Jeanette Hayes-Klug

Infectious prions in pre-clinical deer and transmission of chronic wasting disease solely by environmental exposure.

Key to understanding the epidemiology and pathogenesis of prion diseases, including chronic wasting disease (CWD) of cervids, is determining the mode of transmission from one individual to another. We have previously reported that saliva and blood from CWD-infected deer contain sufficient infectious prions to transmit disease upon passage into naïve deer. Here we again use bioassays in deer to show that blood and saliva of pre-symptomatic deer contain infectious prions capable of infecting naïve deer and that naïve deer exposed only to environmental fomites from the suites of CWD-infected deer acquired CWD infection after a period of 15 months post initial exposure. These results help to further explain the basis for the facile transmission of CWD, highlight the complexities associated with CWD transmission among cervids in their natural environment, emphasize the potential utility of blood-based testing to detect pre-clinical CWD infection, and could augur similar transmission dynamics in other prion infections.

B Cells and Platelets Harbor Prion Infectivity in the Blood of Deer Infected with Chronic Wasting Disease

ABSTRACT Substantial evidence for prion transmission via blood transfusion exists for many transmissible spongiform encephalopathy (TSE) diseases. Determining which cell phenotype(s) is responsible for trafficking infectivity has important implications for our understanding of the dissemination of prions, as well as their detection and elimination from blood products. We used bioassay studies of native white-tailed deer and transgenic cervidized mice to determine (i) if chronic wasting disease (CWD) blood infectivity is associated with the cellular versus the cell-free/plasma fraction of blood and (ii) in particular if B-cell (MAb 2-104+), platelet (CD41/61+), or CD14+ monocyte blood cell phenotypes harbor infectious prions. All four deer transfused with the blood mononuclear cell fraction from CWD+ donor deer became PrPCWD positive by 19 months postinoculation, whereas none of the four deer inoculated with cell-free plasma from the same source developed prion infection. All four of the deer injected with B cells and three of four deer receiving platelets from CWD+ donor deer became PrPCWD positive in as little as 6 months postinoculation, whereas none of the four deer receiving blood CD14+ monocytes developed evidence of CWD infection (immunohistochemistry and Western blot analysis) after 19 months of observation. Results of the Tg(CerPrP) mouse bioassays mirrored those of the native cervid host. These results indicate that CWD blood infectivity is cell associated and suggest a significant role for B cells and platelets in trafficking CWD infectivity in vivo and support earlier tissue-based studies associating putative follicular B cells with PrPCWD. Localization of CWD infectivity with leukocyte subpopulations may aid in enhancing the sensitivity of blood-based diagnostic assays for CWD and other TSEs.

Aerosol Transmission of Chronic Wasting Disease in White-Tailed Deer

ABSTRACT While the facile transmission of chronic wasting disease (CWD) remains incompletely elucidated, studies in rodents suggest that exposure of the respiratory mucosa may be an efficient pathway. The present study was designed to address this question in the native cervid host. Here, we demonstrate aerosol transmission of CWD to deer with a prion dose >20-fold lower than that used in previous oral inoculations. Inhalation of prions may facilitate transmission of CWD and, perhaps, other prion infections.

Mucosal immunization with an attenuated Salmonella vaccine partially protects white-tailed deer from chronic wasting disease

Prion disease is a unique category of illness, affecting both animals and humans, in which the underlying pathogenesis is related to a conformational change of a normal, self-protein called PrP(C) (C for cellular) to a pathological and infectious conformer known as PrP(Sc) (Sc for scrapie). Bovine spongiform encephalopathy (BSE), a prion disease believed to have arisen from feeding cattle with prion contaminated meat and bone meal products, crossed the species barrier to infect humans. Chronic wasting disease (CWD) infects large numbers of deer and elk, with the potential to infect humans. Currently no prionosis has an effective treatment. Previously, we have demonstrated we could prevent transmission of prions in a proportion of susceptible mice with a mucosal vaccine. In the current study, white-tailed deer were orally inoculated with attenuated Salmonella expressing PrP, while control deer were orally inoculated with vehicle attenuated Salmonella. Once a mucosal response was established, the vaccinated animals were boosted orally and locally by application of polymerized recombinant PrP onto the tonsils and rectal mucosa. The vaccinated and control animals were then challenged orally with CWD-infected brain homogenate. Three years post CWD oral challenge all control deer developed clinical CWD (median survival 602 days), while among the vaccinated there was a significant prolongation of the incubation period (median survival 909 days; p=0.012 by Weibull regression analysis) and one deer has remained CWD free both clinically and by RAMALT and tonsil biopsies. This negative vaccinate has the highest titers of IgA in saliva and systemic IgG against PrP. Western blots showed that immunoglobulins from this vaccinate react to PrP(CWD). We document the first partially successful vaccination for a prion disease in a species naturally at risk.

Mother to Offspring Transmission of Chronic Wasting Disease in Reeves’ Muntjac Deer

The horizontal transmission of prion diseases has been well characterized in bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD) of deer and elk and scrapie of sheep, and has been regarded as the primary mode of transmission. Few studies have monitored the possibility of vertical transmission occurring within an infected mother during pregnancy. To study the potential for and pathway of vertical transmission of CWD in the native cervid species, we used a small cervid model–the polyestrous breeding, indoor maintainable, Reeves’ muntjac deer–and determined that the susceptibility and pathogenesis of CWD in these deer reproduce that in native mule and white-tailed deer. Moreover, we demonstrate here that CWD prions are transmitted from doe to fawn. Maternal CWD infection also appears to result in lower percentage of live birth offspring. In addition, evolving evidence from protein misfolding cyclic amplification (PMCA) assays on fetal tissues suggest that covert prion infection occurs in utero. Overall, our findings demonstrate that transmission of prions from mother to offspring can occur, and may be underestimated for all prion diseases.

Susceptibility of domestic cats to chronic wasting disease

ABSTRACT Domestic and nondomestic cats have been shown to be susceptible to feline spongiform encephalopathy (FSE), almost certainly caused by consumption of bovine spongiform encephalopathy (BSE)-contaminated meat. Because domestic and free-ranging nondomestic felids scavenge cervid carcasses, including those in areas affected by chronic wasting disease (CWD), we evaluated the susceptibility of the domestic cat (Felis catus) to CWD infection experimentally. Cohorts of 5 cats each were inoculated intracerebrally (i.c.) or orally (p.o.) with CWD-infected deer brain. At 40 and 42 months postinoculation, two i.c.-inoculated cats developed signs consistent with prion disease, including a stilted gait, weight loss, anorexia, polydipsia, patterned motor behaviors, head and tail tremors, and ataxia, and the cats progressed to terminal disease within 5 months. Brains from these two cats were pooled and inoculated into cohorts of cats by the i.c., p.o., and intraperitoneal and subcutaneous (i.p./s.c.) routes. Upon subpassage, feline CWD was transmitted to all i.c.-inoculated cats with a decreased incubation period of 23 to 27 months. Feline-adapted CWD (FelCWD) was demonstrated in the brains of all of the affected cats by Western blotting and immunohistochemical analysis. Magnetic resonance imaging revealed abnormalities in clinically ill cats, which included multifocal T2 fluid attenuated inversion recovery (FLAIR) signal hyperintensities, ventricular size increases, prominent sulci, and white matter tract cavitation. Currently, 3 of 4 i.p./s.c.- and 2 of 4 p.o. secondary passage-inoculated cats have developed abnormal behavior patterns consistent with the early stage of feline CWD. These results demonstrate that CWD can be transmitted and adapted to the domestic cat, thus raising the issue of potential cervid-to-feline transmission in nature.

MUCOSAL IMMUNIZATION TO PREVENT CHRONIC WASTING DISEASE (CWD) IN DEER

P4-204 NEURO-PEPTIDE TREATMENT WITH CEREBROLYSIN IMPROVES THE SURVIVAL OF NEURAL STEM CELL GRAFTS IN THE APP TRANSGENIC MODEL OFALZHEIMER’S DISEASE Edward Rockenstein, Paula Desplats, Michael Mante, Jazmin Florio, Scott Anderson, Dieter Meier, Stefan Winter, Anthony Adame, Herbert Moessler, Eliezer Masliah, University of California San Diego, La Jolla, California, United States; University of California San Diego, La Jolla, California, United States; University of California San Diego, La Jolla, California, United States; University of California San Diego, La Jolla, California, United States; Ever Neuro Pharma GmbH, Unterach, Austria; Ever Neuro Pharma GmbH, Unterach, Austria; Ever Neuro Pharma GmbH, San Diego, California, United States; University of California San Diego, La Jolla, California, United States. Contact e-mail: erockenstein@ucsd.edu

Active immune intervention for prionoses in deer

micromolar range. We previously reported that the drug showed neuroprotective potential against amyloid toxicity in mice. In particular, ANAVEX2-73 attenuated the oxidative stress, caspases induction, cellular loss and learning and memory deficits observed in mice one week after the icv injection of an oligomeric preparation of amyloid beta-(25-35) peptide (Abeta 25-35) (Villard et al., J Psychopharmacol 2011). More recently, we reported that ANAVEX2-73 blocked the Abeta 25-35 -induced P-Akt decrease and P-GSK-3beta increase, indicating activation of the PI3K neuroprotective pathway (Lahmy et al., Soc Neurosci Abstr 2011). As a result, ANAVEX2-73 attenuated, in the dose-range tested, the hyperphosphorylation of Tau on physiological epitopes (AT-8 antibody clone) and on pathological epitopes (AT-100 clone). ANAVEX273 also decreased the Abeta 25-35 -induced endogenous Abeta 1-42 seeding. The drug may thus act as a disease-modifying agent not only protecting brain cells from toxicity but also contributing to decrease Tau pathology and amyloid load. Methods:We here analyzed the impact of administration schedule and combination with donepezil on the efficacy of the drug. Results: ANAVEX2-73 (0.1, 0.3 mg/kg ip), administered once a day between day -7 and day -1 before Abeta 25-35 (day 0), blocked the Abeta 25-35 -induced memory deficits (spontaneous alternation in the Y maze and passive avoidance response) and lipid peroxidation in the hippocampus 7 days after Abeta 25-35. ANAVEX2-73 (0.3 mg/kg ip) was also effective when it was administered once a day between day 7 and day 13 after Abeta 25-35 (on day 0), on memory deficits and lipid peroxidation increase measured 14 days after Abeta 25-35. In combination with donepezil (0.25, 0.5 mg/kg ip), the effects appeared synergistic, since the combination of the lowest non-effective doses (0.25 donepezil + 0.1 ANAVEX2-73) led to a significant protection on all parameters examined. Combination studies with memantine are in progress. Conclusions: These results showed a good preventing and restorating efficacy for the compound in the Abeta 25-35 model in mice and showed a preserved efficacy in combination with the reference acetylcholinesterase inhibitor.