Transmissible Spongiform Encephalopathies in Sheep: Experimental Determination of Genetic Susceptibility and Interspecies Transmission

Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations

2019

Transmissible spongiform encephalopathies in sheep: Experimental determination of genetic susceptibility and interspecies transmission

Eric Cassmann Iowa State University

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Recommended Citation Cassmann, Eric, "Transmissible spongiform encephalopathies in sheep: Experimental determination of genetic susceptibility and interspecies transmission" (2019). Graduate Theses and Dissertations. 17418. https://lib.dr.iastate.edu/etd/17418

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Transmissible spongiform encephalopathies in sheep: Experimental determination of genetic susceptibility and interspecies transmission

Eric David Cassmann

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Veterinary Pathology

Program of Study Committee: Justin Greenlee, Co-major Professor Jodi Smith, Co-major Professor M. Heather West Greenlee Shankumar Mooyottu Joseph Haynes

The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred.

Iowa State University Ames, Iowa 2019

DEDICATION

This dissertation is dedicated to

Samantharae Isaiah Gabriel John

iii

TABLE OF CONTENTS Page

ACKNOWLEDGMENTS ...... v

ABSTRACT ...... vii

GENERAL INTRODUCTION AND LITERATURE REVIEW ...... 1 Dissertation Organization ...... 1 Literature Review ...... 1 Abstract ...... 1 Background ...... 2 Transmission and Pathogenesis of Scrapie in Sheep ...... 7 Determinants of Susceptibility and Resistance of Sheep to the Scrapie Agent ...... 9 Strains of the Scrapie Agent ...... 10 Atypical Scrapie ...... 12 Diagnostic and Research Methods for Studying the Scrapie Agent ...... 13 Interspecies Transmission of the Scrapie Agent ...... 16 Transmission of Non-Scrapie TSE Agents to Sheep ...... 18 Scrapie Eradication in the United States ...... 20 Summary ...... 20 References ...... 22

SHEEP WITH THE HOMOZYGOUS LYSINE-171 PRION PROTEIN GENOTYPE ARE RESISTANT TO CLASSICAL SCRAPIE AFTER EXPERIMENTAL ORONASAL INOCULATION...... 37 Abstract ...... 37 Introduction ...... 38 Materials and Methods ...... 40 Results ...... 43 Discussion ...... 47 References ...... 50 Figures ...... 54 Tables ...... 59

SHEEP ARE SUSCEPTIBLE TO THE AGENT OF TME BY INTRACRANIAL INOCULATION AND HAVE EVIDENCE OF INFECTIVITY IN LYMPHOID TISSUES...... 62 Abstract ...... 62 Introduction ...... 63 Results ...... 65 Discussion ...... 69 Materials and Methods ...... 72 References ...... 79 Figures ...... 83 Tables ...... 88 iv

GENERAL CONCLUSIONS ...... 90 General Discussion ...... 90 Future Directions ...... 92

ACKNOWLEDGMENTS

I would like to express my deepest appreciation to my committee co-chairs, Drs.

Justin Greenlee and Jodi Smith and my committee members, Heather West Greenlee,

Shankumar Mooyottu, and Joseph Haynes for their guidance throughout the course of my graduate training. I am especially grateful to Dr. Justin Greenlee and Dr. Heather

Greenlee of the Greenlee lab at the National Animal Disease Center. The completion of this dissertation would not have been possible without their support and encouragement as a scientist. Their thoughtful suggestions and experience were critical to my success. I am also grateful to Dr. Jodi Smith for being instrumental in my neuropathology training, giving me unwavering guidance, constructive criticism, and lending her extensive knowledge.

Thanks should also go to Drs. Jesse Hostetter, Michael Yaeger, and Mark

Ackermann for previously serving on my committee. These individuals and numerous others including Drs. Amanda Fales-Williams, Claire Andreasen, Shannon Hostetter,

Heather Flaherty, Austin Viall, Tomislav Jelesijević, and Shannon McClelland also contributed to my training in pathology, and I am grateful for that! I would also like to thank my friends, colleagues, the department faculty and staff for making my time at

Iowa State University a wonderful experience.

I would like to acknowledge the assistance of the following USDA technicians:

Leisa Mandell, Trudy Tatum, Kevin Hassall, Joe Lesan, Robyn Kokemuller, Martha

Church, Ginny Montgomery, and Judy Stasko. These individuals were instrumental to the research projects, and their extensive knowledge was helpful for troubleshooting laboratory techniques. vi

I also wish to thank Dr. John Melville (Doc) for initially cultivating my interest in research. His support has been firm for nearly 15 years. Also, Dr. Al Jergens provided me with critical exposure to veterinary research early in my veterinary medical education, and his support and encouragement has been very important to me.

Last, but certainly not least, I would like to extend my deepest gratitude to my wife, Samantharae, for giving me unconditional support during my graduate training. Her support never wavered, and I could not have done this without it. Thank you! I am also grateful to my parents, David and Lisa, and my brother, Christopher, for believing in me and always being supportive. vii

ABSTRACT

Transmissible spongiform encephalopathies (TSE) are a group of infectious diseases caused by a misfolded protein. Naturally occurring TSEs are found in numerous species including sheep, cattle, and mink. The naturally occurring TSE of sheep is called scrapie. Scrapie is one of the oldest described TSEs, and it is caused by a misfolded protein (PrPSc) that initiates an autocatalytic conversion of an endogenous host protein

(PrPC). The mammalian PRNP gene encodes PrPC, and sheep are variably susceptible to disease based on naturally occurring polymorphisms in the PRNP gene at codons 136,

154, and 171. The selection of resistant genotypes is essential to scrapie eradication programs. Currently there are polymorphisms in some breeds of sheep that have not been thoroughly investigated vis-à-vis their contribution to scrapie susceptibility. Herein we describe the contribution of lysine at codon 171 (K171) of the PRNP gene towards resistance to classical scrapie. Using sheep, the natural host, as model to evaluate genetic susceptibility, we experimentally infected sheep with different allelic polymorphisms at codon 171. Orally inoculated sheep with the homozygous lysine 171 (KK171) genotype did not develop scrapie or harbor any detectable PrPSc. In the second study encompassing this work, we sought to evaluate the etiology of transmissible mink encephalopathy

(TME), a naturally occurring TSE of mink. TME is a foodborne TSE of farmed mink, and earlier theories suggested that classical scrapie or atypical BSE may have been the source of prions to mink. The etiology of prion diseases is best understood by experimental transmission between species. To determine if scrapie is the source of TME, we examined the susceptibility and disease phenotype of sheep inoculated with the TME agent. We determined that only intracranially inoculated sheep are susceptible, and the viii disease phenotype in sheep is different from classical scrapie. Primarily, sheep inoculated with the TME agent do not develop immunoreactivity for PrPSc in their lymphoid tissue; whereas, it is known that sheep with classical scrapie have prominent lymphoid accumulation of PrPSc. Cumulatively, this research expands the current knowledge of ovine genetic susceptibility to scrapie and indicates that classical scrapie is not likely to be the source of TME. Practically, the knowledge presented in this work is immediately applicable to designing or updating genotype-based scrapie eradication plans.

GENERAL INTRODUCTION AND LITERATURE REVIEW

Dissertation Organization

This dissertation is arranged in the alternative journal paper format, and it is divided into three manuscripts. In this first chapter, a detailed review of relevant scientific literature is presented. The literature review was prepared for submission as a review for the American

Journal of Veterinary Research. The second chapter was published as an infectious disease- original research article in the journal Veterinary Pathology. The third chapter was prepared for submission as an original research paper to Frontiers in Veterinary Science. The figures and tables cited within the text appear at the end of their respective chapters.

Literature Review

The pathogenesis, detection, and control of scrapie in sheep

Modified from a review prepared for submission to the American Journal of Veterinary Research

Eric Cassmanna,b and Justin Greenleea aVirus and Prion Research Unit, National Animal Disease Center, Agricultural Research

Service, United States Department of Agriculture, Ames, IA, USA bOak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy and the U.S. Department of Agriculture

Abstract

Sheep scrapie is a fatal neurologic disease that is caused by an infectious misfolded protein called a prion. The prion protein is encoded by an endogenous gene, PRNP, and it 2 resides in high concentrations within the central nervous system. A broad range of functions for the prion protein have been uncovered, but the entire range of function is undetermined.

In the misfolded conformation, prion accumulation results in neurodegeneration. There are several naturally occurring polymorphisms in the PRNP gene, and there is a strong correlation between disease susceptibility and the PRNP genotype. The cornerstone of many scrapie eradication programs is selection of scrapie resistant genotypes. The transmission of scrapie often occurs between sheep in the periparturient period when lambs are the most susceptible due to higher densities of gut-associated lymphoid tissue. Subsequently, prions are disseminated to the lymphoid system and spread to the central nervous system. The shedding of prions occurs prior to the onset of clinical signs. In contrast to classical scrapie, atypical scrapie is a spontaneous form of scrapie that occurs in individual older animals within a flock. It is not considered to be naturally infectious. This review outlines the current modalities for diagnosing scrapie in sheep and the techniques for studying its host range and pathogenesis. Scrapie transmission to other species including deer has been demonstrated experimentally as well as the transmission of non-scrapie prion diseases to sheep. Finally, the effectiveness of the United States scrapie eradication program is reviewed.

Background

The History of Prion Diseases and the Protein Only Theory

Transmissible spongiform encephalopathies (TSEs) are protein misfolding diseases that lead to fatal neurodegeneration.1 The misfolded protein, denoted PrPSc, is derived from the endogenous cellular prion protein, PrPC.2 There are many naturally occurring prion diseases of animals and humans: scrapie in sheep and goats,3 kuru4,5 and Creutzfeldt-Jakob disease in humans,6 bovine spongiform encephalopathy in cattle,7,8 chronic wasting disease 3 in cervids,9,10 feline spongiform encephalopathy in cats,11 transmissible mink encephalopathy in mink,12,13 and, recently described, camel prion disease in dromedary camels.14 Scrapie is the oldest recorded prion disease; references to scrapie date back at least 300 years.15 In 18th century England during the agricultural and textile revolution, scrapie was a politico- economic concern. The disease was even discussed in the British House of Commons in

1775 due to mounting concerns about the potential economic effects on the merchant trade of woolen products.16 There are also well-described clinical signs of scrapie that appear in a

German text from the same time period.17 However, it wasn’t until the end of the 19th century that the archetypal pathologic vacuolation (spongiform encephalopathy) was first documented.18

Initial attempts at experimental transmission of scrapie did not yield positive results, because insufficient time was allowed for incubation. The first successful transmission was published in the 1930’s after animals were observed for a prolonged period after inoculation.19 Even with successful demonstration of transmissibility, the etiologic agent of scrapie was still unknown; however, the experiments suggested that the agent of scrapie was not a “typical” pathogen. Ionizing radiation, extreme heat, and chemicals known to deactivate viruses and bacteria failed to inactive the agent of scrapie.20-22 This was the first step towards a “protein only” theory. In 1967, J.S. Griffith overtly proposed that a protein, and not an organism containing nucleic acid, was the etiologic agent of scrapie.23 Suitable to the central dogma of biology, he posed several hypotheses by which a protein could be the scrapie agent. In one, he correctly speculated that “the protein” could be one that the animal was “genetically equipped to make, but not in that [deleterious] form”. Such a gene was first described by Dickinson and colleagues in 1968. By back crossing F1 progeny with their 4 donor parent, they demonstrated an autosomal gene without dominance that was linked to the duration of scrapie incubation in mice.24 The gene was aptly named “sinc” for “scrapie incubation”. Within a decade-and-a-half of J.S Griffith’s published hypotheses, Stanley

Prusiner coined the term “prion” (pronounced pree-on) in his preeminent manuscript outlining his hypotheses on the etiology of scrapie.1 The most revolutionary concept was a protein-only theory that advocated the existence of an infectious protein. Since purification of the scrapie agent had not been accomplished yet, he also cautiously proposed that nucleic acid might exist undetected within the core of a tightly packed resilient protein coat. Later in the same year, Prusiner, Boulton, and colleagues isolated and purified scrapie prions.25 The protein isolated was approximately 27,000 to 30,000 daltons, and as such, was often referred to as PrP27-30 in early studies. PrP27-30 is the main component of the PK-resistant core of

PrPSc.26

Hoping to identify viral mRNA in scrapie infected brains, Chesebro et al.27 designed an oligonucleotide probe corresponding to the translated mRNA sequence of PrP27-30.

Using the probe, a cDNA clone was obtained from scrapie infected brain. The isolated sequence correctly translated back to the amino acid sequence of PrP27-30. Concurrently, the researchers also derived the same sequence from normal control mice and hamsters, indicating that PrP27-30 was most likely derived from an endogenous gene. Other work was performed contemporarily that reported similar findings.28 In addition to mice and hamsters, humans were also found to have a gene encoding PrP27-30 (PRNP). PrP27-30 was detected in both scrapie infected and normal animals, but the detection of the protein from normal brains was lost after the application of proteinase K (PK). The protein only theory was given ever-increasing credibility after susceptibility to scrapie was eliminated in PrP-/- mice.29 5

Further convincing evidence was added by the creation of de novo infectious prions recombinantly expressed in E. coli.30,31 These recombinant de novo prions that exhibited attributes of pathogenic PrPSc were used to effectively reproduce disease in mice. Taken together, these data provide overwhelming evidence that prion infectivity and disease is related to conformational alterations in the host’s prion protein.

Normal Cellular Prion Protein (PrPC)

The cellular prion protein (PrPC) is a glycoprotein approximately 231 amino acid residues long32 that is fixed to the cell’s plasma membrane via phosphatidylinositol, a

33 2+ glycolipid anchor. There are well-conserved octapeptide repeats that bind Cu in the NH2- terminus.34 In sheep, PRNP mRNA transcripts are found at the highest levels in the thalamus and cerebrum followed by the cerebellum, spinal cord, spleen, other lymphoid tissues, brain stem, gastrointestinal tract, and reproductive organs.35

Many functions of PrPC have been elucidated, but the entire range of functions is not known. A prion gene knockout in mice is sublethal, and there are no obvious phenotypic changes.36 However, there is a broad range of testable phenotypic changes that indicate that the activity of PrPC is diverse. For example, PrPC deficient mice have abnormal spatial cognitive abilities that are rescuable by reintroduction of PrPC to neurons.37 Other physiologic roles of PrPC include synaptic function,38 murine uterine decidualization,39 and sleep regulation.40 Physiologic activities of PrPC in the immune system include host-pathogen interactions,41-43 T cell to dendritic cell immunosynapse formation,44 and negative regulation of phagocytosis.45 There also appear to be important neuroprotective functions of PrPC that are related to its binding capabilities. A fragment of the N-terminal portion of PrPC is capable of binding to neurotoxic β-amyloid oligomers thereby abating their deleterious effects.46,47 6

Other protective effects are exerted by reducing free radical damage to neurons. The prion protein decreases oxidative damage by affecting superoxide dismutase activity.48-51

Additionally, by binding Cu2+,52 PrPC abates hydroxyl radical (OH•) creation by preventing free Cu2+ from participating in the Fenton reaction. Given the role of free Cu2+ in the creation of reactive oxygen species, the association of high concentrations of PrPC around the neuronal synapse,53 where copper is also released,54 is not surprising. In fact, the response of

C synaptic activity to hydrogen peroxide (H2O2) correlates with the amount of neuronal PrP expression.53

Prion Protein Structure and Misfolding

Prion diseases result from the accumulation of a misfolded form of the normal cellular prion protein.2 The cellular prion protein and the scrapie prion have distinct structural characteristics evident in their secondary and tertiary structures. PrPC is comprised of 40% α- helical and 3% β-helical folds; however, PrPSc is folded into a parallel left-handed β-helical structure containing a 30% α-helical and 40% β-helical conformation.55,56 Individual β-sheets organize to form a helical pattern that is purported to associate in a three or four-rung β- solenoid that polymerizes into amyloid fibrils.57,58 There are at least two proposed models for

PrPSc autocatalytic propagation (reviewed by Aguzzi and colleagues).59 The refolding model assumes that an initial conversion event is precluded by an energy barrier; however, once

PrPSc is introduced or the energy barrier to conversion is breached, transformation of PrPC into PrPSc ensues. Another model called the “seeding model” asserts that PrPSc and PrPC exist in thermodynamic equilibrium between the two forms. In this model, PrPSc begins to 7 aggregate when a highly ordered monomeric PrPSc, the seed, stabilizes and recruits more monomeric PrPSc to form larger aggregates.

Aggregation of misfolded proteins in the brain is a pathologic feature of prion disease, but exactly how prion disease results in neurodegeneration is less clear. Direct neurotoxicity, indirect toxicity, dysregulation of the unfolded protein response (UPR), and loss of the normal neuroprotective functions of PrPC are hypothesized mechanisms that trigger neurodegeneration. Direct toxicity of PrPSc to neurons is likely refuted by the lack of neuronal pathology in PrPC depleted mice inoculated with PrPSc.60,61 On the other hand, there is evidence that indirect toxicity and dysregulation of the cellular UPR provoke neurodegenerative changes.62-65 Interestingly, the prototypical vacuolar change associated with spongiform encephalopathies is not a prerequisite to clinical neurologic signs.66

Transmission and Pathogenesis of Scrapie in Sheep

The main route of transmission in naturally occurring classical scrapie is through the oral route,67 and exposure at birth seems to be a major risk factor in developing disease.

Contact with infected fetal membranes in the periparturient period or contamination of the environment by this material contributes to transmission.68,69 Nursing lambs can also be exposed to PrPSc through colostrum and milk.70,71 After birth, environmental contamination serves as a reservoir for infectious PrPSc resulting in horizontal transmission of scrapie.72 In addition to reproductive tract-associated shedding, PrPSc is shed in the saliva,73,74 urine,75,76 and feces77 of infected sheep. Shedding during the preclinical stage of disease78 is of particular concern for the transmission of scrapie. Additionally, both PK resistant and PK susceptible conformers of PrPSc are known to be infectious.79 8

The pathogenesis of natural scrapie infection can be separated in three phases: GALT invasion, lymphatic invasion and spread, and neuroinvasion.66,80 After oral exposure, PrPSc crosses the mucosal border at the palatine tonsil, distal jejunum, and ileum. In the small intestine PrPSc accumulates in Peyer’s patches,81 and M cells are a critical entry point for

PrPSc.82 After entry, the movement of prions from M cells to the Peyer’s patch is facilitated by conventional dendritic cells expressing the chemokine receptor CXCR5.83 The presence of prions within the Peyer’s patches is essential for disease development,84 and there is initial replication of PrPSc in the follicular dendritic cells (FDCs) that express PrPC.85 FDC deficient models indicate that there is a marked decrease in susceptibility to scrapie infection; however, FDC-independent pathways of neuroinvasion also appear to exist.86-88

During the asymptomatic incubation period, PrPSc disseminates throughout the lymphoid system.66,81 The spread of PrPSc from the palatine tonsil and Peyer’s patches to the medial retropharyngeal and mesenteric lymph nodes occurs through afferent drainage of lymph. It is less clear how PrPSc accumulates in other organs like the spleen that does not receive afferent lymphatic drainage, but hematogenous spread is a likely route.

Hematogenous spread of PrPSc is supported by observations of early neuroinvasion to the circumventricular organs that lack a blood-brain barrier.89 Furthermore, hematogenous dissemination is supported by research that demonstrates infectious prions in the blood of sheep.90-93

The initial site of neuroinvasion is the enteric nervous system (ENS). In an experimental sheep scrapie model, invasion of the ENS did not occur until after widespread

PrPSc lymphoid dissemination.66 Infection of ENS neurons could occur at multiple levels.

The first possibility is through submucosal fine nerve fibers in close proximity to mucosal 9 epithelium and lacteals.94 Alternatively, active cell transport or drainage of PrPSc from

Peyer’s patches to the ENS could occur.95 The latter is supported by studies documenting the preferential accumulation of PrPSc in the ENS adjacent to Peyer’s patches.81,96 After entering the ENS, PrPSc moves retrograde along efferent axons of parasympathetic and sympathetic neurons to the medulla oblongata or the thoracic spinal cord, respectively.80

In cases of natural infection, PrPSc does not stimulate a humoral response owing to the identical amino acid sequences of PrPC and PrPSc.97,98 Since T-cells process antigen as linear epitopes, they cannot discriminate between peptide sequences of PrPSc and PrPC; therefore, antigen presenting cells are deprived of necessary T-cell costimulatory signals required to elicit robust humoral immunity. There is evidence that complement components interact with

PrPSc, and membrane attack complexes localize in scrapie infected brains.99-101 These mechanisms proposedly contribute to neurodegeneration. Additionally, complement meditated endocytosis may contribute to the movement of PrPSc to lymphoid tissues by dendritic cells expressing CD35 (complement receptor 1).102

Determinants of Susceptibility and Resistance of Sheep to the Scrapie Agent

Susceptibility to the classical scrapie agent is determined by the host PrP genotype, exposure age, volume of infectious dose, and route of infection. In sheep there are several naturally occurring polymorphisms in the prion gene. There are three main sites or codons that have the greatest impact on susceptibility: 136, 154, and 171.103-111 Valine at codon 136

(V136), arginine at 154 (R154), and glutamine at 171 (Q171) are associated with susceptibility to scrapie. Conversely, the following amino acid polymorphisms confer relative resistance: alanine (A136), histidine (H154), and arginine (R171). The polymorphic codons 136 and 171 appear to have a greater effect than codon 154 on ultimate disease 10 vulnerability.107,112 A lysine (K) amino acid substitution at codon 171 is seen in Dorper,

Barbados, Barbados/St Croix, Suffolk crosses, and some Mediterranean breeds.113-115

Epidemiologic studies reveal that sheep carrying this allele are infrequently scrapie positive in natural settings.114 Experimentally, heterozygous QK171 genotype sheep have prolonged incubation periods after intracerebral inoculation.116 The relative resistance in decreasing order of common PrP genotypes observed in natural scrapie cases is ARR/ARR > ARR/ARQ

> ARQ/ARQ > ARR/VRQ > ARQ/VRQ > VRQ/VRQ.117 While the ARR/ARR genotype is highly resistant to scrapie, there are at least three cases of naturally occurring scrapie in homozygous ARR sheep documented around the world.118,119

The age at first exposure affects susceptibility to scrapie. Statistical modeling from a large flock indicated that sheep younger than 24-months-old at the time of exposure had three times the risk for developing scrapie compared to older sheep.120 Since Peyer’s patches are required for lymphoreticular uptake of scrapie in the intestine,84 the age related effect on susceptibility is likely due to decreasing GALT density. The percentage of ileal Peyer’s patches averages 49.8-60.3% in 0-3 month-old lambs; in sheep older than 18 months, the average percentage of ileal Peyer’s patches is 0-7%.121 As previously described, FDC are also associated with early scrapie pathogenesis. An age-related decrease in the FDC network within Peyer’s patches also occurs.122 Together these findings offer a compelling explanation for the observed age-related susceptibility phenomenon.

Strains of the Scrapie Agent

The concept of prion strains refers to the distinct pathologic characteristics conferred by different isolates of a TSE agent. Dissimilar strains can result in observable differences in attack rates, incubation periods, clinical signs, brain vacuolation, molecular patterns, and 11 immunolabeling patterns.123-125 A widely utilized and accepted method for differentiating

TSE strains is by comparing the degree of brain vacuolation and the incubation periods in inbred mice.126-128 PrPSc immunolabeling patterns (PrPSc profiling) can be used to differentiate strains of scrapie in sheep without passage in mice.125,129,130

In the United States, different strains of scrapie have been identified from natural field cases. Field isolates 13-7 and x124 are recognized as separate strains based on differences in their PrPSc immunolabeling profiles in sheep and attack rates in C57BL/6 mice.131 Worldwide, strain typing techniques in inbred mice have identified at least 15 distinct strains of classical scrapie.132 There are also at least three distinct PrP glycoform types documented for classical scrapie isolates from archival scrapie samples in the UK.133 In one manuscript, these glycoform types were separated into distinct categories, A, B, and C, based on their relative mobilities and glycoform ratios observed by western blotting.

Glycoform type A was derived from experimental and natural cases of scrapie, and type B was observed only from natural cases. The observed differences between glycoforms A and

B from scrapie cases provides evidence that there are naturally occurring scrapie strains. The type C glycoform was an experimental isolate named CH1641; it is similar to the glycoform signature of C-BSE in sheep.133,134 However, despite their biochemical similarity, C-BSE and

CH1641 scrapie are distinguishable upon transmission to mice. This suggests that biochemical analysis should be used in conjunction with other methods to distinguish strain characteristics.

There are multiple theories for how different strains arise in a host. Two recognized theories are the “cloud hypothesis” and the “deformed template theory”. According to the cloud hypothesis, isolates are comprised of a heterogeneous mixture of PrPSc conformations 12

(strains), and over time a permissive conformer arises to become the predominant variant.135,136 Prevailing conformers are host selected by differences in the replication environment. This varies from the deformed template theory that assumes there is initially a predominant conformer instead of a mixture.137 After changes in the environment, trial and error seeding events generate a new dominant conformer. It is hypothesized that this difference in transmissibility can be accounted for by the existence of multiple conformers

(strains) within an isolate.138-140 These models require that each conformer has a distinct capability to replicate given different environmental parameters, such as a different host species.

Atypical Scrapie

In 1998 a novel type of scrapie called Nor98 or atypical scrapie was identified in sheep.141 Atypical scrapie differs from classical scrapie since it is considered a spontaneously occurring prion disease that typically occurs in a single older animal within a flock.142 The distribution of brain lesions is also distinct in atypical scrapie. Vacuolation and PrPSc occurs in the cortex of the cerebellum and cerebrum;141,143 whereas, in classical scrapie, lesions are concentrated in the medulla oblongata, especially the dorsal motor nucleus of the vagus nerve. Another prominent difference is that atypical scrapie does not result in detectable lymphoid accumulation of PrPSc. Interestingly, cases of experimental atypical scrapie with undetectable PrPSc by immunoassay were found to contain infectious PrPSc in lymphoid tissue, muscles, and peripheral nervous tissue.144 These results were determined by positive bioassays in transgenic mice expressing sheep prion protein. The next section discusses the use of laboratory techniques including mouse bioassays for studying prion diseases.

Diagnostic and Research Methods for Studying the Scrapie Agent

The current standard for identifying PrPSc utilizes anti-PrP antibodies. Many antibodies are available and recognize different conformational or linear epitopes of PrPSc.

Typically, antibodies are generated by injecting a PrP-/- animal with PrPC followed by selecting neutralizing antibodies. The use of anti-PrP antibodies can be applied to various immunoassay techniques including western blot (WB or immunoblot), enzyme-linked immunoassay (ELISA), immunohistochemistry (IHC), immunocytochemistry and immunoprecipitation. Usually digestion with PK is a necessary step for antibody discrimination between PrPC and PrPSc. After PK digestion, PrPC is undetectable, and the resistant core of PrPSc (PrP27-30) remains intact for immunodetection.26 Owing to the recognition of unique conformational epitopes, there are some antibodies that selectively bind PrPSc without first digesting PrPC with PK.145

Accumulation of PrPSc in the brain is detectable with IHC after death; however, PrPSc is also detectable in the lymphoid tissue of a high number of scrapie affected animals (98%) that have neuropathological changes.146 Antemortem diagnostics to detect preclinical scrapie utilize immunohistochemistry on lymphoid tissue samples including tonsils,147 third eyelid mucosa,148 and recto-anal mucosa.149 The acquisition of tonsillar biopsies in live animals requires a honed skillset and may require anesthesia. Given this difficulty, a more accessible biopsy site from the third eyelid conjunctiva that utilizes local analgesia was developed as an antemortem test. Biopsy of the third eyelid reportedly yields a minimum number of lymphoid follicles in roughly 80% of samples that corresponds to an estimated test sensitivity of 85-

90%.150 A drawback of the eyelid biopsy technique is that older animals tend to have less third eyelid lymphoid tissue. Furthermore, lymphoid tissue does not regenerate at this location precluding repeated sampling.150 Considering the number of lymphoid follicles per 14 biopsy, rectal mucosa is superior to both tonsil and third eyelid conjunctiva.149 Another advantage of rectal mucosa biopsies is that this site can be biopsied multiple times without a decrease in the number of follicles obtained, and anesthesia or sedation is not required. For comparison to other methods, 87% of rectal biopsies had at least 10 follicles present, which corresponded to a false negative rate of about 9%. In addition to the use of IHC, rectal mucosa biopsies can be used in tandem with enzyme immunoassay.151 Negative test results to any antemortem test for scrapie should be revaluated multiple times at the herd and individual level owing to influences by PrP genotype, scrapie strain, level of infection, and incubation period time-point.

Bioassays using mice expressing transgenic PrPC are useful for testing the transmission efficacy of the scrapie agent. For example, Tg338 mice overexpress an ovine

VRQ transgene,152,153 so these mice can be used to analyze sheep scrapie isolates. The effect of the species barrier on cross-species transmission is nullified by using a transgenic mouse.

Furthermore, transgenic mice often overexpress PrPC. This allows prion diseases to be propagated in vivo more rapidly with greater sensitivity than in a natural host that may take years under normal conditions. For example, for the classical scrapie strain x124 derived from an VRQ/ARR sheep, the incubation time in intranasally inoculated sheep with the

VRQ/ARQ genotype is 9.9 to 13.9 months.131 Tg338 mice will succumb to the same inoculum in approximately 76 days [internal unpublished data, USDA ARS, Ames, IA]. The rapid incubation of prion diseases in transgenic mouse models allows experimentation to be performed at faster and less expensive rates than studies in the natural host.

Mouse models are also widely used for strain typing prion diseases. For this technique, the degree of vacuolation in predefined grey matter and white matter locations is 15 plotted to compare strains.127 Wild type inbred mice, C57b, RIII, and VM are regularly utilized; however, due to species barrier effects, low transmission attack rates can preclude comparisons with inbred mice. As an alternative, using transgenic mice that express the host species’ PrPC can result in higher attack rates and shorter incubations.153,154 For example, scrapie strain 13-7 has a 5.9% (1/17) attack rate in C57BL/6 mice on primary passage,131 but in Tg338 mice, the attack rate is 100% (17/17) [internal unpublished data, USDA ARS,

Ames, IA].

In vitro assays have been developed to identify prions for more rapid diagnostic and experimental purposes. Protein misfolding cyclic amplification (PMCA) is conceptually analogous to PCR amplification of DNA. For this method, a sample containing prions is added to a brain homogenate from a normal animal and subjected to alternating steps of incubation and sonication.155 Small amounts of undetectable prions are eventually amplified to detectable levels by western blotting. An improved method (rPrP-PMCA) using PK sensitive recombinant PrP (rPrPsen), decreased the assay time from 3 weeks to a couple of days.156 By adding PrPSc, rPrPsen is converted to detectable rPrPres (PK resistant) via sonication.

Quaking induced conversion (QuIC) is an alternative to PMCA that employs shaking instead of sonication.157 The original QuIC technique identified prions from a sample within a day; however, the original detection method relied on laborious western blotting. An improved version of QuIC called real-time QuIC (RT-QuIC) exploits the fluorescence of thioflavin T interacting with amyloid fibrils.158,159 The fluorescence-based assay utilizes a plate reader and measures the intensity of fluorescence in response to amyloid formation. By establishing a standard curve, this technique has been adapted to quantify amount of prions in 16 a sample.160 A more recently described variation of QuIC called endpoint-QuIC (EP-QuIC) utilizes a thermomixer instead of a plate reader to perform shaking.161 The same research group later reported distinct differences in the detection thresholds between conventional RT-

QuIC and EP-QuIC at various time intervals.162 The utility of RT-QuIC and ELISA-coupled

PMCA extends beyond diagnostic detection of PrPSc. RT-QuIC has also been used to discriminate prion strains.163,164

The stability of folded prions varies between some TSE strains, and by denaturing prions with increasing concentrations of guanidine hydrochloride (GdnHCl), strains can be segregated into distinct groups.165 After GdnHCl denaturation, the relative amount of prion remaining is expressed in terms of relative absorbance as determined by ELISA.166,167 One example of this technique’s application is the its ability to distinguish isolates of scrapie in sheep.168

Interspecies Transmission of the Scrapie Agent

The transmission of TSEs between animals of different species is often impeded by a species barrier that is related to differences between the host and donor PRNP sequence. A prime example of a robust species barrier is seen with dogs. Dogs are highly resistant to prion infection and have not been shown to be susceptible to TSEs in vivo.169 This example epitomizes the “species barrier” concept. Contrariwise, there are instances of natural TSE transmission between species. Two examples would be the transmission of C-BSE to humans,170-172 and likely, L-BSE to mink.173,174 Experimentally, interspecies transmission studies are used to evaluate host ranges and the origins of TSEs. The results inform policy decisions regarding biosecurity, food security, and the use/disposal of specified risk material. 17

Experimental transmission of scrapie to cattle is sometimes precluded by a significant species barrier. Cattle appear to be resistant to a U.S. derived scrapie strain by the oral route.175 However, cattle are susceptible to intracerebral inoculation.176,177 Cattle that develop scrapie do not exhibit spongiform change and mainly have PrPSc immunolabeling visible within neurons. Notably, this disease phenotype in scrapie affected cattle does not resemble

BSE in cattle, refuting the hypothesis that scrapie is the etiologic agent of BSE.

North American white-tailed deer are susceptible to the agent of classical scrapie by intracerebral and oral inoculation routes.178,179 Interestingly, deer intranasally inoculated with scrapie, simulating a natural route of exposure, accumulated PrPSc with distinct molecular phenotypes on western blot. Depending on the brain region examined, the deer had a lower unglycosylated band similar to the original scrapie inoculum or a higher unglycosylated band similar to CWD isolates.180 Elk are also susceptible to the scrapie agent after experimental intracranial inoculation.181 Scrapie-affected elk exhibit a PrPSc immunophenotype similar to scrapie inoculum and distinct from cervids with CWD, indicating that the origin of CWD in elk is not likely directly from sheep with scrapie.

There are several other experiments aimed at determining the host range of scrapie.

For example, classical scrapie has been successfully transmitted to raccoons via intracerebral inoculation;182 although, they are resistant to atypical scrapie.183 Swine are poorly susceptible to the agent of scrapie, and in intracerebrally inoculated swine, the disease phenotype is different than in sheep with scrapie.184 In a study of pigs intracranially inoculated with scrapie, no positive animals were detected at market weight by traditional diagnostic methods

(ELISA, IHC, and WB), but 5/10 aged pigs (51 months old) were positive by one or more traditional diagnostic techniques.184 No orally inoculated pigs had detectable PrPSc by 18 traditional methods, but a mouse bioassay did demonstrate some mice with evidence of PrPSc accumulation after inoculation with brain material.

The zoonotic potential of scrapie has been investigated. Generally, the likelihood of zoonosis is considered to be low, since there are no reported human TSE cases with scrapie compatible phenotypes.185 There are, however, comparative models that suggest zoonotic potential. For example, Cassard et al. demonstrated that a subset of transgenic mice overexpressing human prion protein were susceptible to various strains of classical scrapie; albeit, there was a notable species barrier in place considering that the mice overexpressed

PrPC and the attack rates were low.186 Non-human primates including various new world and old world monkeys have been susceptible to the scrapie agent, often after a significantly long incubation period.5,187,188

Transmission of Non-Scrapie TSE Agents to Sheep

Sheep are susceptible to several TSEs originating in other species. In the early

1990’s, experimental transmission of classical BSE to sheep was prompted by investigations into the origins of BSE in cattle. Sheep were shown to be susceptible to C-BSE by both oral and intracranial inoculation.189 However, there are notable differences in the distribution of

C-BSE in sheep compared to cattle. In sheep the spleen is infectious; whereas, in cattle the spleen does not harbor infectious PrPSc.190,191 Furthermore, C-BSE inoculated sheep have a variety of other infectious tissues including the distal ileum (ENS), Peyer’s patches, spleen, liver, mesenteric lymph nodes, prescapular lymph nodes, tonsils, and thymus.192 Sheep are most susceptible to C-BSE prior to weaning.193 Further investigations into the presence of

BSE within small ruminant populations demonstrate that BSE can spread within a closed population of sheep by either in utero or perinatal exposure.194 Although experimental 19 evidence demonstrates that sheep could be a reservoir for BSE, widespread surveillance within the BSE epizootic region has failed to substantiate this concern.195

Attempted transmission of atypical BSE agents to sheep has had variable success depending on the inoculation route. Intracranial inoculation of the atypical BSE (L-BSE) agent in sheep results in disease, but sheep are not susceptible orally. Sheep affected by L-

BSE lack peripheral lymphoreticular PrPSc, but they have detectable PrPSc in peripheral nervous tissues including various ganglia and neuromuscular spindles.196 Sheep PRNP genotype influenced their susceptibility to intracranial inoculation with the L-BSE agent.

Sheep with the ARR/ARR genotype did not developed disease, but sheep with VRQ/VRQ,

VRQ/ARQ, and ARQ/ARQ genotypes were susceptible. Subsequent passage of sheep adapted L-BSE prions to sheep resulted in a substantial decrease in the incubation period, suggesting enhanced adaptation of L-BSE to sheep.

Transmission of the chronic wasting disease agent from mule deer (CWDMD) to sheep has been demonstrated after intracranial inoculation of sheep with VRQ/ARQ and

ARQ/ARQ genotypes.197 A single sheep with the VRQ/ARQ genotype, developed neurologic signs at 35 months post-inoculation. This sheep had spongiform encephalopathy and widespread lymphoid accumulation of PrPSc throughout the body including the tonsils, lymph nodes, and spleen. One subclinical sheep with the ARQ/ARQ genotype had spongiform change and PrPSc accumulation in the brain and tonsils that was noted at the termination of the study (72 months post-inoculation). Sheep with a more resistant genotype, ARQ/ARR, did not have detectable PrPSc or spongiform change.

Scrapie Eradication in the United States

There is an ongoing effort to eradicate scrapie in the United States. The U.S. scrapie eradication program focuses on several primary areas including genetic susceptibly testing, selective breeding of rams with a resistant genotype, an indemnification program, and surveillance.198 The components of surveillance are regulatory scrapie slaughter surveillance

(RSSS), non-slaughter surveillance (on-farm and trace investigations), and the scrapie free flock certification program (SFCP).199 The surveillance program aims to test a minimum of

40,000 animals (sheep and goats) each year on farms and through RSSS. While scrapie is not completely eradicated, the program has seen considerable success in the past twenty years.

According to the 2017 annual report, the incidence of positive cull sheep at slaughter between 2003 and 2017 has decreased by 99%.200 Furthermore, infected and source flocks decreased dramatically from 2006 to 2016.

Summary

Scrapie is a naturally occurring and fatal neurologic disease of sheep that is caused by a misfolded protein called a prion. The tendency to transmit disease between individual sheep is influenced by the donor and host’s prion protein genotype. Sheep with the ARR/ARR genotype are considered to be relatively resistant; whereas, sheep that have a VRQ/VRQ genotype are highly susceptible to classical scrapie. Antemortem diagnosis of scrapie can provide valuable information to confirm suspected positive cases, but negative results should be interpreted with caution and in light of repeated sampling. One aim of future work is toward improved methods for preclinical detection. Another area of future work will be to continue probing historical and novel scrapie strains for characteristics that help elucidate their origins, pathogenesis, and host ranges. Although experimental interspecies transmission 21 of prion agents is sometimes possible, it is generally considered unlikely to occur under natural conditions. The U.S. scrapie eradication program has demonstrated successful results with a substantial decrease in cases throughout the program’s tenure.

Acknowledgements

Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the Department of Agriculture. The Department of Agriculture is an equal- opportunity provider and employer.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This work was funded in its entirety by congressionally appropriated funds to the United

States Department of Agriculture, Agricultural Research Service. The funders of the work did not influence study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This work was supported in part by an appointment to the Agricultural Research Service

(ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of 22

Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by

ORAU under DOE contract number DE-SC0014664. All opinions expressed in this paper are the author's and do not necessarily reflect the policies and views of USDA, ARS, DOE, or

ORAU/ORISE.

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SHEEP WITH THE HOMOZYGOUS LYSINE-171 PRION PROTEIN

GENOTYPE ARE RESISTANT TO CLASSICAL SCRAPIE AFTER

EXPERIMENTAL ORONASAL INOCULATION

Modified from a manuscript published in Veterinary Pathology

Vet Pathol 2019; 56(3): 409-417 © The Author(s) 2018 DOI: 10.1177/0300985818817066

E.D. Cassmann1, S.J. Moore2, J.D. Smith1, and J.J. Greenlee2

1Department of Veterinary Pathology, Iowa State University, Ames, IA, USA (EDC)

2Virus and Prion Research Unit, National Animal Disease Center, Agricultural Research

Service, United States Department of Agriculture, Ames, IA, USA (SJM, JDS, JJG)

Dr. Smith’s present address is Department of Veterinary Pathology, College of Veterinary

Medicine, Iowa State University, Ames, IA 50011.

Abstract

Scrapie is a fatal neurodegenerative disease of sheep resulting from the accumulation of a misfolded form of the prion protein (PrPSc). Polymorphisms in the host prion protein gene (PRNP) can affect susceptibility to the scrapie agent. Lysine (K) at codon 171 of PRNP is an inadequately characterized, naturally occurring polymorphism in sheep. We inoculated

Barbado sheep with PRNP genotypes QQ171, QK171 or KK171 by either the intracranial

(IC, n=2-7 per genotype) or oronasal (ON, n=5 per genotype) routes with a scrapie isolate to investigate the effect of lysine at codon 171 on susceptibility. When neurologic signs were 38 observed or at the end of the experiment (70 months post-inoculation (MPI)), sheep were necropsied and tissue collected for histopathologic, immunohistochemical, enzyme immunoassay and western blot examination for PrPSc. All genotypes of sheep developed scrapie after IC inoculation. After ON inoculation, sheep with the QK171 genotype had prolonged incubation periods compared to the QQ genotype. During the experiment, 2/5 of the ON-inoculated QK genotype sheep developed neurologic signs and had PrPSc in the brain.

The other 3/5 sheep were asymptomatic at 70 MPI, but had detectable PrPSc in peripheral tissues. None of the ON-inoculated sheep of the KK171 genotype developed signs or had detectable PrPSc. Our experiments demonstrate that sheep with the KK171 genotype are resistant to scrapie via oronasal exposure and that sheep with the QK171 genotype have prolonged incubation relative to QQ171 sheep. The K171 prion protein allele may be useful to enhancing scrapie resistance in certain breeds of sheep.

Introduction

Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative diseases that affect multiple species. Scrapie is the naturally occurring TSE of sheep and goats.18,24,29 The causative agent is a misfolded form of the prion protein (PrPSc) which is self-replicating and infectious.7,28 Sheep are susceptible via natural exposure to PrPSc both vertically from mother to offspring and horizontally between unrelated animals in a flock.8,30

The normal cellular prion protein (PrPC) is encoded by an endogenous gene, PRNP.27

Allelic variation in PRNP codons 136, 154 and 171 produces amino acid polymorphisms in

C 5,9,10 PrP that correlate with varying degrees of scrapie susceptibility in sheep. Alanine (A)

136 and arginine (R) 171 lend relative resistance while valine (V) 136 and glutamine (Q) 171 correlate with an increased risk for developing scrapie. Arginine 154 has a minor association 39 with scrapie susceptibility. Ultimately, disease susceptibility is influenced primarily by the

136/171 haplotype with a distinct influence imparted by codon 171.4,5,11,21 The

A136R154R171/ARR and ARQ/ARR haplotypes are associated with relative resistance while

ARQ/ARQ is linked to scrapie susceptibility.4,15 Homozygosity for Q171 increases susceptibility whereas a survival advantage exists with heterozygosity (e.g. QR171).21,34

The effect of common amino acid polymorphisms at codon 171 (Q171, R171) on

14,21,32 scrapie susceptibility has been investigated. However, little is known about the contribution of lysine (K) other than the presence of naturally occurring scrapie affecting heterozygous K171 sheep in Greek and Italian sheep.1,6 K171 has a relatively higher incidence in some Mediterranean breeds and also occurs in other breeds seen in the U.S. including Barbado, Barbado/St Croix, Dorper and Suffolk crosses.2,17 Non-random sampling of flocks in the U.S. has also identified the polymorphism in American Blackbelly, Black

Hawaiian, Painted Desert, and Texas Dall sheep (D. Sutton and D. Norden, personal communication, unpublished data USDA APHIS, 2017). For regulatory purposes, the current scrapie eradication program in the U.S. considers sheep with Q, H, and K at position 171 equally susceptible to scrapie.3 Therefore, determining the contribution of K171 to scrapie susceptibility could impact breeding programs by allowing retention of otherwise desirable rams.

We previously reported a prolonged incubation period in heterozygous QK171

Barbado sheep intracranially (IC) inoculated with the scrapie agent.16 The purpose of the current study was to compare the relative susceptibility of sheep with PRNP encoding

QQ171, QK171, or KK171 after either IC or oronasal (ON) inoculation with a US-derived scrapie isolate. 40

Materials and Methods

Animal procedures

Animals for this experiment were sourced from a known scrapie-free flock housed at the USDA National Animal Disease Center (NADC) in Ames, IA. Approval from the

Institutional Animal Care and Use Committee was obtained prior to conducting this experiment (protocol number 3893). PRNP genotypes were sequenced by PCR using previously described methods.15 Amino acid sequences of the prion protein were predicted from the genotypes; they were determined to be homozygous at other potentially polymorphic sites M112, G127, A136, M137, S138, L141, R151, R154, M157, N176, H180,

Q189, T195, T196, R211, Q220, and R223. For this study, genotype polymorphisms were documented at codon 171: QQ171, QK171, or KK171. The experimental groups comprised sheep with QK and KK genotypes while the QQ genotype served as a positive control. Sheep in the IC experiment were inoculated at 11 months old except the 2 positive controls (QQ) that were retired breeding ewes (24 and 59 months of age). All lambs in the ON experiment were inoculated at approximately 3 months of age.

Scrapie isolates and animal procedures

A classical scrapie isolate from the continental United States (No. 13-7) was serially passaged four times through ARQ/ARQ Suffolk sheep prior to use as an inoculum in the current experiment.19 Brain tissue was mechanically ground, 100 µg/ml of gentamicin was added, and the final homogenate was prepared as a 10% (w/v) solution with phosphate- buffered saline. Lambs from each genotype group were experimentally infected as described below. 41

The procedure for intracranial inoculation has been described.16,20 Briefly, the sheep were sedated with xylazine and the dorsal frontal region was surgically prepared prior to making a 1 cm midline incision caudal to the junction of the parietal and frontal bones. A 1 mm hole was drilled through the underlying bone. A 22-gauge spinal needle was inserted through the hole to the ventral aspect of the calvarium where 1 ml of inoculum was injected slowly as the needle was withdrawn. The skin incision was closed with tissue glue. Oronasal inoculation was performed as previously described.25 Briefly, the head was elevated slightly, and 1 ml of inoculum was deposited into the right nostril via a needle-less syringe.

All inoculated sheep were housed in biosafety level 2 facilities for two weeks following exposure to scrapie. After this period, the sheep were moved to outside pens at the

National Animal Disease Center. Animals were fed a pelleted growth and alfalfa hay ration.

Sheep were monitored daily for clinical signs consistent with scrapie. Necropsy was performed when unequivocal clinical signs occurred or at the pre-determined end of the study (70 months post-infection).

Sample collection and processing

At necropsy, duplicate tissue samples were collected and stored in 10% buffered neutral formalin (globes in Bouin’s fixative) or frozen. Specifically, tissues were collected from brain, spinal cord, pituitary, trigeminal ganglia, eyes, sciatic nerve, third eyelid, palatine tonsil, pharyngeal tonsil, lymph nodes (mesenteric, retropharyngeal, prescapular and popliteal), spleen, esophagus, forestomaches, intestines, rectal mucosa, thymus, liver, kidney, urinary bladder, pancreas, salivary gland, thyroid gland, adrenal gland, trachea, lung, turbinate, nasal planum, heart, tongue, masseter, diaphragm, triceps brachii, biceps femoris, and psoas major. 42

Microscopic and immunohistochemical evaluation

To study the accumulation of PrPSc in tissue, bright-field microscopy was used in combination with immunohistochemistry. Paraffin-embedded tissues were sectioned at their optimum thickness (brain 4 µm, lymphoid 3 µm, other 5 µm). The presence of PrPSc and the pathologic phenotype (immunolabeling pattern and distribution) were evaluated by application of a cocktail containing two monoclonal antibodies (F89/160.1.5 and F99/97.6.1) each used at a concentration of 5 µg/ml utilizing an automated processor.25 Descriptions of the PrPSc immunolabeling patterns have been published previously.12,31 Hematoxylin and eosin (HE)-stained sections of brain were evaluated for evidence of spongiform change in each animal.

Western blot

To evaluate the molecular characteristics of PrPSc, western blot detection was performed on the frozen sections of brainstem at the level of the obex from representative samples of each genotype and inoculation group. The assay was performed as previously described16 utilizing monoclonal antibody P4 (0.1 µg/ml; R-Biopharm, Inc., Marshall, MI), a biotinylated anti-mouse secondary antibody (0.1 µg/ml; Biotinylated anti-mouse IgG,

Amersham Biosciences, USA, Waltham, MA), and streptavidin horseradish-peroxidase conjugate (dilution 1∶10,000; Streptavidin horseradish-peroxidase conjugate, Amersham

Biosciences, Waltham, MA).16 A chemiluminescent detection system was used to acquire images.

Enzyme immunoassay

A commercially available enzyme immunoassay (HerdChek®, IDEXX Laboratories

Inc., Westbrook, ME) was used to detect the presence of PrPSc in brainstem at the level of the obex, retropharyngeal lymph node and palatine tonsil. Assays were conducted according to kit instructions except that the samples were prepared as a 20% (w/v) tissue homogenate.

Cut-off numbers were determined with a negative control per kit instructions; values greater than the mean optical density of negative controls + 0.180 were considered positive.

Definitions

To analyze experimental data, the following definitions were utilized. The attack rate was defined as the percentage of sheep from an experimental group that had detectable PrPSc in any tissue. Incubation period was defined as the amount of time that elapsed between inoculation and euthanasia at the onset of clinical signs.

Results

Attack rate and incubation period

All of the IC-inoculated sheep developed clinical disease and had detectable PrPSc in the brainstem based on immunohistochemistry (100% attack rate). Differences in the incubation period were associated with route of inoculation and genotype at codon 171.

Among the IC-inoculated sheep, the mean incubation periods for QQ, QK and KK genotypes were 8.8, 26.6 and 49.4 months post-inoculation (MPI), respectively (Table 1; Figure 1).

The attack rates for sheep with QQ, QK and KK genotypes inoculated via the ON route were 100%, 100% and 0%, respectively. The QQ and QK genotypes of ON-inoculated sheep with neurologic signs had mean incubation periods of 21.6 and 52.7 months 44 respectively (Table 1; Figure 2). At the experimental endpoint (70 MPI), none of the sheep with the KK genotype had developed clinical signs.

PrPSc accumulation in intracranially inoculated sheep

To investigate the microscopic changes in the brain, HE-stained sections of brain were evaluated for spongiform change. Spongiform change occurred in the brainstem of all

IC-inoculated sheep. Immunolabeled sections of brain, spinal cord and peripheral tissues were examined for PrPSc accumulation. PrPSc accumulation occurred throughout the central nervous system in all IC-inoculated sheep. Differences were observed in the PrPSc accumulation patterns between genotypes and were most prominent in the frontal cortex

(Figures 3-8) and cerebellum.

In the neocortex of sheep with the KK and QK genotype (Figures 3-4, 6-7), PrPSc was found primarily within the white matter and associated with extracellular accumulation of

PrPSc in glial cells (stellate and perivacuolar immunolabeling types). In sheep with the KK genotype, PrPSc accumulation in neocortical gray matter was characterized by moderately dense aggregated, granular and plaque-like deposits that were most commonly present in the deeper cortical layers (external pyramidal through the polymorphic layer). In contrast, in sheep with the QQ genotype (Figures 5, 8) most of the PrPSc was present in the gray matter with stellate, linear and aggregated deposits particularly in the molecular and internal pyramidal layers. In the gray matter of sheep with the QK genotype, intraneuronal, intraglial and granular immunolabeling types were most prominent in the internal pyramidal layer.

There was also a patchy distribution of linear type immunolabeling throughout the neocortex.

The patterns of PrPSc accumulation in the cerebella of sheep with the QK or KK genotypes were similar to each other and different to those of sheep of the QQ genotype. 45

Similar to what was observed in the neocortex, white matter accumulation of PrPSc was rare in the cerebella of sheep with the QQ genotype and common in sheep with the QK and KK genotypes. Patterns of PrPSc accumulation in the granular and molecular layers were similar across all genotypes with moderate but variable amounts of granular, intraneuronal and intraglial PrPSc in the granular cell layer and scant punctate, granular and stellate deposits within the molecular layer. There was a small amount of intraneuronal PrPSc within Purkinje cells.

Spinal cord sections (cervical, thoracic and lumbar) were immunoreactive for PrPSc in all sheep. PrPSc accumulation occurred primarily in the gray matter with increasing white matter involvement in QK and QQ genotypes. Immunolabeling patterns included intraneuronal, granular and intraglial.

There was limited peripheral accumulation of PrPSc in intracranially inoculated sheep as evidenced by immunohistochemistry. Only 3/7 sheep with the QK genotype had detectable PrPSc within the retropharyngeal lymph nodes (RPLN) compared to 2/2 of the QQ genotype sheep (Table S1). In sheep with the KK genotype, 1/5 sheep accumulated PrPSc in the RPLN; however, immunoreactivity was present only as a few scattered positive cells in a single follicle. A single sheep from the QK group had PrPSc within a lymphoid follicle in the spleen. None of the IC-inoculated sheep had PrPSc within the palatine tonsil, pharyngeal tonsils or the rectal mucosal associated lymphoid tissue by means of IHC; however, one sheep with a QQ genotype had detectable PrPSc by EIA in the palatine tonsil.

PrPSc accumulation in oronasally inoculated sheep

To assess the pathological changes and PrPSc accumulation in sheep inoculated by the

ON route, we evaluated HE-stained and immunolabeled sections of brain, spinal cord and 46 peripheral tissues by light microscopy. At the end of experiment (70 months), all the sheep with the KK genotype were euthanized without developing clinical signs suggestive of scrapie. No immunoreactivity for PrPSc was detected within the CNS of any sheep with the

KK genotype as compared to 2/5 of the sheep with the QK genotype and 5/5 of the QQ

(Table S1). These same animals had spongiform change in their brainstems. Overall, the pathologic phenotype (PrPSc pattern and distribution) was similar (Figures 9-10), but differences in the amount of PrPSc deposition within genotypes (intragenotypic) and between genotypes (intergenotypic) were observed. This was prominently displayed by more intense stellate labeling in the superficial cortex of sheep with the QQ genotype. The intragenotypic difference was observed in sheep with a QQ genotype; there was a positive correlation between incubation period and amount of PrPSc deposition. The intergenotypic differences were most evident in the neocortex. PrPSc deposition was greater in sheep with the QQ genotype compared to the QK genotype despite a longer incubation period for the QK genotype.

There was no evidence of accumulation of PrPSc in any non-CNS tissues from sheep with the KK genotype. In contrast, QK and QQ genotypes had variably distributed PrPSc within the gastrointestinal (myenteric and submucosal ganglionic plexuses), lymphoreticular and peripheral nervous systems (Table 2 and Figures 11-14).

Western blot

Molecular profiles obtained from a western blot of brain homogenates from each sheep were identical to each other and the donor inoculum (Figure 15). Homogenized brain material from the ON-inoculated sheep with a KK genotype was negative for PrPSc.

Discussion

In this study, we assessed the effect of lysine at codon 171 of PRNP on the susceptibility of sheep to the scrapie agent. We demonstrated that, in our challenge model, the KK171 genotype confers resistance to the scrapie agent via ON inoculation and a prolonged incubation period after IC inoculation. Our results also reveal a prolonged incubation period in sheep with a heterozygous PRNP genotype (QK) as compared to the QQ genotype when inoculated via both the IC and ON routes.

We utilized ON inoculation to simulate natural exposure to scrapie and found that sheep with the KK genotype are resistant (0% attack rate) similar to orally inoculated sheep with the ARR/ARR genotype.13,23 The reason for comparable susceptibility between these amino acid polymorphisms, lysine (K171) and arginine (R171), could be attributable to similarities between the functional groups in their side chains. The amino (lysine) and guanidino (arginine) groups are both basic and have similar isoelectric points (9.7 and 10.8).

By comparison, glutamine (Q) has a non-basic nitrogen side chain that contains an amide functional group and has an isoelectric point of 5.7.33

After ON inoculation, prolonged incubation periods occurred in sheep with the heterozygous QK genotype compared to sheep with the QQ171 genotype. Only 2/5 of the sheep with the QK171 genotype developed neurologic signs and accumulated PrPSc in the brain. The remaining 3/5 sheep accumulated PrPSc in peripheral tissues but did not develop neurologic signs before the experimental endpoint at 70 MPI. The mean incubation period in

QK171 genotype sheep with clinical signs (52.7 MPI) was much longer than the mean incubation period observed in sheep with the QQ genotype (21.6 MPI). The presence of PrPSc in peripheral tissues of all ON-inoculated sheep of the QK genotype indicates that they are susceptible to the classical scrapie agent (strain 13-7) despite a prolonged incubation period. 48

These findings suggest that the resistance imparted by the QK171 genotype is similar but weaker than the QR171 genotype. For example, orally inoculated Cheviot lambs with the

ARQ/ARR genotype exhibited prolonged incubation times compared to the ARQ/ARQ genotype.13 This is similar to the difference between ARQ/ARK and ARK/ARK in the present study; however, differences in experimental design involving scrapie strain (13-7 vs.

RBP1) and inoculum volume (0.1 g once vs. 1 g repeated for 5 days) make direct comparisons between these studies difficult. We previously used the No. 13-7 scrapie agent to conduct an oral challenge of neonatal Suffolk lambs of the QR genotype, but there was no evidence of transmission.15 The present study used identical methods with respect to inoculum volume and scrapie strain, but we observed a 100% attack rate in the QK genotype.

This suggests that heterozygous arginine may have a stronger effect on resistance than lysine.

That hypothesis is consistent with recently published investigations of naturally occurring scrapie in herds with relatively high frequencies of ARK haplotypes. The authors reported that K171 was the second most resistant allele after arginine (R>K>H>Q).6

Evaluation of IHC stained tissues from IC-inoculated sheep revealed intergenotypic differences in PrPSc phenotypes (pattern and distribution). White matter immunolabeling was largely present in the KK and QK genotype contrasting a relative absence in sheep with the

QQ genotype. In sheep with the QK genotype, neocortical immunolabeling resembled an amalgamation of the KK and QQ phenotypes. It is unclear to what extent serial passage in

ARQ/ARQ genotype sheep influenced these differences, and it’s possible that a QK genotype-adapted strain would impart a different phenotype. Since PrPSc in scrapie-infected sheep is predominantly composed of the susceptible allelic protein isoform,22,26 we expected that even in IC-inoculated animals the QK group’s pathologic phenotype would more closely 49 resemble the QQ phenotype, because Q171 is more susceptible to conversion. Instead we observed a “combined” phenotype. This finding suggests that a combination of PrPSc containing both K and Q at codon 171 was present. Quantification of the PrPSc allotype composition in these sheep would be useful to further evaluate differential expression of protein isoform fractions between genotype and inoculation groups.

The results of the present study indicate that sheep with a homozygous lysine-171

(KK171) genotype are resistant to scrapie via oronasal exposure in our challenge model, and that sheep with the heterozygous genotype (QK171) have prolonged incubation periods.

These findings may be useful in designing breeding programs to enhance scrapie resistance in breeds of sheep that carry the K171 prion protein allele.

Acknowledgements

We thank Martha Church, Kevin Hassall, Joe Lesan, Leisa Mandell, and Trudy

Tatum for providing technical support to this project. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the Department of Agriculture. The

Department of Agriculture is an equal-opportunity provider and employer.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This research was funded in its entirety by congressionally appropriated funds to the

United States Department of Agriculture, Agricultural Research Service. The funders of the work did not influence study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Figures

Figure 1. A survival plot of Barbado sheep that were intracranially inoculated with the No. 13-7 scrapie agent. The survival curves for the KK, QK and QQ genotypes are illustrated by the short dash, long dash and solid lines, respectively.

Figure 2. A survival plot of Barbado sheep that were oronasally inoculated with the No. 13-7 scrapie agent. The survival curves for the KK, QK and QQ genotypes are illustrated by the short dash, long dash and solid lines, respectively.

Figures 3-8. Scrapie, intracranial inoculation, neocortex, sheep. Immunohistochemistry for PrPSc, monoclonal antibodies F89/160.1.5 and F99/97.6.1. Figure 3 and 6. Sheep with a KK171 genotype have prominent glial-associated stellate immunolabeling in the white matter. In the gray matter, there are aggregated, granular and plaque-like deposits in deeper cortical layers. Figure 4 and 7. Sheep with a QK171 genotype have similar white matter immunolabeling to the KK genotype: stellate and perivacuolar immunolabeling types. The cortex is characterized by intraneuronal, intraglial and granular immunolabeling types predominantly in the internal pyramidal layer. Linear immunolabeling is present within the internal granular and external pyramidal layers. Figure 5 and 8. Sheep with a QQ171 genotype predominantly have gray matter labeling types: stellate, linear and aggregated deposits primarily in the molecular and internal pyramidal layers. In contrast to sheep with a K171 allele, white matter immunolabeling is largely absent.

Figures 9-10. Scrapie, oronasal inoculation, neocortex, sheep. Immunohistochemistry for PrPSc, monoclonal antibodies F89/160.1.5 and F99/97.6.1. Figure 9. A sheep with the QK171 genotype has scattered transcortical stellate (arrowheads) and punctate immunolabeling. There is also punctate, linear (arrow) and mild intraglial immunolabeling in the pyramidal layers. The white matter contains stellate and punctate immunolabeling types. Figure 10. A sheep with the QQ171 genotype contains similar immunolabeling types as the QK genotype; however, stellate immunolabeling is present more broadly affecting the deep cortical layers. Granular accumulations predominate over the punctate type.

Figures 11-14. Scrapie, oronasal inoculation, sheep. Immunohistochemistry for PrPSc, monoclonal antibodies F89/160.1.5 and F99/97.6.1. Figure 11. The pharyngeal tonsil from a sheep with the QK171 genotype contains of PrPSc immunolabeling. There was no detectable PrPSc in the CNS of this sheep. Figure 12. The trigeminal ganglion contains intraneuronal PrPSc from a sheep with the QK171 genotype. Figure 13. A sheep with the QQ171 genotype has detectable PrPSc in the neuromuscular spindle (cross-section) of the masseter muscle. Figure 14. The ileum of a sheep with the QQ171 genotype contains PrPSc within neurons of the myenteric plexus.

Figure 15. Western blot of brain homogenates from scrapie inoculated sheep developed with anti-PrPSc monoclonal antibody P4. Lane key: M, molecular mass marker. Lanes 1-3, intracranial inoculation: KK (1), QK (2), QQ (3). Lanes 4-6, oronasal inoculation: KK (4), QK (5), QQ (6). Lane 7, inoculum scrapie strain 13-7. Lane 8, negative control.

Tables

Table 1. Summary of results by genotype of Barbado sheep inoculated with US scrapie isolate 13-7. Brainstem Immunohistochemistry (PrPSc) Mean Genotype Inoculation Clinical Attack Histopath n IP Immunoblot Brainstem Lymphoida PRNP 171 Route Disease Rate (SE) (MPI) KK 5 IC 5/5 49.4 100% 5/5 + 5/5 1/7 QK 7 IC 7/7 26.6 100% 7/7 + 7/7 3/7 QQ 2 IC 2/2 8.8 100% 2/2 + 2/2 2/2 KK 5 ON 0/5 ND 0% 0/5 - 0/5 0/5 QK 5 ON 2/5 52.7 100% 2/5 + 2/5 5/5 QQ 5 ON 5/5 21.6 100% 5/5 + 5/5 5/5 Abbreviations: IC, intracranial; ON, oronasal; IP, incubation period; MPI, months post-inoculation; SE, spongiform encephalopathy; a

ND, no disease. pharyngeal tonsil + palatine tonsil + retropharyngeal lymph node. 59

Table 2. Non-CNS accumulation of PrPSc in oronasally inoculated Barbado sheep by genotype.a Tissue AA KK AA QK AA QQ

Lymphoid Palatine tonsil - + + Pharyngeal tonsil - + + Retropharyngeal LN - + + Spleen - + + Mesenteric LN - - + Prescapular LN - + + Popliteal LN - + + RAMALT - + +

Gastrointestinal Forestomachs - - + Small Intestines - + +

Nervous System Retina - + + Trigeminal ganglia - + +

Other Adrenal gland - + + Masseter muscle - - + Pituitary gland - + + aDesignated by + if at least 1 animal per genotype group had detectable PrPSc in that tissue.

Supplemental Table 1. Microscopic and enzyme immunoassay findings in different tissues of Barbado sheep inoculated with U.S. classical scrapie isolate 13-7 by intracranial or oronasal routes. Brainstem Immunohistochemistry (PrPSc) Genotype Inoculation Incubation Clinical Exp No. Ear Tag Histopath (SE) EIA Brainstem PHT PAL RPLN PRNP 171 Route Period (MPI) Scrapie A 1 977 KK IC 55.4 Y + + + - - + 2 913 KK IC 44.1 Y + + + n/d - - 3 915 KK IC 46.2 Y + + + - - - 4 919 KK IC 46.2 Y + + + - - - 5 924 KK IC 55.4 Y + + + - - - 6 978 QK IC 27.6 Y + + + - - - 7 918 QK IC 27.4 Y + + + - - - 8 912 QK IC 23.4 Y + + + - - - 9 916 QK IC 24.6 Y + + + - - + 10 925 QK IC 33.3 Y + + + - - - 11 923 QK IC 28.4 Y + n/d + - - + 12 922 QK IC 21.4 Y + n/d + - - +

13 248 QQ IC 6.9 Y + + + - - + 61

14 51 QQ IC 10.7 Y + + + - IS + B 15 1004 KK ON n/a N ------16 1006 KK ON n/a N ------17 1013 KK ON n/a N ------18 1014 KK ON n/a N ------19 1020 KK ON n/a N ------20 1008 QK ON n/a N - - - - - + 21 1005 QK ON n/a N - - - - + + 22 1009 QK ON n/a N - - - + - - 23 1023 QK ON 40.7 Y + + + + + - 24 1007 QK ON 64.7 Y + + + + + + 25 1010 QQ ON 21.9 Y + + + - + + 26 1019 QQ ON 21.4 Y + + + - + + 27 1002 QQ ON 19.8 Y + + + + IS + 28 1012 QQ ON 19.4 Y + + + + + + 29 1021 QQ ON 25.6 Y + + + + + + Abbreviations: IC, intracranial; ON, oronasal; MPI, months post-inoculation; SE, spongiform encephalopathy; EIA, enzyme immunoassay; PHT, pharyngeal tonsil; PAL, palatine tonsil; RPLN, retropharyngeal lymph node; n/a, not applicable in non-clinical animals at experimental end-point (70 MPI); n/d, test not performed; IS, insufficient sample.

SHEEP ARE SUSCEPTIBLE TO THE AGENT OF TME BY

INTRACRANIAL INOCULATION AND HAVE EVIDENCE OF INFECTIVITY IN

LYMPHOID TISSUES

Modified from a manuscript prepared for submission to Frontiers in Veterinary Science

E.D. Cassmanna,b, S.J. Moorea,b, J.D. Smithc, and J.J. Greenleea

aVirus and Prion Research Unit, National Animal Disease Center, Agricultural Research

(USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664. cDepartment of Veterinary Pathology, Iowa State University, Ames, IA, USA

Abstract

Transmissible mink encephalopathy (TME) is a food borne prion disease.

Epidemiological and experimental evidence suggests similarities between the agent of TME and L-BSE. This experiment demonstrates the susceptibility of four different genotypes of sheep to the agent of TME by intracranial inoculation. The four genotypes of sheep used in this experiment had polymorphisms corresponding to codons 136 and 171 of the prion gene:

VV136QQ171, AV136QQ171, AA136QQ171, and AA136QR171. All intracranially inoculated sheep without comorbidities (15/15) developed clinical scrapie and had detectable PrPSc by immunohistochemistry, western blot, and enzyme immunoassay (EIA). The mean incubation periods in TME infected sheep correlated with their relative genotypic susceptibility. There 63 was peripheral distribution of PrPSc in the trigeminal ganglion and neuromuscular spindles; however, unlike classical scrapie and C-BSE in sheep, ovine TME did not accumulate in the lymphoid tissue. To rule out the presence of infectious, but proteinase K susceptible PrPSc, the lymph nodes of two sheep genotypes, VV136QQ171 and AA136QQ171, were bioassayed in transgenic ovinized mice. None of the mice (0/32) inoculated by the intraperitoneal route had detectable PrPSc by EIA. Interestingly, mice intracranially inoculated with RPLN tissue from a VV136QQ171 sheep were EIA positive (3/17) indicating that sheep inoculated with TME harbor infectivity in their lymph nodes. Western blot analysis demonstrated similarities in the migration patterns between ovine TME and the bovine TME inoculum. Overall, these results demonstrate that sheep are susceptible to the agent of TME, and that the tissue distribution of

PrPSc in TME infected sheep is distinct from classical scrapie.

Introduction

Transmissible spongiform encephalopathies (TSE) are infectious fatal neurodegenerative diseases caused by a misfolded form of the prion protein (PrPSc) (1, 2).

There are numerous naturally occurring prion diseases in animals including bovine spongiform encephalopathy (BSE), scrapie in small ruminants, chronic wasting disease

(CWD) in cervids, transmissible mink encephalopathy (TME), Creutzfeldt-Jakob disease

(CJD) in humans, and camel prion disease (3-6). Genetically susceptible animals develop disease spontaneously or after exposure to contaminated feedstuffs, environments, or infected animals.

Despite the infectious nature of PrPSc, there is often a hardy species barrier that precludes cross-species transmission (7). Nevertheless, food-borne prion diseases may infect humans and have economically devastating consequences on trade. Beginning in the mid- 64

1980’s and peaking in 1996, the UK experienced an epizootic of BSE (classical BSE or C-

BSE) that evidenced the zoonotic potential of prion diseases (8). A variant of Creutzfeldt-

Jakob disease (vCJD) was identified in a subset of humans exposed to the agent of C-BSE in contaminated beef products (9-12). Due to the fatal consequences associated with zoonotic prions within the human food supply, substantial investigation has aimed at determining the origin of C-BSE. While the etiology of the original C-BSE case remains uncertain, it has been determined that feedback of BSE infected bovine carcasses likely amplified the outbreak (8, 13).

In addition to C-BSE, atypical forms of BSE (L-type and H-type) occur spontaneously in older cattle at a much lower frequency (4). Atypical BSE types have distinct molecular phenotypes relative to C-BSE that are demonstrable by a lower or higher molecular mass of the unglycosylated PrPSc glycoform (14). One standing theory for the origin of C-BSE is its emergence from atypical BSE and subsequent transmission between cattle. This is supported by the emergence of classical BSE phenotypes after serial transmission of H-BSE strains in wild type mice (15, 16).

Like C-BSE, transmissible mink encephalopathy (TME) is considered to be a foodborne TSE. In a 1985 outbreak of TME in Stetsonville, WI, a TSE-affected downer cow may have been the source of prion disease in infected mink. Subsequent investigations revealed a lack of strain de-adaptation after transmission of TME between cattle and mink, reinforcing the hypothesis of a common origin (5). Furthermore, bovine TME (bTME) is phenotypically similar to L-BSE which also suggests that a downer cow may have been the source of TSE to the mink (17). This scenario supports the theory of naturally occurring interspecies transmission. With the aim of further characterizing the host range of the TME agent, we 65 investigated the susceptibility and disease phenotype of bovine passaged TME in sheep.

Since TME has previously been demonstrated as similar to L-BSE, we also discuss the similarities between ovine TME (oTME) and previously published ovine L-BSE.

Results

Sheep were susceptible to the agent of TME by intracranial inoculation

In order to test the susceptibility of sheep to TME, we intracranially inoculated 17 sheep that had four different PRNP polymorphisms. All experimental sheep in the intracranial inoculation group developed scrapie-like neurologic disease except for two sheep. These two sheep were culled due to intercurrent disease at 10 months post- inoculation. Neither of the culled sheep had detectable PrPSc in the CNS or peripheral tissues.

Excluding animals with intercurrent disease that were removed from the study early, the attack rate was 100% (15/15) and the mean incubation periods were 29, 38, 40, and 52 months for VVQQ, AVQQ, AAQQ, and AAQR genotypes, respectively. A survival plot for the intracranial inoculation group is illustrated in Figure 1.

Intracranially inoculated sheep developed spongiform encephalopathy and had widespread PrPSc accumulation in the brain

Hematoxylin and eosin stained sections of brain were evaluated for spongiform change in each animal. All genotypes of intracranially inoculated sheep had diffuse spongiform encephalopathy except for two sheep that were euthanized at 10 months post- inoculation (MPI) due to intercurrent disease (Table 1).

A cocktail of two antibodies, F89/160.1.5 and F99/97.6.1, directed against PrPSc was used to assess the distribution and immunolabeling types of PrPSc in the brain. There were 66 similar PrPSc immunolabeling profiles in the cerebrum and brainstem of each genotype characterized by particulate, intraglial, stellate, perineuronal and linear types. The sheep with an AAQR genotype varied by having more prominent linear and perineuronal labeling types in the neocortex (Figure 2A). This genotype also contained areas with vascular plaque-like accumulations and coalescing PrPSc (Figure 2B).

The cerebellar PrPSc immunolabeling patterns were similar between all genotypes except AAQR with grey matter particulate and intraglial labeling in the granule cell layer accompanied by particulate, perineuronal and linear in the molecular layer (Figure 2C).

There was minimal white matter PrPSc labeling in the cerebellum. The AAQR genotype had overall less immunolabeling; however, when present, there were patchy areas of intraglial, perineuronal, intraneuronal and particulate labeling patterns (Figure 2D).

The cervical, thoracic, and lumbar spinal cords contained PrPSc in all genotypes.

There was diffuse accumulation of PrPSc in the grey matter which was concentrated primarily in the dorsal horn. Immunolabeling types included particulate, intraglial, intraneuronal, and sometimes perineuronal.

Immunoreactivity was present in the peripheral nervous system but not lymphoid tissues

In order to evaluate peripheral tissue for the presence of PrPSc, we applied PrPSc specific antibodies, F89/160.1.5 and F99/97.6.1, to formalin fixed paraffin embedded tissue sections. We detected PrPSc in peripheral tissues of all four genotypes (Table 2) of sheep.

Various sheep of the VVQQ, AVQQ and AAQQ genotypes had PrPSc within the neuromuscular spindles of the masseter (Figure 3A) and psoas major muscles. Except for the

AAQR genotype, most sheep also had detectable immunolabeling of PrPSc in the trigeminal 67 ganglia (Figure 3B). Accumulation of PrPSc in the trigeminal ganglia and neuromuscular spindles is similar to the distribution of L-BSE in sheep (18). All genotypes of sheep had retinal PrPSc accumulation. A single sheep from the AAQR genotype group had PrPSc labeling in the myenteric plexus of the small intestinal enteric nervous system. Other tissues had variable PrPSc accumulation including the sciatic nerve, adrenal medulla and pituitary gland. Unlike classical scrapie and C-BSE in sheep, there was no immunohistochemical detection of PrPSc in lymphoreticular tissue.

Sheep were not susceptible to the agent of TME by oronasal inoculation

We assessed the susceptibility of sheep to TME agent after oronasal exposure. After incubation periods of 58 to 69 months, none of the oronasally inoculated animals (0/12) had spongiform encephalopathy nor was PrPSc detectable by IHC in brainstem at the level of the obex. The retropharyngeal lymph node, palatine tonsil, pharyngeal tonsil, and small intestines were also negative for PrPSc by IHC. Misfolded prion proteins were not detected in the brainstems nor retropharyngeal lymph nodes by enzyme immunoassay (EIA).

The molecular profiles of brain tissues from sheep or cattle with TME can be distinguished by western blot using P4 antibody

We were able to discriminate oTME from bTME. We analyzed the banding patterns of oTME using two separate antibodies that can discriminate scrapie from C-BSE and L-BSE

(19-21). Both oTME and bTME were immunoreactive for Sha31 (Figure 4). With the P4 antibody, oTME was immunoreactive; however, bTME was not immunoreactive.

In order to further characterize the molecular profile of oTME, we compared it to other relevant prion strains of sheep and cattle using the Sha31 antibody (Figure 5). Different 68

TSE strains vary by their molecular banding pattern (molecular weight) of the diglycosylated, monoglycosylated, and unglycosylated PrPSc fragments (22). All samples for this blot were acetone precipitated, and the sample amounts were adjusted to obtain optimal banding densities. Overall, the banding pattern of oTME resembled the inoculum, third passage bTME (also seen in Figure 4 using non-enriched samples). The molecular profile of

L-BSE was similar to both ovine and cattle passaged TME isolates. Scrapie had a distinct banding pattern compared to the TME and L-BSE samples. We did not observe intergenotypic differences in the molecular properties of oTME.

Enzyme immunoassays for misfolded proteins were positive in samples from the brains of intracranially inoculated sheep

As a rapid screening method for PrPSc, we used EIA to test the brainstem and lymph nodes of all experimental sheep. All sheep in the ON inoculation experiment were negative for PrPSc by EIA of the retropharyngeal lymph nodes and the brainstem at the level of the obex. A single sheep (#816, AVQQ) in the IC inoculation study had low positive O.D.

(0.597) from the retropharyngeal lymph node. It was later determined to be negative by IHC.

Excluding two sheep that died 10 MPI, all sheep (15/15) had positive brainstem samples

(range O.D. 1.739 – 4.00). All positives were confirmed by IHC.

Lymph nodes from TME-infected sheep are infectious.

In order to rule out the possibility of undetectable infectious PrPSc within the retropharyngeal lymph nodes (RPLNs) of sheep with TME (VVQQ and AAQQ genotypes), we performed bioassays in ovinized (Tg338) mice. Four experimental groups of mice were inoculated with RPLN from VVQQ or AAQQ genotype sheep by either the IC or 69 intraperitoneal (IP) route. Three mice IC inoculated with RPLN from sheep #935 (VVQQ) were positive as detected by EIA with an O.D. >4.0. None of the mice (0/18) inoculated with

RPLN homogenate from sheep #808 (AAQQ) and none of the mice (0/32) intraperitoneally inoculated developed clinical signs or had detectable PrPSc.

Discussion

In the context of prion diseases, the purpose of interspecies transmission studies is to determine plausible host ranges and investigate the origins of TSE strains. This study demonstrates the susceptibility of sheep to the agent of TME by intracranial inoculation. Our findings corroborate some similarities between TME and L-BSE. These findings are congruent with previous research implicating L-BSE as the source of TME, at least the TME strain from the 1985 Stetsonville, WI outbreak (5, 17).

The tissue distribution of PrPSc in sheep with TME is similar to L-BSE in sheep.

Sheep intracranially inoculated with L-BSE have accumulation of PrPSc in peripheral ganglia and neuromuscular spindles; however, they lack lymphoreticular PrPSc accumulation (18,

23). Similar to sheep challenged with L-BSE, all genotypes of sheep in the current study, except AAQR sheep, had immunolabeling of peripheral neuromuscular spindles and trigeminal ganglia. None of the sheep with TME had detected PrPSc in lymphoid tissue by

IHC. This observation is discordant to the findings of lymphoreticular accumulation of PrPSc in sheep with C-BSE (24) and scrapie (25). One sheep (#816; AAQQ) had a low positive

EIA result from a RPLN sample. Subsequent evaluation by IHC did not detect PrPSc in that tissue. However, in some TSE strains, including atypical scrapie, peripheral tissues are negative by conventional PrPSc immunodetection, but they harbor infectivity that is demonstrable by mouse bioassay (26). In this study, we examined the infectivity of RPLNs 70 from two sheep genotypes (VVQQ and AAQQ) using a transgenic mouse bioassay. We detected infectivity in the RPLN from sheep #935 (VVQQ). None of the mice inoculated with RPLN homogenate from a sheep with the AAQQ genotype (#808) had a positive EIA result. For further characterization, subsequent passage of PrPSc from these positive bioassay mice is ongoing. Additionally, a bioassay testing infectivity of RPLN from sheep #816 with the AVQQ genotype (low positive EIA result and negative IHC) is currently underway; these results will be reported separately.

The western blot migration patterns of oTME were identical for all sheep genotypes.

We also compared oTME to other TSE strains that affect cattle and sheep. In both the non- enriched and acetone precipitated western blots, oTME resembled the inoculum, bTME. In the acetone precipitated blot, we compared multiple TSE strains of ruminants. The TME strains were similar to L-BSE, and all strains were distinct from classical scrapie in sheep.

We further scrutinized the molecular characteristics of oTME by staining with the N- terminus antibody, P4. Differential binding of P4 near the protease cleavage site has been used to discriminate scrapie from some BSE strains (18-21). Briefly, classical scrapie is immunoreactive with the P4 antibody while L-BSE and C-BSE do not react. Ovine L-BSE is detectable with the P4 antibody, and therefore has a scrapie-like detectability (18). We observed that our inoculum (third pass bTME) was not detectable with the P4 antibody (C-

BSE/L-like), but oTME samples did react with P4 (scrapie-like). The gain of N-terminal antibody binding in oTME but not bTME suggests that host species PrP has some influence on P4 antibody binding.

The susceptibility of sheep to cattle passaged TME is similar to previous work. The transmission of TME directly from mink (Idaho strain) to sheep (intracranially) was 71 previously documented (27); however, since that experiment occurred prior to the advent of

PRNP genotyping techniques and the widespread use of anti-PrPSc IHC detection, it lacked sheep genotype and IHC information. In the present study, all genotypes of sheep were susceptible to IC inoculation, and the incubation period was associated with the genotype.

The duration of the incubation period by genotype (VVQQ < AVQQ < AAQQ < AAQR) was consistent with the susceptibility of sheep to classical scrapie infection (28).

We were unable to produce prion disease in sheep after oronasal inoculation with the

TME agent from cattle. The possibility of oral transmission cannot be unequivocally rejected given the small sample size in our study (12 sheep). Other variables that influence susceptibility include the volume of inoculum and the age of inoculated animals (29, 30). For example, sheep older than 2-3 months old have reduced susceptibility to oral infection with

BSE (31). An age-dependent decrease in susceptibility to prion disease corresponds closely with ileal Peyer’s patch involution in sheep (29). The volume of the inoculum is also a factor.

In this study, we used 0.1 gram (1 ml of 10% w/v solution) of inoculum once, which has effectively transmitted scrapie orally to sheep in previous studies (32-34). Other studies have successfully used different volumes of scrapie inoculum in sheep: 1 gram repeated for 5 days

(35). However, there are other potential natural routes of infection that were not evaluated in the present study. For example, intralingual transmission of TME in hamsters is 100,000- times more efficient than the oral route (36). Based on that research, it is hypothesized that epithelial defects or abrasions in the tongue could predispose livestock to oral prion infection.

Failed oral transmission of TME to sheep is similar to failed oral transmission of L-

BSE to sheep (18). However, concern for interspecies transmission should not be readily dismissed; the lack of transmission to one species may not accurately predict other species 72 barriers. Case-in-point, L-BSE does not orally transmit to sheep, but L-BSE has been orally transmitted to non-human primates (37) and humanized PrP transgenic mice (38, 39). These findings indicate the potential for L-BSE zoonosis. Consequently, there should remain a concern for human and livestock exposure to the agents of L-BSE and TME, which may actually be the same agent. The susceptibility of humanized transgenic mice to oTME is underway and will be reported in a future manuscript.

The results of the present study indicate that sheep are susceptible to the agent of

TME. Unlike classical scrapie in sheep, the agent of TME does not appear in lymphoid tissues. Instead non-lymphoid peripheral accumulation of PrPSc occurs similar to the reported distribution of experimental L-BSE in sheep. In this respect, our findings support a common origin with L-BSE. Future research utilizing a transgenic mouse model would be useful to further discriminate the observed phenotypes.

Materials and Methods

Animal procedures

Experimental animals were sourced from a known scrapie-free flock housed at the

USDA National Animal Disease Center (NADC) in Ames, IA. Approval from the

Institutional Animal Care and Use Committee was procured prior to conducting this experiment (protocol number ARS-2871). PRNP genotypes were sequenced using previously described methods (34). Amino acid sequences of the prion protein were predicted from the genotypes; they were determined to be homozygous at other known polymorphic sites G127,

M137, S138, L141, R151, R154, M157, N176, H180, Q189, T195, T196, R211, Q220, and

R223. Two sheep in the intracranial inoculation group were heterozygous MT112.

Experimental groups were composed of sheep with polymorphisms at codons 136 and 171; 73

there were four genotypes included in this experiment: VV136QQ171 (VVQQ), AV136QQ171

(AVQQ), AA136QQ171 (AAQQ), and AA136QR171 (AAQR).

Inoculum preparation

Due to insufficient availability of the original mink derived TME isolate, a bovine

TME isolate was used for this experiment. In a previous experiment, the agent of transmissible mink encephalopathy was passaged two times in cattle via intracranial inoculation (40). The inoculum for this experiment was passaged a third time and then prepared from brainstem at the level of the obex. The final inoculum was prepared as a 10%

(w/v) homogenate.

Animal inoculation

Experimental inoculation of sheep was performed by two routes: oronasal and intracranial. The number of sheep for each genotype inoculated intracranially was VVQQ, n=5; AVQQ, n=4; AAQQ, n=4; and AAQR, n=4. The procedure for intracranial inoculation was performed as previously described (25, 41, 42) using 1 ml of brain homogenate.

Oronasal inoculation also was performed as previously described and demonstrated to be effective for the transmission of the scrapie agent (32, 33). Briefly, the head was elevated slightly, and 1 ml of the inoculum was deposited into the right nostril via a needle-less syringe. The number of sheep inoculated oronasally for each genotype was VVQQ, n=4;

AVQQ, n=4; and AAQQ, n=4. Oronasally inoculated sheep were 3 months old at the time of inoculation. 74

Following inoculation, all sheep were housed for two weeks in a biosafety level 2 facility. Subsequently, sheep were housed outside in pens at the National Animal Disease

Center where they were fed a pelleted growth and alfalfa hay ration.

The sheep were examined for signs of clinical scrapie on a daily basis. When unequivocal neurologic signs were noted, spontaneous intercurrent death occurred, or the experimental endpoint was reached, necropsy was performed on each animal.

Sample collection

The procedure for collection of samples was performed similar to other experiments

(42). At necropsy, duplicate tissue samples were collected and stored in 10% buffered neutral formalin (globes in Bouin’s fixative) or frozen. Tissues collected included brain, spinal cord, pituitary, trigeminal ganglia, eyes, sciatic nerve, third eyelid, palatine tonsil, pharyngeal tonsil, lymph nodes (mesenteric, retropharyngeal, prescapular and popliteal), spleen, esophagus, forestomaches, intestines, rectal mucosa, thymus, liver, kidney, urinary bladder, pancreas, salivary gland, thyroid gland, adrenal gland, trachea, lung, turbinate, nasal planum, heart, tongue, and muscles (masseter, diaphragm, triceps brachii, biceps femoris, and psoas major).

Microscopic and immunohistochemical evaluation

For the evaluation of spongiform change, hematoxylin and eosin (HE) stained sections of cerebrum, brainstem and cerebellum from corresponding levels at the forebrain, basal nuclei, thalamus, midbrain, pons, medulla oblongata and obex were assessed by brightfield microscopy and scored as positive or negative. 75

To study the accumulation of PrPSc in tissue, bright-field microscopy was used in combination with immunohistochemistry (IHC) similar to previously described methods (25,

41-43). Briefly, paraffin-embedded tissues were sectioned at their optimum thickness (brain

4 µm, lymphoid 3 µm, other 5 µm). The presence of PrPSc and the pathologic phenotype

(immunolabeling pattern and distribution) were evaluated by application of a cocktail containing two monoclonal antibodies (F89/160.1.5 and F99/97.6.1) each used at a concentration of 5 µg/ml utilizing an automated processor (Ventana Nexes, Ventana Medical

Systems, Inc., Tucson, AZ). Immunohistochemical labeling patterns for PrPSc were evaluated. Explanations of the immunolabeling patterns of PrPSc have been published previously in detail (44-46).

Western blot

We evaluated the molecular characteristics of PrPSc using western blot detection on frozen sections of brain. Representative samples of each genotype and inoculation group were assessed and compared to selected TSEs of interest. Acetone precipitation was used for the blot comparing multiple TSE strains. The tissue volume equivalent (mg eq) loaded for scrapie, ovine TME, bovine TME, and L-BSE was 0.3 mg, 1.5 mg, 1.5 mg, and 1.5 mg, respectively. Acetone precipitation was performed as follows. Proteinase K treated brain homogenates were mixed with four volumes of -20 °C acetone and incubated 1.5 hours at

−20 °C. The samples were pelleted (15,000 rcf for 10 min at 10 °C). The supernatant was discarded, and the pellet was resuspended in 1x LDS loading buffer containing 1 μL of β- mercaptoethanol. Protein gel electrophoresis and subsequent immunoblotting procedures were performed as previously described (41) utilizing murine monoclonal antibodies Sha31

(diluted to a final concentration of 0.15 µg/ml; Cayman Chemical Company, Ann Arbor, MI) 76 or P4 (diluted to a final concentration of 0.1 µg/ml; R-Biopharm, Inc., Marshall, MI), a biotinylated anti-mouse secondary antibody (diluted to a final concentration of 0.1 µg/ml;

Biotinylated anti-mouse IgG, Amersham Biosciences, USA), and streptavidin conjugated to horseradish-peroxidase (diluted to a final concentration of 0.1 µg/ml; Streptavidin horseradish-peroxidase conjugate, Amersham Biosciences, USA). For detection and image acquisition, a chemiluminescent and chemifluorescent (HRP) substrate (Pierce ECL-Plus,

ThermoFisher Scientific, Waltham, MA) was used in conjunction with a gel imaging system

(G:BOX Chemi-XT4, Syngene, Frederick, MD).

Enzyme immunoassay

A commercially available enzyme immunoassay (HerdChek®, IDEXX Laboratories

Inc., Westbrook, ME) was used to screen for the presence of PrPSc in brainstem at the level of the obex and the retropharyngeal lymph node. Assays were conducted according to kit instructions except that the samples were prepared as a 20% (w/v) tissue homogenate. Cut- off numbers were determined with a negative control per kit instructions; values greater than the mean optical density (O.D.) of negative controls + 0.180 were considered positive for the purposes of screening samples.

Transgenic mouse bioassays

In order to rule out the possibility of undetectable infectious PrP within peripheral lymphoid tissue, we performed a bioassay with transgenic mice expressing ovine PRNP

(Tg338). The RPLN of sheep with VVQQ or AAQQ genotypes were included in the bioassay (sheep 935 and 808, respectively). Thirty-two ovinized PRNP transgenic mice

(Tg338) (47) were inoculated IP with 100 l of 1% w/v RPLN homogenate: eighteen with 77

VVQQ inoculum and fourteen with AAQQ inoculum. Separate groups of mice were also IC inoculated: seventeen with VVQQ inoculum and eighteen with AAQQ inoculum. Mice were monitored daily for the appearance of clinical signs. At the time of death or at the end of study, brains were tested for PrPSc by EIA. The attack rate was defined as the percentage of mice from an experimental group that had a positive EIA result. Incubation periods (IPs) were expressed as the mean number of days post-inoculation (days PI) for mice with positive

EIA results. To determine the attack rate, mice dying from intercurrent disease or without clinical signs that survived 2 SD less than the mean IP of positive mice were excluded.

Survival analyses

Survival analyses for all experiments were performed using Graphpad Prism 7

(GraphPad Software, San Diego, CA). Animals were censored only if they were negative and died from intercurrent disease before the experimental end-point.

Definitions

To analyze experimental data, the following definitions were utilized in agreement with previous reports (42). In the sheep experiment, the attack rate was defined as the percentage of animals from an experimental group that had detectable PrPSc in any tissue.

Incubation period (IP) was defined as the amount of time that elapsed between inoculation and euthanasia at the onset of clinical signs.

Acknowledgements

We thank Martha Church, Kevin Hassall, Joe Lesan, Leisa Mandell, Rylie Frese, and

Trudy Tatum for providing technical support to this project. We thank Hubert Laude for 78 providing the transgenic Tg338 mice used for bioassay. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the Department of Agriculture. The

Department of Agriculture is an equal-opportunity provider and employer.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This research was funded in its entirety by congressionally appropriated funds to the

This research was supported in part by an appointment to the Agricultural Research

Service (ARS) Research Participation Program administered by the Oak Ridge Institute for

Science and Education (ORISE) through an interagency agreement between the U.S.

Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664. All opinions expressed in this paper are the author's and do not necessarily reflect the policies and views of USDA,

ARS, DOE, or ORAU/ORISE.

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Figures

Figure 1. Survival plot of sheep inoculated intracranially with the agent of TME. The shortest survival time was observed in sheep with the VVQQ genotype indicated by a solid black line.

Figure 2 A-D. Photomicrographs of brain regions from sheep that were inoculated intracranially with the agent of TME. Immunohistochemistry for PrPSc, monoclonal antibodies F89/160.1.5 and F99/97.6.1. A, PrPSc in the frontal neocortex from a sheep with an AAQR genotype. In this genotype there is diffuse particulate that is denser in neocortical layers IV and V. There are also prominent linear (arrow) and perineuronal (arrowhead) immunolabeling patterns. B, In the cerebrum from sheep with the AAQR genotype, there are vascular plaque-like accumulations (arrow) of PrPSc. The arrowhead indicates an area of coalescing particulate immunolabeling. C, Cerebellum from a sheep with an AVQQ genotype. In the granular layer there is particulate and intraglial immunolabeling, and in this image, linear type PrPSc (arrow) is evident in the molecular layer. The immunolabeling types present are typical of the VVQQ, AVQQ and AAQQ genotypes. D, Cerebellum from a sheep with the AAQR genotype. Overall there is less PrPSc immunolabeling. In the selected image there is primarily diffuse particulate with intermittent intraglial (arrow) PrPSc.

Figure 3 A-B. Photomicrographs of PrPSc in peripheral tissues from sheep that were inoculated intracranially with the agent of TME. Immunohistochemistry for PrPSc, monoclonal antibodies F89/160.1.5 and F99/97.6.1. A, There is immunolabeling of PrPSc within the neuromuscular spindles of the masseter muscle of sheep #962 (VVQQ). B, There is intraneuronal immunolabeling of PrPSc in the trigeminal ganglion of sheep #834 (AVQQ). 86

Figure 4. Western blots of brain samples from sheep that were inoculated intracranially with the bovine passaged TME agent (bTME). The blots were immunolabeled with either the Sha31 (top) or P4 (bottom) antibody. Sha31, The antibody Sha31 was used on brain homogenates from sheep with the AVQQ genotype. The banding patterns are similar to the inoculum (bTME). P4, The antibody P4 is immunoreactive with brain homogenates from sheep with the AAQQ genotype. The inoculum, bTME, does not bind with the P4 antibody.

Figure 5. Western blot comparing ovine TME (oTME) to other ruminant TSE types. The blot was immunolabeled with Sha31 anti-PrP antibody after acetone precipitation. Ovine TME is similar to 3rd passage bTME which resembles L-BSE. Scrapie has a distinct migration pattern.

Tables

Table 1. Experimental findings in sheep intracranially inoculated with TME agent.

Sc Immunohistochemistry (PrP ) Brainstem Incubation Peripheral Ear Genotype Trigeminal Period Brainstem PHT PAL RPLN RAMALT Muscle ENS SE EIA Tag PRNP Ganglia (MPI) NMS 962 VVQQ 32 + + - - - - M - + + 935 VVQQ 27 + + - - - - P - + + 811 VVQQ 32 + + - - - - M - + + 945 VVQQ 10a - NA ------957 VVQQ 24 + + ------+ NA 816 AVQQ 28 + + ------+ + 818 AVQQ 61 + ------+ + 88 834 AVQQ 33 + + - - - - M - + + 841 AVQQ 31 + NA ------+ + 808 AAQQ 26 + NA - - - NA NA - + + 815 AAQQ 53 + + ------+ + 835 AAQQ 55 + + - - - - P + + + 936 AAQQ 26 + NA ------+ + 833 AAQR 50 + ------+ + 951 AAQR 51 + ------+ + 955 AAQR 10a ------970 AAQR 56 + ------+ + PNRP = prion protein gene; MPI = months post-inoculation; PHT = pharyngeal tonsil; PAL = palatine tonsil; RPLN = retropharyngeal lymph node; RAMALT = Recto-anal mucosa associated lymphoid tissue; NMS = neuromuscular spindle; ENS = enteric nervous system; SE = spongiform encephalopathy; EIA = enzyme immunoassay; IC = intracranial; NA = not available; M = masseter; P = psoas major a Culled or died from intercurrent disease

Table 2. Immunolabeling of PrPSc in tissue organized by genotype of intracranially inoculated Suffolk sheep.

VVQQ AVQQ AAQQ AAQR #Positive / n #Positive / n #Positive / n #Positive / n Total number of sheep 4/5a 4/4 4/4 3/4a Excluding intercurrent disease 4/4 4/4 4/4 3/3 Mean IP of negative sheep (days) 10 10 Mean IP of positive sheep (days) 29 38 40 52 Tissue Muscle Masseter + + 0 0 Psoas major + 0 + 0

Nervous: Non-CNS Retina + + + + Trigeminal ganglia + + + 0 Sciatic nerve + 0 0 0 Adrenal medulla + + 0 0 Myenteric plexus (SI) 0 0 + 0 CNS Cerebrum + + + + Cerebellum + + + + Brainstem + + + + Spinal cord + + + + Lymphoreticular 0 0 0 0 Other Pituitary gland + + + + a Sheep culled or died from intercurrent disease; IP = incubation period; SI = small intestine + = Positive; 0 = Negative 90

GENERAL CONCLUSIONS

General Discussion

There are practical outcomes of TSE research. First, the origins of naturally occurring prion diseases can be studied by interspecies transmission experiments. Second, experimental

TSE research is important to evaluating genetic contributions to prion disease susceptibility.

In sheep there are numerous polymorphisms in the prion protein gene (PRNP). The work presented in the first half of this dissertation evaluates the susceptibility of sheep with a

Q171K PRNP polymorphism to infection with classical scrapie. Previous research indicated that sheep with a QK171 genotype have a prolonged incubation period after intracranial inoculation [1]. We hypothesized that sheep with the K171 genotype would be relatively resistant to classical scrapie after a more natural route of inoculation. The results of this study demonstrate that sheep with the homozygous KK171 genotype are resistant to classical scrapie when inoculated with the same parameters that produced disease in less susceptible sheep genotypes (QK171 and QQ171). Our findings agree with recently published epidemiologic data that describes a low prevalence of natural scrapie in Greek dairy sheep with the QK171 haplotype and no cases in homozygous KK171 [2]. The results of our study have immediate effects on scrapie eradication programs worldwide and domestically. An increased prevalence of the K171 allele is documented in some flocks from Italy and Greece

[2, 3], and there is a relatively higher incidence of K171 in some Mediterranean sheep breeds. The overall prevalence of the K171 allele in the United States is not known, but non- random sampling on some farms indicates the K171 genotype is present in American

Blackbelly, Black Hawaiian, Painted Desert, and Texas Dall sheep [D. Sutton and D.

Norden, personal communication, unpublished data USDA APHIS, 2017]. Currently, the 91

United States scrapie eradication program uses selective breeding of genetically resistant genotypes to increase scrapie resistance at the individual and herd level. The documentation of KK171 resistance in the present research could be invaluable to a small producer with this genotype identified within their flock.

Interspecies transmission experiments are critical for understanding the etiology and host ranges of naturally occurring TSEs. The second half of this work demonstrated that sheep are susceptible to the agent of transmissible mink encephalopathy via intracranial inoculation. Sheep were not susceptible to oronasal inoculation at the inoculation doses received. Given that sheep are permissible to infection via intracranial inoculation, it is possible that higher doses of infectious material or alternate inoculation routes, such as intralingual, could result in successful transmission. The purpose of intracranial inoculation is twofold: giving us a positive control and demonstrating the capability of host PrPSc amplification. Collectively, the absence of oral transmission and positive intracranial transmission demonstrates a high species barrier; sheep are relatively resistant to the TME agent.

Characterization of the disease phenotype in sheep with TME revealed at least two critical points. First, TME in sheep is distinct from classical scrapie in sheep. This signifies that scrapie is not likely to be the source of TME, at least not the Stetsonville, WI outbreak from where our TME sample was obtained. Second, if a case of TME in sheep occurred naturally, differentiation from scrapie via molecular glycosylation patterns and peripheral

PrPSc distribution would be possible. In this experiment, we did not have material from L-

BSE infected sheep to compare directly to ovine TME; however, we observed that the peripheral distribution of PrPSc in our sheep was similar to published work on sheep with L- 92

BSE [4]. This is interesting because research has previously indicated a similar phenotype between L-BSE and TME [5]. As originally proposed by Marsh et al. [6], the present research supports atypical BSE as the origin of TME in Stetsonville, WI as opposed to sheep scrapie. Interestingly, at the time Marsh made this observation, atypical BSE had not yet been described.

Future Directions

Future directions for this work include further testing the host ranges of TME and directly comparing TME strains to L-BSE in cattle. This work is ideally performed in a mouse model for two reasons. First, the incubation period of TSEs in mice is faster than livestock species. Secondly, the comparison of spongiform changes in mice permits the differentiation of TSE strains [7]. This research would give further credibility to the theory that TME originated from a cow with L-BSE. If we assume that this premise is true, the comparison of multiple interspecies transmissions of the TME agent is analogous to testing the permissiveness of L-BSE to cross species barriers.

References

1. Greenlee, J.J., et al., Prolonged incubation time in sheep with prion protein containing lysine at position 171. J Vet Diagn Invest, 2012. 24(3): p. 554-8.

2. Boukouvala, E., et al., The association between 171 K polymorphism and resistance against scrapie affection in Greek dairy sheep. Small Ruminant Research, 2018. 161: p. 51-56.

3. Acutis, P.L., et al., Low frequency of the scrapie resistance-associated allele and presence of lysine-171 allele of the prion protein gene in Italian Biellese ovine breed. J Gen Virol, 2004. 85(10): p. 3165-72.

4. Simmons, M.M., et al., L-BSE experimentally transmitted to sheep presents as a unique disease phenotype. Vet Res, 2016. 47(1): p. 112. 93

5. Baron, T., et al., Phenotypic similarity of transmissible mink encephalopathy in cattle and L-type bovine spongiform encephalopathy in a mouse model. Emerg Infect Dis, 2007. 13(12): p. 1887-94.

6. Marsh, R.F., et al., Epidemiological and experimental studies on a new incident of transmissible mink encephalopathy. J Gen Virol, 1991. 72 ( Pt 3): p. 589-94.

7. Fraser, H. and A.G. Dickinson, Scrapie in mice. Agent-strain differences in the distribution and intensity of grey matter vacuolation. J Comp Pathol, 1973. 83(1): p. 29-40.