Caprine PRNP polymorphisms N146S and Q222K are associated with proteolytic cleavage of PrPC.

Expression of the cellular prion protein (PrPC) is crucial for the development of prion diseases. Amino acid changes in PrPC or a reduced amount of PrPC may modulate disease resistance. The relative abundance of C1, a natural α-cleavage fragment of PrPC, was previously found to be associated with a resistant PRNP genotype in sheep. Goats are another small ruminant where classical scrapie susceptibility is under strong genetic control. In this study, we assessed PrPC in goats for the existence of similar associations between PrPC fragments and genotype. Brain tissue homogenates from scrapie-free goats with wild type PRNP or polymorphisms (I142M, H143R, N146S, or Q222K) were deglycosylated prior to immunoblot for assessment of the relative abundance of the C1 fragment of PrPC. The presence of K222 or S146 alleles demonstrated significantly different relative levels of C1 compared to that observed in wild type goats, which suggests that the genotype association with C1 is neither unique to sheep nor exclusive to the ovine Q171R dimorphism.

Pathogen-mediated selection in free-ranging elk populations infected by chronic wasting disease.

Pathogens can exert a large influence on the evolution of hosts via selection for alleles or genotypes that moderate pathogen virulence. Inconsistent interactions between parasites and the host genome, such as those resulting from genetic linkages and environmental stochasticity, have largely prevented observation of this process in wildlife species. We examined the prion protein gene (PRNP) in North American elk (Cervus elaphus nelsoni) populations that have been infected with chronic wasting disease (CWD), a contagious, fatal prion disease, and compared allele frequency to populations with no history of exposure to CWD. The PRNP in elk is highly conserved and a single polymorphism at codon 132 can markedly extend CWD latency when the minor leucine allele (132L) is present. We determined population exposure to CWD, genotyped 1,018 elk from five populations, and developed a hierarchical Bayesian model to examine the relationship between CWD prevalence and PRNP 132L allele frequency. Populations infected with CWD for at least 30-50 y exhibited 132L allele frequencies that were on average twice as great (range = 0.23-0.29) as those from uninfected populations (range = 0.04-0.17). Despite numerous differences between the elk populations in this study, the consistency of increase in 132L allele frequency suggests pathogen-mediated selection has occurred due to CWD. Although prior modeling work predicted that selection will continue, the potential for fitness costs of the 132L allele or new prion protein strains to arise suggest that it is prudent to assume balancing selection may prevent fixation of the 132L allele in populations with CWD.

Antiprion Activity of DB772 and Related Monothiophene- and Furan-Based Analogs in a Persistently Infected Ovine Microglia Culture System.

The transmissible spongiform encephalopathies are fatal neurodegenerative disorders characterized by the misfolding of the native cellular prion protein (PrP(C)) into the accumulating, disease-associated isoform (PrP(Sc)). Despite extensive research into the inhibition of prion accumulation, no effective treatment exists. Previously, we demonstrated the inhibitory activity of DB772, a monocationic phenyl-furan-benzimidazole, against PrP(Sc) accumulation in sheep microglial cells. In an effort to determine the effect of structural substitutions on the antiprion activity of DB772, we employed an in vitro strategy to survey a library of structurally related, monothiophene- and furan-based compounds for improved inhibitory activity. Eighty-nine compounds were screened at 1 µM for effects on cell viability and prion accumulation in a persistently infected ovine microglia culture system. Eleven compounds with activity equivalent to or higher than that of DB772 were identified as preliminary hit compounds. For the preliminary hits, cytotoxicities and antiprion activities were compared to calculate the tissue culture selectivity index. A structure-activity relationship (SAR) analysis was performed to determine molecular components contributing to antiprion activity. To investigate potential mechanisms of inhibition, effects on PrP(C) and PrP(Sc) were examined. While inhibition of total PrP(C) was not observed, the results suggest that a potential target for inhibition at biologically relevant concentrations is through PrP(C) misfolding to PrP(Sc) Further, SAR analysis suggests that two structural elements were associated with micromolar antiprion activity. Taken together, the described data provide a foundation for deeper investigation into untested DB compounds and in the design of effective therapeutics.

Primary transmission of chronic wasting disease versus scrapie prions from small ruminants to transgenic mice expressing ovine or cervid prion protein.

Development of mice expressing either ovine (Tg338) or cervid (TgElk) prion protein (PrP) have aided in characterization of scrapie and chronic wasting disease (CWD), respectively. Experimental inoculation of sheep with CWD prions has demonstrated the potential for interspecies transmission but, infection with CWD versus classical scrapie prions may be difficult to differentiate using validated diagnostic platforms. In this study, mouse bioassay in Tg338 and TgElk was utilized to evaluate transmission of CWD versus scrapie prions from small ruminants. Mice (≥5 per homogenate) were inoculated with brain homogenates from clinically affected sheep or goats with naturally acquired classical scrapie, white-tailed deer with naturally acquired CWD (WTD-CWD) or sheep with experimentally acquired CWD derived from elk (sheep-passaged-CWD). Survival time (time to clinical disease) and attack rates (brain accumulation of protease resistant PrP, PrPres) were determined. Inoculation with classical scrapie prions resulted in clinical disease and 100 % attack rates in Tg338, but no clinical disease at endpoint (>300 days post-inoculation, p.i.) and low attack rates (6.8 %) in TgElk. Inoculation with WTD-CWD prions yielded no clinical disease or brain PrPres accumulation in Tg338 at endpoint (>500 days p.i.), but rapid onset of clinical disease (~121 days p.i.) and 100 % attack rate in TgElk. Sheep-passaged-CWD resulted in transmission to both mouse lines with 100 % attack rates at endpoint in Tg338 and an attack rate of ~73 % in TgElk with some culled due to clinical disease. These primary transmission observations demonstrate the potential of bioassay in Tg338 and TgElk to help differentiate possible infection with CWD versus classical scrapie prions in sheep and 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.

Classical scrapie prions are associated with peripheral blood monocytes and T-lymphocytes from naturally infected sheep.

BACKGROUND: Classical scrapie is a transmissible spongiform encephalopathy (TSE) that affects sheep and goats. Our previous bioassay studies in lambs revealed that scrapie prions could be detected in association with peripheral blood monocular cells (PBMC), B lymphocytes and platelet-rich plasma fractions. In the present study, bioassay in lambs was again used to determine if scrapie prions are associated with the other two subsets of PBMC, monocytes and T lymphocytes. RESULTS: PBMC, monocytes and T lymphocytes were isolated from two preclinically affected VRQ/VRQ sheep naturally infected with classical ovine scrapie and intravenously transfused into VRQ/VRQ lambs post-weaning. As determined using standard immunohistochemistry for scrapie, abnormal isoforms of prion protein were detected in lymphoid tissues of lambs inoculated with PBMC (4/4 recipient lambs), monocytes (2/5) and T lymphocytes (1/4). Prion protein misfolding activity was detected by serial protein misfolding cyclic amplification (sPMCA) in PBMC from monocyte and T lymphocyte recipient sheep in agreement with antemortem rectal biopsy results, but such prion protein misfolding activity was not detected from other recipients. CONCLUSIONS: These findings show that scrapie prions are associated with monocytes and T lymphocytes circulating in the peripheral blood of sheep naturally infected with classical scrapie. Combined with our previous findings, we can now conclude that all three major subsets of PBMC can harbor prions during preclinical disease and thus, present logical targets for development of a sensitive assay to detect scrapie prions. In this regard, we have also demonstrated that sPMCA can be used to detect scrapie prions associated with PBMC.

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 goat's placenta. Thus, relevant to mixed-herd management and scrapie-eradication efforts worldwide, we determined if the goat's 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-naïve 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 goat's placenta as a risk for horizontal transmission to sheep and other goats.

Differential immunoreactivity of goat derived scrapie following in vitro misfolding versus mouse bioassay.

The protein misfolding cyclic amplification (PMCA) assay allows for detection of prion protein misfolding activity in tissues and fluids from sheep with scrapie where it was previously undetected by conventional western blot and immunohistochemistry assays. Studies of goats with scrapie have yet to take advantage of PMCA, which could aid in discerning the risk of transmission between goats and goats to sheep. The aim of the current study was to adapt PMCA for evaluation of scrapie derived from goats. Diluted brain homogenate from scrapie-infected goats (i.e., the scrapie seed, PrP(Sc)) was subjected to PMCA using normal brain homogenate from ovinized transgenic mice (tg338) as the source of normal cellular prion protein (the substrate, PrP(C)). The assay end-point was detection of the proteinase K-resistant misfolded prion protein core (PrP(res)) by western blot. Protein misfolding activity was consistently observed in caprine brain homogenate diluted 10,000-fold after 5 PMCA rounds. Epitope mapping by western blot analyses demonstrated that PrP(res) post-PMCA was readily detected with an N-terminus anti-PrP monoclonal antibody (P4), similar to scrapie inoculum from goats. This was in contrast to limited detection of PrP(res) with P4 following mouse bioassay. The inverse was observed with a monoclonal antibody to the C-terminus (F99/97.6.1). Thus, brain homogenate prepared from uninoculated tg338 served as an appropriate substrate for serial PMCA of PrP(Sc) derived from goats. These observations suggest that concurrent PMCA and bioassay with tg338 could improve characterization of goat derived scrapie.

PrPres in placental tissue following experimental transmission of atypical scrapie in ARR/ARR sheep is not infectious by Tg338 mouse bioassay.

Nor98-like atypical scrapie is a sporadic disease that affects the central nervous system of sheep and goats that, in contrast to classical scrapie, is not generally regarded as naturally transmissible. However, infectivity has been demonstrated via bioassay not only of brain tissue but also of certain peripheral nerves, lymphoid tissues, and muscle. This study examines placental tissue, a well characterized route of natural transmission for classical scrapie. Further, this study was conducted in sheep homozygous for the classical scrapie resistant ARR genotype and is the first to characterize the transmission of Nor98-like scrapie between homozygous-ARR sheep. Nor98-like scrapie isolated from a United States ARR/ARR sheep was transmitted to four ARR/ARR ewes via intracerebral inoculation of brain homogenate. These ewes were followed and observed to 8 years of age, remained non-clinical but exhibited progression of infection that was consistent with Nor98-like scrapie, including characteristic patterns of PrPSc accumulation in the brain and a lack of accumulation in peripheral lymphoid tissues as detected by conventional methods. Immunoblots of placental tissues from the infected ewes revealed accumulation of a distinct conformation of PrPres, particularly as the animals aged; however, the placenta showed no infectivity when analyzed via ovinized mouse bioassay. Taken together, these results support a low risk for natural transmission of Nor98-like scrapie in ARR/ARR sheep.

Theileria equi RAP-1a and RAP-1b proteins contain immunoreactive epitopes and are suitable candidates for vaccine and diagnostics development.

Theileria equi is an obligate intracellular protozoan parasite that causes severe hemolytic anaemia in most equid species. Similar to other apicomplexan parasites, T. equi contains rhoptries whose contents have been implicated in host cell invasion and formation of the parasitophorous vacuole that is crucial for survival of the species within cells. Despite their importance, the composition of T. equi rhoptries and their role(s) in host cell invasion remain unexplored. To gain insight into these issues, we evaluated the expression, immunogenicity, and functional roles of two T. equi rhoptry-associated proteins abbreviated as RAP-1a and RAP-1b. The full-length RAP-1a protein was expressed to perform the analysis but our efforts to express the full-length RAP-1b protein failed due to an unknown reason. We therefore generated synthetic immunogenic peptides that map onto the N- and C-termini of the RAP-1b protein as an alternative approach. Our findings show that both proteins are expressed in the extracellular and intra-erythrocytic merozoite stages of T. equi. Serological analyses show that T. equi-infected horses mount antibody responses that recognise both proteins and correlate with a decrease in T. equi load in both acutely and persistently infected horses. In vitro neutralisation studies show that the T. equi RAP-1a protein contains neutralisation-sensitive epitopes as antibodies developed against the protein significantly inhibited the parasites from invading equine erythrocytes. Conversely, antibodies developed against the RAP-1b synthetic peptides did not neutralise parasite invasion, showing that the protein regions on which the peptides were based are not required for T. equi invasion. Overall, the data shows that T. equi rhoptries and their contents are involved in invasion of host cells and supports T. equi RAP-1 proteins as candidates for developing novel serodiagnosis tools and vaccines.

Theileria parasites subvert E2F signaling to stimulate leukocyte proliferation.

Intracellular pathogens have evolved intricate mechanisms to subvert host cell signaling pathways and ensure their own propagation. A lineage of the protozoan parasite genus Theileria infects bovine leukocytes and induces their uncontrolled proliferation causing a leukemia-like disease. Given the importance of E2F transcription factors in mammalian cell cycle regulation, we investigated the role of E2F signaling in Theileria-induced host cell proliferation. Using comparative genomics and surface plasmon resonance, we identified parasite-derived peptides that have the sequence-specific ability to increase E2F signaling by binding E2F negative regulator Retinoblastoma-1 (RB). Using these peptides as a tool to probe host E2F signaling, we show that the disruption of RB complexes ex vivo leads to activation of E2F-driven transcription and increased leukocyte proliferation in an infection-dependent manner. This result is consistent with existing models and, together, they support a critical role of E2F signaling for Theileria-induced host cell proliferation, and its potential direct manipulation by one or more parasite proteins.

Tissue inhibitor of metalloproteinase-1 and interleukin-10 in serum from naïve and scrapie infected sheep.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) and interleukin-10 (IL-10) were identified as potential biomarkers for ovine scrapie in a mouse model. The development of novel diagnostic methods to identify pre-clinical scrapie-infected animals is needed. In this study, ELISA was used to assess TIMP-1 and IL-10 levels in 158 serum samples from naïve and preclinical scrapie-infected sheep. Young (≤ 18 months) naïve sheep had significantly lower TIMP-1 levels compared with old (≥ 20 months) naïve and old infected sheep (P<0.04). Young naïve sheep had lower IL-10 than old naïve sheep (P<0.001). Both cytokines tended to have lower levels in young naïve sheep compared to infected sheep but this did not reach significance. A larger sample size will be helpful in determining the potential of these cytokines as a diagnostic tool.

Up-regulated expression of spherical body protein 2 truncated copy 11 in Babesia bovis is associated with reduced cytoadhesion to vascular endothelial cells.

The factors involved in gain or loss of virulence in Babesia bovis are unknown. Spherical body protein 2 truncated copy 11 (sbp2t11) transcripts in B. bovis were recently reported to be a marker of attenuation for B. bovis strains. Increased cytoadhesion of B. bovis-infected red blood cells (iRBC) to vascular endothelial cells is associated with severe disease outcomes and an indicator of parasite virulence. Here, we created a stable B. bovis transfected line over-expressing sbp2t11 to determine whether up-regulation of sbp2t11 is associated with changes in cytoadhesion. This line was designated sbp2t11up and five B. bovis clonal lines were derived from the sbp2t11up line by limiting dilution for characterisation. We compared the ability of iRBCs from the sbp2t11up line and its five derivative clonal lines to adhere to bovine brain endothelial cells, using an in vitro cytoadhesion assay. The same lines were selected for in vitro cytoadhesion and the levels of sbp2t11 transcripts in each selected line were quantified. Our results demonstrate that up-regulation of sbp2t11 is accompanied by a statistically significant reduction in cytoadhesion. Confirmed up-regulation of sbp2t11 in B. bovis concomitant with the reduction of iRBC in vitro cytoadhesion to bovine brain endothelial cell is consistent with our previous finding that up-regulation of sbp2t11 is an attenuation marker in B. bovis and suggests the involvement of sbp2t11 transcription in B. bovis virulence.

Quantification of O-GlcNAc protein modification in neutrophils by flow cytometry.

Observations of intracellular O-linked beta-N-acetylglucosamine (O-GlcNAc) protein modification are primarily performed by Western blot or immunofluorescence microscopy. The goal of this study was to develop a flow cytometric-based assay for O-GlcNAc signaling and thus provide a more quantitative and rapid method to facilitate clinical analyses. Isolated peripheral blood neutrophils were stimulated with fMLF after adherence to glass cover slips. Cells in suspension were treated with either fMLF or PMA. Unstimulated cells served as controls. Neutrophils were fixed with formaldehyde and permeabilized with cold methanol before intracellular O-GlcNAc staining. Cells on cover slips were analyzed by fluorescence microscopy, and suspension cell data were acquired by flow cytometry. O-GlcNAc protein modification was increased following neutrophil stimulation with either 100 nM fMLF or 10 nM PMA. Increases were detected following either treatment using both flow cytometry and fluorescence microscopy. The time necessary for the completion of staining, data acquisition, and analysis was considerably less using flow cytometry. In addition, flow cytometry allows for the analysis of a substantially greater number of cells. Neutrophil protein modifications by O-GlcNAc are rapidly detected using flow cytometry and provide information similar to that observed using fluorescence microscopy.

Low-volume goat milk transmission of classical scrapie to lambs and goat kids.

The risk of classical scrapie transmission in small ruminants is highest during the neonatal period with the placenta recognized as a significant source of infection. Milk has also been identified as a source of scrapie with sheep-to-sheep transmission occurring after neonatal consumption of as little as 1-2 liters of milk; concurrent mastitis due to small ruminant lentivirus (SRLV) infection may be associated with increased scrapie transmission via milk in sheep. In contrast, goat-to-sheep transmission has been documented only after prolonged consumption of >30 liters of milk. The goal of the current study was to assess transmission of scrapie to goat kids and lambs following low volume, short duration consumption of milk from infected goats. Milk from two does (female goats) with pre-clinical scrapie was fed to four goat kids (≤4.5 L each) and four lambs (~3.7 L each) beginning ~24 hours after birth. Scrapie transmission was detected in three sheep as early as 18 months post inoculation; transmission was also detected in two goats but not until postmortem analyses at 33 months post inoculation. Each milk donor goat also had naturally-acquired infection with SRLV. Different degrees of lymphohistiocytic inflammation and PrPSc accumulation were observed in mammary gland tissues of the donors, which appeared to associate with transmission of scrapie via milk. Thus, similar to the risks of milk transmission of scrapie from sheep, even limited exposure to milk from goats can pose significant risk for scrapie transmission to both goat kids and lambs.

Glucocorticoid modulation of Bcl-2 family members A1 and Bak during delayed spontaneous apoptosis of bovine blood neutrophils.

Neutrophils are critical for innate immune defense against microbial invasion but can also cause inflammatory tissue damage if their life span is not tightly regulated. Antiinflammatory glucocorticoids delay spontaneous apoptosis in human, rodent, and bovine neutrophils, but mechanisms involved are unknown. We hypothesized here that glucocorticoids delay neutrophil apoptosis by altering expression of key Bcl-2 apoptosis regulatory proteins, A1 and Bak, via activation of the cell's glucocorticoid receptors. To test this hypothesis, isolated bovine blood neutrophils were exposed to dexamethasone with and without glucocorticoid receptor antagonism (RU486) and aged ex vivo over 0-24 h for assessment of various spontaneous apoptosis pathway indicators and A1 and Bak abundance. Results show that dexamethasone preserved neutrophil mitochondrial membrane integrity, delayed caspase-9 activation, and reduced the rate of spontaneous apoptosis. Also, dexamethasone increased A1 and decreased Bak mRNA abundance. RU486 pretreatment of the cells abrogated each of these dexamethasone effects. Dexamethasone-induced increases in A1 mRNA were reflected in A1 protein increases, which also were observed in circulating neutrophils of dexamethasone-treated animals. Bak protein decreases were observed in neutrophils of the dexamethasone-treated animals but not in isolated neutrophils, suggesting that stimuli additional to (and perhaps regulated by) glucocorticoid are required to affect Bak protein expression changes in neutrophils. Collectively, our results are unique in demonstrating a mechanism behind glucocorticoid regulation of spontaneous apoptosis and implicate steroid receptor activation and subsequent regulation of A1 and Bak as contributors to mitochondrial membrane stability, reduced caspase-9 activity, and delayed apoptosis in bovine neutrophils exposed to glucocorticoids.

Differences in leukocyte differentiation molecule abundances on domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis) neutrophils identified by flow cytometry.

Although both domestic sheep (DS) and bighorn sheep (BHS) are affected by similar respiratory bacterial pathogens, experimental and field data indicate BHS are more susceptible to pneumonia. Cross-reactive monoclonal antibodies (mAbs) for use in flow cytometry (FC) are valuable reagents for interspecies comparative immune system analyses. This study describes cross-reactive mAbs that recognize leukocyte differentiation molecules (LDMs) and major histocompatibility complex antigens on DS and BHS leukocytes. Characterization of multichannel eosinophil autofluorescence in this study permitted cell-type specific gating of granulocytes for evaluating LDMs, specifically on neutrophils, by single-label FC. Evaluation of relative abundances of LDMs by flow cytometry revealed greater CD11a, CD11b, CD18 (ß2 integrins) and CD 172a (SIRPα) on DS neutrophils and greater CD14 (lipopolysaccharide receptor) on BHS neutrophils. Greater CD25 (IL-2) was identified on BHS lymphocytes following Concavalin A stimulation. While DS and BHS have similar total peripheral blood leukocyte counts, BHS have proportionately more neutrophils.

A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation.

To assess scrapie infectivity associated with caprine-origin tissues, bioassay can be performed using kids, lambs or transgenic mice expressing caprine or ovine prion (PRNP) alleles, but the incubation periods are fairly long. Although several classical ovine scrapie prion permissive cell lines with the ability to detect brain-derived scrapie prion have been available, no classical caprine scrapie permissive cell line is currently available. Therefore, the aims of this study were to generate a rabbit kidney epithelial cell line (RK13) stably expressing caprine wild-type PRNP (cpRK13) and then to assess permissiveness of cpRK13 cells to classical caprine scrapie prion propagation. The cpRK13 and plasmid control RK13 (pcRK13) cells were incubated with brain-derived classical caprine scrapie inocula prepared from goats or ovinized transgenic mice (Tg338, express ovine VRQ allele) infected with caprine scrapie. Significant PrP(Sc) accumulation, which is indicative of scrapie prion propagation, was detected by TSE ELISA and immunohistochemistry in cpRK13 cells inoculated with classical caprine scrapie inocula. Western blot analysis revealed the typical proteinase K-resistant 3 PrP(res) isoforms in the caprine scrapie prion inoculated cpRK13 cell lysate. Importantly, PrP(Sc) accumulation was not detected in similarly inoculated pcRK13 cells, whether by TSE ELISA, immunohistochemistry, or western blot. These findings suggest that caprine scrapie prions can be propagated in cpRK13 cells, thus this cell line may be a useful tool for the assessment of classical caprine prions in the brain tissues of goats.

PRNP variants in goats reduce sensitivity of detection of PrP(Sc) by immunoassay.

Diagnostic analyses often employ single antibody systems but are potentially limited by epitope sequence variation. United States regulatory testing for scrapie primarily uses antibody F99/97.6.1 for immunohistochemistry (IHC) of the prion protein associated with scrapie (PrP(Sc)). Whereas the epitope bound by F99/97.6.1 is highly conserved in sheep, a polymorphism in caprine PRNP results in a glutamine to lysine change at codon 222 and affects PrP detection. This study evaluated the performance of immunoassays (Western blot and IHC) in the presence of PRNP polymorphisms observed in U.S. goat populations. Effects of naturally occurring caprine prion protein alterations at codons 142, 143, 146, 154, or 222 were first evaluated using bacterially expressed recombinant normal cellular prion protein (rec-PrP(C)) and commercially available antibodies (F99/97.6.1, F89/160.1.5, L42, and SAF84). Detection of rec-PrP(C) using F89/160.1.5 was reduced by alterations at 142 and 143; this was also observed in brain PrP(C) from goats expressing these PRNP variants. Effect of allelic variation at 222 was confirmed by Western blot with F99/97.6.1. No differences were observed with L42 or SAF84. IHC of brain demonstrated reduced signal with F89/160.1.5 in animals heterozygous at 143. Decreasing F89/160.1.5 titers were used to demonstrate the impact of PrP(Sc) immunolabeling in preclinical goats and as a surrogate for F99/97.6.1 detection in 222 variants. In the absence of epitope-relevant knowledge of individual goat PRNP, a multi-antibody approach or an antibody that binds an invariant site may provide a more robust immunoassay of PrP(Sc) in classical scrapie, thus reducing the likelihood of false-negative results due to allelic variation.