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S U M M A R YWe assessed three different visualization systems used routinely in research and diagnosis of transmissable spongiform encephalopathies (TSEs) to demonstrate whether the methodology applied to immunohistochemical (IHC) examination may alter the results concerning detection of prion protein (PrP sc ) in the lymphoreticular system (LRS): avidin-biotin-peroxidase (Vectastain ABC kit; Vector), Envision (DAKO), and catalyzed signal amplification (CSA; DAKO). The study aimed to determine which of these showed the highest sensitivity, with the hope of providing an accurate tool for pathogenesis and preclinical diagnosis research in TSEs. Histological sections from palatine tonsils, spleen, GALT (ileum and ileocecal valve), and lymph nodes from sheep belonging to a Spanish scrapie-positive flock were processed by IHC using L42 as primary antibody. As substrate chromogen, diaminobenzidine was used, and all slides were subjectively assessed by light microscopy. A further study using an image analyzer software system was carried out to confirm that the conclusion provided by microscopic examination was objective. The CSA system showed the highest sensitivity in all cases, increasing both variables assessed: the number of follicles that were PrP sc -positive was detected as well as the intensity of immunostaining in each of them.

S U M M A R YAlthough detection of the abnormal isoform of prion protein (PrP sc ), the specific feature of transmissable spongiform encephalopathies (TSEs), has been previously demonstrated on formalin-fixed autolytic tissue, no samples with autolysis as severe as tested here (i.e., liquid state) have previously been tested. It is inevitable that a small but significant proportion of brains, especially in summer due to delays in postmortem examination, undergo an extremely severe autolysis that makes samples unsuitable for diagnosis by conventional techniques. In this study, 25 bovine samples were diagnosed by applying immunocytochemistry on the corresponding liquid fraction. Four additional portions of brainstem (positive and negative sheep and cattle) were subjected to one of the autolysis regimens at 56C or environmental conditions for up to 80 days and were analyzed with the same methodology. No abnormal protein could be detected in any of the control animals. PrP sc accumulation was observed by immunocytochemistry in all cases that were positive by either immunohistochemistry on the corresponding filtrates or by Prionics Western blotting, showing an excellent agreement between the methodology assessed and these routine techniques. The results of this study demonstrate immunocytochemistry as a useful tool for diagnosis in liquid-state samples, solving a most relevant problem in BSE and scrapie epidemiology.

Environmental exposure to metal appears to enhance susceptibility to Transmissible Spongiform Encephalopathies (TSEs); however, published data are not conclusive. The current study focuses on assessing the effects of copper depletion and/or manganese enhancement in the diet on susceptibility to Scrapie and this disease progression. The degree of spongiosis was the highest in the animals that received a copper- depleted diet. These observations suggest that this diet contributes to the Scrapie lesions and to the worsening of the condition in animals that have been inoculated with Scrapie. The highest intensities of GFAP immunostaining were also associated with the copper- depleted diet. Dietary supplementation with manganese had a negative effect on neuronal counts. In conclusion, this study demonstrates that certain environmental factors may aggravate neuropathological Scrapie lesions. This is consistent with reports from other neurodegenerative diseases where some metalloenzymes play a pivotal protector role against the oxidative stress associated with pathogenesis.

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