Quantitative trait loci linked to PRNP gene controlling health and production traits in INRA 401 sheep

In this study, the potential association of PrP genotypes with health and productive traits was investigated. Data were recorded on animals of the INRA 401 breed from the Bourges-La Sapinière INRA experimental farm. The population consisted of 30 rams and 852 ewes, which produced 1310 lambs. The animals were categorized into three PrP genotype classes: ARR homozygous, ARR heterozygous, and animals without any ARR allele. Two analyses differing in the approach considered were carried out. Firstly, the potential association of the PrP genotype with disease (Salmonella resistance) and production (wool and carcass) traits was studied. The data used included 1042, 1043 and 1013 genotyped animals for the Salmonella resistance, wool and carcass traits, respectively. The different traits were analyzed using an animal model, where the PrP genotype effect was included as a fixed effect. Association analyses do not indicate any evidence of an effect of PrP genotypes on traits studied in this breed. Secondly, a quantitative trait loci (QTL) detection approach using the PRNP gene as a marker was applied on ovine chromosome 13. Interval mapping was used. Evidence for one QTL affecting mean fiber diameter was found at 25 cM from the PRNP gene. However, a linkage between PRNP and this QTL does not imply unfavorable linkage disequilibrium for PRNP selection purposes

Optimization of RT-QuIC for detection of seeding activity in preclinical blood samples from prion-infected sheep.

Contrasting the knowledge about prion diseases or TSEs in sheep, only a very limited number of strain typing studies are available in goats. Two cases deriv- ing from the zoonotic bovine BSE epidemic were however detected in goats. During 2004–2012, over 70 TSE goat brain samples were collected from seven European countries and evaluated for TSE type/strain variation. A selection of these materials was chosen for in-depth analysis based on various criteria: tissue quality, genotype, broad geographical distribution, potential type variation...

Prions in Milk from Ewes Incubating Natural Scrapie

Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrPSc accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 µg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species

Four types of scrapie in goats differentiated from each other and bovine spongiform encephalopathy by biochemical methods

Scrapie in goats has been known since 1942, the archetype of prion diseases in which only prion protein (PrP) in misfolded state (PrPSc) acts as infectious agent with fatal consequence. Emergence of bovine spongiform encephalopathy (BSE) with its zoonotic behaviour and detection in goats enhanced fears that its source was located in small ruminants. However, in goats knowledge on prion strain typing is limited. A European-wide study is presented concerning the biochemical phenotypes of the protease resistant fraction of PrPSc (PrPres) in over thirty brain isolates from transmissible spongiform encephalopathy (TSE) affected goats collected in seven countries. Three different scrapie forms were found: classical scrapie (CS), Nor98/atypical scrapie and one case of CH1641 scrapie. In addition, CS was found in two variants—CS-1 and CS-2 (mainly Italy)—which differed in proteolytic resistance of the PrPres N-terminus. Suitable PrPres markers for discriminating CH1641 from BSE (C-type) appeared to be glycoprofile pattern, presence of two triplets instead of one, and structural (in)stability of its core amino acid region. None of the samples exhibited BSE like features. BSE and these four scrapie types, of which CS-2 is new, can be recognized in goats with combinations of a set of nine biochemical parameters

Quantitative trait loci linked to PRNP gene controlling health and production traits in INRA 401 sheep

In this study, the potential association of PrP genotypes with health and productive traits was investigated. Data were recorded on animals of the INRA 401 breed from the Bourges-La Sapinière INRA experimental farm. The population consisted of 30 rams and 852 ewes, which produced 1310 lambs. The animals were categorized into three PrP genotype classes: ARR homozygous, ARR heterozygous, and animals without any ARR allele. Two analyses differing in the approach considered were carried out. Firstly, the potential association of the PrP genotype with disease (Salmonella resistance) and production (wool and carcass) traits was studied. The data used included 1042, 1043 and 1013 genotyped animals for the Salmonella resistance, wool and carcass traits, respectively. The different traits were analyzed using an animal model, where the PrP genotype effect was included as a fixed effect. Association analyses do not indicate any evidence of an effect of PrP genotypes on traits studied in this breed. Secondly, a quantitative trait loci (QTL) detection approach using the PRNP gene as a marker was applied on ovine chromosome 13. Interval mapping was used. Evidence for one QTL affecting mean fiber diameter was found at 25 cM from the PRNP gene. However, a linkage between PRNP and this QTL does not imply unfavorable linkage disequilibrium for PRNP selection purposes

Neuroimmune connections in ovine pharyngeal tonsil: potential site for prion neuroinvasion

Recent studies have proved the possible implication of nasal associated lymphoid tissues, mainly the pharyngeal tonsil, in prion pathogenesis. However, the mechanisms of this neuroinvasion are still being debated. To determine the potential sites for prion neuroinvasion inside the ovine pharyngeal tonsil, the topography of neurofilaments heavy (200 kDa) (NFH), neurofilaments light (70 kDa) (NFL) and glial fibrillar acidic protein (GFAP) was semi-quantitatively analysed inside the different compartments of the tonsil. The results showed that the most innervated areas were the interfollicular area and the connective tissue located beneath the respiratory epithelium. Even if the germinal centre of the lymphoid follicles was poorly innervated, the existence of rare follicular dendritic cell-nerve synapses inside the germinal centre indicates that this mechanism of neuroinvasion is possible but unlikely to be unique. The host PRNP genotype did not influence the pattern of innervation in these different tonsil compartments, unlike age: an increase of nerve endings in a zone of high trafficking cells beneath the respiratory epithelium occurred with ageing. A minimal age-related increase of innervation inside the lymphoid follicles was also observed. An increase in nerve fibre density around the lymphoid follicles, in an area rich in mobile cells able to transport PrPd, could ensure a more efficient infectivity, not in the early phase but in the advanced phase of lymphoinvasion after amplification of PrPd, or could act as direct site of entry during neuroinvasion

PrP^res detection in PMCA reactions seeded with white blood cells (WBC) from BSE infected and healthy sheep.

<p>WBC from BSE orally challenged and TSE free control ARQ/ARQ sheep were homogenised and used to seed PMCA reactions. Brain homogenate from ovine PrP transgenic mouse (ARQ variant) was used as PMCA substrate. Each sample was submitted to up to 6 rounds of amplification. Resulting PMCA products were analyzed by Western Blot (WB) for the presence of abnormal PK resistant PrP (PrP^res -antibody Sha31 epitope YEDRYYRE). On each gel a classical scrapie isolate (PK digested) was used as positive control (WB control). (<b>A</b>) In BSE orally challenged sheep (ARQ/ARQ), WBC prepared from blood collected at different time points (indicated as months post inoculation: mpi) of the incubation period were tested. The first clinical signs developed at 20 mpi. (<b>B</b>) BSE affected sheep (3 different individuals-20 mpi) and TSE free controls sheep (breed, genotype and age matched) were submitted to up to 6 PMCA rounds to check the specificity of the amplification.</p

Red blood cell and inhibition of PMCA vCJD amplification.

<p>(A) Two vCJD brain homogenate (human) dilution series (1/10 dilution, 10⁻² to 10⁻⁹) were prepared (aliquots of 450 µL). Each aliquot also contained 50 µL of PMCA buffer (series 1: ▿) or Red blood cell lysate (human) (series 2:▾). A third series (series 3: ) was prepared starting from a 10⁻² dilution of the vCJD brain homogenate (900 µL) in which 100 µL of red blood cell lysate have been added. In each aliquot (450 µL) of dilution series 3, 50 µL of PMCA buffer were added. The three dilution series were then used to seed PMCA reactions (7 µL of seed) in which ovine PrP expressing mice (ARQ variant) was used as substrate (63 µL). Five successive rounds of PMCA were performed. After each round PrP^res detection was carried out in PMCA reactions by Western blot (Sha31 anti PrP monoclonal antibody: epitope: YEDRYYRE, amino acid 145–152). Ten unseeded controls (○) were included in the experiment. The results of the PMCA amplification after each round are presented in graph (<b>B</b>). WB corresponding to the fifth round of amplification is presented as an illustration (<b>C</b>).</p

PrP^res in PMCA reactions seeded with WBC or BC from a vCJD affected patients and healthy controls.

<p>(<b>A</b>) WBCs from a French vCJD affected patient (vCJD-WBC) and healthy controls (H-WBC) were used to seed serial PMCA amplification (six rounds). PMCA controls included unseeded reactions (no seed). (<b>B</b>) Similarly, nine human buffy coat samples (received from the MRC Prion Unit (London, UK) were used to seed serial PMCA amplification (six rounds). The panel included three vCJD affected patients (sample 1, 3 and 8) and six healthy controls. A vCJD brain homogenate (10%, 10⁻⁸ diluted) was used as positive amplification control. In all cases brain homogenate from ovine PrP transgenic mice (ARQ variant) was used as substrate. PMCA products were analyzed by Western blot (WB) for the presence of abnormal PK resistant PrP (PrP^res -antibody Sha31 epitope YEDRYYRE). On each gel a classical scrapie isolate (PK digested) was used as positive control (WB control).</p

Analytical sensitivity of endogenous vCJD agent PMCA detection in the blood of affected patients.

<p>1/10 dilution series (in PMCA buffer) were prepared using (<b>A</b>) WBC homogenate from one French vCJD affected patient and (<b>B</b>) buffy coat homogenates from two UK vCJD affected patients. These dilutions series were used to seed serial PMCA amplifications (up to 6 rounds) using brain homogenate from ovine PrP transgenic mouse (ARQ variant) as substrate. After each round, PMCA products were analyzed by Western Blot (WB) for the presence of abnormal PK resistant PrP (PrP^res - antibody Sha31 epitope YEDRYYRE). On each gel a classical scrapie isolate (PK digested) was used as positive control (WB control). (<b>A</b>) WBC homogenate from the French vCJD affected patient (Hu vCJD WBC) and healthy patients (H-WBC) were submitted to three amplification rounds. Each dilution was tested in duplicate. The equivalent whole blood amount used to seed the reactions is indicated in the figure. (<b>B</b>) PrP^res WB detection after the first (white circle), the second (grey circle), the third (black circle) and the sixth (white triangle) round of PMCA. Reactions were unseeded (no seed) or seeded with serial 1/10 dilution of the nine BC samples provided by the MRC unit (London, UK). (<b>C</b>) WBC from the French vCJD affected patient was tested either alone or after pooling with WBC from 11 (p12), 23 (p24), 47 (p48) or 95 (p96) healthy controls. The WBC homogenates used to prepare pools were equivalent to 50 µL of starting whole blood. Reactions seeded with WBC from healthy controls (H-WBC) were included as controls.</p