types in the peripheral lymphoid system was explored using a range of mice with specific immunological defects. Infection of these mice with several experimental TSE strains showed that the presence of mature FDCs was also important for the pathogenesis of the strains tested. Clinical cases of vCJD have been confined almost exclusively to young adults, although the reasons behind this apparent age-related susceptibility are not fully understood. The capacity of the immune system to mediate immune responses to pathogens declines with age as a result of impaired lymphocyte and FDC function. As FDCs are critically involved in the pathogenesis of many TSEs, including vCJD, it was hypothesised that an aging immune system may impair disease pathogenesis. Peripheral infection of senescent mice failed to produce clinical disease during lifespan, although evidence of disease transmission, was detected in a proportion of aged mice. These findings demonstrate that this inefficient disease transmission, as a consequence of age, may lead to considerable levels of sub-clinical disease within the population. Finally the influence of immune system stimulation, by the generation of a humoral immune response, on peripheral TSE pathogenesis was investigated. These findings demonstrated that immunisation can influence pathogenesis, but only during the early stages of infection prior to spread to the CNS. These data imply that modulation of the immune system does not alter TSE pathogenesis once disease has been initiated in the CNS. Finally, these studies have found some preliminary evidence that TSE infection may induce FDC activation suggesting that TSE infection may influence the immune response. Together, these data show that a functional immune system and specifically, the presence of mature FDCs, are central to the pathogenesis of peripherally acquired TSE infections.

Darbo tikslas – atlikti Baltijos šalių vietinių avių veislių: Estijos baltagalvių, Estijos juodgalvių, Estijos Ruhnu, Latvijos tamsiagalvių, Lietuvos vietinių šiurkščiavilnių, Lietuvos juodgalvių prioninio baltymo genetinės įvairovės tyrimus. Darbo uždaviniai: 1. Ištirti prioninio baltymo geno polimorfizmą 134, 157 ir 171 kodonuose, nustatyti šio geno alelius bei jų paplitimą Baltijos šalių vietinėse avių veislėse; 2. Nustatyti prioninio baltymo genotipų dažnius Baltijos šalių vietinių avių veislėse; 3. Identifikuoti prioninio baltymo genotipus pagal rizikos susirgti skrepi liga grupes, Baltijos šalių vietinių avių veislėse; 4. Sumodeliuoti atsparių skrepi ligai bandų formavimą pagal nustatytus prioninio baltymo genotipus bei jų paplitimo dažnį Baltijos šalių vietinėse avių veislėse. Pirmą kartą Baltijos šalių avių vietinėse veislėse: Estijos baltagalvių, Estijos juodgalvių, Estijos Ruhnu, Latvijos tamsiagalvių, Lietuvos vietinių šiurkščiavilnių, Lietuvos juodgalvių – buvo genotipuotas prioninio baltymo genas, identifikuoti šio geno aleliai, nustatytas jų paplitimas bei identifikuoti genotipai, pagal rizikos susirgti skrepi liga grupes. Atlikti prognoziniai skaičiavimai, siekiant suformuoti atsparių skrepi ligai avių bandas. / The aim of the work - to research prion protein gene(PrP) genetic diversity of local Baltic sheep in such breeds as Estonian whitehead, blackhead, and Ruhnu, Latvian darkhead, and Lithuanian coarsewool native, and Lithuanian blackface. Tasks of the research: 1. To investigate Prion protein gene polymorphism in 134, 157 and 171 codons, identify the gene alleles and their prevalence in the Baltic countries of the local native breeds of sheep; 2. To determine Prion protein genotype frequencies in the Baltic local native breeds of sheep; 3. To identify Prion protein genotypes according to the risk to contract scrapie disease in the native Baltic breeds of sheep; 4. To simulate scrapie resistant flocks according to the Prion protein genotypes and their prevalence rate in the native Baltic breeds of sheep. It is first time in the Baltics that the local sheep breed (Estonian whitehead, Estonian blackhead, Estonian Ruhnu, Latvian darkhead, Lithuanian coarsewool native, Lithuanian blackface) Prion protein gene was genotiped, gene alleles identified in their prevalence and genotypes classified into groups according to the risk to contract scrapie. The calculations were carried out in order to distinguish the groups of sheep resistant to scrapie.

As encefalopatias espongiformes transmissíveis (EETs), também conhecidas como doenças do príon, ocorrem tanto nos animais como no homem, são responsáveis por doenças transmissíveis e hereditárias e provocam lesões degenerativas no cérebro. A presença de uma forma anormal da proteína (PrPsc) no tecido encefálico e linforreticular é característica peculiar das EETs. Essa proteína é altamente insolúvel e resistente à degradação por proteases e se deposita eventualmente sob forma de placas amilóides na substância cinzenta. Tais placas provocam a morte maciça de neurônios e células gliais, causando vacuolização intensa no tecido afetado. Este trabalho consistiu na otimização da técnica de imuno-histoquímica para proteína priônica no tecido nervoso e no tecido linforreticular, como método diagnóstico de EETs em bovinos, ovinos e caprinos. Além disso, verificou-se a eficiência da utilização de folículos linfóides coletados por biopsia de terceira pálpebra e mucosa retal para o diagnóstico de scrapie em ovinos e caprinos. Além disso, verificou-se a eficiência da utilização de folículos linfóides coletados por biopsia de terceira pálpebra e mucosa retal para o diagnóstico de scrapie em ovinos e caprinos. Entre janeiro de 2005 e janeiro de 2008 foram realizados exames de imuno-histoquímica e coloração por hematoxilina-eosina em amostras de tecidos de 5571 ruminantes (4829 bovinos, 1 bubalino, 708 ovinos e 33 caprinos) obtidas em necropsias, biopsias e materiais enviados para diagnóstico em formalina 10% ou em blocos de parafina. Os anticorpos monoclonais anti-príon F89/160.1.5 e F99/97.6.1 foram utilizados na técnica de imuno-histoquímica. Todas as amostras de bovinos e a do bubalino analisadas foram negativas para PrPSc. Entre os 741 ovinos e caprinos pesquisados, foram diagnosticados 81 animais positivos, 16 suspeitos e, em 100 animais, o material foi insuficiente para o diagnóstico. Foram realizadas cinco repetições da imuno-histoquímica para cada amostra positiva para PrPSc. O diagnóstico por imuno-histoquímica no tecido linforreticular foi considerado positivo quando o material apresentava um número de no mínimo quatro folículos linfóides, com centros germinativos. Este trabalho inclui o primeiro diagnóstico positivo de scrapie na espécie caprina no Brasil, com marcação imuno-histoquímica nas tonsilas, terceira pálpebra e linfonodo. A marcação pela imuno-histoquímica em quatro ovinos da raça Santa Inês, todos com diagnóstico positivo nas tonsilas e linfonodos e um deles também com depósito de PrPSc na terceira pálpebra, surge como primeiro diagnóstico da doença nessa raça no Brasil. Os resultados considerados suspeitos ou insuficientes observados nas amostras de biopsias ou enviadas por terceiros indicam que há ajustes a serem estudados, especialmente quanto à coleta das amostras. O envio de material de vários órgãos linfóides possibilitaria a confirmação da presença ou não do agente em pelo menos dois deles, diminuindo o número de casos considerados suspeitos e o risco de falsos positivos. O envio de amostras de biopsia de tonsila, terceira pálpebra e mucosa retal, conjuntamente, reduziria o número de casos suspeitos e eliminaria grande parte, senão a totalidade dos casos com material insuficiente para diagnóstico. / Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, occur in animals and humans and are responsible for transmissible and inherited disorders that cause fatal degenerations of the brain. A peculiar characteristic of TSEs is the conversion of a host-encoded glycoprotein termed prion protein (PrP), from a normal cellular form, PrPC, to an abnormally folded isoform, PrPSc, present in the nervous and lymphoreticular tissues. The prion protein PrPSc is highly insoluble and resistant to the proteases degradation, eventually leading its accumulation, forming amyloid protein plates deposition in the brain gray substance. These plates cause massive death of neurons and glial cells, producing intense vacuolation in the affected tissue. In this work, the immunohistochemical procedures were optimizated for prion protein in nervous and lymphoreticular tissues to provide diagnosis of TSEs in bovine, sheep and goat, and also to verify the efficiency of lymphoid follicles from third eyelid and rectal mucosa biopsies for the scrapie diagnosis in sheep and goats. Between January, 2005 and January, 2008 immunohistochemistry examinations and hematoxylin-eosin staining were accomplished in tissue samples from 5571 ruminants (4829 bovines, 1 water buffalo, 708 sheep and 33 goats), obtained from necropsies and biopsies, and samples of 10% formalin-fixed or paraffin embedded tissues submitted by other laboratories. The monoclonal antibodies anti-PrP F89/160.1.5 and F99/97.6.1 were used in the immunohistochemical procedures. All the samples from cattle and the water buffalo analyzed were negative to PrPSc. Among the 741 sheep and goats, there were 81 positive and 16 suspect samples, and 100 of them were insufficient to test. Five immunohistochemical repetitions were performed for each positive PrPSc sample. The immunohistochemistry diagnosis in the lymphoreticular tissues was considered positive when the material presented at least four lymphoid follicles with germinal centers. This work includes the first positive case in goats in Brazil (through immunohistochemical labelling in tonsils, third eyelid and lymph node) and the first four cases in Santa Inês sheep, by the same technique. The results considered as suspect or of insufficient sample indicate that some aspects associated with sample collection need further adjustment. The submission of samples from several lymphoid organs would probably make possible the confirmation or not of the agent presence in at least two of them, reducing the number of suspect and false positive cases. Simultaneous submission of biopsy samples from tonsil, third eyelid and rectal mucosa together could reduce the number of suspicious cases, besides of eliminating great part or even the totally of the insufficient samples.

As encefalopatias espongiformes transmissíveis (EETs), também conhecidas como doenças do príon, ocorrem tanto nos animais como no homem, são responsáveis por doenças transmissíveis e hereditárias e provocam lesões degenerativas no cérebro. A presença de uma forma anormal da proteína (PrPsc) no tecido encefálico e linforreticular é característica peculiar das EETs. Essa proteína é altamente insolúvel e resistente à degradação por proteases e se deposita eventualmente sob forma de placas amilóides na substância cinzenta. Tais placas provocam a morte maciça de neurônios e células gliais, causando vacuolização intensa no tecido afetado. Este trabalho consistiu na otimização da técnica de imuno-histoquímica para proteína priônica no tecido nervoso e no tecido linforreticular, como método diagnóstico de EETs em bovinos, ovinos e caprinos. Além disso, verificou-se a eficiência da utilização de folículos linfóides coletados por biopsia de terceira pálpebra e mucosa retal para o diagnóstico de scrapie em ovinos e caprinos. Além disso, verificou-se a eficiência da utilização de folículos linfóides coletados por biopsia de terceira pálpebra e mucosa retal para o diagnóstico de scrapie em ovinos e caprinos. Entre janeiro de 2005 e janeiro de 2008 foram realizados exames de imuno-histoquímica e coloração por hematoxilina-eosina em amostras de tecidos de 5571 ruminantes (4829 bovinos, 1 bubalino, 708 ovinos e 33 caprinos) obtidas em necropsias, biopsias e materiais enviados para diagnóstico em formalina 10% ou em blocos de parafina. Os anticorpos monoclonais anti-príon F89/160.1.5 e F99/97.6.1 foram utilizados na técnica de imuno-histoquímica. Todas as amostras de bovinos e a do bubalino analisadas foram negativas para PrPSc. Entre os 741 ovinos e caprinos pesquisados, foram diagnosticados 81 animais positivos, 16 suspeitos e, em 100 animais, o material foi insuficiente para o diagnóstico. Foram realizadas cinco repetições da imuno-histoquímica para cada amostra positiva para PrPSc. O diagnóstico por imuno-histoquímica no tecido linforreticular foi considerado positivo quando o material apresentava um número de no mínimo quatro folículos linfóides, com centros germinativos. Este trabalho inclui o primeiro diagnóstico positivo de scrapie na espécie caprina no Brasil, com marcação imuno-histoquímica nas tonsilas, terceira pálpebra e linfonodo. A marcação pela imuno-histoquímica em quatro ovinos da raça Santa Inês, todos com diagnóstico positivo nas tonsilas e linfonodos e um deles também com depósito de PrPSc na terceira pálpebra, surge como primeiro diagnóstico da doença nessa raça no Brasil. Os resultados considerados suspeitos ou insuficientes observados nas amostras de biopsias ou enviadas por terceiros indicam que há ajustes a serem estudados, especialmente quanto à coleta das amostras. O envio de material de vários órgãos linfóides possibilitaria a confirmação da presença ou não do agente em pelo menos dois deles, diminuindo o número de casos considerados suspeitos e o risco de falsos positivos. O envio de amostras de biopsia de tonsila, terceira pálpebra e mucosa retal, conjuntamente, reduziria o número de casos suspeitos e eliminaria grande parte, senão a totalidade dos casos com material insuficiente para diagnóstico. / Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, occur in animals and humans and are responsible for transmissible and inherited disorders that cause fatal degenerations of the brain. A peculiar characteristic of TSEs is the conversion of a host-encoded glycoprotein termed prion protein (PrP), from a normal cellular form, PrPC, to an abnormally folded isoform, PrPSc, present in the nervous and lymphoreticular tissues. The prion protein PrPSc is highly insoluble and resistant to the proteases degradation, eventually leading its accumulation, forming amyloid protein plates deposition in the brain gray substance. These plates cause massive death of neurons and glial cells, producing intense vacuolation in the affected tissue. In this work, the immunohistochemical procedures were optimizated for prion protein in nervous and lymphoreticular tissues to provide diagnosis of TSEs in bovine, sheep and goat, and also to verify the efficiency of lymphoid follicles from third eyelid and rectal mucosa biopsies for the scrapie diagnosis in sheep and goats. Between January, 2005 and January, 2008 immunohistochemistry examinations and hematoxylin-eosin staining were accomplished in tissue samples from 5571 ruminants (4829 bovines, 1 water buffalo, 708 sheep and 33 goats), obtained from necropsies and biopsies, and samples of 10% formalin-fixed or paraffin embedded tissues submitted by other laboratories. The monoclonal antibodies anti-PrP F89/160.1.5 and F99/97.6.1 were used in the immunohistochemical procedures. All the samples from cattle and the water buffalo analyzed were negative to PrPSc. Among the 741 sheep and goats, there were 81 positive and 16 suspect samples, and 100 of them were insufficient to test. Five immunohistochemical repetitions were performed for each positive PrPSc sample. The immunohistochemistry diagnosis in the lymphoreticular tissues was considered positive when the material presented at least four lymphoid follicles with germinal centers. This work includes the first positive case in goats in Brazil (through immunohistochemical labelling in tonsils, third eyelid and lymph node) and the first four cases in Santa Inês sheep, by the same technique. The results considered as suspect or of insufficient sample indicate that some aspects associated with sample collection need further adjustment. The submission of samples from several lymphoid organs would probably make possible the confirmation or not of the agent presence in at least two of them, reducing the number of suspect and false positive cases. Simultaneous submission of biopsy samples from tonsil, third eyelid and rectal mucosa together could reduce the number of suspicious cases, besides of eliminating great part or even the totally of the insufficient samples.

As encefalopatias espongiformes transmissíveis (EETs), também conhecidas como doenças do príon, ocorrem tanto nos animais como no homem, são responsáveis por doenças transmissíveis e hereditárias e provocam lesões degenerativas no cérebro. A presença de uma forma anormal da proteína (PrPsc) no tecido encefálico e linforreticular é característica peculiar das EETs. Essa proteína é altamente insolúvel e resistente à degradação por proteases e se deposita eventualmente sob forma de placas amilóides na substância cinzenta. Tais placas provocam a morte maciça de neurônios e células gliais, causando vacuolização intensa no tecido afetado. Este trabalho consistiu na otimização da técnica de imuno-histoquímica para proteína priônica no tecido nervoso e no tecido linforreticular, como método diagnóstico de EETs em bovinos, ovinos e caprinos. Além disso, verificou-se a eficiência da utilização de folículos linfóides coletados por biopsia de terceira pálpebra e mucosa retal para o diagnóstico de scrapie em ovinos e caprinos. Além disso, verificou-se a eficiência da utilização de folículos linfóides coletados por biopsia de terceira pálpebra e mucosa retal para o diagnóstico de scrapie em ovinos e caprinos. Entre janeiro de 2005 e janeiro de 2008 foram realizados exames de imuno-histoquímica e coloração por hematoxilina-eosina em amostras de tecidos de 5571 ruminantes (4829 bovinos, 1 bubalino, 708 ovinos e 33 caprinos) obtidas em necropsias, biopsias e materiais enviados para diagnóstico em formalina 10% ou em blocos de parafina. Os anticorpos monoclonais anti-príon F89/160.1.5 e F99/97.6.1 foram utilizados na técnica de imuno-histoquímica. Todas as amostras de bovinos e a do bubalino analisadas foram negativas para PrPSc. Entre os 741 ovinos e caprinos pesquisados, foram diagnosticados 81 animais positivos, 16 suspeitos e, em 100 animais, o material foi insuficiente para o diagnóstico. Foram realizadas cinco repetições da imuno-histoquímica para cada amostra positiva para PrPSc. O diagnóstico por imuno-histoquímica no tecido linforreticular foi considerado positivo quando o material apresentava um número de no mínimo quatro folículos linfóides, com centros germinativos. Este trabalho inclui o primeiro diagnóstico positivo de scrapie na espécie caprina no Brasil, com marcação imuno-histoquímica nas tonsilas, terceira pálpebra e linfonodo. A marcação pela imuno-histoquímica em quatro ovinos da raça Santa Inês, todos com diagnóstico positivo nas tonsilas e linfonodos e um deles também com depósito de PrPSc na terceira pálpebra, surge como primeiro diagnóstico da doença nessa raça no Brasil. Os resultados considerados suspeitos ou insuficientes observados nas amostras de biopsias ou enviadas por terceiros indicam que há ajustes a serem estudados, especialmente quanto à coleta das amostras. O envio de material de vários órgãos linfóides possibilitaria a confirmação da presença ou não do agente em pelo menos dois deles, diminuindo o número de casos considerados suspeitos e o risco de falsos positivos. O envio de amostras de biopsia de tonsila, terceira pálpebra e mucosa retal, conjuntamente, reduziria o número de casos suspeitos e eliminaria grande parte, senão a totalidade dos casos com material insuficiente para diagnóstico. / Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, occur in animals and humans and are responsible for transmissible and inherited disorders that cause fatal degenerations of the brain. A peculiar characteristic of TSEs is the conversion of a host-encoded glycoprotein termed prion protein (PrP), from a normal cellular form, PrPC, to an abnormally folded isoform, PrPSc, present in the nervous and lymphoreticular tissues. The prion protein PrPSc is highly insoluble and resistant to the proteases degradation, eventually leading its accumulation, forming amyloid protein plates deposition in the brain gray substance. These plates cause massive death of neurons and glial cells, producing intense vacuolation in the affected tissue. In this work, the immunohistochemical procedures were optimizated for prion protein in nervous and lymphoreticular tissues to provide diagnosis of TSEs in bovine, sheep and goat, and also to verify the efficiency of lymphoid follicles from third eyelid and rectal mucosa biopsies for the scrapie diagnosis in sheep and goats. Between January, 2005 and January, 2008 immunohistochemistry examinations and hematoxylin-eosin staining were accomplished in tissue samples from 5571 ruminants (4829 bovines, 1 water buffalo, 708 sheep and 33 goats), obtained from necropsies and biopsies, and samples of 10% formalin-fixed or paraffin embedded tissues submitted by other laboratories. The monoclonal antibodies anti-PrP F89/160.1.5 and F99/97.6.1 were used in the immunohistochemical procedures. All the samples from cattle and the water buffalo analyzed were negative to PrPSc. Among the 741 sheep and goats, there were 81 positive and 16 suspect samples, and 100 of them were insufficient to test. Five immunohistochemical repetitions were performed for each positive PrPSc sample. The immunohistochemistry diagnosis in the lymphoreticular tissues was considered positive when the material presented at least four lymphoid follicles with germinal centers. This work includes the first positive case in goats in Brazil (through immunohistochemical labelling in tonsils, third eyelid and lymph node) and the first four cases in Santa Inês sheep, by the same technique. The results considered as suspect or of insufficient sample indicate that some aspects associated with sample collection need further adjustment. The submission of samples from several lymphoid organs would probably make possible the confirmation or not of the agent presence in at least two of them, reducing the number of suspect and false positive cases. Simultaneous submission of biopsy samples from tonsil, third eyelid and rectal mucosa together could reduce the number of suspicious cases, besides of eliminating great part or even the totally of the insufficient samples.

Ein Ziel dieser Arbeit war die Entwicklung eines differentialdiagnostischen Tests (FLI-Test), der die Abgrenzung einer BSE- von einer Scrapieinfektion durch die direkte Untersuchung des Hirnstammmaterials ermöglicht. Bei einem Teil der dabei untersuchten deutschen klassi-schen Scrapiefälle wurde diese Charakterisierung zusätzlich im bis dahin zur Differenzierung verwendeten klassischen Mausbioassay durchgeführt, um die Ergebnisse aus dem FLI-Test zu verifizieren und um die vorhandenen Scrapieisolate weitergehend zu charakterisieren. Im zweiten Teil dieser Arbeit wurden die biochemischen Eigenschaften atypischer deutscher Scrapieisolate analysiert und ihre Infektiosität anhand von Übertragungsversuchen auf drei Wildtypmauslinien und eine transgene Mauslinie beurteilt. Darüber hinaus wurden diese Isolate dem klassischen BSE-Isolat gegenüber gestellt.