Guillaume Tabouret

PrPSc accumulation in myocytes from sheep incubating natural scrapie.

Because variant Creutzfeldt–Jakob disease (vCJD) in humans probably results from consumption of products contaminated with tissue from animals with bovine spongiform encephalopathy, whether infectious prion protein is present in ruminant muscles is a crucial question. Here we show that experimentally and naturally scrapie-affected sheep accumulate the prion protein PrPSc in a myocyte subset. In naturally infected sheep, PrPSc is detectable in muscle several months before clinical disease onset. The relative amounts of PrPSc suggest a 5,000-fold lower infectivity for muscle as compared to brain.

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.

Serine protease activity in excretory-secretory products of Oestrus ovis (Diptera: Oestridae) larvae.

The sheep bot fly, Oestrus ovis, is a very common myiasis of nasal and sinus cavities of sheep and goats causing severe welfare and production implications. As the viability of O. ovis adult flies strictly depends on larval abilities to assimilate and to stock nutrients from the host, it was necessary to investigate proteolytic activities in larval excretory/secretory products (ESP). ESP of O. ovis larvae degrade mucosal and plasmatic components such as mucin, albumin or immunoglobulin G. A preliminary biochemical characterization, using substrate gel analysis and inhibitor sensitivity, demonstrated the presence of at least six major serine proteases (molecular weights from 20 to 100 kDa), mainly trypsin-like, secreted in the digestive tube of larvae. Their involvement in larval trophic activity and evasion from the host immune response is further discussed as O. ovis excretory/secretory serine proteases could represent potential vaccinal targets.

Relevance of oral experimental challenge with classical scrapie in sheep.

Oral inoculation is currently considered as the best approach to mimic natural TSE contamination in ruminants. In this study, we compared the timing of abnormal prion protein (PrP(Sc)) dissemination and accumulation in the organism of susceptible sheep either orally inoculated or naturally infected with classical scrapie. Both animal groups shared a similar PrP(Sc) dissemination scheme and accumulation dynamics in lymphoid tissues. However, orally challenged animals displayed an earlier neuro-invasion and a dramatically shorter incubation period than naturally exposed sheep. No differences were observed between the groups with regards to the neuro-invasion route. These results unambiguously indicate that oral inoculation can have an impact on both the earliness of neuro-invasion and the incubation period. They also support the statement that oral inoculation is a relevant model for investigating transmissible spongiform encephalopathy pathogenesis. Nevertheless, data obtained under such experimental conditions should be used with some caution.

Response to dietary-induced energy restriction in dairy sheep divergently selected for resistance or susceptibility to mastitis.

Dairy ruminants experiencing a severe postpartum negative energy balance (NEB) are considered to be more susceptible to mastitis. Although the genetic variability of mastitis resistance is well established, the biological basis of the link between energy metabolism and resistance is mostly unknown. The aim of this study was to characterize the effect of NEB on metabolism and immune response according to the genetic background for mastitis resistance or susceptibility. Forty-eight ewes from high and low somatic cell score (SCS) genetic lines were allocated to 2 homogeneous subgroups 2 wk after lambing: one group (NEB) received an energy-restricted diet to cover 60% of their energy requirements, and the other group received a control (positive energy balance: PEB) diet. Both diets met the protein requirements. After 10 d on either the NEB or PEB diet, all ewes were injected with a Pam3CSK4/MDP solution in one half-udder to induce an inflammatory response. The ewes were monitored for milk production, somatic cell count (SCC), body weight (BW), body condition score (BCS), and blood metabolites. Differential milk cell counts were determined by flow cytometry. Plasma concentrations of glucose, insulin, nonesterified fatty acids (NEFA), β-hydroxybutyrate (BHB), and triiodothyronine were determined. Energy restriction resulted in an increased fat:protein ratio in milk and decreased milk yield, BW, and BCS. The NEB ewes had significantly higher NEFA and BHB and lower plasma glucose concentrations than PEB ewes, reflecting a mobilization of body reserves and ketone body synthesis. High-SCS ewes had a higher SCS than low-SCS throughout the experiment, except after the inflammatory challenge, which resulted in similar SCS in all 4 groups. A noteworthy interaction between genetic background and diet was evidenced on metabolic parameters and BW. Indeed, high-SCS ewes subjected to NEB showed greater decrease in BW and increased NEFA and BHB concentrations compared with low-SCS ewes. Thus, NEB in early lactation led to extensive mobilization of body reserves and intense ketone body synthesis in mastitis-susceptible sheep. These results reinforce the hypothesis of a genetic association between mastitis susceptibility and energy metabolism and open the way to further studies on the biological basis for this association.

Whole blood transcriptome analysis reveals potential competition in metabolic pathways between negative energy balance and response to inflammatory challenge.

Negative Energy Balance (NEB) is considered to increase susceptibility to mastitis. The objective of this study was to improve our understanding of the underlying mechanisms by comparing transcriptomic profiles following NEB and a concomitant mammary inflammation. Accordingly, we performed RNA-seq analysis of blood cells in energy-restricted ewes and control-diet ewes at four different time points before and after intra mammary challenge with phlogogenic ligands. Blood leucocytes responded to NEB by shutting down lipid-generating processes, including cholesterol and fatty acid synthesis, probably under transcriptional control of SREBF 1. Furthermore, fatty acid oxidation was activated and glucose oxidation and transport inhibited in response to energy restriction. Among the differentially expressed genes (DEGs) in response to energy restriction, 64 genes were also differential in response to the inflammatory challenge. Opposite response included the activation of cholesterol and fatty acid synthesis during the inflammatory challenge. Moreover, activation of glucose oxidation and transport coupled with the increase of plasma glucose concentration in response to the inflammatory stimuli suggested a preferential utilization of glucose as the energy source during this stress. Leucocyte metabolism therefore undergoes strong metabolic changes during an inflammatory challenge, which could be in competition with those induced by energy restriction.