Location: Animal Disease Research
2021 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
This is the final report for the project 2090-32000-035-000D which will terminate in October 2021. The new project, titled “Leveraging Host-pathogen Interactions into Intervention Strategies to Combat Bacterial Pathogens of Small Ruminants” is currently undergoing NP103 OSQR review. A summary of results for all the expiring project sub-objectives are described below.
In support of Objective 1, research continued to determine variations in the prion protein that affect transmission and detection of classical scrapie infection in small ruminants. We completed tissue collections from the last of the timed culls in support of Sub-Objective 1.2, a three-year incubation study to determine the effects of a mutation in the prion protein in goats exposed to classical scrapie at birth. Evaluation of tissues from goats that only express the G127 form of the prion protein (i.e., goats genetically homozygous for G127) confirmed disease transmission to all animals. Infection in these goats resulted in a significant accumulation of prions (infectious, misfolded prion protein) in the rectal mucosa by 18 months of age, confirming a high inoculum dose of prions. Disease transmission also occurred in goats expressing both G127 and S127 forms of the prion protein (i.e., genetically heterozygous goats). However, prion accumulations in these goats' rectal mucosa and brain were considerably reduced and significantly delayed. These results demonstrate that goats heterozygous for the S127 variation are at risk of classical scrapie infection and predict these animals will be challenging to detect by surveillance aimed at goats 18 months to three years of age.
The results under Objective 1 confirm that variation in this region of the prion protein—T112 in sheep (Sub-objective 1.1) and S127 in goats (Sub-objective 1.2)—does not protect small ruminants from classical scrapie but instead only slows the progress of the disease. The mechanism that slows disease but does not protect against transmission is unknown. It may be that certain variations in this region confer resistance of the protein to prion-induced misfolding. Our earlier findings (Sub-objective 1.1) support this hypothesis since we found no evidence for infection in sheep expressing only the T112 variation (i.e., genetically homozygous for the T112 variation) despite residing within flocks experiencing natural transmission of classical scrapie. If this is true, progression of the disease would be slowed in heterozygous sheep since essentially half the pool of prion protein is not susceptible to misfolding. To pursue this hypothesis further, three goats homozygous for the S127 variation were inoculated at birth and monitored for infection. Still, they remain healthy at more than two years of age. We also collected fecal and urine samples from all Sub-objective 1.2 study goats to assess the comparative risk posed by the slow form of infection experienced in S127 heterozygous goats.
In support of Objective 2, research continued developing two amplification methods—the serial protein misfolding cyclic amplification (sPMCA) assay and the real-time quaking-induced conversion (RT-QuIC) assay, for the sensitive detection of prions in various sample types (Sub-objective 2.2). Both assays detect the misfolding of prion protein supplied as the substrate to the reaction. The misfolding response in these assays is self-perpetuating such that only very few prion particles are needed to initiate the process. To develop blood as a convenient and safe diagnostic sample, we collected blood from sheep and goats during the disease's late preclinical and early clinical stages. Blood was collected into different anti-coagulants and then separated into the mononuclear cell and plasma components. Although small ruminant blood mononuclear cells are consistently associated with infectious prions, detection by sPMCA and RT-QuIC assays was variable. Spiking known amounts of prions into amplification assays revealed significant inhibition of the misfolding response by constituents of this cellular blood component. We fractionated mononuclear cells into their subcellular components using either nitrogen cavitation or controlled hypotonic lysis to investigate further which fractions associate with prions and inhibit the misfolding response. We also investigated the effect of heme on the misfolding response since the collection and manipulation of blood and vascularized tissues invariably release heme from red blood cells. Heme was a potent inhibitor of the misfolding response in sPMCA and RT-QuIC. Overcoming heme contamination and inhibition will be vital to optimizing all diagnostic samples for these amplification assays. We also made progress in optimizing samples of rectal mucosa for use in amplification assays. The rectal mucosa is attractive as a diagnostic sample since this tissue is accessible in live animals by biopsy and harbors infectious prions relatively early in classical scrapie infection of small ruminants and chronic wasting disease of cervids. The results demonstrate that the additional step of collagen digestion is not necessary, producing no clear advantage over simple collagen fiber removal after tissue disruption and centrifugation. Removing this extra step reduces the cost and time of preparing rectal mucosa for analysis. In contrast, the addition of selective precipitation to sample preparation resulted in enhanced detection sensitivity. This outcome resulted from the combined benefits of prion enrichment and exclusion of tissue inhibitory factors. Progress was also made in optimizing the assay buffers used in RT-QuIC for rectal mucosa samples. The use of sodium iodide has improved detection for specific tissue types and different protein misfolding diseases. For the detection of prions in rectal mucosal samples, however, the use of sodium iodide reduced the sensitivity of the assay. It only produced a minor improvement in the rate of false-positive reactions. Research was conducted on the potential use of ocular structures in the surveillance of scrapie in small ruminants. Immunohistochemistry was performed and revealed accumulation of prions in the retina is not restricted to the optic nerve region but is dependent on stage of infection. Techniques to prepare the retina and cornea for amplification assay were perfected. Retina, cornea and aqueous humor was tested for prions by RT-QuIC. Surprisingly prions were detected only in the retina of infected animals despite the high innervation of the cornea and an abundance of normal prion protein present in aqueous humor.
In support of Sub-objective 2.3, tissues were collected from the last of the first-generation lambs born to sheep resistant to classical scrapie but infected with Nor98-like scrapie, an atypical form of scrapie in small ruminants. Despite some evidence for prion-like material in the placentas associated with these lambs' births, we have found no immunohistologic evidence for atypical scrapie infection in these animals, some of which were seven years of age at postmortem examination. Subsequent bioassay of the placentas associated with prion-like material has failed to detect atypical scrapie prions. These findings are consistent with epidemiological studies that describe Nor98 and Nor98-like scrapie as a sporadic but not naturally transmissible disease of sheep and goats.
Significant progress was also made in the subordinate project 58-2090-1-001 which endeavors to optimize and validate an RT-QuIC protocol for diagnosing chronic wasting disease in white-tailed deer. A pre-analytical procedure for preparing rectal mucosal samples and the buffer conditions of the assay were determined. The optimized protocol has now been applied to over 150 samples to which we are blind to disease status. Collaborators not blind to the disease status of the samples have indicated that the assay's apparent sensitivity relative to immunohistologic results is encouraging for continued development. The experiments and findings of this subordinate project support our efforts to similarly optimize the RT-QuIC assay for diagnosing classical scrapie by rectal biopsy in small ruminants.
Accomplishments
1. Genetic mechanisms protecting small ruminants from scrapie. Scrapie is a uniformly fatal disease of small ruminants that results in export losses valued at over $20 million. Numerous genetic variations in the prion protein of goats are known, but only two confer strong resistance to scrapie. ARS researchers in Pullman, Washington, have identified two prion protein fragmentation patterns associated with these resistant genotypes. The pattern associated with one of the resistant genotypes in goats was the same as that of the only known resistance genotype in sheep. The fragmentation observed in goats bearing the other disease-resistant genotype was novel and indicated a second mechanism for genetic resistance to this devastating disease.
Review Publications
Massa, A.T., Mousel, M.R., Herndon, M.K., Herndon, D.R., Murdoch, B.M., White, S.N. 2021. Genome-wide histone modifications and CTCF enrichment predict gene expression in sheep macrophages. Frontiers in Genetics. 11. Article 612031. https://doi.org/10.3389/fgene.2020.612031.
Madsen-Bouterse, S.A., Stewart, P., Williamson, H., Schneider, D.A., Goldmann, W. 2021. Caprine PRNP polymorphisms N146S and Q222K are associated with proteolytic cleavage of PrPC. Genetics Selection Evolution. 53. Article 52. https://doi.org/10.1186/s12711-021-00646-x.
