David A. Osborn

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.

Evaluation of Wildlife Warning Reflectors for Altering White-Tailed Deer Behavior Along Roadways

Abstract We evaluated the behavioral responses of white-tailed deer (Odocoileus virginianus) to 4 colors of wildlife warning reflectors (red, white, blue-green, and amber) that are purported to reduce the incidence of deer–vehicle collisions. We observed white-tailed deer behaviors relative to roads before and after installation of wildlife warning reflectors using a forward-looking infrared camera during 90 observation nights. We concluded that wildlife warning reflectors were ineffective in changing deer behavior such that deer–vehicle collisions might be prevented.

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.

Volatile compounds from interdigital gland of male white-tailed deer (Odocoileus virginianus)

Interdigital secretions were collected from eight male white-tailed deer of various ages. Analysis of volatiles was performed by gas chromatography-mass spectrometry with a modified headspace technique. Forty-six volatile compounds were found including alkanes, arenes, aldehydes, ketones, aliphatic acids, esters, pyrroles, furans, and sulfur compounds. Eleven occurred in higher concentrations (P≤0.10) in dominant (≥3.5-year-old) than in subordinate (≥1.5-year-old) animals. Dominant males typically have higher serum testosterone levels, and fatty acids and esters fluctuate with sebum production, which is under hormonal control. Therefore, these compounds may reflect testosterone levels and act as chemical signals indicating the presence of a dominant male. Interdigital volatiles also may act as generalized scent trail markers.

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.

EFFECTIVENESS OF ANTAGONISTS FOR TILETAMINE-ZOLAZEPAM/XYLAZINE IMMOBILIZATION IN FEMALE WHITE-TAILED DEER

A combination of tiletamine-zolazepam/xylazine (TZ/X) is effective in the chemical immobilization of white-tailed deer (Odocoileus virginianus); however, the lengthy duration of immobilization may limit its usefulness. From October to November 2002, 21 captive female deer were assigned randomly to an α2 antagonist treatment to reverse xylazine-induced sedation (seven does per group). All deer were given 220 mg of TZ (4.5±0.4 mg/kg) and 110 mg of X (2.2±0.2 mg/kg) intramuscularly (IM). Antagonist treatments were either 200 mg of tolazoline (4.0±0.4 mg/kg), 11 mg of atipamezole (0.23±0.02 mg/kg), or 15 mg of yohimbine (0.30±0.02 mg/kg) injected, half intravenously and half subcutaneously, 45 min after the IM TZ/X injection. In addition, 10 other deer (five per group) were immobilized as before and then given tolazoline (200 mg) after 45 min, with either a carrier (dimethyl sulfoxide [DMSO]) or carrier (DMSO) plus flumazenil (5 mg) to reverse the zolazepam portion of TZ. Mean times from antagonist injection until a deer raised its head were different for α2 antagonist treatments (P = 0.02). Times were longer for yohimbine (62.3±42.7 min) than for either atipamezole (24.3±17.1 min) or tolazoline (21.3±14.3 min). Mean times from antagonist injection until standing were not different (P=0.15) among yohimbine (112.0±56.4 min), atipamezole (89.7±62.8 min), or tolazoline (52.6±37.2 min). A sedation score based on behavioral criteria was assigned to each deer every 30 min for 5 hr. On the basis of sedation scores, tolazoline resulted in a faster and more complete reversal of immobilization. Flumazenil treatment did not affect recovery.

Volatile Compounds from the Forehead Region of Male White-Tailed Deer (Odocoileus virginianus)

Secretions produced by sebaceous and apocrine glands of cervids may be important in identifying individuals, establishing dominance, and signaling sexual readiness. The secretions from these glands are transferred to the hair for both lubrication and scent communication via forehead rubbing. We collected hair samples from the forehead and back of 10 male white-tailed deer (Odocoileus virginianus) of various ages and analyzed them with gas chromatography–mass spectrometry to determine age-related differences. Fifty-seven compounds were identified, including alkanes, arenes, aldehydes, ketones, aliphatic alcohols, terpenes, terpene alcohols, and phenols. Although forehead apocrine glands of dominant deer become more active during the breeding season, we found that concentrations of eight compounds found on the forehead hair were higher in subordinate deer, while only one was higher in dominant deer. Subordinate deer may have higher concentrations of these compounds because they rub less frequently than dominant deer. Additionally, only five forehead hair volatiles differed in concentration from those taken from the back hair. This seems to indicate that an increase in forehead glandular activity may take place concurrently with an increase in general integumentary glandular activity. The variation in hair volatiles among individuals also may be indicative of an individual-specific odor that could aid in identification.

Use of two pregnancy-associated glycoprotein RIA's for pregnancy diagnosis in white-tailed deer

Analysis of blood for a protein associated with pregnancy has been suggested as a reliable alternative to traditional pregnancy tests for deer. However, the accuracy of this technique has not been tested throughout gestation in white-tailed deer (Odocoileus virginianus). In addition, quantification of protein relative to stage of gestation and fetal number has not been reported. To address these needs, sera collected from 6 captive white-tailed does before breeding, throughout gestation, and following parturition were tested by radioimmunoassays (RIA) developed using glycoproteins isolated from domestic cattle and sheep. These RIAs provide the first quantitative pregnancy-associated glycoprotein (PAG) tests for deer. In our study, PAG levels increased throughout pregnancy and declined linearly following parturition. Beyond mid-gestation, PAG levels reflected in utero fetal number. The bovine-PAG (bPAG) assay was 100% accurate beyond day 65 of gestation. The ovine-PAG (oPAG) assay detected pregnancy by mean day 22 of gestation and was 100% accurate beyond day 32. Our oPAG-RIA is the most effective pregnancy test reported for white-tailed deer.

A COMPARISON OF CARFENTANIL/XYLAZINE AND TELAZOL®/XYLAZINE FOR IMMOBILIZATION OF WHITE-TAILED DEER

From October 2001 to January 2002, captive free-ranging white-tailed deer (Odocoileus virginianus) were immobilized with a combination of carfentanil citrate and xylazine hydrochloride. From this study, we selected a dose of carfentanil/xylazine for the purpose of comparing immobilization parameters and physiologic effects with those of a combination of tiletamine and zolazepam (Telazol®) and xylazine. Animals were initially given intramuscular injections of 10 mg xylazine and one of four doses of carfentanil (i.e., 0.5, 1.0, 1.5, and 2.0 mg). A carfentanil dose of 1.2 mg (x̄±SD=23.5±3.2 μg/kg) and 10 mg xylazine (0.2±0.03 mg/kg) were selected, based on induction times and previously published reports, to compare with a combination of 230 mg of Telazol® (4.5±0.6 mg/kg) and 120 mg xylazine (2.3±0.3 mg/kg). Time to first observable drug effects and to induction were significantly longer for deer treated with carfentanil/xylazine than with Telazol®/xylazine (P<0.01). Hyperthermia was common in deer immobilized with carfentanil/xylazine, but heart rate, respiration rate, and hemoglobin saturation were within acceptable levels. Degree of anesthesia of deer immobilized with Telazol®/xylazine was superior to deer immobilized with carfentanil/xylazine. The combination of 120 mg of naltrexone hydrochloride and 6.5 mg of yohimbine hydrochloride provided rapid and complete reversal (1.9±1.1 min) of carfentanil/xylazine immobilization. Animals immobilized with Telazol®/xylazine had long recovery times with occasional resedation after antagonism with 6.5 mg of yohimbine. The combination of carfentanil and xylazine at the doses tested did not provide reliable induction or immobilization of white-tailed deer even though drug reversal was rapid and safe using naltrexone and yohimbine.