Location: Animal Disease Research
2017 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 both objectives, which fall under National Program 103, Component 7 – Transmissible Spongiform Encephalopathies (TSEs). The project specifically addresses research needs identified under Problem 7C – Diagnostics, Detection and Prevention, but also the related needs identified under Problem 7A – Pathobiology of Prion Strains and 7B – Genetics of Prion Disease Susceptibility.
The aim of experiments under Objective 1 is to determine the influence of genetic variation in sheep and goats on our ability to diagnose classical scrapie infection using currently approved assay protocols and standard tissues. Under Objective 1.1, we made significant progress in developing the archived sample dataset with which we will determine the impact of the prion protein gene (PRNP) M112T polymorphism on immunohistochemical detection of classical scrapie infection in sheep. Immunohistochemistry is the assay most commonly used to conduct postmortem surveillance for infected animals. This study utilizes a collection of archived brain and lymph node samples from ~2,500 sheep obtained postmortem as a part of field investigations. From this archive, the PRNP genotypes could be validated for 1,125 sheep. Immunohistochemical detection and measurement of accumulated disease-associated prion protein (PrP-Sc) was initiated on brain and lymph node samples from these sheep. Significant progress was made in a study to determine the impact of the PRNP G127S polymorphism on immunohistochemical detection of classical scrapie disease in goats (Objective 1.2). This study utilizes a natural infection model whereby genetically-defined newborn goats are exposed by the oral route to a standardized dose of scrapie prions found in the placenta. Large pools of cotyledon homogenates were prepared from the placentas of a natural case of goat scrapie. The PrP-Sc in each homogenate was characterized by immunohistochemistry and standard western blot. Goat does and bucks were selected by genotyping and, at the end of this first year of controlled breeding, sixteen goat kids were produced and orally inoculated with scrapie-positive homogenate.
Regarding antemortem or live-animal diagnosis, immunohistochemistry applied to biopsies of the rectal mucosa is the method preferred for detecting classical scrapie infection. A study under Objective 2.1 was initiated to answer a recent question of whether repeating the biopsy procedure, as may be necessary to obtain a proper sample or to monitor progression of infection, might reduce detection of PrP-Sc in the rectal mucosa. Rectal tissues from sheep and goats have been obtained through postmortem examinations at various time points after previous biopsy, and immunohistochemical processing on these and other archived samples has been initiated.
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 and goats, it is likely that final eradication will require development of assays with greatly enhanced sensitivity for earlier stages of infection. In this regard, we made significant progress under Objective 2.2 to develop highly sensitive, high-throughput assays suitable for use in veterinary diagnostic laboratories. To conduct assay development for use with standard (brain and lymph node) and novel (e.g., blood) sample types, large standardized pools of homogenates were created from the brain of infected and non-infected sheep. The PrP-Sc in each homogenate was characterized by immuno-histochemistry and western blot, and transgenic mice have been inoculated to further characterize the scrapie strain and prion titer in the homogenates. For development of a blood-based assay, a standardized protocol was established and has been used to collect, process and archive samples of blood from sheep and goats clinically affected with classical scrapie. These homogenates and blood samples are being used to optimize novel high-sensitivity detection methods for PrP-Sc. We have initiated experiments to optimize use and detection of a critical biotinylated-DNA marker, by determining immuno-qPCR reaction conditions and by testing two mechanisms for release of avidin-bound marker. As an alternative method to immuno-PCR, we established a collaboration with the TSE/Prion Biochemistry Section at the National Institute of Allergy and Infectious Disease (NIAID) Rocky Mountain Laboratories, which pioneered for human prion disease an ultra-sensitive prion protein misfolding assay known as the quaking-induced conversion (QuIC) assay. Experiments have been initiated to adapt the QuIC assay to blood and blood components using samples we have processed from sheep and goats naturally infected with classical scrapie. In addition, postmortem samples of skin from sheep and goats were provided for experiments initiated to develop a novel skin-based QuIC assay method for detection of classical scrapie infection.
In addition to our efforts on classical scrapie, significant progress was made under Objective 2.3 which aims to improve detection of an atypical strain of scrapie, known in U.S. sheep and goats as Nor98-like scrapie. To characterize the infection, the presence of PrP-Sc (Nor98) in the brains and lymph nodes from four Nor98-inoculated ewes was determined by immunohistochemistry, standard scrapie western blot, and by a western blot protocol optimized for detection of atypical scrapie. In addition, a formalin-fixed brain sectioning protocol was established and used to begin region-specific quantification of PrP-Sc (Nor98) accumulation. Toward development of high-sensitivity detection methods, large standardized brain homogenate pools were created from one of the experimentally-infected sheep and genotype-matched non-infected sheep. Inoculations of transgenic mice were initiated to determine the brain titer of Nor98-like scrapie prions. To determine if Nor98-like scrapie is naturally transmissible, we have also prepared homogenate pools from the placentas of these experimentally infected sheep. The presence of PrP-Sc (Nor98) has been tested by immunohistochemistry and the standard and modified western blot assays. Further, inoculation of transgenic mice with these homogenates was initiated as the currently most sensitive method for detecting infectivity. In addition, we continue to monitor and breed the progeny of these experimentally infected ewes. Since Nor98-like scrapie has a prolonged incubation of six or more years, tissues were collected from the first progeny achieving 7-years of age. Analysis for the accumulation of PrP-Sc (Nor98) has been initiated. In collaboration with Rocky Mountain Laboratories, we have initiated postmortem collection of brain and cerebrospinal fluid to adapt a version of the QuIC assay originally optimized to differentiate classical and Nor98-like strains of scrapie in the brains of sheep. These samples were also collected from the progeny of Nor98-inoculated sheep.
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. In addition to blood collections from the general flock population, we began the selection of ewes and rams that will be used to establish a small PRNP-defined breeding and tissue donor flock.
Accomplishments
1. Optimized biopsy procedure for the diagnosis of scrapie in sheep and goats. Diagnosis of scrapie in live animals is commonly determined from biopsies of the rectal mucosa but the diagnostic quality of such biopsies can be variable, especially in goats. ARS researchers in Pullman, Washington, conducted a comparative study to determine which factors impact the diagnostic quality of such biopsies. Both procedural and animal-related factors were identified. An age-related steady decline in the density of rectal mucosal follicles was a major limiting factor identified which did not differ between sheep and goats and was not affected by the biopsy site location. This study provides information critical to improving the consistent collection and processing of diagnostically useful samples from sheep and goats.
2. Inoculated goats with one copy of either the S146 or K222 allele of the PRNP gene show no scrapie beyond 6 years of age. ARS scientists in Pullman, Washington, led a project in collaboration with Washington State University and Texas Agrilife Research, where goats with either zero or one copy of the S146 or K222 allele at the goat PRioN Protein (PRNP) gene were inoculated with scrapie within 24 hours of birth. While control goats with no copies of either S146 or K222 developed clinical scrapie at average ages of less than 2 years, goats with one copy showed no clinical scrapie at much more advanced ages. Specifically, goats with one copy of S146 have remained free of scrapie clinical signs for an average of 7.4 years, and goats with one copy of K222 have remained free of scrapie clinical signs for an average of 6.7 years. These results demonstrate that goats with these alleles of the PRNP gene have remained free of scrapie for periods approximating or exceeding the productive lifespans of many commercial goats. These data support exploration of selective breeding for goat with these alleles to reduce clinical scrapie, and the experiment is ongoing to determine the full post-inoculation disease-free periods and to examine implications for scrapie transmission.
Review Publications
Tuggle, C.K., Giuffra, E., White, S.N., Clarke, L., Zhou, H., Ross, P.J., Acloque, H., Reecy, J.M., Archibald, A., Boichard, M., Chamberlain, A., Cheng, H.H., Crooijmans, R., Delany, M., Groenen, M.A., Hayes, B., Lunney, J.K., Plastow, G.S., Silverstein, J., Song, J., Watson, M. 2016. GO-FAANG meeting: A gathering on functional annotation of animal genomes. Animal Genetics. 47(5):528-533.
Cinar, M., Mousel, M.R., Herrmann-Hoesing, L.M., Taylor, J.B., White, S.N. 2016. Ovar-DRB1 haplotypes *2001 and *0301 are associated with sheep growth and ewe lifetime prolificacy. Gene. 595(2):187-192.
