In Vitro and In Vivo Evidence towards Fibronectin's Protective Effects against Prion Infection.

A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrPSc, in the central nervous system of prion-affected humans and animals. PrPSc is also found in peripheral tissues, raising concerns about the potential transmission of pathogenic prions through human food supplies and posing a significant risk to public health. Although muscle tissues are considered to contain levels of low prion infectivity, it has been shown that myotubes in culture efficiently propagate PrPSc. Given the high consumption of muscle tissue, it is important to understand what factors could influence the establishment of a prion infection in muscle tissue. Here we used in vitro myotube cultures, differentiated from the C2C12 myoblast cell line (dC2C12), to identify factors affecting prion replication. A range of experimental conditions revealed that PrPSc is tightly associated with proteins found in the systemic extracellular matrix, mostly fibronectin (FN). The interaction of PrPSc with FN decreased prion infectivity, as determined by standard scrapie cell assay. Interestingly, the prion-resistant reserve cells in dC2C12 cultures displayed a FN-rich extracellular matrix while the prion-susceptible myotubes expressed FN at a low level. In agreement with the in vitro results, immunohistopathological analyses of tissues from sheep infected with natural scrapie demonstrated a prion susceptibility phenotype linked to an extracellular matrix with undetectable levels of FN. Conversely, PrPSc deposits were not observed in tissues expressing FN. These data indicate that extracellular FN may act as a natural barrier against prion replication and that the extracellular matrix composition may be a crucial feature determining prion tropism in different tissues.

Primary cilia presence and implications in bladder cancer progression and invasiveness.

Urothelial bladder cancer is the tenth most common cancer worldwide. It is divided into muscle and non-muscle invading bladder cancer. Primary cilia have been related to several cancer hallmarks such as proliferation, epithelial-to-mesenchymal transition (EMT) or tumoral progression mainly through signaling pathways as Hedgehog (Hh). In the present study, we used immunohistochemical and ultrastructural techniques in human tissues of healthy bladder, non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) to study and clarify the activation of epithelial-to-mesenchymal transition and Hedgehog signaling pathway and the presence of primary cilia. Thus, we found a clear correlation between EMT and Hedgehog activation and bladder cancer stage and progression. Moreover, we identified the presence of primary cilia in these tissues. Interestingly, we found that in NMIBC, some ciliated cells cross the basement membrane and localized in lamina propria, near blood vessels. These results show a correlation between EMT beginning from urothelial basal cells and primary cilia assembly and suggest a potential implication of this structure in tumoral migration and invasiveness (likely in a Hh-dependent way). Hence, primary cilia may play a fundamental role in urothelial bladder cancer progression and suppose a potential therapeutic target.

Evidence of p75 Neurotrophin Receptor Involvement in the Central Nervous System Pathogenesis of Classical Scrapie in Sheep and a Transgenic Mouse Model.

Neurotrophins constitute a group of growth factor that exerts important functions in the nervous system of vertebrates. They act through two classes of transmembrane receptors: tyrosine-kinase receptors and the p75 neurotrophin receptor (p75NTR). The activation of p75NTR can favor cell survival or apoptosis depending on diverse factors. Several studies evidenced a link between p75NTR and the pathogenesis of prion diseases. In this study, we investigated the distribution of several neurotrophins and their receptors, including p75NTR, in the brain of naturally scrapie-affected sheep and experimentally infected ovinized transgenic mice and its correlation with other markers of prion disease. No evident changes in infected mice or sheep were observed regarding neurotrophins and their receptors except for the immunohistochemistry against p75NTR. Infected mice showed higher abundance of p75NTR immunostained cells than their non-infected counterparts. The astrocytic labeling correlated with other neuropathological alterations of prion disease. Confocal microscopy demonstrated the co-localization of p75NTR and the astrocytic marker GFAP, suggesting an involvement of astrocytes in p75NTR-mediated neurodegeneration. In contrast, p75NTR staining in sheep lacked astrocytic labeling. However, digital image analyses revealed increased labeling intensities in preclinical sheep compared with non-infected and terminal sheep in several brain nuclei. This suggests that this receptor is overexpressed in early stages of prion-related neurodegeneration in sheep. Our results confirm a role of p75NTR in the pathogenesis of classical ovine scrapie in both the natural host and in an experimental transgenic mouse model.

Serological and shedding patterns after Coxiella burnetii vaccination in the third gestation trimester in dairy cows.

This study sought to assess the effects of an inactivated phase I vaccine against Coxiella burnetii at the start of the third trimester of gestation on serological profiles, bacterial shedding patterns and subsequent reproductive performance in dairy cows. Cows were randomly assigned to a control (n = 78) or a vaccinated (n = 78) group on days 171-177 of gestation. Samples of placenta and colostrums at parturition, vaginal fluid, faeces, milk (PCR identification) and blood (anti-C. burnetii antibody detection) were obtained on the day of treatment and on days 91-97 post partum, and also on parturition day and weekly on days 1-7, 8-14, 15-21, 22-28 and 29-35 post partum in a subset of 70 animals. By Kaplan-Meier survival analysis, no significant effect of vaccination was detected on any of the reproductive variables studied. According to the odds ratio, C. burnetii shedding on days 171-177 of gestation was highly correlated with seropositivity against C. burnetii (OR = 9.1), while vaccination was not linked to reduced shedding of the bacterium. In shedders compared to others, the likelihood of pregnancy to first AI decreased and increased by factors of 0.26 and 16.1 on days 1-35 and 91-97 post partum, respectively. In conclusion, when administered at the start of the third trimester of pregnancy, the inactivated C. burnetii phase I vaccine failed to reduce bacterial shedding.

Melanin-Based Nanoparticles for Lymph Node Tattooing: Experimental, Histopathological and Ultrastructural Study.

In breast cancer, Targeted Axillary Dissection (TAD) allows for the selective excision of the sentinel lymph node (SLN) during primary tumor surgery. TAD consists of the resection of labelled SLNs prior to neoadjuvant chemotherapy (NACT). Numerous clinical and preclinical studies have explored the use of carbon-based colloids for SLN tattooing prior to NACT. However, carbon vectors show varying degrees of inflammatory reactions and, in about one fifth of cases, carbon particles migrate via the lymphatic pathway to other nodes, causing the SLN to mismatch the tattooed node. To overcome these limitations, in this study, we explored the use of melanin as a staining endogenous pigment. We synthesized and characterized melanin-loaded polymeric nanoparticles (Mel-NPs) and used them to tattoo lymph nodes in pig animal models given the similarity in the size of the human and pig nodes. Mel-NPs tattooed lymph nodes showed high identification rates, reaching 83.3% positive identification 16 weeks after tattooing. We did not observe any reduction in the identification as time increased, implying that the colloid is stable in the lymph node tissue. In addition, we performed histological and ultrastructural studies to characterize the biological behavior of the tag. We observed foreign-body-like granulomatous inflammatory responses associated with Mel-NPs, characterized by the formation of multinucleated giant cells. In addition, electron microscopy studies showed that uptake is mainly performed by macrophages, and that macrophages undergo cellular damage associated with particle uptake.

No detectable precolostral antibody response in calves born from cows with cotyledons positive for Coxiella burnetii by quantitative PCR.

Samples from 45 dams (milk/colostrum, faeces, vaginal fluid and blood on days 171-177 of gestation and at parturition, and cotyledons at parturition) and their calves (blood collected before colostrum intake and weekly until days 29-35) were analysed to examine the vertical transmission of Coxiella burnetii and links between shedding and seropositivity. All calves were born C. burnetii seronegative. Only those born to seropositive dams seroconverted following colostrum intake. Logistic regression analyses indicated that the likelihood of dam seropositivity was 21 and 4.85 times higher for multiparous than for primiparous (65.6% vs. 8.3%, P = 0.006) and for prepartum shedding cows (75% vs. 38.2%, P = 0.03) compared to the remaining animals, respectively. In conclusion, the results of this study indicate no detectable precolostral antibody response in calves born from dams with cotyledons positive for C. burnetii by qPCR. In order to analyse the possibility of persistent infection due to immunotolerance to an early in utero infection, further studies will need to test for C. burnetii DNA. In addition, in the present study multiparous cows showed a significantly higher seroprevalence than primiparous cows and heifers, colostral antibodies were efficiently transferred to newborn calves, and there was a link between bacterial shedding on days 171-177 of gestation and Coxiella seropositivity of the dam.

Spheresomes are the main extracellular vesicles in low-grade gliomas.

Cancer progression and its impact on treatment response and prognosis is deeply regulated by tumour microenvironment (TME). Cancer cells are in constant communication and modulate TME through several mechanisms, including transfer of tumour-promoting cargos through extracellular vesicles (EVs) or oncogenic signal detection by primary cilia. Spheresomes are a specific EV that arise from rough endoplasmic reticulum-Golgi vesicles. They accumulate beneath cell membrane and are released to the extracellular medium through multivesicular spheres. This study describes spheresomes in low-grade gliomas using electron microscopy. We found that spheresomes are more frequent than exosomes in these tumours and can cross the blood-brain barrier. Moreover, the distinct biogenesis processes of these EVs result in unique cargo profiles, suggesting different functional roles. We also identified primary cilia in these tumours. These findings collectively contribute to our understanding of glioma progression and metastasis.

Involvement of astrocytes in transmissible spongiform encephalopathies: a confocal microscopy study.

Astroglial proliferation associated with pathological prion protein (PrPsc) deposition is widely described in Transmissible Spongiform Encephalopathies (TSEs). However, little is known of the actual role played by glia in their pathogenesis. The aim of the study has been to determine whether PrPsc is located exclusively in neurons or in both neurons and glial cells present in the central nervous system in a natural Scrapie model. Samples of cerebellum from 25 Scrapie sheep from various flocks were sectioned. Following epitope retrieval with formic acid, proteinase K and heat treatment, primary antibody L42 and primary antibodies against glial fibrillary acidic protein were applied as prion- and astrocytic-specific markers, respectively. For visualization, a suitable mixture of fluorochrome-conjugated secondary antibodies was used. Relevant controls were processed in the same manner. As determined by confocal microscopy, PrPsc deposits co-localized with glial cells in all samples. Our results suggest that these cells can sustain active prion propagation, in agreement with similar findings from other studies of primary cell cultures and inoculated mice. Furthermore, despite ongoing debate regarding whether varied TSE sources show differences in their tropism for different cell lineages in the brains of affected animals, no differences in co-localization results were seen.

Changes induced by natural scrapie in the calretinin-immunopositive cells and fibres of the sheep cerebellar cortex.

Calretinin (CR)-immunopositive cells and fibres in the cerebellar cortex (vermal archicerebellum and neocerebellum) of scrapie-affected, ARQ/ARQ, Rasa Aragonesa breed sheep were studied in comparison with healthy, young and aged, ARQ/ARQ, Rasa Aragonesa animals and with Manchega breed sheep. The scrapie-affected sheep showed signs of both cellular involution and hypertrophic/hyperimmunoreactive responses in all neuronal subtypes; the distribution of the neuronal subtypes in the archi- and neocerebellum, however, did not change compared with controls. The results suggest that the different CR expression and/or CR content of cerebellar cortical neurons in scrapie-affected sheep are more related to their specific functions than any neuroprotective response. The reduction in the cell density of some CR-immunopositive neuronal subsets (i.e. unipolar brush cells) is contradictory to the supposed neuroprotective role of the calcium binding protein CR. However, the hyperimmunoreactivity of many CR-immunopositive neuronal subsets (e.g. the Purkinje cells) suggests the involvement of an over-expression of CR (transitory or restricted to selected neurons) as an adaptative mechanism to fight against the neurodegeneration caused by this prion disease. The changes in the number of immunopositive cells and the hypertrophic/hyperimmunoreactive response seen in scrapie-affected and aged sheep suggests that some different and some similar mechanisms are at work in this disease and aging.

Liver tissue remodeling following ablation with irreversible electroporation in a porcine model.

Irreversible electroporation (IRE) is a method of non-thermal focal tissue ablation characterized by irreversibly permeabilizing the cell membranes while preserving the extracellular matrix. This study aimed to investigate tissue remodeling after IRE in a porcine model, especially focusing on the extracellular matrix and hepatic stellate cells. IRE ablation was performed on 11 female pigs at 2,000 V/cm electric field strength using a versatile high-voltage generator and 3 cm diameter parallel-plate electrodes. The treated lobes were removed during surgery at 1, 3, 7, 14, and 21 days after IRE. Tissue remodeling and regeneration were assessed by histopathology and immunohistochemistry. Throughout the treated area, IRE led to extensive necrosis with intact collagenous structures evident until day 1. From then on, the necrosis progressively diminished while reparative tissue gradually increased. During this process, the reticulin framework and the septal fibrillar collagen remained in the necrotic foci until they were invaded by the reparative tissue. The reparative tissue was characterized by a massive proliferation of myofibroblast-like cells accompanied by a complete disorganization of the extracellular matrix with the disappearance of hepatic architecture. Hepatic stellate cell markers were associated with the proliferation of myofibroblast-like cells and the reorganization of the extracellular matrix. Between 2 and 3 weeks after IRE, the lobular architecture was almost completely regenerated. The events described in the present study show that IRE may be a valid model to study the mechanisms underlying liver regeneration after extensive acute injury.

Stromal cells of giant cell tumor of bone show primary cilia in giant cell tumor of bone.

Giant cell tumor of bone (GCTB) is a locally aggressive primary bone neoplasm composed by tumoral stromal cells (SCs) and a reactive component that consists of monocytic/histiocytic cells that give rise by fusion to osteoclast-like multinucleated cells. Recently, specific Histone 3.3 mutations have been demonstrated in SCs of GCTB. Many of the pathways related to bone proliferation and regulation depend on the primary cilium, a microtubule-based organelle that protrudes outside the cell and acts as a sensorial antenna. In the present work, we aimed to study the presence and role of primary cilia in GCTB. Ultrastructural, immunohistochemical, and immunofluorescence studies were performed in order to demonstrate, for the first time, that the primary cilium is located in spindle-shaped SCs of GCTB. Moreover, we showed Hedgehog (Hh) signaling pathway activation in these cells. Hence, primary cilia may play a relevant role in GCTB tumorogenesis through Hh signaling activation in SCs. RESEARCH HIGHLIGHTS: Transmission electron microscopy allows describing and differentiating cellular subpopulations in giant cell tumor of bone (GCTB). The primary cilium is present in some tumoral stromal cells of GCTB. Hedgehog signalling is activated in these cells.

Classical and Atypical Scrapie in Sheep and Goats. Review on the Etiology, Genetic Factors, Pathogenesis, Diagnosis, and Control Measures of Both Diseases.

Prion diseases, such as scrapie, are neurodegenerative diseases with a fatal outcome, caused by a conformational change of the cellular prion protein (PrPC), originating with the pathogenic form (PrPSc). Classical scrapie in small ruminants is the paradigm of prion diseases, as it was the first transmissible spongiform encephalopathy (TSE) described and is the most studied. It is necessary to understand the etiological properties, the relevance of the transmission pathways, the infectivity of the tissues, and how we can improve the detection of the prion protein to encourage detection of the disease. The aim of this review is to perform an overview of classical and atypical scrapie disease in sheep and goats, detailing those special issues of the disease, such as genetic factors, diagnostic procedures, and surveillance approaches carried out in the European Union with the objective of controlling the dissemination of scrapie disease.

SARS-CoV-2 Outbreak on a Spanish Mink Farm: Epidemiological, Molecular, and Pathological Studies.

Farmed minks have been reported to be highly susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and may represent a risk to humans. In this study, we describe the first outbreak of SARS-CoV-2 occurred on a mink farm in Spain, between June and July 2020, involving 92,700 animals. The outbreak started shortly after some farm workers became seropositive for SARS-CoV-2. Minks showed no clinical signs compatible with SARS-CoV-2 infection throughout the outbreak. Samples from 98 minks were collected for histopathological, serological, and molecular studies. Twenty out of 98 (20.4%) minks were positive by RT-qPCR and 82 out 92 (89%) seroconverted. This finding may reflect a rapid spread of the virus at the farm with most of the animals overcoming the infection. Additionally, SARS-CoV-2 was detected by RT-qPCR in 30% of brain samples from positive minks. Sequencing analysis showed that the mink sequences were not closely related with the other mink SARS-CoV-2 sequences available, and that this mink outbreak has its probable origin in one of the genetic variants that were prevalent in Spain during the first COVID-19 epidemic wave. Histological studies revealed bronchointerstitial pneumonia in some animals. Immunostaining of viral nucleocapsid was also observed in nasal turbinate tissue. Farmed minks could therefore constitute an important SARS-CoV-2 reservoir, contributing to virus spread among minks and humans. Consequently, continuous surveillance of mink farms is needed.

Variability in disease phenotypes within a single PRNP genotype suggests the existence of multiple natural sheep scrapie strains within Europe.

Variability of pathological phenotypes within classical sheep scrapie cases has been reported for some time, but in many instances it has been attributed to differences in the PRNP genotype of the host. To address this issue we have examined by immunohistochemistry (IHC) and Western blotting (WB) for the disease-associated form of the prion protein (PrP(d)), the brains of 23 sheep from five European countries, all of which were of the same ARQ/ARQ genotype. As a result of IHC examinations, sheep were distributed into five groups with different phenotypes and the groups were the same regardless of the scoring method used, 'long' or 'short' PrP(d) profiling. The groups made did not respond to the geographical origin of the cases and did not correlate with the vacuolar lesion profiles, which showed a high individual variability. Discriminatory IHC and WB methods coincided to detect a 'CH1641-like' case but otherwise correlated poorly in the classification of disease phenotypes. No other polymorphisms of the PRNP gene were found that could account for the pathological differences, except perhaps for a sheep from Spain with a mutation at codon 103 and a unique pathological phenotype. Preliminary evidence indicates that those different IHC phenotypes correlate with distinct biological properties on bioassay, suggesting that they are indicative of strain diversity. We therefore conclude that natural scrapie strains exist and that they can be revealed by detailed pathological examinations, which can be harmonized between laboratories to produce comparable results.

Effects of an anti-gonadotrophin releasing hormone vaccine on the morphology, structure and function of bull testes.

Castration reduces aggressive and sexual behaviour and provides better carcass quality in bull calves. Vaccination against gonadotrophin-releasing hormone (GnRH) is used as an alternative to surgical castration for the purposes of reducing pain and distress in the animals. Currently, no anti-GnRH vaccine has been authorized for use in cattle in the European Union. The aim of the present study was to assess the effect of an anti-GnRH swine-specific vaccine (Improvac®, Zoetis, USA) on the morphology, structure and function of bull testes. Animals were vaccinated at days 1, 21 and 104 of the experimental period and were classified based on their live weight into the following two groups: LIGHT (172.9 ±â€¯30.00 kg) and HEAVY (323.8 ±â€¯37.79 kg). The scrotal circumference was measured on day 1 and prior to slaughter (day 164). At slaughter, the sperm motility and concentration in the caudae epididymis were assessed. Testes were weighed, measured and examined using ultrasound, and then tissue samples were collected and fixed in formalin. Histological and immunohistochemical studies were performed on the testes to measure the diameter of the seminiferous tubules and assess the testicular cell populations. The results revealed that suppression of testicular development was associated with the use of the Improvac® vaccine, which resulted in a smaller size of the testes and impaired spermatid production. However, the effect of Improvac® was more pronounced and consistent in calves vaccinated at a low live weight than at a heavy live weight, which suggested that vaccination is more effective when calves are vaccinated before or early during puberty. However, testes from calves vaccinated at a low live weight were more prone to the development of intraluminal concretions in the seminiferous tubules.

Calretinin-immunopositive cells and fibers in the cerebellar cortex of normal sheep.

Calretinin (CR)-immunopositive cells and fibers in the cerebellar cortex (vermal archicerebellum--lobules X and IX--and neocerebellum--lobules VIIb and VIII) of two and 4-year-old Manchega and Rasa Aragonesa sheep were studied. CR-immunoreactivity was seen in subsets of all neurons and afferent fibers described in the cerebellar cortex. Generally, immunopositive cells were seen in very high densities in lobules X and IX, and in low density in lobule VIIb. Apparently, all unipolar brush cells were CR-immunopositive and showed a greater variety of shape than had been reported in other species. CR-immunoreactivity of Purkinje cells was either absent or varied from low to medium intensity. Few granule cell perikarya were immunostained (<5%) but a large number of their axons were CR-immunopositive. Subsets of stellate and basket cells were CR-immunoreactive--quite different to what is seen in most of mammalian species. Strongly CR-immunopositive mossy and climbing fibers, isolated or grouped, were observed in all lobules. Although we found neither a difference in CR-immunoreactivity between the two breds of sheep, nor between the two ages examined, we observed important differences in CR-immunoreactivity between sheep and other mammalian species. Our observation of neuronal clusters and groups of fibers with very high CR-immunopositivity supports the idea of a heterogeneous species-specific functional organization for the cerebellar cortex within an apparent homogeneous histological structure maintained throughout mammalian evolution. The results also suggest that the varied levels of CR expression may be related to the specific functions of these immunopositive neurons and fibers rather than to a general neuroprotective role played by calretinin in the cerebellar cortex.

Bovine spongiform encephalopathy in Sweden: an H-type variant.

Bovine spongiform encephalopathy (BSE) had never been detected in Sweden until 2006, when the active surveillance identified a case in a 12-year-old cow. The case was an unusual form, because several molecular features of the protease-resistant prion protein (PrP(res)) were different from classical BSE. The differences included higher susceptibility for proteinase K, higher molecular weight of the PrP(res) bands, affinity to the N-terminus-specific antibodies 12B2 and P4, and peculiar banding pattern with antibody SAF84 showing an additional band at the 14 kDa position. The molecular characteristics were in accordance to previous descriptions of H-type BSE. This report shows that a range of Western blot techniques and antibodies can be applied to confirm H-type BSE and further describes that the ratio of the amounts of PrPres#1 and PrPres#2, after deglycosylation, depends on the antibody used during processing. Immunohistochemistry on sections of medulla at the level of the obex applying antibodies with epitopes covering a broad range of the PrP sequence showed accumulation of disease-specific PrP (PrP(d)) in the gray matter. Fine punctate deposition in the neuropil was the most predominant type and was more severe in BSE target nuclei. The types of PrP(d) deposition are described in comparison with classical BSE. PrP-gene sequencing showed 6 copy octarepeat alleles and no abnormalities. It is postulated that the disease had a spontaneous origin, rather than having had been acquired in the BSE epidemic.

Changes in the expression pattern of the nitrergic system of ovine cerebellum affected by scrapie.

The constitutive and inducible isoforms of nitric oxide synthase (NOS) and the end-product of nitration, nitrotyrosine, were analyzed by immunohistochemistry, Western blotting, and enzymatic activity in sheep at different stages of the prion disease, scrapie. Four groups were studied: 1) nonaffected (control), 2) preclinical, 3) clinical, and 4) terminal. Constitutive neuronal NOS (nNOS) was the most abundant isoform present in cerebellar neurons of the sheep. Expression of nNOS increased in preclinical animals but diminished in the late stages of the disease. The Purkinje cells that usually are not immunoreactive for this protein became immunopositive in the clinical phase. In unaffected sheep, the inducible isoform (iNOS) was slightly positive in the Purkinje cells. As the disease progressed, the immunoreactivity of Purkinje neurons for iNOS increased. At the final stages, numerous iNOS-positive microglial cells were found in the molecular layer. There was a basal level of protein nitration in the cerebellum of unaffected sheep, especially in the molecular layer. As the disease progressed, the distal prolongations of the Purkinje cells and the astroglia became immunoreactive for nitrotyrosine. Our results suggest that the nitrergic system reacts to the progression of spongiform diseases and may be part of their pathogenesis mechanism.

Protein misfolding cyclic amplification corroborates the absence of PrPSc accumulation in placenta from foetuses with the ARR/ARQ genotype in natural scrapie.

Ovine scrapie is a worldwide spread prion disease that is transmitted horizontally under field conditions. Placenta from scrapie-infected ewes is an important source of infection, since this tissue can accumulate high amounts of PrPSc depending on the foetal genotype. Therefore, placentas carrying susceptible foetuses can accumulate PrPSc but there is not PrPSc accumulation in presence of foetuses with at least one ARR haplotype. In scrapie eradication programs, ARR/ARR males are used for breeding to increase the resistant progeny and reduce the horizontal transmission of the disease through the placenta. The development of highly sensitive techniques, that allow the detection of minimal amounts of PrPSc, has caused many secretions/excretions and tissues that had previously been deemed negative to be relabeled as positive for PrPSc. This has raised concerns about the possible presence of minimal amounts of PrPSc in placentas from ARR foetuses that conventional techniques had indicated were negative. In the present study we examined 30 placentas from a total of 23 gestations; 15 gestations resulted from naturally ARQ/ARQ scrapie-infected ewes mated with ARR/ARR rams. The absence of PrPSc in placentas carrying the foetal ARR haplotype (n=19) was determined by IDEXX HerdChek scrapie/BSE Antigen EIA Test, Prionics®-Check WESTERN and corroborated by the highly sensitive Protein Misfolding Cyclic Amplification technique (PMCA). By immunohistochemistry, several unspecific stainings that might mislead a diagnosis were observed. The results of the present study support that using ARR/ARR males in scrapie eradication programs efficiently decreases the spreading of the agent in the environment via shed placentas.

Assessment of calcium-binding proteins (Parvalbumin and Calbindin D-28K) and perineuronal nets in normal and scrapie-affected adult sheep brains.

Scrapie is a prion disease in small ruminants that manifests itself with neurological clinical signs amongst which are ataxia and tremors. These signs can be explained partially by an imbalance in central inhibitory innervation. The study of the brain's inhibitory neuronal GABAergic populations and of their extracellular matrix has been used to define, in part, the pathogenesis of human prion diseases and scrapie models in rodents. The brain's distribution of neuronal GABAergic subpopulations has been monitored carefully using, as markers, antibodies against the calcium binding proteins parvalbumin and calbindin D-28K. The distribution of this perineuronal net marker was evaluated by means of affinity histochemistry with W. floribunda agglutinin. These techniques were performed on the brains of nine scrapie-positive sheep and on four infection-free sheep. These animals had undergone previously a clinical follow-up as well as a lesion profile and an immunohistochemical profile of the scrapie-associated prion protein deposition in the brain. The study of calcium-binding proteins revealed an alteration of the parvalbumin positive GABAergic neuronal subpopulation. In scrapie-positive cases, a reduction in stained neuronal perykaria was observed, along with a marked reduction of neurite labelling. This finding was noticeable in regions such as the neocortex, particularly the motor frontal cortex, and was concomitant with a moderate PrPsc deposition and a mild degree of lesion. No changes were observed in the extracellular matrix study. The results of the present study provide a partial explanation for the mechanisms of scrapie clinical signs due to a disturbance of the parvalbumin-positive inhibitory neuronal population.