PrP\u3csup\u3eSc\u3c/sup\u3e accumulation in fetal cotyledons of scrapieresistant lambs is influenced by fetus location in the uterus

Placentae from scrapie-infected ewes have been shown to accumulate PrPSc when the genotype of the fetus is of a susceptible genotype (VRQ/VRQ, ARQ/VRQ or ARQ/ARQ). Cotyledons from fetuses of genotypes ARR/ARR, ARQ/ARR and ARQ/VRR have previously been shown to be resistant to PrPSc accumulation. By using ewes from a naturally infected scrapie flock, cotyledons from fetuses of multiple births of different genotypes were examined. PrPSc was detected in fetal cotyledons of genotype ARQ/ARQ, but not in cotyledons from their dizygotic twin of genotype ARQ/ARR. This confirms earlier reports of single fetuses of these genotypes, but is the first description of such a finding in twin fetuses, one of each genotype. It is also demonstrated that cotyledons from sibling fetuses of genotypes ARQ/VRQ and ARQ/ARQ have different patterns of PrPSc accumulation depending on whether the dam is of genotype ARQ/ARQ or ARQ/VRQ. Lastly, it is shown that cotyledons from fetuses with resistant genotypes are weakly positive for PrPSc when they have shared the same pregnant uterine horn with a fetus of a susceptible genotype with cotyledons positive for the detection of PrPSc. Additionally, a PCR product for the Sry gene, a product specific to males, was found in cotyledons from female fetuses that had shared a uterine horn with a male fetus. This indicates that some sharing of fetal blood occurs between placentomes and fetuses residing in the same uterine horn, which can result in PrPSc accumulation in cotyledons with resistant genotypes

PrP\u3csup\u3eSc\u3c/sup\u3e accumulation in fetal cotyledons of scrapieresistant lambs is influenced by fetus location in the uterus

Placentae from scrapie-infected ewes have been shown to accumulate PrPSc when the genotype of the fetus is of a susceptible genotype (VRQ/VRQ, ARQ/VRQ or ARQ/ARQ). Cotyledons from fetuses of genotypes ARR/ARR, ARQ/ARR and ARQ/VRR have previously been shown to be resistant to PrPSc accumulation. By using ewes from a naturally infected scrapie flock, cotyledons from fetuses of multiple births of different genotypes were examined. PrPSc was detected in fetal cotyledons of genotype ARQ/ARQ, but not in cotyledons from their dizygotic twin of genotype ARQ/ARR. This confirms earlier reports of single fetuses of these genotypes, but is the first description of such a finding in twin fetuses, one of each genotype. It is also demonstrated that cotyledons from sibling fetuses of genotypes ARQ/VRQ and ARQ/ARQ have different patterns of PrPSc accumulation depending on whether the dam is of genotype ARQ/ARQ or ARQ/VRQ. Lastly, it is shown that cotyledons from fetuses with resistant genotypes are weakly positive for PrPSc when they have shared the same pregnant uterine horn with a fetus of a susceptible genotype with cotyledons positive for the detection of PrPSc. Additionally, a PCR product for the Sry gene, a product specific to males, was found in cotyledons from female fetuses that had shared a uterine horn with a male fetus. This indicates that some sharing of fetal blood occurs between placentomes and fetuses residing in the same uterine horn, which can result in PrPSc accumulation in cotyledons with resistant genotypes

Abstract

I am especially grateful to my major advisor, Dr. Timothy V. Baszler, for his mentoring during this journey. I would also like to thank my committee members, Dr. Donald P. Knowles, Dr. Doug R. Call, and Dr. Kelly A. Brayton, who each provided me with excellent guidance. Dr. Katherine O’Rourke and the rest of the prion group at USDA/ARS/ADRU were indispensible, and for that I thank them. My appreciation is extended to those who provided excellent technical support, Bruce Mathison, Katie Mathison, Beth Mathison, and Brad Meyer, which made this work possible. I relied on the support of friends, Gary Haldorson, Kevin Lahmers, and Sunshine Lahmers, and I thank them for their friendship. I also thank my colleagues, Michael Dark and Joshua Daniels, for accompanying me along this marathon and for always providing assistance, both professionally and personally

Biology of PrP\u3csup\u3esc\u3c/sup\u3e accumulation in two natural scrapie-infected sheep flocks

Sheep scrapie is a prion disease that requires interaction of exogenous prions with host prion protein (PrP) supporting prion formation. Disease is associated with deposition of a host-generated conformational variant of PrP, PrPsc, in a variety of tissues, including brain, resulting in fatal spongiform encephalopathy. Efficiency of PrPsc formation is determined by polymorphisms in the PrP-coding sequence. This article adds to previous data of natural sheep scrapie, concentrating on the effect of host genotype and age on PrPsc accumulation patterns during preclinical and clinical disease. Two entire scrapie-infected, predominantly Suffolk-cross, sheep flocks euthanized for regulatory purposes were genotyped and analyzed for PrPsc deposition in various tissues using single- and dual-label immunohistochemistry. Scrapie, as defined by PrPsc deposition, occurred in 13/80 sheep. Preclinical disease was evident in nearly 70% of infected sheep, ranging in age from 14 months to 7 years. PrPsc accumulated systemically in the nervous tissue, various lymphoid tissues, both alimentary tract related and non–alimentary tract related, and the placenta. Clinical neurological illness was always associated with spongiform encephalopathy and PrPsc deposition in the brain. Only 6 of 9 sheep with preclinical scrapie had PrPsc deposition in the brain but widespread PrPsc deposition in peripheral lymphoid tissue, supporting previous data showing peripheral PrPsc accumulation preceding deposition in the brain. PrPsc colocalized with a marker for follicular dendritic cells throughout the lymphoid system. PrPsc also accumulated in the peripheral nervous system, particularly the nervous supply of the gastrointestinal tract. Abundant PrPsc was evident in trophoblast cells of placentomes but not in the endometrium, myometrium, or associated nervous plexus. PrPsc deposits were not observed in the mammary parenchyma or bone marrow. Scrapie susceptibility was defined genetically by PrP codon 171: PrPsc deposition was restricted to PrP genotype AA136RR154QQ171 in 12/13 cases or AV136RR154QQ171 in 1/13 cases. The earliest accumulation was observed in the single VRQ/ARQ heterozygous animal, consistent with the reported high scrapie susceptibility and brief incubation period observed in breeds with predominance of the V136R154Q171 allele. Disease occurred within, as well as independent of, mother– daughter lines, suggesting both maternal and nonmaternal transmission in the flocks

Biology of PrPsc accumulation in two natural scrapie-infected sheep flocks

Sheep scrapie is a prion disease that requires interaction of exogenous prions with host prion protein (PrP) supporting prion formation. Disease is associated with deposition of a host-generated conformational variant of PrP, PrPsc, in a variety of tissues, including brain, resulting in fatal spongiform encephalopathy. Efficiency of PrPsc formation is determined by polymorphisms in the PrP-coding sequence. This article adds to previous data of natural sheep scrapie, concentrating on the effect of host genotype and age on PrPsc accumulation patterns during preclinical and clinical disease. Two entire scrapie-infected, predominantly Suffolk-cross, sheep flocks euthanized for regulatory purposes were genotyped and analyzed for PrPsc deposition in various tissues using single- and dual-label immunohistochemistry. Scrapie, as defined by PrPsc deposition, occurred in 13/80 sheep. Preclinical disease was evident in nearly 70% of infected sheep, ranging in age from 14 months to 7 years. PrPsc accumulated systemically in the nervous tissue, various lymphoid tissues, both alimentary tract related and non-alimentary tract related, and the placenta. Clinical neurological illness was always associated with spongiform encephalopathy and PrPsc deposition in the brain. Only 6 of 9 sheep with preclinical scrapie had PrPsc deposition in the brain but widespread PrPsc deposition in peripheral lymphoid tissue, supporting previous data showing peripheral PrPsc accumulation preceding deposition in the brain. PrPsc colocalized with a marker for follicular dendritic cells throughout the lymphoid system. PrPsc also accumulated in the peripheral nervous system, particularly the nervous supply of the gastrointestinal tract. Abundant PrPsc was evident in trophoblast cells of placentomes but not in the endometrium, myometrium, or associated nervous plexus. PrPsc deposits were not observed in the mammary parenchyma or bone marrow. Scrapie susceptibility was defined genetically by PrP codon 171: PrPsc deposition was restricted to PrP genotype AA136RR154QQ171 in 12/13 cases or AV136RR154QQ171 in 1/13 cases. The earliest accumulation was observed in the single VRQ/ARQ heterozygous animal, consistent with the reported high scrapie susceptibility and brief incubation period observed in breeds with predominance of the V136R154Q171 allele. Disease occurred within, as well as independent of, mother-daughter lines, suggesting both maternal and nonmaternal transmission in the flocks

PrPSc Is Not Detected in Peripheral Blood Leukocytes of Scrapie-Infected Sheep: Determining the Limit of Sensitivity by Immunohistochemistry

Peripheral blood leukocytes (PBLs) from scrapie-infected sheep were evaluated for the presence of PrP Sc by using dissociated retropharyngeal lymph node (DRLN) cells and immunohistochemistry (IHC). PrP Sc -positive cells were detected in 2.05% ± 0.28% of 3 × 10 6 DRLN cells, but were not detected in 3 × 10 6 PBLs from scrapie-infected sheep. Titration of DRLN cells mixed with PBLs showed that IHC detects a minimum of 0.00205% or 60 PrP Sc -positive cells in 3 × 10 6 PBLs

Biology of PrP sc

Sheep scrapie is a prion disease that requires interaction of exogenous prions with host prion protein (PrP) supporting prion formation. Disease is associated with deposition of a host-generated conformational variant of PrP, PrPsc, in a variety of tissues, including brain, resulting in fatal spongiform encephalopathy. Efficiency of PrPsc formation is determined by polymorphisms in the PrP-coding sequence. This article adds to previous data of natural sheep scrapie, concentrating on the effect of host genotype and age on PrPsc accumulation patterns during preclinical and clinical disease. Two entire scrapie-infected, predominantly Suffolk-cross, sheep flocks euthanized for regulatory purposes were genotyped and analyzed for PrPsc deposition in various tissues using single- and dual-label immunohistochemistry. Scrapie, as defined by PrPsc deposition, occurred in 13/80 sheep. Preclinical disease was evident in nearly 70% of infected sheep, ranging in age from 14 months to 7 years. PrPsc accumulated systemically in the nervous tissue, various lymphoid tissues, both alimentary tract related and non-alimentary tract related, and the placenta. Clinical neurological illness was always associated with spongiform encephalopathy and PrPsc deposition in the brain. Only 6 of 9 sheep with preclinical scrapie had PrPsc deposition in the brain but widespread PrPsc deposition in peripheral lymphoid tissue, supporting previous data showing peripheral PrPsc accumulation preceding deposition in the brain. PrPsc colocalized with a marker for follicular dendritic cells throughout the lymphoid system. PrPsc also accumulated in the peripheral nervous system, particularly the nervous supply of the gastrointestinal tract. Abundant PrPsc was evident in trophoblast cells of placentomes but not in the endometrium, myometrium, or associated nervous plexus. PrPsc deposits were not observed in the mammary parenchyma or bone marrow. Scrapie susceptibility was defined genetically by PrP codon 171: PrPsc deposition was restricted to PrP genotype AA136RR154QQ171 in 12/13 cases or AV136RR154QQ171 in 1/13 cases. The earliest accumulation was observed in the single VRQ/ARQ heterozygous animal, consistent with the reported high scrapie susceptibility and brief incubation period observed in breeds with predominance of the V136R154Q171 allele. Disease occurred within, as well as independent of, mother-daughter lines, suggesting both maternal and nonmaternal transmission in the flocks

Validation of a Commercially Available Monoclonal Antibody-Based Competitive-Inhibition Enzyme-Linked Immunosorbent Assay for Detection of Serum Antibodies to Neospora caninum in Cattle

A previously described monoclonal antibody (MAb)-based competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was modified to optimize performance, and the assay was validated in various defined cattle populations for detection of serum antibody to Neospora caninum , a major cause of bovine abortion. Modifications to the cELISA included capturing native N. caninum antigen with a parasite-specific MAb (MAb 5B6-25) and directly conjugating the competitor MAb (MAb 4A4-2), with both MAbs binding different epitopes of a conserved, immunodominant 65-kDa tachyzoite surface antigen. The assay was validated using three serum sets, a “gold standard” set of 184 cow sera defined by fetal histopathology and N. caninum immunohistochemistry and by maternal N. caninum indirect fluorescence assay (IFA) at a 1:200 serum dilution, a relative standard set of 330 cow sera defined by IFA alone, and a set of 4,323 cow sera of unknown N. caninum status. A test cutoff of 30% inhibition was identified. The diagnostic sensitivity was 97.6%, and diagnostic specificity was 98.6% for the gold standard abortion-defined sera. The diagnostic sensitivity was 96.4%, and diagnostic specificity was 96.8% for the relative standard IFA-defined sera. Testing of the 4,323 bovine sera of unknown N. caninum status revealed a distinct bimodal distribution and steep sigmoid frequency curve with only 1.8% of samples within 5% of the test cutoff, indicating a sharp discrimination between test-positive and test-negative samples. In summary, the modified N. caninum cELISA provided a simple, rapid, and versatile method to accurately identify N. caninum infection status in cattle using a single cutoff value