Location: Animal Disease Research
2018 Annual Report
Objectives
Objective 1: Determine the effects of the PRNP genotype on current diagnostic test assay accuracy in sheep and goats with scrapie.
Subobjective 1.1: Determine the association of M112T polymorphism with the density and distribution of PrP-Sc in an archived set of brain and lymphoid tissues of sheep from U.S. surveillance program.
Subobjective 1.2: Determine the effect of G127S polymorphism on the temporal spread of PrP-Sc from the gut to the brain in goats.
Objective 2: Develop improved methods for antemortem detection of PrP-Sc in sheep and goats with scrapie.
Subobjective 2.1: Determine the effect of prior biopsy on the kinetics and distribution of PrP-Sc accumulation in the RAMALT of sheep and goats.
Subobjective 2.2: Develop a sensitive, high-throughput assay (immuno-quantitative PCR; immuno-qPCR) suitable for use in veterinary diagnostic laboratories for detection of PrP-Sc in sheep with classical scrapie.
Subobjective 2.3: Determine the suitability of the immuno-qPCR for detection of PrP-Sc(Nor98) in brain, peripheral tissues, and placentas from sheep with Nor98.
Approach
Objective 1 will support eradication efforts by addressing the unknown effects of specific prion protein gene (PRNP) polymorphisms on current diagnostic test performance. Previous work with chronic wasting disease demonstrates that certain PRNP polymorphisms prolong disease incubation and negatively impact diagnostic detection in white-tailed deer. In the current project, two polymorphisms that prolong scrapie incubation in small ruminants and which are common in U.S. livestock will be studied: M112T in sheep and G127S in goat. For sheep, a large validated tissue archive is available to test the hypotheses that the M112T polymorphism (1) affects the probability of detecting PrP-Sc in tissues collected during postmortem surveillance, and (2) the relative quantity and distribution of PrP-Sc accumulating within positive tissues. A similar archive does not exist for goats, thus an inoculation study will be conducted using goats of known genotypes to determine if the G127S polymorphism affects the kinetics of PrP-Sc accumulation in peripheral lymphoid tissues and brain.
Objective 2 aims to improve upon methods of scrapie detection in small ruminants by addressing the unknown effects of previous biopsy on subsequent diagnosis by biopsy of the rectal mucosa, and by producing a higher throughput assay with improved diagnostic sensitivity that might expedite eradication of classical scrapie in the U.S., be adapted to blood-based detection, and improve etiological understanding of atypical (Nor98) scrapie. With regard to rectal biopsy, data from deer suggests prior biopsy may reduce disease detection in subsequent biopsies. This knowledge gap in sheep and goats will be addressed by determining the effect of first biopsy at 1 year of age on the diagnostic quality of the lymphoid tissue remaining after 1 and 2 years healing time. Development of a higher throughput, higher sensitivity diagnostic will be based on detecting total PrP-Sc (proteinase-sensitive and proteinase-resistant) using methods already in use in veterinary diagnostic laboratories in the U.S. The hybrid assay to be developed (immuno-qPCR) couples the specificity and convenience of a well validated, proteinase-free plate binding assay with the high sensitivity and rapid turnaround of real-time PCR. The hybrid assay will be first adapted to tissues collected during postmortem surveillance and sensitivity compared to prion titer as determined by transgenic mouse assay. The hybrid assay will then be applied to the components of blood to which prions are most frequently associated. Finally, this project aims to adapt the immuno-qPCR assay to enhance detection of PrP-Sc(Nor98) and to apply immuno-qPCR and standard transgenic mouse bioassay to determine the infection status of progeny born to Nor98-infected ewes.
Progress Report
Progress was made on infectious disease research on domestic sheep and goats, outcomes of which include two genetic tests newly available to goat producers to help protect their herds from classical scrapie infection.
Objective 1: The aim of experiments is to determine the influence of genetic variation in sheep and goats on surveillance efforts to discover cases of classical scrapie infection as early as possible. Surveillance is most commonly conducted by applying immunohistochemistry to samples of brain and lymphoid tissue collected postmortem.
Sub-objective 1.1: A collection of archived brain and lymph node samples obtained from approximately 2,500 sheep were assessed. Of 1,119 sheep selected for study, 262 bore at least one allele of the M112T polymorphism of the prion protein gene (PRNP). Analyses were completed to test for associations of this and other PRNP polymorphisms with scrapie infection. Measurements of tissue accumulation of disease-associated prion protein (PrPSc) were completed and analyzed to determine the effects of PRNP polymorphisms on scrapie diagnosis by immunohistochemistry.
Sub-objective 1.2: Selected goat breeding was completed and twenty-seven genetically-defined newborn goats were exposed by the oral route to a standardized dose of scrapie prions found in the placenta. The purpose of this study is to determine the impact of the G127S polymorphism of PRNP on detection of classical scrapie infection by immunohistochemistry in goats.
Significant progress regarding genetic resistance to classical scrapie in goats has resulted in two new DNA tests. Progress included publication of a peer-reviewed journal article demonstrating strong resistance under experimental challenge, fulfillment of multiple outgoing agreements with partner organizations involved in commercialization, and publication of an article to facilitate Technology Transfer of DNA testing to the goat producer community. The two DNA tests are now commercially available in the U.S. and enable goat producers to selectively breed goats with strong resistance to classical scrapie. Customers include the largest dairy and meat goat organizations in the U.S., which have both arranged discounts on this DNA testing for their members. This technology will protect goat operations from classical scrapie and its lethal consequences, and it will assist in scrapie eradication by providing a genetic means to protect goats such as has been available for sheep for many years. This will prevent goats from being a reservoir for classical scrapie and will advance international trade by accelerating U.S. scrapie-free status to open export markets.
Objective 2: The goal is to improve the sensitivity of diagnostic testing for scrapie infection. Immunohistochemistry is most commonly applied to biopsies of the rectal mucosa to diagnose classical scrapie infection in living sheep and goats.
Sub-objective 2.1: The effects of the first biopsy on the diagnostic accuracy of a second biopsy, as may be necessary to obtain a proper sample or to monitor progression of infection, is being tested. Accordingly, second year collection and processing of rectal tissues from sheep and goats was completed at prescribed time points after first biopsy, and measurement of PrPSc accumulation in these samples is underway. While application of approved diagnostics such as immunohistochemistry on biopsies of the rectal mucosa has achieved great reductions in the number of new cases of classical scrapie in U.S. sheep, it is likely that final eradication will require development of assays with greatly enhanced sensitivity for earlier stages of infection.
Sub-objective 2.2: Substantial progress was made in developing highly sensitive, high-throughput assays for classical scrapie infection. Second year progress included further development of immuno-PCR and quaking-induced conversion (QuIC) assays, the latter in collaboration with the TSE/Prion Biochemistry Section at the National Institute of Allergy and Infectious Disease (NIAID) Rocky Mountain Laboratories. For immuno-PCR, experiments to adapt the anti-prion antibody F99/97 to a PK-independent commercial enzyme-linked immunosorbent assay (ELISA) kit were not successful. However, considerable progress was made by, instead, adapting DNA marker-assisted detection to the anti-prion antibody provided with this ELISA kit. This success was similarly accomplished for a PK-dependent commercial ELISA kit. Experiments were begun using these two ELISA platforms to optimize DNA marker linkage, release and detection protocols as applied to brain and lymph node samples (Sub-objective 2.2.1). In support of novel assay development, second-year efforts were completed to create a standardized large pool of brain homogenate from sheep infected with classical scrapie. This included inoculations and monitoring of transgenic mice to complete the titration series and assessing classical scrapie infection in culled mice by immunoassay. In the related effort to develop a blood-based assay (Sub-objective 2.2.3), additional blood samples from clinically affected sheep and goats were collected using the protocol standardized in year-one. An enhanced Quaking Induced Conversion Assay (eQuIC) was tested using blood plasma from sheep. Experiments were also conducted to test equivalency of different column purification and regeneration procedures to lessen the production cost of recombinant prion protein—a critical component of QuIC assays.
Sub-objective 2.3: The goal is to improve detection of an atypical strain of scrapie referred to as Nor98-like scrapie, and to understand its potential for natural transmission. Under Sub-objective 2.3.1, inoculations of transgenic mice were completed to determine the presence of atypical scrapie infectivity in placental tissues of experimentally infected ewes in which prion protein with atypical biochemical characteristics has been observed. To confirm atypical scrapie infection and for comparative purposes, inoculations of transgenic mice with brain homogenates from these same ewes were begun. This included mouse inoculations to determine the atypical scrapie infectious titer of a large pool of brain homogenate. Collaborator pathologists from the University of Georgia have completed analysis of fixed brain samples to characterize region-specific accumulation of PrP-Sc (Nor98) in these ewes. To detect natural transmission of atypical scrapie, postmortem collection of tissues from seven-year-old first-generation progeny of these experimentally inoculated ewes were completed and biochemical and immunoassays for the accumulation of PrP-Sc (Nor98) were initiated. This included initiation of experiments to detect and differentiate classical and Nor98-like strains of scrapie in the cerebrospinal fluid of sheep by QuIC assay.
In collaboration with the ARS U.S. Sheep Experiment Station in Dubois, Idaho, the experiments and collaborations associated with this project were supplied with scrapie-naïve tissues from sheep. This critical need was supported by continued efforts to maintain of a small breeding flock of PRNP-defined tissue donors.
In addition to progress on prion infections of sheep and goats, major progress was also made regarding infection caused by Coxiella burnetii, the zoonotic pathogen responsible for coxiellosis in domestic ruminants and Q fever in human beings. Prior data have suggested a strong host genetic component in shedding of C. burnetii from domestic goats. As a first step toward identification of underlying host genes responsible, samples were collected from a large goat herd with an abortion due to C. burnetii. These samples enable quantification of C. burnetii shedding and empower genome scans for major genes involved in presence/absence and quantitative variation in C. burnetii shedding.
Using genetics to select livestock for improved resilience or resistance to infections will improve production and human health but may change animals’ immune response adversely. There are no live animal tests to monitor the variability of the innate and adaptive immune responses in domestic sheep or goats. A technique to quantify variability of the innate and adaptive immune response that was developed for cattle was successfully modified for use in sheep and goats. Sheep and goats demonstrated variability in antibody production and skin thickness, indicators of adaptive and innate immune responses, when injected with different foreign antigens. Therefore, this technique may be used to monitor the variability of immune responses in sheep and goats when selecting for increased resilience or resistance to pathogens including C. burnetii.
Accomplishments
1. Demonstrated strong genetic resistance to classical scrapie in goats. Scrapie is a transmissible spongiform encephalopathy of goats, and previous data showed that goats with only one copy of specific prion amino acid substitutions (either NS146 or QK222) can extend the disease-free periods after the infection. ARS researchers in Pullman, Washington, in collaboration with Washington State University and Texas Agrilife Research, investigated whether the extended disease-free periods of experimentally inoculated goats would exceed 6 years, which represents the productive lifetimes of most commercial goats. The results showed all control goats without either amino acid substitution developed clinical scrapie by an average of 2 years of age, but the NS146 or QK222 goats remained clinically disease-free for an average period exceeding 6 years, suggesting both the NS146 and QK222 substitutions each confer strong resistance to classical scrapie exceeding the commercial lifetimes of most goats. Genetic selection of scrapie-resistant goats is a great alternative control strategy. Collaboration with external organizations have made this genetic test commercially available and producers now can selectively breed goats with strong resistance to classical scrapie.
2. Multiple genetic factors are associated with the infection of sheep brain cell by scrapie prions. Scrapie is a fatal brain disorder in sheep and goats for which the mechanisms of cellular infection are poorly understood. Using a model cell culture system of scrapie prion infection, ARS researchers in Pullman, Washington, in collaboration with scientists at Washington State University and the University of Georgia, studied the expression of multiple genes across several clones of a sheep brain cell line that varied in permissiveness to infection. Permissiveness was associated with altered expression of multiple genes, potentially involving several cellular pathways. Altered gene expression, however, did not result in significant functional alterations to proliferation rates of cells or activities of extracellular matrix metalloproteinases. This demonstrates the importance of functional studies to substantiate the impact on cellular pathways implicated by altered gene expression profiles. The knowledge gained from this study can be used to enhance the use of this model cell culture system as a sensitive tool for detecting scrapie prions and to guide mechanistic and therapeutic investigations.
Review Publications
Oliveira, R.D., Mousel, M.R., Pabilonia, K.L., Highland, M.A., Taylor, J.B., Knowles Jr, D.P., White, S.N. 2017. Domestic sheep show average coxiella burnetii seropositivity generations after a sheep-associated human Q fever outbreak and lack detectable shedding by placental, vaginal, and fecal routes. PLoS One. 12(11):e0188054. https://doi.org/10.1371/journal.pone.0188054.
Monello, R.J., Galloway, N.L., Powers, J.G., Madsen-Bouterse, S.A., Edwards, H., Wood, M., O'Rourke, K., Wild, M.A. 2017. Pathogen-mediated selection in free-ranging elk populations infected by chronic wasting disease. Proceedings of the National Academy of Sciences. 114(46):12208-12212. https://doi.org/10.1073/pnas.1707807114.
Cinar Ulas, M., Schneider, D.A., Waldron, D.F., O'Rourke, K., White, S.N. 2018. Goats singly heterozygous for PRNP S146 or K222 orally inoculated with classical scrapie at birth show no disease at ages well beyond six years. Journal of Animal Breeding and Genetics. 233:19-24.
Dinkel, K.D., Schneider, D.A., Muñoz-Gutiérrez, J.F., Mcelliott, V.R., Stanton, J.B. 2017. Correlation of cellular factors and differential scrapie prion permissiveness in ovine microglia. Virus Research. 240:69-80.
