Pierre Sarradin

Early accumulation of PrPSc in gut-associated lymphoid and nervous tissues of susceptible sheep from a Romanov flock with natural scrapie

The immune system is known to be involved in the early phase of scrapie pathogenesis. However, the infection route of naturally occurring scrapie and its spread within the host are not entirely known. In this study, the pathogenesis of scrapie was investigated in sheep of three PrP genotypes, from 2 to 9 months of age, which were born and raised together in a naturally scrapie-affected Romanov flock. The kinetics of PrP(Sc) accumulation in sheep organs were determined by immunohistochemistry. PrP(Sc) was detected only in susceptible VRQ/VRQ sheep, from 2 months of age, with an apparent entry site at the ileal Peyers patch as well as its draining mesenteric lymph node. At the cellular level, PrP(Sc) deposits were associated with CD68-positive cells of the dome area and B follicles before being detected in follicular dendritic cells. In 3- to 6-month-old sheep, PrP(Sc) was detected in most of the gut-associated lymphoid tissues (GALT) and to a lesser extent in more systemic lymphoid formations such as the spleen or the mediastinal lymph node. All secondary lymphoid organs showed a similar intensity of PrP(Sc)-immunolabelling at 9 months of age. At this time-point, PrP(Sc) was also detected in the autonomic myenteric nervous plexus and in the nucleus parasympathicus nervi X of the brain stem. These data suggest that natural scrapie infection occurs by the oral route via infection of the Peyers patches followed by replication in the GALT. It may then spread to the central nervous system through the autonomic nervous fibres innervating the digestive tract.

Cases of scrapie with unusual features in Norway and designation of a new type, Nor98

Five cases of scrapie with unusual features have been diagnosed in Norway since 1998. The affected sheep showed neurological signs dominated by ataxia, and had the PrP genotypes homozygous A136 H154 Q171/ A136 H154 Q171 or heterozygous A136 HI54Q171/A136 R154 Q171, which are rarely associated with scrapie. Brain histopathology revealed neuropil vacuolisation essentially in the cerebellar and cerebral cortices; vacuolation was less prominent in the brainstem, and no lesions were observed at the level of the obex. The deposits of PrPSc were mainly in the cortex of the cerebellum and cerebrum, and no PrPSc was detectable by immunohistochemistry and ELISA in the lymphoid tissues investigated. Western blot analysis showed that the glycotype was different from other known scrapie strains and from the BSE strain. From a diagnostic point of view, these features indicate that this type of scrapie, designated Nor98, could have been overlooked and may be of significance for sampling in scrapie surveillance programmes.

BSE agent signatures in a goat

SIR, – One of the concerns about BSE is the potential presence of the agent in small ruminants, sheep and goats, as well as cattle. With the objective of documenting this, seven French laboratories have analysed 438 brain samples from confirmed cases of TSE in sheep and goats. These comprised

Compartmentalization of Prion Isoforms Within the Reproductive Tract of the Ram

Abstract Cellular prion protein (PrpC) is a glycoprotein usually associated with membranes via its glycosylphosphatidylinositol (GPI) anchor. The trans-conformational form of this protein (PrpSC) is the suggested agent responsible for transmissible neurodegenerative spongiform encephalopathies. This protein has been shown on sperm and in the reproductive fluids of males. Antibodies directed against the C-terminal sequence near the GPI-anchor site, an N-terminal sequence, and against the whole protein showed that the Prp isoforms were compartmentalized within the reproductive tract of the ram. Immunoblotting with the three antibodies showed that the complete protein and both N- and C-terminally truncated and glycosylated isoforms are present within cauda epididymal fluid and seminal plasma. Moreover, we demonstrate that in these fluids, the PrpC isoforms are both in a soluble state as well as associated with small membranous vesicles (epididymosomes). We also report that only one major glycosylated 25 kDa C-terminally truncated PrpC isoform is associated with sperm from the testis, cauda epididymis, and semen, and this form is also present in the sperm cytoplasmic droplets that are released during maturation. In sperm, this C-terminal truncated form was found to be associated with membrane lipid rafts present in the mature sperm, suggesting a role for it in the terminal stages of sperm maturation.

Mapping PrPSc Propagation in Experimental and Natural Scrapie in Sheep with Different PrP Genotypes

Twenty-one orally inoculated and seven naturally infected sheep with scrapie were examined for PrPSc in peripheral tissues and in the central nervous system (CNS), using immunohistochemistry. In the inoculated group, VRQ (valine at codon 136, arginine at codon 154 and glutamine at codon 171)/VRQ sheep generally had a greater accumulation of the pathologic form of prion protein (PrPSc) in peripheral tissues, as compared with VRQ/ARQ (alanine at codon 136, arginine at codon 154, and glutamine at codon 171) animals at corresponding time points after inoculation. PrPSc was not detected in the ileal Peyers patch, the spleen, the superficial cervical lymph node, and peripheral nervous tissues of several inoculated VRQ/ARQ animals. All inoculated VRQ/VRQ sheep, but only one of eight inoculated VRQ/ARQ animals, were PrPSc-positive in the CNS. Thus, the propagation of PrPSc seemed slower and more limited in VRQ/ARQ animals. Tissue and cellular localization of PrPSc suggested that PrPSc was disseminated through three different routes. PrPSc-positive cells in lymph node sinuses and in lymphatics indicated spreading by lymph. The sequential appearance of PrPSc in the peripheral nervous system and the CNS, with satellite cells as early targets, suggested the periaxonal transportation of PrPSc through supportive cells. Focal areas of vascular amyloid-like PrPSc in the brain of five sheep, suggested the hematogenous dissemination of PrPSc. There was a poor correlation between the amount of PrPSc in the CNS and clinical signs. One subclinically affected sheep showed widespread PrPSc accumulation in the CNS, whereas three sheep had early clinical signs without detectable PrPSc in the CNS. A VV136 (homozygous for valine at codon 136) sheep inoculated with ARQ/ARR (alanine at codon 136, arginine at codon 154, and arginine at codon 171) tissue succumbed to disease, demonstrating successful heterologous transmission. Less susceptible sheep receiving VRQ/VRQ or ARQ/ARR material were PrPSc-negative by immunohistochemistry, enzyme-linked immunosorbent assay, and western blot.

Schmallenberg virus experimental infection of sheep.

Since late 2011, a novel orthobunyavirus, named Schmallenberg virus (SBV), has been implicated in many cases of severely malformed bovine and ovine offspring in Europe. In adult cattle, SBV is known to cause a mild transient disease; clinical signs include short febrile episodes, decreased milk production and diarrhoea for a few days. However, the knowledge about clinical signs and pathogenesis in adult sheep is limited. In the present study, adult sheep of European domestic breeds were inoculated with SBV either as cell culture grown virus or as virus with no history of passage in cell cultures. Various experimental set-ups were used. Sampling included blood collection at different time points during the experimental period and selected organ material at autopsy. Data from this study showed, that the RNAemic period in sheep was as short as reported for cattle; viral genome was detectable for about 3-5 days by real-time RT-PCR. In total, 13 out of 30 inoculated sheep became RNAemic, with the highest viral load in animals inoculated with virus from low cell culture passaged or the animal passaged material. Contact animals remained negative throughout the study. One RNAemic sheep showed diarrhoea for several days, but fever was not recorded in any of the animals. Antibodies were first detectable 10-14 days post inoculation. Viral RNA was detectable in spleen and lymph nodes up to day 44 post inoculation. In conclusion, as described for cattle, SBV-infection in adult sheep predominantly results in subclinical infection, transient RNAemia and a specific antibody response. Maintenance of viral RNA in the lymphoreticular system is observed for an extended period.

Astrocytes accumulate 4-hydroxynonenal adducts in murine scrapie and human Creutzfeldt-Jakob disease.

Scrapie-infected mice are considered a model for study in prion diseases, which are characterized by the progressive accumulation in the brain of an abnormal isoform (PrPsc) of the normal cellular prion protein (PrPc). Increasing data suggest that the neurodegenerative process in prion diseases may result, at least partially, from a defect in antioxidant function, but so far in vivo oxidative stress remains poorly documented. We report here that 4-hydroxynonenal, a lipid peroxidation by-product, forms protein adducts in brains of scrapie-infected mice and of Creutzfeldt-Jakob disease affected patients. In scrapie mice, studies on the progression of PrPsc accumulation, glial activation, ubiquitin deposition, and 4-HNE adduct formation allowed us to conclude the late occurrence of oxidative damage in the course of the disease. Massive 4-HNE accumulation was identified in astrocytes, but not in neurons or microglial cells. These findings suggest an important oxidative stress (and subsequent lipid peroxidation) in astrocytes, with possible consequences on their neuronal trophic function.

Experimental reproduction of bluetongue virus serotype 8 clinical disease in calves.

Cattle are commonly subclinically infected following natural or experimental infection with bluetongue virus (BTV). The introduction of BTV serotype 8 (BTV-8) in Europe has been characterized by the manifestation of clinical signs in infected cattle. In order to study the pathogenesis of BTV-8 in this host, an animal model able to reproduce the clinical manifestations of the disease is required. In this work, two calves were subcutaneously and intravenously injected with a low passage cell-adapted strain of BTV-8. Both calves showed typical bluetongue clinical signs, including pyrexia, ocular discharge, conjunctivitis, oral mucosal congestion, development of ulcers and necrotic lesions on the lips and tongue, submandibular oedema, coronitis and oedema of the coronet and pastern region. A score was assigned depending on the severity of the lesions and a total clinical score was calculated for each animal daily and at the end of the experiment. Both calves became viraemic 24h post-infection and seroconversion occurred between 7 and 11 days P.I. In this study we present the development of a protocol of infection in calves able to reproduce the severity of the lesions observed with BTV-8 in field conditions.

Original Findings Associated with Two Cases of Bovine Papular Stomatitis

ABSTRACT Bovine papular stomatitis virus was isolated from two calves in an animal house with biosafety level 3 confinement. The hypotheses on the origin of the infection, the interesting features of the partial amino acid sequences of the major envelope viral protein, and the importance of diagnostic tools available for animal diseases that are not listed by the World Organization for Animal Health (OIE) are discussed.

Two alternative inocula to reproduce bluetongue virus serotype 8 disease in calves

The aim of this study was to investigate the consequences in calves of two forms of inocula alternative to the use of wild type infectious blood. Two groups of five calves were infected with low cell-passaged virus and infectious blood issued from one animal passage of the same strain. A longitudinal study was implemented and characterised by clinical standardised observations, haematology, BTV RNA detection and viral isolation from blood, detection of serogroup and neutralising antibodies, cytokine expression and post-mortem examination 46 days post-infection (PI). Both tested inocula were able to reproduce clinical expression of the disease, in the bloodstream viral genome was detected until the end of the experiment while virus isolation was possible between days 7 and 31 PI. Humoral immune response developed earlier in calves infected with low cell-passaged virus, while in both groups a massive antibody production was confirmed by the immune balance between IL-4 and IFN-γ expression. Both tested inocula are presented as valid alternative to the use of wild type infectious blood in the study of the pathogenesis of BTV-8 or the efficacy of current and future vaccines.