Laurie A. Baeten

A Natural Case of Chronic Wasting Disease in a Free-ranging Moose (Alces alces shirasi)

Chronic wasting disease (CWD) was diagnosed in a free-ranging moose (Alces alces shirasi) killed by a hunter in Jackson County, Colorado, USA, in September 2005. The diagnosis was based upon immunohistochemistry (IHC) demonstrating the presence of accumulations of CWD-associated prion protein (PrPCWD) in tissue sections of medulla oblongata at the level of the obex (dorsal motor nucleus of the vagus) and in retropharyngeal lymph node (RPLN); additional testing by IHC revealed deposits of PrPCWD in multiple sections of medulla oblongata and cervical spinal cord as well as palatine tonsil and submandibular lymph node tissues. Western blot confirmed the presence of PrPCWD in RPLN and tonsil tissue. The PrPCWD also was detected via enzyme-linked immunosorbent assay of RPLN tissue. Spongiform encephalopathy was observed in sections of the brainstem and cervical spinal cord, although no clinical signs were noted by the hunter who killed the animal. The affected moose was homozygous for methionine at codon 209 of the prion protein coding region. In October 2006, two additional free-ranging moose were diagnosed with CWD. Epidemiology and implications of CWD in moose remain to be determined.

Persistent Bovine Viral Diarrhea virus Infection in Wild Cervids of Colorado

Bovine viral diarrhea virus (BVDV) is a significant viral pathogen of domestic cattle. Worldwide, there is evidence of BVDV exposure and infection in wild ungulates; however, the frequency and significance of such events are unknown. To determine the prevalence and distribution of Colorado deer, elk, and moose persistently infected (PI) with BVDV, a cross-sectional study was conducted using full-thickness ear tissue samples collected from animals presented to the Colorado Division of Wildlife for chronic wasting disease surveillance in the 2005–2006 hunting season. Tissue from 5,597 harvested animals (2,934 mule deer, 2,516 elk, 141 white-tailed deer, and 6 moose) was paraffin-embedded and stained for BVDV using immunohistochemistry. A single adult male mule deer had BVDV antigen in the skin; staining distribution was consistent with that seen in PI cattle. Skin and lymph node were also positive for viral RNA by polymerase chain reaction, and the virus was determined to be a type 1. The prevalence of BVDV PI cervids in Colorado is very low. However, the identification of a naturally infected adult PI animal in the wild suggests that the virus infects free-ranging populations. The source of the BVDV is unknown and is assumed to be spillover from cattle or maintenance within wildlife populations. Consideration of a potential wild animal reservoir is important in the design and implementation of BVDV management practices in cattle.

Leptospirosis and tularaemia in raccoons (Procyon lotor) of Larimer Country, Colorado.

Raccoons (Procyon lotor) are commonly implicated as carriers of many zoonotic pathogens. The purpose of this cross‐sectional study was to look for Leptospira interrogans and Francisella tularensis in opportunistically sampled, free‐ranging raccoons of Larimer Country, Colorado, USA. Sixty‐five animals were included in the study and testing consisted of gross post‐mortem examination, histopathology, and both immunohistochemistry and PCR for L. interrogans and F. tularensis. No significant gross lesions were identified and the most common histological lesions were lymphoplasmacytic interstitial nephritis and pulmonary silicosis; rare periportal hepatitis, splenic lymphoid hyperplasia and small pulmonary granulomas were also identified. Of 65 animals, 20 (30%) were positive for Leptospira on IHC but only one by PCR. Animals with inflammation in their kidneys were seven times more likely to be positive for Leptospira than animals without inflammation. The severity of inflammation was variable but often mild with minimal associated renal pathology. One animal was positive for Francisella on both IHC and PCR; IHC staining was localized to histiocytic cells within a pulmonary granuloma. In Colorado the significance and epidemiology of Leptospira is poorly understood. The high prevalence of infection in raccoons in this study population suggests that this species may be important in the regional epidemiology or could be used to estimate risk to domestic animals and humans. Identification of a single Francisella positive animal is significant as this is an uncommon disease in terrestrial animals within the state; the apparently higher prevalence in this peridomestic species implies that raccoons may be good indicators of the pathogen in the region. The results of this study suggest that raccoons may serve as effective sentinels for both Leptospira and Francisella in the state of Colorado. Further studies are needed to better characterize the prevalence and epidemiology of both organisms within the region.

New Records of Hair Follicle Mites (Demodecidae) from North American Cervidae

Individuals of three species of cervids, with varying degrees of alopecia, were examined for ectoparasites: Rocky Mountain elk (Cervus elaphus nelsoni) and mule deer (Odocoileus hemionus hemionus) in Colorado and white-tailed deer (Odocoileus virginianus) in South Dakota. Hair follicle mites were recovered and identified as Demodex kutzeri, a species originally described from the European red deer (Cervus elaphus, from Austria) and the sika deer (Cervus nippon pseudaxis, captive in Germany). These findings expand the geographic range of D. kutzeri to North America and extend its host range to include the genus Odocoileus. Thus, the host range for D. kutzeri spans two subfamilies of cervids. Additionally, D. kutzeri was identified in material from a white-tailed deer collected in South Carolina in 1971, indicating this parasite has been present, but unrecognized, on US cervids for some time.

Use of Macrocyclic Lactones in Cattle in the USA

The use of macrocyclic lactones has become the main stay for the treatment of endo- and ectoparasites in the cattle industry. Here we review those drugs that are currently approved for use in cattle in the United States. The general efficacy, tissue distribution and toxicity of each drug formulation are discussed. Included is a discussion regarding the current status for nematode anthelmintic resistance in cattle populations within the United States. Also provided is a current summary of ecological effects of macrocyclic lactones residues in manure.

A Rapid Field Test for Sylvatic Plague Exposure in Wild Animals

Abstract Plague surveillance is routinely conducted to predict future epizootics in wildlife and exposure risk for humans. The most common surveillance method for sylvatic plague is detection of antibodies to Yersinia pestis F1 capsular antigen in sentinel animals, such as coyotes (Canis latrans). Current serologic tests for Y. pestis, hemagglutination (HA) test and enzyme-linked immunosorbent assay (ELISA), are expensive and labor intensive. To address this need, we developed a complete lateral flow device for the detection of specific antibodies to Y. pestis F1 and V antigens. Our test detected anti-F1 and anti-V antibodies in serum and Nobuto filter paper samples from coyotes, and in serum samples from prairie dogs (Cynomys ludovicianus), lynx (Lynx canadensis), and black-footed ferrets (Mustela nigripes). Comparison of cassette results for anti-F1 and anti-V antibodies with results of ELISA or HA tests showed correlations ranging from 0.68 to 0.98. This device provides an affordable, user-friendly tool that may be useful in plague surveillance programs and as a research tool.

IMMUNOLOGICAL AND CLINICAL RESPONSE OF COYOTES (CANIS LATRANS) TO EXPERIMENTAL INOCULATION WITH YERSINIA PESTIS

Abstract Multiple publications have reported the use of coyotes (Canis latrans) in animal-based surveillance efforts for the detection of Yersinia pestis. Coyotes are likely exposed via flea bite or oral routes and are presumed to be resistant to the development of clinical disease. These historic data have only been useful for the evaluation of the geographic distribution of Y. pestis in the landscape. Because the canid immunologic response to Y. pestis has not been thoroughly characterized, we conducted experimental inoculation of captive-reared, juvenile coyotes (n = 8) with Y. pestis CO92 via oral or intradermal routes. We measured the humoral response to Y. pestis fraction 1 capsular protein (anti-F1) and found a significant difference between inoculation groups in magnitude and duration of antibody production. The anti-F1 titers in animals exposed intradermally peaked at day 10 postinoculation (PI; range = 1∶32 to 1∶128) with titers remaining stable at 1∶32 through week 12. In contrast, orally inoculated animals developed higher titers (range = 1∶256 to 1∶1,024) that remained stable at 1∶256 to 1∶512 through week 6. No clinical signs of disease were observed, and minimal changes were noted in body temperature, white blood cell counts, and acute phase proteins during the 7 days PI. Gross pathology was unremarkable, and minimal changes were noted in histopathology at days 3 and 7 PI. Rechallenge at 14 wk PI via similar dosage and routes resulted in marked differences in antibody response between groups. Animals in the orally inoculated group produced a striking increase in anti-F1 titers (up to 1∶4,096) within 3 days, whereas there was minimal to no increase in antibody response in the intradermal group. Information gathered from this experimental trial may provide additional insight into the spatial and temporal evaluation of coyote plague serology.

Leptospirosis and tularaemia in raccoons (Procyon lotor) of Larimer County, [corrected] Colorado.

Raccoons (Procyon lotor) are commonly implicated as carriers of many zoonotic pathogens. The purpose of this cross‐sectional study was to look for Leptospira interrogans and Francisella tularensis in opportunistically sampled, free‐ranging raccoons of Larimer Country, Colorado, USA. Sixty‐five animals were included in the study and testing consisted of gross post‐mortem examination, histopathology, and both immunohistochemistry and PCR for L. interrogans and F. tularensis. No significant gross lesions were identified and the most common histological lesions were lymphoplasmacytic interstitial nephritis and pulmonary silicosis; rare periportal hepatitis, splenic lymphoid hyperplasia and small pulmonary granulomas were also identified. Of 65 animals, 20 (30%) were positive for Leptospira on IHC but only one by PCR. Animals with inflammation in their kidneys were seven times more likely to be positive for Leptospira than animals without inflammation. The severity of inflammation was variable but often mild with minimal associated renal pathology. One animal was positive for Francisella on both IHC and PCR; IHC staining was localized to histiocytic cells within a pulmonary granuloma. In Colorado the significance and epidemiology of Leptospira is poorly understood. The high prevalence of infection in raccoons in this study population suggests that this species may be important in the regional epidemiology or could be used to estimate risk to domestic animals and humans. Identification of a single Francisella positive animal is significant as this is an uncommon disease in terrestrial animals within the state; the apparently higher prevalence in this peridomestic species implies that raccoons may be good indicators of the pathogen in the region. The results of this study suggest that raccoons may serve as effective sentinels for both Leptospira and Francisella in the state of Colorado. Further studies are needed to better characterize the prevalence and epidemiology of both organisms within the region.

EFFECT OF STORAGE TIME AND STORAGE CONDITIONS ON ANTIBODY DETECTION IN BLOOD SAMPLES COLLECTED ON FILTER PAPER

Abstract Using filter paper to collect blood from wildlife for antibody analysis can be a powerful technique to simplify the collection, transport, and storage of blood samples. Despite these advantages, there are limited data that detail how long these samples can be stored and how storage conditions affect antibody longevity. We used blood samples collected on filter paper from coyotes experimentally infected with Yersinia pestis to determine optimum sample storage conditions over time. Blood samples collected on filter paper were stored for 454 d or more in four groups: 1) at ambient temperature and at ambient relative humidity, 2) at ambient temperature with desiccant, 3) at 4 C with desiccant, and 4) at −20 C with desiccant. Samples stored at 4 C or −20 C with desiccant had detectable antibody for a longer period of time than the samples stored at room temperature.

Standardized guinea pig model for Q fever vaccine reactogenicity

Historically, vaccination with Coxiella burnetii whole cell vaccines has induced hypersensitivity reactions in humans and animals that have had prior exposure to the pathogen as a result of infection or vaccination. Intradermal skin testing is routinely used to evaluate exposure in humans, and guinea pig hypersensitivity models have been developed to characterize the potential for reactogenicity in vaccine candidates. Here we describe a refinement of the guinea pig model using an alternate vaccine for positive controls. An initial comparative study used viable C. burnetii to compare the routes of sensitizing exposure of guinea pigs (intranasal vs intraperitoneal), evaluation of two time points for antigen challenge (21 and 42 days) and an assessment of two routes (intradermal and subcutaneous) of challenge using the ruminant vaccine Coxevac as the antigenic control. Animals sensitized by intraperitoneal exposure exhibited slightly larger gross reactions than did those sensitized by intranasal exposure, and reactions were more pronounced when skin challenge was performed at 42 days compared to 21 days post-sensitization. The intradermal route proved to be the optimal route of reactogenicity challenge. Histopathological changes at injection sites were similar to those previously reported and a scoring system was developed to compare reactions between groups receiving vaccine by intradermal versus subcutaneous routes. Based on the comparative study, a standardized protocol for assessment of vaccine reactogenicity in intranasally-sensitized animals was tested in a larger confirmatory study. Results suggest that screens utilizing a group size of n = 3 would achieve 90% power for detecting exposure-related reactogenic responses of the magnitude induced by Coxevac using either of two outcome measures.