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While neurochemical metabolite modifications, determined by different techniques, have been diffusely reported in human and mice brains affected by transmissible spongiform encephalopathies (TSEs), this aspect has been little studied in the natural animal hosts with the same pathological conditions so far. Herein, we investigated, by high resolution (1)H NMR spectroscopy and multivariate statistical data analysis, the brain metabolite profile of sheep exposed to a scrapie agent in a naturally affected flock. On the basis of clinical examinations and western blotting analysis for the pathological prion protein (PrP(Sc)) in brain tissues, sheep were catalogued as not infected (H), infected with clinical signs (S), and infected without clinical signs (A). By discriminant analysis of spectral data, comparing S vs. H, we found a different metabolite distribution, with inosine, cytosine, creatine, and lactate being higher in S than in H brains, while the branched chain amino acids (leucine, isoleucine, and valine), phenylalanine, uracil, tyrosine, gamma-amino butyric acid, total aspartate (aspartate + N-acetyl aspartate) being lower in S. By a soft independent modelling of class analogy approach, 1 out of 3 A samples was assigned to class H. Furthermore, A brains were found to be higher in choline and choline-containing compounds. By means of partial least squares regression, an excellent correlation was found between the PrP(Sc) amount and the (1)H NMR metabolite profile of infected (S and A) sheep, and the metabolite mostly correlated with PrP(Sc) was alanine. The overall results, obtained using different chemometric tools, were able to describe a brain metabolite profile of infected sheep with and without clinical signs, compared to healthy ones, and indicated alanine as a biomarker for PrP(Sc) amounts in scrapie brains.

Atypical and classical scrapie-infected sheep brain tissue was monolaterally injected into the tonsils of lambs to investigate their role as a prion entry point. We first detected classical PrP Sc within the inoculated tonsil and in the ipsilateral retropharyngeal lymph node at 3 months postinoculation (p.i.). At 7 months p.i., PrP Sc colonized other lymphoid tissues bilaterally, including ileal Peyer's patches. The earliest PrP Sc deposition within the brain was ipsilaterally observed at 9 months p.i. in the substantia reticularis of the medulla oblongata. At 12 months p.i., PrP Sc deposition was present bilaterally in the nucleus parasympathicus nervi vagi, as well as in the intermediolateral cell column of the thoracolumbar spinal cord. No PrP Sc was detected in the lambs inoculated with atypical scrapie. These findings suggest that neuroinvasion may naturally occur from the tonsil after a widespread prion replication within the lymphoid tissues during classical scrapie only, thus mimicking the pathogenesis after oral ingestion.

Follicular dendritic cells (FDCs) residing within ileal Peyer's patches (PPs) are of crucial relevance for sheep scrapie early pathogenesis and subsequent scrapie prion neuroinvasion. In this study, ileal PP follicles were significantly more numerous in lambs than in adult Sarda breed sheep, with significant differences being also found in lymphoid follicle area, perimeter and FDC density. Furthermore, PrPd deposition within ileal PPs and host's PrP genotype did not significantly influence these parameters. We conclude that age significantly affects FDC density in ileal PPs from Sarda breed ovines, independently from host's scrapie status and PrP genotype.

In this study, we investigated the susceptibility to scrapie of Sarda breed sheep carrying the genotype ARQ/ARQ with additional polymorphisms at the PRNP gene. To do this, we examined 256 scrapie-affected sheep and 320 flock-mate negative controls from 24 flocks. Logistic regression analysis demonstrated that sheep carrying the ARQ/ARQ genotype with additional dimorphisms had lower risk of becoming scrapie affected when compared with those with ARQ/ARQ(wildtype) genotype. ARQ/ARQ genotypes that were detected with heterozygous or homozygous p.Asn176Lys and p.Met137Thr dimorphisms were associated with the lowest susceptibility to the disease. A significant lower risk was also associated with the p.Arg154His dimorphism, while p.Leu141Phe had a protective effect that was not statistically significant.

Ileal Peyer's patches (PPs) are involved early during sheep scrapie infection. This study qualitatively and semi-quantitatively evaluated ileal tract and PP innervation in 29 Sarda ovines of different age, PrP genotype and scrapie status. A prominent network of fibres was detected within PPs, mainly located in interfollicular lymphoid and stromal components. Intrafollicular fibres were rarely observed, with no apparent differences between scrapie-free and scrapie-affected animals, or among ovines carrying different PrP genotypes. In adult sheep, independent of their scrapie status, nerve fibres could be detected infrequently, close to the follicle-associated epithelium. Fibres were also detected within newly formed follicles and intrafollicular microgranulomas.

Concerns have been raised about the possibility that the bovine spongiform encephalopathy (BSE) agent could have been transmitted to sheep populations via contaminated feedstuffs. The objective of our study was to investigate the suitability of molecular strain typing methods as a surveillance tool for studying scrapie strain variations and for differentiating PrP Sc from sheep scrapie, BSE, and sheep BSE. We studied 38 Italian sheep scrapie cases from 13 outbreaks, along with a British scrapie case, an experimental ovine BSE, and 3 BSE cases, by analyzing the glycoform patterns and the apparent molecular masses of the nonglycosylated forms of semipurified, proteinase-treated PrP Sc . Both criteria were able to clearly differentiate sheep scrapie from BSE and ovine experimental BSE. PrP Sc from BSE and sheep BSE showed a higher glycoform ratio and a lower molecular mass of the nonglycosylated form compared to scrapie PrP Sc . Scrapie cases displayed homogeneous PrP Sc features regardless of breed, flock, and geographic origin. The glycoform patterns observed varied with the antibody used, but either a monoclonal antibody (MAb) (F99/97.6.1) or a polyclonal antibody (P7-7) was able to distinguish scrapie from BSE PrP Sc . While more extensive surveys are needed to further corroborate these findings, our results suggest that large-scale molecular screening of sheep populations for BSE surveillance may be eventually possible.

The genotype of the host plays a crucial role in the pathogenesis of transmissible spongiform encephalopathies (TSEs). In this respect, the most important factor is represented by the gene of the prion protein (PrP). The present work summarizes the currently available knowledge on the genetic basis of TSEs focusing, in particular, on sheep scrapie. Interest in this disease has grown markedly following the discovery of bovine spongiform encephalopathy, both for scientific and health reasons. In Italy, specific research grants from the Ministry of Health and the National Research Council (CNR), together with cooperation between the Istituto Superiore di Sanità and the Istituti Zooprofilattici Sperimentali, have allowed us to study the PrP genotype and to investigate the genetic susceptibility to scrapie in the most important Italian sheep breeds, with special reference to Sarda, Comisana and Massese. The PrP genotype in relation to scrapie susceptibility was also studied in goats of Ionica breed.

Several PrP gene polymorphisms modulate sheep scrapie susceptibility. Recently, an increase of scrapie outbreaks has been reported in Italy. A vaccine containing sheep brain homogenate was used in most of the outbreaks. We investigated PrP gene polymorphisms in scrapie-affected and clinically healthy Sarda breed sheep from a flock exposed to the aforementioned vaccine, and in affected Sarda sheep from unexposed flocks. All affected animals were (Gln/Gln)171 homozygous. Moreover, we observed no variation for Ala136 and a new polymorphism (Lys to Asn) at codon 176. Our findings confirm the correlation between scrapie and (Gln/Gln)171 in breeds with no variation for Ala136.

Prion diseases are fatal neurodegenerative disorders affecting many mammals, ovine scrapie being the archetypal prion disease. Several independent studies in murine and cell-based models of scrapie have highlighted the presence of a link between prion generation and lipid alterations; yet, no data on natural disease are available. In this study we investigated levels of total lipids and cholesterol as well as profiles of fatty acids in brain homogenates from symptomatic and asymptomatic scrapie-infected sheep vs. healthy sheep, all belonging to the same flock. Lipid extracts were analyzed by means of gas chromatography and high performance liquid chromatography. Data of fatty acids were submitted to multivariate statistical analysis to give a picture of the brain lipid profiles of sheep. Interestingly, results revealed abnormalities in the brain fatty acid unsaturation of infected/symptomatic animals. Significant reduction of monoene 18:1 n-9 was detected in brain lipids from infected/symptomatic sheep, as compared to healthy and infected/asymptomatic animals, and this alteration occurred in combination with a significant increase in 18:0 level. The unsupervised Principal Component Analysis showed that infected/symptomatic and healthy sheep samples lie in two different regions of the plot, infected/asymptomatic lie mostly next to healthy. The increase of cerebral saturated fatty acids provides a rough indication of presumed alterations in lipid raft domains of nervous cells during scrapie, suggesting that they may exist in a notable viscous liquid-ordered state. Such physicochemical alteration would have a profound impact on the raft thermodynamic properties, its spatial organization, and signal transduction, all potentially relevant for prion generation.

In sheep scrapie, pathological prion protein (PrP(Sc)) deposition occurs in the lymphoreticular and central nervous systems. We investigated PrP(Sc) distribution in scrapie-affected sheep showing simultaneous evidence of chronic lymphofollicular, lymphoproliferative/non-lymphofollicular, and/or granulomatous inflammations in their mammary gland, lung, and ileum. To do this, PrP(Sc) detection was carried out via immunohistochemistry and Western Blotting techniques, as well as through inflammatory cell immunophenotyping. Expression studies of gene coding for biological factors modulating the host's inflammatory response were also carried out. We demonstrated that ectopic PrP(Sc) deposition occurs exclusively in the context of lymphofollicular inflammatory sites, inside newly formed and well-organized lymphoid follicles harboring follicular dendritic cells. On the contrary, no PrP(Sc) deposition was detected in granulomas, even when they were closely located to newly formed lymphoid follicles. A significantly more consistent expression of lymphotoxin α and β mRNA was detected in lymphofollicular inflammation compared to the other two types, with lymphotoxin α and β signaling new lymphoid follicles' formation and, likely, the occurrence of ectopic PrP(Sc) deposition inside them. Our findings suggest that, in sheep co-affected by scrapie and chronic inflammatory conditions, only newly formed lymphoid follicles provide a suitable micro-environment that supports the scrapie agent's replication in inflammatory sites, with an increased risk of prion shedding through body secretions/excretions.

Prions are misfolded proteins that are infectious and naturally transmitted, causing a fatal neurological disease in humans and animals. Prion shedding routes have been shown to be modified by inflammation in excretory organs, such as the kidney. Here, we show that sheep with scrapie and lentiviral mastitis secrete prions into the milk and infect nearly 90% of naïve suckling lambs. Thus, lentiviruses may enhance prion transmission, conceivably sustaining prion infections in flocks for generations. This study also indicates a risk of prion spread to sheep and potentially to other animals through dietary exposure to pooled sheep milk or milk products.

The placenta is important in the horizontal transmission of the aetiological agent in scrapie-affected sheep. It has been demonstrated that the placentas of fetuses carrying the dimorphism Q171R of the PRNP gene is resistant to pathological prion protein (PrP(Sc)) accumulation in the placenta. To test whether other PRNP polymorphisms are associated with a lack of placental PrP(Sc) deposition, we carried out a study on 26 naturally and 11 experimentally scrapie-affected ewes with or without clinical signs. PrP(Sc) was detected in the placenta of ARQ/ARQ(wild type) fetuses by Western blot and immunohistochemical analysis, but not in ARQN(176)/ARQK(176) or, as expected, ARQ/ARR samples. Furthermore, three of four AL(141)RQ/AF(141)RQ placentas were also PrP(Sc) negative, suggesting that the dimorphism at codon 141 may also mediate placental deposition of PrP(Sc). This finding demonstrates for the first time that fetal PRNP polymorphisms, other than those at codon 171, are associated with the lack of placental deposition of PrP(Sc).

The pathological isoform of the prion protein (PrP(res)) can serve as a marker for prion diseases, but more practical tests are needed for preclinical diagnosis and sensitive detection of many prion infections. Previously we showed that the quaking-induced conversion (QuIC) assay can detect sub-femtogram levels of PrP(res) in scrapie-infected hamster brain tissue and distinguish cerebral spinal fluid (CSF) samples from normal and scrapie-infected hamsters. We now report the adaptation of the QuIC reaction to prion diseases of medical and agricultural interest: human variant Creutzfeldt-Jakob disease (vCJD) and sheep scrapie. PrP(res)-positive and -negative brain homogenates from humans and sheep were discriminated within 1-2 days with a sensitivity of 10-100 fg PrP(res). More importantly, in as little as 22 h we were able to distinguish CSF samples from scrapie-infected and uninfected sheep. These results suggest the presence of prions in CSF from scrapie-infected sheep. This new method enables the relatively rapid and sensitive detection of human CJD and sheep scrapie PrP(res) and may facilitate the development of practical preclinical diagnostic and high-throughput interference tests.

This study aimed at identifying genes that could mark scrapie infection in the central nervous system of sheep. We used the subtractive suppressive hybridization (SSH) technique on brain samples from sheep healthy or clinically affected by scrapie. Following subtraction, several discrete differential bands appeared between the two reciprocally subtracted samples. These bands were cloned and sequenced, allowing identifying the genes COX1, CHN1, PPP2CA, LRFN5, CAMK2A and RABEPK. Two of the genes identified, CHN1 and RABEPK, appear to locate inside a QTL region known to modulate prion disease incubation time in mice, and LRFN5 maps inside a QTL region identified in sheep. Furthermore, CHN1 and RABEKP showed new unreported differential splicing.

Prions have been documented in extra-neuronal and extra-lymphatic tissues of humans and various ruminants affected by Transmissible Spongiform Encephalopathy (TSE). The presence of prion infectivity detected in cervid and ovine blood tempted us to reason that kidney, the organ filtrating blood derived proteins, may accumulate disease associated PrPSc. We collected and screened kidneys of experimentally, naturally scrapie-affected and control sheep for renal deposition of PrPSc from distinct, geographically separated flocks. By performing Western blot, PET blot analysis and immunohistochemistry we found intraepithelial (cortex, medulla and papilla) and occasional interstitial (papilla) deposition of PrPSc in kidneys of scrapie-affected sheep. Interestingly, glomerula lacked detectable signals indicative of PrPSc. PrPSc was also detected in kidneys of subclinical sheep, but to significantly lower degree. Depending on the stage of the disease the incidence of PrPSc in kidney varied from approximately 27% (subclinical) to 73.6% (clinical) in naturally scrapie-affected sheep. Kidneys from flocks without scrapie outbreak were devoid of PrPSc. Here we demonstrate unexpectedly frequent deposition of high levels of PrPSc in ovine kidneys of various flocks. Renal deposition of PrPSc is likely to be a pre-requisite enabling prionuria, a possible co-factor of horizontal prion-transmission in sheep.

The enteric nervous system (ENS) probably plays a dominant role in sheep scrapie pathogenesis, but little is known about the cell types involved. We investigated the ileal myenteric and submucosal plexuses of four naturally and four orally experimentally scrapie-affected ARQ/ARQ Sarda sheep, as well as those of 12 healthy-control Sarda sheep carrying different PrP genotypes. All scrapie-affected animals, euthanized at clinical-disease end stage, showed PrPd deposition within enteric glial cells (EGCs) and calbindin-immunoreactive (CALB-IR) and neuronal nitric oxide synthase (nNOS)-IR neurons. Whole-mount investigations revealed no significant differences between the densities of total, CALB-IR and nNOS-IR neurons in scrapie-affected versus healthy sheep, irrespective of PrP genotype. Our results suggest that EGCs and CALB-IR and nNOS-IR neurons are probably involved in the pathogenesis of natural and oral experimental sheep scrapie. Furthermore, the infectious agent may be less pathogenic towards ENS neurons than it is towards central nervous system neurons.

The pathogenesis of natural scrapie in Sarda breed sheep was investigated in 1050 asymptomatic and 49 sick sheep from scrapie-affected flocks. Central and peripheral nervous system, along with lymphoreticular system (LRS) tissues, were subjected to immunohistochemistry (IHC) and Western-blotting (WB) for detection of pathological isoform of the prion protein (PrP(Sc)). A total of 69 of the 1050 clinically healthy sheep were found to be infected with scrapie, with PrP(Sc) being detected in both the central nervous system (CNS) and enteric nervous system (ENS) plexuses of 60 of the sheep, while IHC and WB yielded evidence of (PrP(Sc)) deposition only in lymphoid tissues of the remaining 9 clinically healthy sheep. PrP(Sc) was also detected in the CNS, as well as in ENS plexuses from all of the 49 clinically affected sheep. Nevertheless, 18 of the 69 clinically healthy animals (26%, 17 ARQ/ARQ and 1 ARQ/AHQ sheep), along with 3 ARQ/ARQ sheep (6%) of the clinically affected group, showed no IHC or WB evidence of PrP(Sc) in lymphoid tissues, but PrP(Sc) could be still detected in their CNS and ENS plexuses. The study demonstrates dual CNS and ENS PrP(Sc) deposition in Sarda sheep with scrapie, in spite of an apparent lack of lymphoid tissue involvement in a number of cases.