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

PrP^Sc distribution in the organism of scrapie incubating animals.

<p>Groups of 4 ARQ/VRQ and 4 VRQ/VRQ sheep (named a-b-c-d) were killed at different time of the incubation period. Clinical signs occurred at 20 months in VRQ/VRQ animals and at 32 months in ARQ/VRQ. A systematic PrP^Sc detection was realized using immunohistochemistry (8G8 antibody) in a large panel of the collected sheep tissues. PrP^Sc accumulation level was scored according to a semi-quantitative scale: (−) no PrP^Sc, (+) minimal PrP^Sc deposits, (++), (+++) moderate PrP^Sc deposits and (++++) strong PrP^Sc deposits.</p><p><b>LN</b>: Lymph Node; <b>MLN</b>: Mesenteric Lymph Node; <b>PP</b>: Peyer's Patches; <b>ENS</b>: Enteric Nervous System.</p

Infectivity testing in a reference brain sample and colostrum/milk fractions from scrapie incubating ewes.

<p>(A,B) Survival curve in Tg338 mice (transgenic mice over-expressing ovine VRQ PRP allele) intracerebrally inoculated with colostrum (A) and milk (B), collected from ewes incubating scrapie. Samples were first fractionated into cellular pellet (▵), cream (▿), and casein whey (○). An immunoprecipitation of PrP on magnetic beads coated with anti-PrP antibodies was then carried out. Beads from each fraction were inoculated into five or six Tg338 mice. (A) Colostrum fractions from a ewe harbouring mammary ectopic lymphoid follicles associated with Maedi lesions (white symbols) and from a ewe with a healthy mammary gland (black symbols). (B) Milk fractions from the same ewes as in A (black symbols and white symbols) and of the cellular fraction from a second scrapie incubating ewe with a healthy mammary gland (grey symbols). The experiment was terminated after 900 days (normal Tg338 mouse lifespan). Incubation periods have to be compared to those of successive 1/10 dilutions of brain (obex- vertical dotted lines) material from a sheep clinically affected with scrapie. The start point (neat) corresponds to the inoculation of 2.5 µg of brain tissue per mice. (C) Western-blotting (anti-PrP SHa31 antibody) of without (lane 1) and with (lane 2) PK treatment of brain material from a Tg338 mouse inoculated with scrapie positive brain (10⁻³ diluted); (lanes 2–6) PK digested brain material from mice inoculated with milk and colostrum cellular fraction – (lane 3) milk from a ewe with a healthy mammary gland – (lane 4) colostrum from a ewe with a healthy mammary gland – (lane 5) milk from TSE free control – (lane 6) colostrum from a Maedi affected (ectopic lymphoid follicle) ewe. (D) Intracerebral end point titration of a 12.5% obex homogenate, prepared from a terminally scrapie affected sheep (Langlade isolate), in a Tg338 mouse model. This titration allowed the determination of the infectious dose 50 (ID₅₀) of the brain sample (10^6.8 ID₅₀/g), see the text. (E) Variation of the incubation period as a function of the infectious dose inoculated intracerebrally in Tg338 mice (obex – Langlade isolate), see the text.</p

PrP^Sc detection in mammary gland from scrapie-incubating sheep.

<p>(A) PrP^Sc immunolabelling (8G8 monoclonal antibody- DAB brown deposit – bar: 80 µm) in mammary gland from a ewe incubating scrapie (preclinical phase – 15 months old – ARQ/ARQ genotype) and harbouring lympho-proliferative mastitis with ectopic lymphoid follicles (Foll.). In the milk ducts lumen (arrow heads), several PrP^Sc positive cells are identifiable. (B) In mammary gland acini (Aci.), positive PrP^Sc staining can be observed; either associated with cells or distributed as free granules. (C) Double labelling for PrP^Sc (R521 polyclonal serum – black deposits) and CD68 (KiM6 clone – red deposits) indicates that intracellular PrP^Sc in milk ducts and acini lumen is associated with phagocytic cells. (D) PrP^Sc immunolabelling (8G8 anti-PrP antibody – DAB brown deposit- bar: 200 µm) and (E) PET blot (SHa31 antibody – NBT/BCIP black deposits – bar: 200 µm) of two successive mammary gland sections confirmed that material in milk ducts is proteinase K resistant (arrow heads indicate lining).</p

Estimation of infectious titre in colostrum and milk from scrapie incubating ewes with apparently healthy mammary glands or lymphoproliferative mastitis (consecutive to Maedi infection).

<p>For each fraction (cell pellet, casein whey, cream) the quantity of the material submitted to immunoprecipitation process is detailed and linked to the initial volume of colostrum or milk from which it was prepared. In samples for which a 100% attack rate was observed, mean incubation period were used to estimate the infectious titre (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000238#ppat-1000238-g003" target="_blank">Figure 3E</a>). For each considered fraction the infectious titre per ml of starting material was calculated. The global infectious titre per ml of colostrum and milk was finally obtained by adding the value corresponding to each fraction.</p><p>N.A: not available at the moment of writing. *Infectivity was estimated from the only those fractions for which results are available. Consequently the calculated infectious titre/ml of milk is certainly underestimated.</p

Immunoprecipitation of PrP in milk and colostrum.

<p>(A) PrP in milk (▵) and colostrum (○), from a negative control animal and three scrapie incubating sheep (casein whey protein extract following NP40/DOC – 10 min at 37°C treatment). PRP levels were measured before (black symbols) and after (white symbols) immunoprecipitation with antibodies (SHa31, SAF-34, and βS-36). The dosage was performed using a two-site sandwich immunoassay (capture antibody 11C6, tracer antibody Bar-224). The positive threshold of the test (0.040 absorbance units) is symbolised by the dotted line. (B–E) PrP contained in different fractions was immunoprecipitated with Sha31/SAF-34/BS36 immunobeads. After washings, PK in PBS (0 to 10 µg in 50 µL) was added to the beads for 10 min at 37°C. Samples were denatured in laemmli's buffer (25 µL), without β-mercaptoethanol, for 5 min at 100°C. Supernatants were then analysed by western blot. (B) 1.4 mL of casein whey, prepared from colostrum (left four lanes) or milk (right four lanes of the gel), from a scrapie incubating ewe (0942 see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000238#ppat-1000238-t004" target="_blank">Table 4</a>), (C) 1.4 mL of casein whey prepared from a TSE free control milk, (D) 100 µl of scrapie positive 2% brain homogenate or (E) 100 µl of scrapie negative 2% brain homogenate.</p

End-point titration of a brain homogenate (posterior brainstem- 12.5% weight/volume homogenate) in Tg338 mice.

<p>The donor ewe was born and bred in the Langlade Flock. This ewe was at the terminal stage of Scrapie at the moment of culling. Each mouse was intracerebrally inoculated with 20 µl of homogenate. Mice were considered positive when abnormal PrP deposition was detected in brain. Incubation periods are presented as mean+/−SD except for that dilution with which less than 20% of mice were found positive. In that case (*) incubation times of the positive mice are individually presented.</p

PrP^Sc in central nervous system, lympho-reticular system, mammary gland and milk duct lumen of natural scrapie exposed ewes bearing various genotypes at codons 136, 154 and 171 of the PRP gene.

<p>All investigated animals (n = 175) were PCR positive for Maedi lentivirus. Abnormal PrP (PrP^Sc) detection was carried out using Western Blotting (SHa31 anti-PrP antibody), ELISA (TeSeE Sheep and Goat, Bio-Rad) and immunohistochemistry (8G8 anti-PrP antibody). The majority of susceptible genotype sheep (ARQ/VRQ- VRQ/VRQ and ARQ/ARQ) were clinical suspect for scrapie at culling.</p