Katherine I. O'Rourke

A prion protein epitope selective for the pathologically misfolded conformation

Conformational conversion of proteins in disease is likely to be accompanied by molecular surface exposure of previously sequestered amino-acid side chains. We found that induction of β-sheet structures in recombinant prion proteins is associated with increased solvent accessibility of tyrosine. Antibodies directed against the prion protein repeat motif, tyrosine-tyrosine-arginine, recognize the pathological isoform of the prion protein but not the normal cellular isoform, as assessed by immunoprecipitation, plate capture immunoassay and flow cytometry. Antibody binding to the pathological epitope is saturable and specific, and can be created in vitro by partial denaturation of normal brain prion protein. Conformation-selective exposure of Tyr-Tyr-Arg provides a probe for the distribution and structure of pathologically misfolded prion protein, and may lead to new diagnostics and therapeutics for prion diseases.

PRP GENOTYPES AND EXPERIMENTAL SCRAPIE IN ORALLY INOCULATED SUFFOLK SHEEP IN THE UNITED STATES

One-hundred and three Unites States Suffolk sheep were inoculated orally with a scrapie agent preparation and monitored for clinical disease and histopathological lesions characteristic of scrapie. A retrospective study of the polymorphisms at codon 171 of the prion protein (PrP) gene was performed on these sheep. All 63 sheep that developed scrapie during the observation period were homozygous for the glutamine 171 (171-QQ) PrP allele. Twelve 171-QQ sheep failed to develop disease. All 5 sheep homozygous for arginine (171-RR) and all 23 heterozygous (171-QR) sheep remained free of scrapie.

Validation of monoclonal antibody F99/97.6.1 for immunohistochemical staining of brain and tonsil in mule deer ( Odocoileus hemionus ) with chronic wasting disease

A new monoclonal antibody (MAb), F99/97.6.1, that has been used to demonstrate scrapie-associated prion protein PrPsc in brain and lymphoid tissues of domestic sheep with scrapie was used in an immunohistochemistry assay for diagnosis of chronic wasting disease (CWD) in mule deer (Odocoileus hemionus). The MAb F99/97.6.1 immunohistochemistry assay was evaluated in brain and tonsil tissue from 100 mule deer that had spongiform encephalopathy compatible with CWD and from 1,050 mule deer outside the CWD-endemic area. This MAb demonstrated abnormal protease-resistant prion protein (PrPres) in brains of all of the 100 mule deer and in 99 of the 100 tonsil samples. No immunostaining was seen in samples collected from deer outside the endemic area. MAb F99/97.6.1 demonstrated excellent properties for detection of PrPres in fresh, frozen, or mildly to moderately autolytic samples of brain and tonsil. This immunohistochemistry assay is a sensitive, specific, readily standardized diagnostic test for CWD in deer.

Chronic Wasting Disease Prions in Elk Antler Velvet

Residue 226 of cervid prion proteins may be a determinant of CWD pathogenesis.

Preliminary Findings on the Experimental Transmission of Chronic Wasting Disease Agent of Mule Deer to Cattle

To determine the transmissibility of chronic wasting disease (CWD) to cattle and to provide information about clinical course, lesions, and suitability of currently used diagnostic procedures for detection of CWD in cattle, 13 calves were inoculated intracerebrally with brain suspension from mule deer naturally affected with CWD. Between 24 and 27 months postinoculation, 3 animals became recumbent and were euthanized. Gross necropsies revealed emaciation in 2 animals and a large pulmonary abscess in the third. Brains were examined for protease-resistant prion protein (PrPres) by immunohistochemistry and Western blotting and for scrapie-associated fibrils (SAFs) by negative-stain electron microscopy. Microscopic lesions in the brain were subtle in 2 animals and absent in the third case. However, all 3 animals were positive for PrPres by immunohistochemistry and Western blot, and SAFs were detected in 2 of the animals. An uninoculated control animal euthanized during the same period did not have PrPres in its brain. These are preliminary observations from a currently in-progress experiment. Three years after the CWD challenge, the 10 remaining inoculated cattle are alive and apparently healthy. These preliminary findings demonstrate that diagnostic techniques currently used for bovine spongiform encephalopathy (BSE) surveillance would also detect CWD in cattle should it occur naturally.

Antiviral, Anti-glycoprotein and Neutralizing Antibodies in Foals with Equine Infectious Anaemia Virus

Equine infectious anaemia virus is related by genome sequence homology to human immunodeficiency virus, caprine arthritis-encephalitis virus and visna virus. Failure of the host to mount a strong neutralizing response detectable in vitro or to eliminate persistent infection in vivo characterizes lentivirus infections in the natural host. In this study the specificities and neutralizing activity of antibodies induced during experimental infection with equine infectious anaemia virus were investigated using antiviral ELISA, radioimmunoprecipitation and neutralization assays. ELISA antibody titres of 10(5) to 10(6) were demonstrated in samples collected 30 and 60 days after infection. Immunoprecipitation titrations demonstrated that antibody titres to the glycoproteins gp90 and gp45 were 10 to 100 times higher than titres to the internal structural protein, p24. Low levels of neutralizing antibody appeared at 23 to 46 days post-infection. The presence of low levels of neutralizing activity in the presence of high levels of anti-glycoprotein activity suggests that the major immunogenic sites on the viral surface are not sensitive to neutralization.

Preliminary Observations of Genetic Susceptibility of elk (Cervus Elaphus Nelsoni) to Chronic Wasting Disease by Experimental Oral Inoculation

To compare the genetic susceptibility of elk (Cervus elaphus nelsoni) with various alleles of the PRNP gene, which encodes the normal cellular prion protein, to chronic wasting disease (CWD), eight 8-month-old elk calves of 3 genotypes (2 132MM, 2 132LM, and 4 132LL) were orally dosed with CWD-infected brain material from elk. During postinoculation (PI) month 23, both 132MM elk had lost appetite, developed clinical signs of weight loss and central nervous system (CNS) dysfunction, and were euthanized. Two other elk (both 132LM) developed similar clinical signs of disease and were euthanized during PI month 40. All 4 affected elk had microscopic lesions of spongiform encephalopathy (SE), and PrPres, the disease-associated form of the prion protein, was detected in their CNS and lymphoid tissues by use of immunohistochemical (IHC) and Western blot (WB) techniques. These findings indicate that elk with MM and LM at codon 132 are susceptible to orally inoculated CWD. All 4 LL elk are alive at PI year 4 and are clinically normal, which suggests that 132LL elk may have reduced susceptibility to oral infection with CWD-infected material or may have prolonged incubation time.

Experimental Transmission of Chronic Wasting Disease Agent from Mule Deer to Cattle by the Intracerebral Route

This communication reports final observations on experimental transmission of chronic wasting disease (CWD) from mule deer to cattle by the intracerebral route. Thirteen calves were inoculated intracerebrally with brain suspension from mule deer naturally affected with CWD. Three other calves were kept as uninoculated controls. The experiment was terminated 6 years after inoculation. During that time, abnormal prion protein (PrPres) was demonstrated in the central nervous system (CNS) of 5 cattle by both immunohistochemistry and Western blot. However, microscopic lesions suggestive of spongiform encephalopathy (SE) in the brains of these PrPres-positive animals were subtle in 3 cases and absent in 2 cases. Analysis of the gene encoding bovine PRNP revealed homozygosity for alleles encoding 6 octapeptide repeats, serine (S) at codon 46, and S at codon 146 in all samples. Findings of this study show that although PrPres amplification occurred after direct inoculation into the brain, none of the affected animals had classic histopathologic lesions of SE. Furthermore, only 38% of the inoculated cattle demonstrated amplification of PrPres. Although intracerebral inoculation is an unnatural route of exposure, this experiment shows that CWD transmission in cattle could have long incubation periods (up to 5 years). This finding suggests that oral exposure of cattle to CWD agent, a more natural potential route of exposure, would require not only a much larger dose of inoculum but also may not result in amplification of PrPres within CNS tissues during the normal lifespan of cattle.

Preclinical diagnosis of chronic wasting disease in captive mule deer (Odocoileus hemionus) and white-tailed deer (Odocoileus virginianus) using tonsillar biopsy.

The usefulness of tonsillar biopsy on live deer for preclinical diagnosis of the transmissible spongiform encephalopathy chronic wasting disease (CWD) was evaluated. Disease was tracked in a CWD-endemic herd using serial tonsillar biopsies collected at 6 to 9 month intervals from 34 captive mule deer (Odocoileus hemionus) and five white-tailed deer (O. virginianus). Tonsillar biopsies were examined for accumulation of PrP(CWD), the protein marker for infection, using immunohistochemical (IHC) staining. 26/34 (76%) mule deer and 4/5 (80%) white-tailed deer had PrP(CWD) accumulation in tonsillar biopsies; CWD was subsequently confirmed by post-mortem examination in all 30 of these tonsillar-positive deer. Six mule deer with IHC-negative tonsillar biopsies had positive brain and tonsillar IHC staining upon death 12 to 40 months following the last biopsy. PrP(CWD) accumulation in tonsillar biopsy was observed 2 to 20 months before CWD-related death and up to 14 months before onset of clinical signs of CWD. Tonsillar biopsies from 3-month-old mule deer (n=6) were IHC negative, but PrP(CWD) accumulation was detected in tonsillar biopsies from 7/10 mule deer by 19 months of age. Tonsillar biopsy evaluated with IHC staining is a useful technique for the preclinical diagnosis of CWD in live mule deer and white-tailed deer when intensive management approaches are possible.

Pregnancy status and fetal prion genetics determine PrPSc accumulation in placentomes of scrapie-infected sheep.

Ovine scrapie is a fatal neurodegenerative disorder that may be transmitted through exposure to infected uterine and placental tissues. Susceptibility to scrapie is primarily controlled by polymorphisms in the prion protein (PrP) gene. Scrapie in the U.S. Suffolk breed and in many breeds in Europe occurs in sheep homozygous for glutamine (171QQ), but rarely in sheep heterozygous for glutamine and arginine (171QR) or homozygous for arginine (171RR) at codon 171 of the PrP gene. This study demonstrated that accumulation of PrPSc in uterine-placental epithelial cells in the placentome was determined by fetal PrP genotype and the pregnancy status of scrapie-infected ewes. PrPSc was detected in 171QQ placentomes of infected ewes, but not in placentomes of infected ewes pregnant with 171QR conceptuses or in the non-pregnant uterus of infected ewes. The distribution of PrPSc plaques in placentomes was temporally associated with stage of gestation. There was a tendency toward increased size and number of placentomal PrPSc plaques from the endometrial stalk (maternal side) to chorionic plate (fetal side). These results indicate that accumulation of PrPSc is eliminated or reduced to undetectable levels in reproductive and placental tissues if infected ewes are not pregnant or conceive conceptuses with a resistant PrP genotype.