Dongyue Zhuang

Pregnancy status and fetal prion genetics determine PrPSc accumulation in placentomes of scrapie-infected sheep.

Ovine scrapie is a fatal neurodegenerative disorder that may be transmitted through exposure to infected uterine and placental tissues. Susceptibility to scrapie is primarily controlled by polymorphisms in the prion protein (PrP) gene. Scrapie in the U.S. Suffolk breed and in many breeds in Europe occurs in sheep homozygous for glutamine (171QQ), but rarely in sheep heterozygous for glutamine and arginine (171QR) or homozygous for arginine (171RR) at codon 171 of the PrP gene. This study demonstrated that accumulation of PrPSc in uterine-placental epithelial cells in the placentome was determined by fetal PrP genotype and the pregnancy status of scrapie-infected ewes. PrPSc was detected in 171QQ placentomes of infected ewes, but not in placentomes of infected ewes pregnant with 171QR conceptuses or in the non-pregnant uterus of infected ewes. The distribution of PrPSc plaques in placentomes was temporally associated with stage of gestation. There was a tendency toward increased size and number of placentomal PrPSc plaques from the endometrial stalk (maternal side) to chorionic plate (fetal side). These results indicate that accumulation of PrPSc is eliminated or reduced to undetectable levels in reproductive and placental tissues if infected ewes are not pregnant or conceive conceptuses with a resistant PrP genotype.

Elk with a long incubation prion disease phenotype have a unique PrPd profile

The transmissible spongiform encephalopathies (TSEs) invariably result in fatal neurodegeneration and accumulation of PrPd, an abnormal form of the host prion protein PrPc, encoded by the PRNP gene. A naturally occurring polymorphism (methionine/valine) at PRNP codon 129 is associated with variation in relative disease susceptibility, incubation time, clinical presentation, neuropathology, and/or PrPd biochemical characteristics in a range of human TSEs. A methionine/leucine polymorphism at the corresponding site in the Rocky Mountain elk PRNP gene is associated with variation in relative susceptibility and incubation time in the cervid TSE chronic wasting disease. We now report that elk lacking the predisposing 132-methionine allele develop chronic wasting disease after a long incubation period and display a novel PrPd folding pattern.

PrP-C and PrP-Sc at the Fetal-Maternal Interface

Scrapie is a naturally occurring prion (PrP) disease causing a fatal neurodegenerative disorder in sheep and goats. Previous studies suggest that scrapie is transmitted naturally through exposure to the scrapie agent in wasted placentas of infected ewes. This study determined the distribution and biochemical properties of PrP cellular (PrP-C) and the distribution of PrP scrapie (PrP-Sc) in reproductive, placental, and selected fetal tissues and fetal fluids in sheep. Glycosylated, N-terminally truncated, proteinase K-sensitive PrP-C with apparent molecular masses of 23–37 kDa was present in reproductive, placental, and fetal tissues and fetal fluids. PrP-C was low or undetectable in intercotyledonary chorioallantois, amnion, urachus, amniotic fluid, and fetal urine. In pregnant ewes, cotyledonary chorioallantois, allantoic fluid, and caruncular endometrium contained higher levels of PrP-C than did intercaruncular endometrium, myometrium, oviduct, ovary, fetal bladder, or fetal kidney. Caruncular endometrial PrP-C was up-regulated during pregnancy. Despite the wide distribution of PrP-C in reproductive, placental, and selected fetal tissues and fetal fluid, PrP-Sc was detected only in caruncular endometrium and cotyledonary chorioallantois of pregnant scrapie-infected ewes. The embryo/fetus may not be exposed to scrapie in utero because it is separated physically from PrP-positive allantois and chorioallantois by PrP-negative amnion.

Sparse PrP Sc accumulation in the placentas of goats with naturally acquired scrapie

BackgroundDomestic goats (Capra hircus) are a natural and experimental host of scrapie and bovine spongiform encephalopathy, the transmissible spongiform encephalopathies (TSE) of sheep and cattle. Goats are also susceptible to experimental infection with the agents of TSEs of deer and elk (chronic wasting disease) and humans (Creutzfeldt Jakob disease). Distribution of PrPSc, the abnormal prion protein, is similar in the tissues of scrapie-infected sheep and goats but no data are available on the potential shedding of the agent through the placenta, the presumed route of transmission of ovine scrapie. We describe the sparse accumulation of PrPSc in the placentas of goats with naturally acquired classical scrapie in comparison to field cases of classical ovine scrapie.ResultsPrPSc was detected in the shed placentas from a sample of U.S. goats with naturally occurring scrapie, diagnosed by antemortem lymphoid tissue biopsy or identified as high risk progeny of infected dams. PrPSc accumulation patterns in the intact placentome and western blot banding was similar in the caprine and ovine samples. However, levels of PrPSc estimated from ELISA and immunohistochemistry assays were generally lower in goats than in sheep, although wide variation was noted in both species.ConclusionsPrPSc accumulates in the shed placentas of goats with naturally acquired scrapie. Although these levels were low in most caprine samples, the caprine placenta may contribute to prion contamination of kidding facilities and transmission to co-housed sheep or goats.

Abundant PrPCWD in Tonsil from Mule Deer with Preclinical Chronic Wasting Disease

A monoclonal antibody dot-blot assay was used to evaluate detergent lysates of tonsil tissue from mule deer to detect PrPCWD, the marker for the cervid transmissible spongiform encephalopathy chronic wasting disease (CWD). Samples of formalin-fixed brain and tonsil tissues from mule deer were examined for PrPCWD using immunohistochemistry (IHC) with Mab F99/97.6.1, the gold standard for diagnosis of preclinical CWD. The contralateral tonsil from each of the 143 deer was prepared for confirmatory IHC and as a 10% (wt/vol) detergent lysate without purification or enrichment steps for monoclonal antibody dot-blot assay. PrPCWD was detected by dot-blot assay in 49 of 50 samples considered positive by IHC. Forty-eight of the positive samples were evaluated with a quantitative dot-blot assay calibrated with recombinant PrP. Tonsillar PrPCWD concentrations ranged from 34 to 1,188 ng per 0.5 mg starting wet weight of tissue. The abundant PrPCWD in mule deer tonsil will facilitate development and validation of high-throughput screening tests for CWD in large populations of free-ranging deer.

Identification of atypical scrapie in Canadian sheep

Scrapie, a transmissible spongiform encephalopathy of sheep and goats, exists in most small ruminant-producing countries of the world. A novel form of this disease was recently recognized and is known by various names, including Nor98, Nor98-like, and atypical scrapie. Differing from classic scrapie in epidemiology, histopathology, and biochemical characteristics, atypical scrapie cases have been identified throughout Europe and in the United States. Enhanced scrapie surveillance efforts recently identified 3 cases of atypical scrapie in Canada.

Classical scrapie prions in ovine blood are associated with B lymphocytes and platelet-rich plasma

BackgroundClassical scrapie is a naturally occurring transmissible spongiform encephalopathy of sheep and goats characterized by cellular accumulation of abnormal isoforms of prion protein (PrPSc) in the central nervous system and the follicles of peripheral lymphoid tissues. Previous studies have shown that the whole blood and buffy coat blood fraction of scrapie infected sheep harbor prion infectivity. Although PrPSc has been detected in peripheral blood mononuclear cells (PBMCs), plasma, and more recently within a subpopulation of B lymphocytes, the infectivity status of these cells and plasma in sheep remains unknown. Therefore, the objective of this study was to determine whether circulating PBMCs, B lymphocytes and platelets from classical scrapie infected sheep harbor prion infectivity using a sheep bioassay.ResultsSerial rectal mucosal biopsy and immunohistochemistry were used to detect preclinical infection in lambs transfused with whole blood or blood cell fractions from preclinical or clinical scrapie infected sheep. PrPSc immunolabeling was detected in antemortem rectal and postmortem lymphoid tissues from recipient lambs receiving PBMCs (15/15), CD72+ B lymphocytes (3/3), CD21+ B lymphocytes (3/3) or platelet-rich plasma (2/3) fractions. As expected, whole blood (11/13) and buffy coat (5/5) recipients showed positive PrPSc labeling in lymphoid follicles. However, at 549 days post-transfusion, PrPSc was not detected in rectal or other lymphoid tissues in three sheep receiving platelet-poor plasma fraction.ConclusionsPrion infectivity was detected in circulating PBMCs, CD72+ pan B lymphocytes, the CD21+ subpopulation of B lymphocytes and platelet-rich plasma of classical scrapie infected sheep using a sheep bioassay. Combining platelets with B lymphocytes might enhance PrPSc detection levels in blood samples.

The placenta shed from goats with classical scrapie is infectious to goat kids and lambs.

The placenta of domestic sheep plays a key role in horizontal transmission of classical scrapie. Domestic goats are frequently raised with sheep and are susceptible to classical scrapie, yet potential routes of transmission from goats to sheep are not fully defined. Sparse accumulation of disease-associated prion protein in cotyledons casts doubt about the role of the goats placenta. Thus, relevant to mixed-herd management and scrapie-eradication efforts worldwide, we determined if the goats placenta contains prions orally infectious to goat kids and lambs. A pooled cotyledon homogenate, prepared from the shed placenta of a goat with naturally acquired classical scrapie disease, was used to orally inoculate scrapie-naı¨ve prion genotype-matched goat kids and scrapie-susceptible lambs raised separately in a scrapie-free environment. Transmission was detected in all four goats and in two of four sheep, which importantly identifies the goats placenta as a risk for horizontal transmission to sheep and other goats.

Sensitive and specific detection of classical scrapie prions in the brains of goats by real-time quaking-induced conversion.

Real-time quaking-induced conversion (RT-QuIC) is a rapid, specific and highly sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect subinfectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully used to detect PrPSc in various tissues from humans and animals, including sheep, tissues from goats infected with classical scrapie have not yet been tested. Therefore, the aims of the present study were to (1) evaluate whether prion seeding activity could be detected in the brain tissues of goats with scrapie using RT-QuIC, (2) optimize reaction conditions to improve scrapie detection in goats, and (3) compare the performance of RT-QuIC for the detection of PrPSc with the more commonly used ELISA and Western blot assays. We further optimized RT-QuIC conditions for sensitive and specific detection of goat scrapie seeding activity in brain tissue from clinical animals. When used with 200  mM sodium chloride, both full-length sheep rPrPSen substrates (PrP genotypes A136R154Q171 and V136R154Q171) provided good discrimination between scrapie-infected and normal goat brain samples at 10(- )3 dilution within 15  h. Our findings indicate that RT-QuIC was at least 10,000-fold more sensitive than ELISA and Western blot assays for the detection of scrapie seeding activity in goat brain samples. In addition to PRNP WT samples, positive RT-QuIC reactions were also observed with three PRNP polymorphic goat brain samples (G/S127, I/M142 and H/R143) tested. Taken together, these findings demonstrate that RT-QuIC sensitively detects prion seeding activity in classical scrapie-infected goat brain samples.

Transmissibility of caprine scrapie in ovine transgenic mice

BackgroundThe United States control program for classical ovine scrapie is based in part on the finding that infection is typically spread through exposure to shed placentas from infected ewes. Transmission from goats to sheep is less well described. A suitable rodent model for examining the effect of caprine scrapie isolates in the ovine host will be useful in the ovine scrapie eradication effort. In this study, we describe the incubation time, brain lesion profile, glycoform pattern and PrPSc distribution patterns in a well characterized transgenic mouse line (Tg338) expressing the ovine VRQ prion allele, following inoculation with brain from scrapie infected goats.ResultsFirst passage incubation times of caprine tissue in Tg338 ovinized mice varied widely but second passage intervals were shorter and consistent. Vacuolation profiles, glycoform patterns and paraffin-embedded tissue blots from terminally ill second passage mice derived from sheep or goat inocula were similar. Proteinase K digestion products of murine tissue were slightly smaller than the original ruminant inocula, a finding consistent with passage of several ovine strains in previous reports.ConclusionsThese findings demonstrate that Tg338 mice propagate prions of caprine origin and provide a suitable baseline for examination of samples identified in the expanded US caprine scrapie surveillance program.