Location: Animal Disease Research
2020 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
In support of Objective 1, research continued to determine the influence of genetic variations in the prion protein gene of sheep and goats on the occurrence and detection of classical scrapie infection. The sensitivity of current diagnostic assays to detect infection is critical to the success and efficiency of eradication efforts. The sensitivity of early detection through live animal testing depends on the rate and extent of accumulation of disease-associated (misfolded) prion protein in lymphoid tissues. In support of Objective 1, analyses were completed which demonstrate association of a genetic variation, called T112, with classical scrapie infection in sheep. The analysis of data from 1,119 naturally exposed sheep revealed strong negative association of this genetic variation with a positive diagnosis, including no infections detected in 27 sheep having two copies of this variant gene. The study also revealed reduced accumulation of misfolded prion protein in lymphoid tissue, which is expected to negatively impact early detection of infection in sheep bearing this genetic variation, potentially increasing their contamination of the environment and exposure of other animals to infection.
Progress was also made in support of Subobjective 1.2A on the effects of several variations in the prion protein gene of goats on scrapie transmission. In a study of a prion protein gene variation called S127, tissues were examined and scored for the extent of misfolded prion protein accumulation. The results thus far demonstrate a strong, uniform exposure of the goats to scrapie but with markedly reduced accumulation of misfolded prion protein in goats bearing the S127 variation. This challenge study in goats continues but the results already suggest effects consistent with the associations made in sheep bearing a similarly located gene variation (T112). Together these findings support a general concept that variations in this region of the prion protein do not provide strong resistance to infection and may in fact add risk to scrapie containment by delaying detection of infection through reduced accumulation of the disease-associated prion protein in tissues used for early diagnosis. Additional breeding was conducted to increase the number of goats bearing two copies of the S127 variation. Progress was also made regarding two other prion protein gene variations in goats, called S146 and K222. No evidence of disease-associated prion protein accumulation was observed in any of the tissues collected from two more goats bearing the S146 variation despite having lived for 10 years after oral challenge with scrapie. Only two goats remain to be studied from the original challenge group of S146-bearing goats, and they continue to be healthy in their advanced age. Similarly, we previously found no evidence of disease-associated prion protein accumulation in scrapie-challenged goats bearing the K222 variation. In a final effort to detect even the smallest amounts of infectious scrapie agent in these goats, mice highly susceptible to scrapie infection were inoculated with tissue from the longest surviving of K222-bearing goats and remained healthy during this reporting period. A similar study will be conducted in mice when tissues from the two surviving S146-bearing goats become available. Together these studies are providing the information necessary to guide producers in the use genetic selection to prevent the devastating consequences of scrapie infection in their goat herds.
The aims of research under Objective 2 are to improve diagnostic sensitivity and determine modes of transmission. Blood is a sample that is relatively cheap, easy, and safe to collect and store, and which can be repeatedly sampled. Progress was made in adapting two highly sensitive, high-throughput assays to the detection of scrapie prions in samples of blood from sheep and goats. Samples from animals defined by infection stage were provided to collaborating scientists at the National Institute of Allergy and Infectious Disease Rocky Mountain Laboratories. Results indicate detection is enhanced by enrichment of the assay with metal-based extraction of prions from the plasma of blood from sheep. The results however have been variable but possibly reflective of our similar experience in detecting prions in plasma by bioassay. As detected by bioassay, scrapie prions are more frequently detected in association with a fraction of the white blood cells. Indeed, experiments conducted using an ultrasensitive assay during this reporting period did detect prions associated with a fraction of white blood cells collected from sheep and goats. Additional techniques were examined to maximize detection of prions associated with the fractions of blood from sheep and goats. To support the ongoing research to adapt ultrasensitive rapid methods to detection of classical scrapie, additional samples of blood and other biological materials (CSF, aqueous humor, retinas, skin, urine, and feces) were collected from sheep and goats.
Also, in support of Sub-objective 2.3A, progress was made on projects aimed at the detection and possible transmission of an unusual ‘atypical’ form of scrapie. To determine the infectious dose of atypical scrapie in the brain of sheep, and to provide a large pool of defined homogenate for use in developing enhanced detection methods, mice inoculated with atypical scrapie were monitored and tissues collected from those reaching planned endpoints. Investigations regarding the potential for natural transmission of atypical scrapie were continued. Despite evidence for limited accumulation of Nor98-like protein in the placentas obtained from aged ewes infected with atypical scrapie, none have to date proved infectious to highly susceptible mice. Furthermore, first and second-generation sheep born to these atypical scrapie infected dams continue to be healthy with advancing age with no detection of disease-associated prion protein accumulation in tissues collected from first generation progeny that have reached the planned end-point of seven years-of-age. At completion of the study, such results will together suggest the risk of natural transmission of atypical scrapie infection from dam to offspring, even at advanced age, is indeed very low.
The importance of Objective 2 aims was underscored again by a request to help analyze a second case of unusual prion-like material in lymphoid tissue from a healthy young sheep tested as part of the APHIS Regulatory Scrapie Slaughter Surveillance (RSSS) program. These cases are unusual for two reasons. First, lymphoid accumulation of prion-like material in 18-month-old sheep is consistent with the pathobiology of classical, but not Nor98-like, scrapie. Both sheep, however, were genotyped as being highly resistant to infection by classical scrapie. Second, the prion-like material was detected by some but not all anti-prion antibodies tested, suggesting certain parts of the detected material are different than expected for the prion protein produced in these animals. Samples from the first case were inoculated into mice highly susceptible to classical and Nor98-like scrapie. These mice remain healthy and will reach the endpoint in the next reporting period. Transfer of samples from the second case are expected and will be similarly tested, including inoculation into transgenic mice. The second case afforded the opportunity to collect a variety of high-quality samples from flock mates. Using standard methods, accumulation of prion-like material has not been detected in any of these samples, samples which include lymphoid tissue and placenta from sheep related to the second case. High quality DNA has been extracted from relatives to the second case but no variation in the prion protein gene was uncovered that might explain the unusual antibody staining of prion-like material or represent a novel genotype with unknown susceptibility to scrapie infection.
Accomplishments
Review Publications
Koltes, J.E., Cole, J.B., Clemmens, R., Dilger, R.N., Kramer, L.M., Lunney, J.K., Mccue, M.E., Mckay, S., Mateescu, R., Murdoch, B.M., Reuter, R., Rexroad III, C.E., Rosa, G.J.M., Serao, N.V.L., White, S.N., Woodward Greene, M.J., Worku, M., Zhang, H., Reecy, J.M., editors. 2019. A vision for development and utilization of high-throughput phenotyping and big data analytics in livestock. Frontiers in Genetics. 10:1197. https://doi.org/10.3389/fgene.2019.01197.
Silva, M.G., Madsen-Bouterse, S.A., Dassanayake, R.P., Mousel, M., Knowles, D.P. 2019. Tissue inhibitor of metalloproteinase-1 and interleukin-10 in serum from naïve and scrapie infected sheep. Veterinary and Animal Science. 7. https://doi.org/10.1016/j.vas.2019.100056.
