Location: Animal Disease Research
2019 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, which fall under National Program 103, Component 7, Transmissible Spongiform Encephalopathies.
The aim of research under Objective 1 is to determine the influence of variation in the prion protein gene (i.e., polymorphisms of PRNP) on the detection of classical scrapie infection in sheep and goats. The sensitivity of immunohistochemistry to detect infection in samples of brain and lymphoid tissue collected postmortem is critical to the success and efficiency of current eradication efforts.
In support of Sub-Objective 1.1, progress was made on a project to determine in sheep the impact of variation in the gene at position 112 (referred to as PRNP polymorphism M112T). This study utilizes archived data and samples from 1,119 sheep. Analysis demonstrates strong negative association of this genetic variation with diagnosis of classical scrapie in sheep and further suggests this gene variation reduces accumulation of misfolded prion protein (PrPSc), a marker of infection, in lymphoid tissue. Progress was made in acquiring additional samples to strengthen the evaluation of PrPSc accumulation in lymphoid tissues used in diagnosis. Reduced tissue accumulation of PrPSc decreases the likelihood of detecting classical scrapie infection in sheep bearing the M112T genetic variation. The evidence supports the possibility that sheep bearing two copies of this genetic variation (referred to as TT112) are relatively resistant to infection. Progress was also made on research projects to determine the influence of several genetic variations that naturally occur in U.S. goat populations. These projects utilize genetic selection and an inoculation model of natural transmission.
Progress was also made on Sub-objective 1.2 in a study conducted to determine the effects of variation in the goat PRNP gene at position 112 (referred to as G127S) by completing selective breeding and by confirming genotypes and inoculating suitable offspring. Postmortem examinations were conducted on offspring inoculated in previous years of the study, the results of which confirm successful transmission of scrapie infection to the fully susceptible offspring; i.e., transmission of infection was confirmed in those offspring bearing two copies of the normal gene sequence (GG127). Selective breeding has also resulted in offspring bearing two copies of the gene variation (SS127), which allows the first direct testing of this genotype for resistance to infection. Similar research continues on a long-term project involving two other PRNP polymorphisms in goats: Q222K and N146S. Despite very long incubation, PrPSc accumulation has not been detected by standard immunoassay in any of the postmortem tissues of goats bearing one copy of the normal and one copy of the variant gene (either QK222 or NS146). As a final effort to detect presence of infection, transgenic mice highly susceptible to infection were inoculated with tissue samples from QK222 goats with the longest incubation times. At this point in time, four NS146 bearing goats continue to survive with no clinical evidence of infection. In addition to this progress, we adapted a buccal (“cheek”) cell collection method to a rapid, codon-specific genotyping method to overcome difficulties genotyping from blood samples of new born animals. A similar method was developed for hair bulb samples but which proved most consistent in adult animals, hair bulb size possibly limiting consistent success in newborns.
In support of Sub-objective 2.2, progress was made on developing highly sensitive, high-throughput assays for classical scrapie infection. Postmortem tissue collection and analyses by immunoassay and survival analysis were completed on all transgenic mice inoculated with classical scrapie, thus providing gold-standard characterization of classical scrapie samples. In addition, brain samples from three scrapie-positive and three negative sheep were identified and prepared for use in assay optimizations. After adapting DNA marker-assisted detection to the antibodies of two commercial platforms, the power of each modified assay to discriminate positive and negative conditions was first tested utilizing kit-provided internal controls and then using the newly created panel of sheep brain samples. Efforts also continued to adapt to a variety of sample types the quaking-induced conversion (QuIC) assay, which detects scrapie prions by virtue of their propensity to cause misfolding of the prion protein. The QuIC assay requires production, purification and proper folding of the substrate—a recombinant prion protein. Two forms of recombinant prion protein were fully processed using a procedure modified to improve efficiency and reduce cost. The new substrates were tested in ‘real time’ QuIC (RT-QuIC) using samples of brain and cerebrospinal fluid (CSF) from sheep and goats with and without classical scrapie. Though misfolding activity in brain and CSF were readily detected, each with high sensitivity, misfolding activity was also detected intermittently in negative controls. Subsequent quality control testing of each substrate batch revealed low but unacceptable frequencies of ‘spontaneous’ misfolding, suggestive of imperfect re-folding during production. Assistance to overcome this matter is being provided by collaborators in the TSE/Prion Biochemistry Section at the National Institute of Allergy and Infectious Disease (NIAID) Rocky Mountain Laboratories. In addition, NIAID collaborators continued testing modifications of the QuIC assay to enhance its utility for use with blood plasma samples. A method using magnetic beads coated with a conformation-specific antibody was too variable for continued testing. An alternative metal-based method, however, shows promise and so additional plasma samples were collected to provide for further assay development. Other blood components and biological materials (CSF, aqueous humor, retinas, skin, urine and feces) were collected from sheep and goat cases of classical scrapie. Based on results from a mouse model of classical scrapie infection, blood levels of two cytokines (tissue inhibitor of metalloproteinase-1 and interleukin-10) were tested as potential biomarkers in the serum of sheep. Unfortunately, neither serum marker proved useful in detecting preclinical infections.
Progress under Sub-objective 2.3 was also made on projects to improve diagnostics and understanding of transmission for an atypical form of scrapie that does not produce significant accumulation of misfolded PrP in tissues other than the brain. Since the placenta is a primary source of infectious prions in cases of classical scrapie, samples of placenta or brain from four cases of atypical scrapie were inoculated into transgenic mice in order to determine if the placenta could also be a significant source of atypical scrapie prions. For comparison, mice were also inoculated with placenta or brain samples from sheep with classical scrapie and from uninfected sheep. Postmortem tissues were collected from all mice culled to date. With regard to the various samples of placenta, infectivity has only been detected to date in samples from sheep with classical scrapie. To determine if atypical scrapie is naturally transmitted to lambs, seven-year-old offspring of the originally infected ewes were culled and tissues collected and tested by biochemical and immuno-assays. To date, these analyses have not provided evidence for natural transmission to any offspring, even after allowing for very long incubation in the offspring. As a final measure to detect natural transmission of this atypical form of scrapie, samples from these and other offspring will be eventually tested for prion protein misfolding activity once the QuIC substrate issue is resolved or a suitable alternative method established.
The collective importance of the projects under Objective 2 was underscored by a request from the coordinator of the National Scrapie Eradication Program to begin a study of an unusual case of sheep scrapie discovered through the Regulatory Scrapie Slaughter Surveillance program. This case involves a sheep considered genetically resistant to classical scrapie. While such sheep remain susceptible to atypical forms of scrapie, these scrapie forms result in significant accumulation of misfolded prion protein (PrPSc) most commonly in ‘old’ sheep and only in the brain. In this unusual case, the sheep was only about 18 months old and significant PrPSc accumulation was detected in its lymphoid tissue. Progress has been made on this request beginning with receipt of the remaining samples from National Veterinary Services Laboratories. The samples were re-embedded in paraffin and completely thin sectioned into enumerated slide sets. Representative thin sections from these original samples and from similarly prepared positive and negative control samples were studied by histochemistry and immunohistochemistry. Other thin sections were prepared and tested in vitro for protein misfolding activity or inoculated into transgenic mice for the detection and characterization of any associated infectivity.
Accomplishments
1. Milk from goats with scrapie is highly infectious. The continued presence of scrapie in the U.S. costs sheep producers an estimated $20 million annually, especially in unrealized exports. Since animals harbor infection for several years before developing clinical signs, it is imperative for eradication efforts to identify and mitigate transmission risks. ARS researchers at Pullman, Washington, have determined that milk from infected goats can be highly infectious to newborn lambs and goat kids. Critically important was that transmission occurred after newborns consumed only about one gallon of milk. This knowledge informs producers that even limited exposure of newborn lambs and goat kids to scrapie-infected goat’s milk can result in their flock acquiring scrapie and helps disease investigators better assess sources of flock exposure.
Review Publications
Madsen-Bouterse, S.A., Highland, M.A., Dassanayake, R.P., Zhuang, D., Schneider, D.A. 2018. Low-volume goat milk transmission of classical scrapie to lambs and goat kids. PLoS One. 13(9):e0204281. https://doi.org/10.1371/journal.pone.0204281.
Murphy Jr, T.W., Stewart, W.C., Notter, D.R., Mousel, M.R., Lewis, G.S., Taylor, J.B. 2019. Evaluation of Rambouillet, Polypay, and Romanov–White Dorper x Rambouillet ewes mated to terminal sires in an extensive rangeland production system: Body weight and wool characteristics. Journal of Animal Science. 97(4):1568-1577. https://doi.org/10.1093/jas/skz070.
Notter, D.R., Mousel, M.R., Leeds, T.D., Lewis, G.L., Taylor, J.B. 2018. Effects of rearing triplet lambs on ewe productivity, lamb survival and performance, and future ewe performance. Journal of Animal Science. 96(12):4944-4958. https://doi.org/10.1093/jas/sky364.
