Location: Animal Disease Research2012 Annual Report
1a. Objectives (from AD-416):
Objective 1: Determine whether goats are a transmission reservoir for ovine scrapie by developing and validating diagnostic methods for detecting goat scrapie. Determine the genetic predisposition and transmission route(s) of goat scrapie. Subobjective 1.1: Improve eradication efforts by developing improved methods for antemortem scrapie diagnosis. Subobjective 1.2: Determine if placenta and milk from goats are potential sources of scrapie to sheep. Objective 2: Develop methods to mitigate infectivity of soil-associated prions by screening soil microbes for potential candidates for bioremediation.
1b. Approach (from AD-416):
Scrapie is a complex and rare disorder affecting outbred farm animals held under a wide variety of husbandry conditions and exposed to an agent for which the transmissible and pathogenic events remain largely unknown. The work described in the research plan is an extension of the previous highly productive studies by this research group, addressing the need for implementation of federal regulations based on the best available science, often in the face of relatively small sample numbers in the natural host. The work includes development of specific management and diagnostic tools and is presented as an integrated series of research objectives. This approach was selected over a hypothesis based approach. After consulting Glass and Hall, the group determined that the work presented in the following plan was best represented by goal statements rather than hypotheses because the work increases the density of data necessary for progress and for support of current and proposed federal regulations. This project addresses only scrapie, the TSE of sheep and goats. Chronic wasting disease (CWD) is the TSE of North America cervids (deer and elk). No live animal work with CWD is included in this project plan since CWD is not endemic in Washington State, the disease appears to be highly communicable, the modes of transmission are unknown, and we do not have suitable biocontainment facilities to conduct CWD studies in large animals.
3. Progress Report:
Regarding progress on characterizing caprine scrapie in sheep and goats: Natural and experimental cases of sheep (ovine)- or goat (caprine)-origin scrapie were monitored for infection and disease progression by biopsy and clinical assessment. Postmortem examinations were conducted on animals developing terminal disease and tissues banked for use in an origin/strain discrimination study. Further, the utility of transgenic mouse bioassay of ovine- and caprine-origin scrapie was determined. Regarding progress on demonstrating typical modes of transmission in mixed sheep and goat operations: Highly susceptible sheep were inoculated with caprine-origin scrapie using a model of natural infection (oral exposure of neonates using placenta or milk derived from a goat with clinical scrapie). Recipients were serially monitored and thus far have confirmed that goat placenta is a risk material for scrapie transmission to sheep. First year sampling and analyses were completed for determining the impact of lactation cycle, local inflammation and small ruminant lentivirus co-infection on prions in milk, and progress has been made in adapting a seeded-conversion assay for detecting misfolded prion proteins in milk. Regarding progress on improving diagnostic testing of goats and effects of prion protein genotype, rectal tissue samples from sheep and goats were collected and analyses initiated. The anticipated results, including the effects of animal age and genotype, will inform optimization of biopsy sampling and processing procedures for use with standard diagnostic assays. To help optimize blood processing for detection of scrapie, bioassay in sheep was used to determine the blood fractions which harbor prions and the effect of starting blood sample volume on detection. To better understand prion distribution in the blood, surface expression of the cellular form of the prion protein was also characterized in blood fractions. As potential alternatives, novel blood borne biomarkers were identified in a proteomic study using a transgenic mouse model of scrapie disease, and transgenic cell lines expressing species-specific prion proteins were created and tested for permissiveness to scrapie infection. Regarding progress on developing methods to mitigate prion contamination of soil, a primary cell line of sheep microglia—a cell type naturally involved in scrapie pathogenesis, was successfully immortalized and found to be permissive to authentic as well as culture-adapted scrapie prions. In addition, a non-subcultivation method was shown to improve culture sensitivity of a widely-used transgenic cell line and a novel synthetic molecule was shown to have anti-prion activity in vitro. The molecular determinants of this novel anti-prion activity are now under study. The progress made indicates the real potential to replace the need for bioassay in sorption and mitigation studies in soil with more timely and efficient culture methods.
1. Prolonged scrapie incubation in goats singly heterozygous for PRNP S146 or K222. Scrapie eradication in sheep is based in part on strong genetic resistance to classical scrapie. Goats may serve as a scrapie reservoir but there has been limited experimental inoculation to confirm strong genetic resistance in goats. ARS researchers at the Animal Disease Research Unit in Pullman, Washington, thus performed the first oral scrapie challenge of goats heterozygous for two PRNP polymorphisms of particular interest. The results confirmed that goats singly heterozygous for S142 or K222 have greatly extended incubation times, indicating a need in scrapie-eradication programs for longer trace-back histories for goats bearing these alleles. Also indicated is a need to assess goats singly homozygous for either of these alleles for enhanced resistance to scrapie infection.
2. Progress toward developing a blood test for detection of the scrapie prion in sheep. The detection of infectious scrapie prions in the blood of some sheep supports development of a blood-based diagnostic test for identification of scrapie infected sheep. Currently, available commercial assays do not possess the sensitivity to detect infectious prion in sheep blood. ARS researchers at the Animal Disease Research Unit in Pullman, Washington, identified components of sheep and goat blood which associate with prion infectivity by using the ultra-sensitive method of transfusion bioassay. The finding that infectious prion can be identified in certain blood fractions of sheep and goats support the development of an assay for regulatory use and this is an important effort in support of the U. S. National Sheep Scrapie eradication effort.
3. Role of genetics in chronic wasting disease of North American cervids. The transmissible spongiform encephalopathies (TSEs) are protein folding disorders in which both relative susceptibility and incubation interval are related to critical single amino acid substitutions in the normal cellular prion protein in deer, elk, sheep and goats. ARS researchers at the Animal Disease Research Unit in Pullman, Washington, and Colorado State University, Fort Collins, Colorado, studied selective breeding for relatively resistant stock, which can be a cost-effective adjunct to conventional disease control programs. In this study, the authors review the evidence for genetic control of susceptibility to the TSE of cervids (chronic wasting disease or CWD) in white tailed deer and Rocky Mountain elk, the two cervid species most commonly farm raised in the US and Canada. This information will be of use to the captive cervid industries and the US Animal Plant Health Inspection Service as they develop control programs for CWD in North America.
4. Cytokine proteins found in the blood are potential biomarkers of preclinical scrapie infection. ARS researchers at the Animal Disease Research Unit in Pullman, Washington, and the University of Washington, Seattle, WA, studied preclinical detection of scrapie infection in live sheep and goats which rely on surrogate detection of disease-associated prion protein, PrP-Sc, in biopsy samples of the rectal mucosa. Methods for detecting PrP-Sc, however, are relatively insensitive and accumulation of PrP-Sc in rectal tissues is slower than that in tissues collected only at postmortem examination. Thus, development of a more sensitive, blood-based test is highly desirable. In this study, the temporal changes in cytokine proteins, in tissues as well as in blood, were globally determined during the course of scrapie infection in transgenic mice, a surrogate model for sheep. The results show that at least two cytokine proteins may represent novel targets for development of blood-based diagnostic testing.
5. Disease-associated prion protein (PrP-TME) in mink. Transmissible mink encephalopathy (TME) is a transmissible spongiform encephalopathy that has occurred as epizootics in farmed mink but which is thought to be from a source extrinsic to mink. Extensive passage of the “Stetsonville isolate” of TME results in the emergence of two prion strains in hamsters but this has not been similarly tested in the natural disease host, mink. Thus, ARS researchers at the Animal Disease Research Unit in Pullman, Washington and the University of Washington, Seattle, Washington, determined the effects of exposure route and extended passage of the Stetsonville isolate in mink. Disease progression, pathology and the biochemical nature of PrP-TME in diseased mink were consistent with the presence of only a single strain. With similarities to other examples of naturally-occurring extrinsic prion diseases, the results support a role for mink as an animal model relevant to extrinsic prion infections in man.
6. Transgenic mice as a model for transmission of scrapie from goats to sheep. Goats are often co-pastured with sheep and in some operations goats are used as milk donors for orphaned lambs. In this project, ARS researchers at the Animal Disease Research Unit in Pullman, Washington, and the University of Washington, Seattle, Washington, studied the risk of introducing scrapie to flocks of sheep from infected goats will be addressed using the natural host and rodent models. In this accomplishment, we assessed the use of a transgenic mouse line expressing the ovine prion gene in bioassay of caprine brain. Mice inoculated with brain homogenates from scrapie infected domestic goats and from an infected reference sheep were evaluated for abnormal prion protein distribution and biochemical characterization and survival times. The mice were susceptible to caprine scrapie, with prion distribution patterns similar to those observed with ovine scrapie. However, shortening of the abnormal protein after passage through mice differed from the ovine pattern, thereby limiting the use of this murine model for certain strain typing experiments.
7. In vitro protein misfolding of goat-derived scrapie. Seeded conversion assays are novel techniques that have provided sensitive detection of misfolded prion protein in sheep with scrapie but such techniques have not been applied to goats. ARS researchers at the Animal Disease Research Unit in Pullman, Washington, have now successfully adapted a technique known as serial protein misfolding cyclic amplification to detect misfolded prion proteins present in the brain of goats with scrapie. The assay yields results in weeks rather than the months to years it generally takes to complete similar studies by bioassay. Furthermore, the adapted technique utilizes material derived from transgenic mice as a more readily available source of substrate than is available from sheep or goats. In addition to these efficiencies, further adaptation of this technique may allow its application to tissues and fluids which could serve to transmit scrapie between animals.
Newsom, D.M., Liggitt, H.D., Orourke, K.I., Zhuang, D., Schneider, D.A., Harrington, R.D. 2011. Cytokine antibody array analysis in brain and periphery of scrapie-infected Tg338 mice. Comparative Immunology Microbiology and Infectious Diseases. 34(5):387-97.