Detection of PrP(CWD) in postmortem rectal lymphoid tissues in Rocky Mountain elk (Cervus elaphus nelsoni) infected with chronic wasting disease.

Preclinical diagnostic tests for transmissible spongiform encephalopathies have been described for mule deer (Odocoileus hemionus), using biopsy tissues of palatine tonsil, and for sheep, using lymphoid tissues from palatine tonsil, third eyelid, and rectal mucosa. The utility of examining the rectal mucosal lymphoid tissues to detect chronic wasting disease (CWD) was investigated in Rocky Mountain elk (Cervus elaphus nelsoni), a species for which there is not a live-animal diagnostic test. Postmortem rectal mucosal sections were examined from 308 elk from two privately owned herds that were depopulated. The results of the postmortem rectal mucosal sections were compared to immunohistochemical staining of the brainstem, retropharyngeal lymph nodes, and palatine tonsil. Seven elk were found positive using the brainstem (dorsal motor nucleus of the vagus nerve), retropharyngeal lymph nodes, and palatine tonsil. Six of these elk were also found positive using postmortem rectal mucosal sections. The remaining 301 elk in which CWD-associated abnormal isoform of the prion protein (PrP(CWD)) was not detected in the brainstem and cranial lymphoid tissues were also found to be free of PrP(CWD) when postmortem rectal mucosal sections were examined. The use of rectal mucosal lymphoid tissues may be suitable for a live-animal diagnostic test as part of an integrated management strategy to limit CWD in elk.     

Evaluation of vaginal implant transmitters in elk (Cervus elaphus nelsoni).

The effects of vaginal implant transmitters for tissue damage after 11 wk in 13 captive adult elk (Cervus elaphus nelsoni) and subsequent reproductive performance in 38 free-ranging elk were evaluated. Vaginal implant transmitters are designed to be shed at parturition and are used to locate birth sites of wild ungulates; however, potential adverse effects of these transmitters on tissues associated with the vaginal walls or subsequent reproductive performance have not been assessed. Vaginal implant transmitters consist of a transmitter encased in inert acrylic with an antenna trailing out the distal end and wings at the proximal end to hold the transmitter in place. Using a laparoscope on sedated captive elk, necrosis or measurable differences in tissue trauma between designs with wing spans of 80 versus 150 mm over an 11-wk trial were not observed. After the captive elk trial, vaginal implant transmitters with 80-mm wings were placed into 38 pregnant wild elk, and 31 live births were documented. Fates of seven calves were not determined, because their transmitters were not shed at the birth site. We recaptured 36 of these cow elk again in fall 2003 or spring 2004, and 32 were pregnant. This study was unable to document any short- or long-term effects of vaginal implant transmitters on reproductive performance of cow elk in captive and free-range environments.     

Experimental transmission of chronic wasting disease (CWD) of elk (Cervus elaphus nelsoni), white-tailed deer (Odocoileus virginianus), and mule deer (Odocoileus hemionus hemionus) to white-tailed deer by intracerebral route

To compare clinical and pathologic findings of chronic wasting disease (CWD) in a natural host, 3 groups (n = 5) of white-tailed deer (WTD) fawns were intracerebrally inoculated with a CWD prion of WTD, mule deer, or elk origin. Three other uninoculated fawns served as controls. Approximately 10 months postinoculation (MPI), 1 deer from each of the 3 inoculated groups was necropsied and their tissues were examined for lesions of spongiform encephalopathy (SE) and for the presence of abnormal prion protein (PrP(d)) by immunohistochemistry (IHC) and Western blot (WB). The remaining deer were allowed to live until they developed clinical signs of the disease which began approximately 18 MPI. By 26 MPI, all deer were euthanatized on humane grounds. Obvious differences in clinical signs or the incubation periods were not observed between the 3 groups of deer given CWD. In 1 of 3 nonclinical deer euthanatized at 10 MPI, minimal microscopic lesions of SE were seen in the central nervous system (CNS) tissues, and PrP(d) was observed by IHC in tissues of all 3 deer. In the clinical deer, CNS lesions of SE and PrP(d) accumulations were more severe and extensive. It is concluded that the 3 sources of CWD prion did not induce significant differences in time to clinical disease or qualitative differences in signs or lesions in WTD. However, this observation does not imply that these CWD agents would necessarily behave similarly in other recipient species.     

Experimental oral transmission of chronic wasting disease to red deer (Cervus elaphus elaphus): Early detection and late stage distribution of protease-resistant prion protein

Chronic wasting disease (CWD), an important emerging prion disease of cervids, is readily transmitted by intracerebral or oral inoculation from deer-to-deer and elk-to-elk, suggesting the latter is a natural route of exposure. Studies of host range susceptibility to oral infection, particularly of those species found in habitats where CWD currently exists are imperative. This report describes the experimental transmission of CWD to red deer following oral inoculation with infectious CWD material of elk origin. At 18 to 20 months post-inoculation, mild to moderate neurological signs and weight loss were observed and animals were euthanized and tested using 3 conventional immunological assays. The data indicate that red deer are susceptible to oral challenge and that tissues currently used for CWD diagnosis show strong abnormal prion (PrP^CWD) accumulation. Widespread peripheral PrP^CWD deposition involves lymphoreticular tissues, endocrine tissues, and cardiac muscle and suggests a potential source of prion infectivity, a means of horizontal transmission and carrier state.     

Field validation and assessment of an enzyme-linked immunosorbent assay for detecting chronic wasting disease in mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus nelsoni).

Tissue samples (n = 25,050 total) from 23,256 mule deer (Odocoileus hemionus), Rocky Mountain elk (Cervus elaphus nelsoni), and white-tailed deer (Odocoileus virginianus) collected statewide in Colorado were examined for chronic wasting disease (CWD) using an enzyme-linked immunosorbent assay developed by Bio-Rad Laboratories, Inc. (brELISA), in a 2-phase study. In the validation phase of this study, a total of 4,175 retropharyngeal lymph nodes (RLN) or obex (OB) tissue samples were examined independently by brELISA and immunohistochemistry (IHC). There were 137 IHC-positive samples and 4,038 IHC-negative samples. Optical density (OD) values from brELISA were classified as "not detected" or "suspect" based on recommended cutoff values during the validation phase. Using IHC-positive cases as known CWD-infected individuals and assuming IHC-negative cases as uninfected, the relative sensitivity of brELISA depending on species ranged from 98.3% to 100% for RLN samples and 92.1% to 93.3% for OB samples; the relative specificity of brELISA depending on species ranged from 99.9% to 100% for RLN samples and was 100% for OB samples. Overall agreement between brELISA and IHC was > or = 97.6% in RLN samples and > or = 95.7% in OB samples of all species where values could be calculated; moreover, mean brELISA OD values were > or = 46X higher in IHC-positive samples than in IHC-negative samples. Discrepancies were observed only in early-stage cases of CWD. Based on the validation phase data, only RLN samples were collected for the field application phase of this study and only samples with brELISA OD values > 0.1 were examined by IHC. Among 20,875 RLN samples screened with brELISA during this second testing phase, 155 of 8,877 mule deer, 33 of 11,731 elk, and 9 of 267 white-tailed deer samples (197 total) had OD values > 0.1 and were further evaluated by IHC to confirm evidence of CWD infection. Of cases flagged for IHC follow-up, 143 of 155 mule deer, 29 of 33 elk, and all 9 white-tailed deer were confirmed positive. Mean (+/- SE) OD values for IHC-positive cases detected during the field application phase were comparable with those measured in RLN tissues during the validation phase. Based on these data, brELISA was determined to be an excellent rapid test for screening large numbers of samples in surveys designed to detect CWD infections in deer and elk populations.     

Effects of supplemental feeding on gastrointestinal parasite infection in elk (Cervus elaphus): preliminary observations

The effects of management practices on the spread and impact of parasites and infectious diseases in wildlife and domestic animals are of increasing concern worldwide, particularly in cases where management of wild species can influence disease spill-over into domestic animals. In the Greater Yellowstone Ecosystem, USA, winter supplemental feeding of Rocky Mountain elk (Cervus elaphus) may enhance parasite and disease transmission by aggregating elk on feedgrounds. In this study, we tested the effect of supplemental feeding on gastrointestinal parasite infection in elk by comparing fecal egg/oocyst counts of fed and unfed elk. We collected fecal samples from fed and unfed elk at feedground and control sites from January to April 2006, and screened all samples for parasites. Six different parasite types were identified, and 48.7% of samples were infected with at least one parasite. Gastrointestinal (GI) nematodes (Nematoda: Strongylida), Trichuris spp., and coccidia were the most common parasites observed. For all three of these parasites, fecal egg/oocyst counts increased from January to April. Supplementally fed elk had significantly higher GI nematode egg counts than unfed elk in January and February, but significantly lower counts in April. These patterns suggest that supplemental feeding may both increase exposure and decrease susceptibility of elk to GI nematodes, resulting in differences in temporal patterns of egg shedding between fed and unfed elk.     

Mortality in Pennsylvania captive elk (Cervus elaphus): 1998-2006.

The necropsy records of 65 elk ranging from 1 day to 13(1/2) years of age were reviewed to determine the primary cause of death of each animal. The animal carcasses were submitted from 22 separate Pennsylvania elk farms over an approximately 8-year period. The most common causes of mortality in animals in which the cause of death was determined were gastrointestinal parasitism (21 cases), pneumonia (7 cases), enterocolitis (5 cases), malnutrition (5 cases), and rumenitis/acidosis (5 cases). The cause of death was undetermined in 4 of the 65 elk. Nematode organisms (20 cases) were the primary parasites identified in cases of mortality owing to gastrointestinal parasitism. Arcanobacterium pyogenes (3 cases), Escherichia coli (3 cases), and Streptococcus sp. (2 cases) were the most commonly isolated bacteria from the lung in the cases of pneumonia. Fungal organisms, consistent with Aspergillus sp. were present histologically within the pulmonary lesions in 5 cases. Bacterial agents associated with enterocolitis included Clostridium perfringens (2 cases), E. coli (1 case), Salmonella Newport (1 case) and Salmonella Thompson (1 case). The majority (56.3%) of the death loss in elk of known ages occurred in animals 1 year of age or less, with 71.4% of the gastrointestinal parasitism, 60.0% of the malnutrition, 60.0% of the enterocolitis, and 57.1% of the pneumonia cases occurring in animals within this age range. In conclusion, gastrointestinal parasitism, pneumonia, malnutrition, enterocolitis and rumenitis/acidosis were considered the most common causes of death in captive elk in this study.     

Evidence of Parelaphostrongylus tenuis infections in free-ranging elk (Cervus elaphus) in southern Ontario

The antemortem detection of a Parelaphostrongylus tenuis infection in a free-ranging wild elk (Cervus elaphus) in southern Ontario is documented. Postmortems on other free-ranging elk that died during 2000-2005 indicated that 59% (17/29) were infected with P. tenuis, based on presence of lesions in the brain.     

A retrospective study of the causes of morbidity and mortality in farmed elk (Cervus elaphus)

A survey of North American farmed elk (Cervus elaphus) producers was performed to determine the causes of sickness and mortality in farmed elk and to estimate mortality rates. Records over a 10-year period from 8 North American veterinary diagnostic pathology laboratories were also examined and summarized. The primary diagnosis for each record was used to classify diseases into categories such as parasitic, infectious, toxicological, and neoplastic. Nonspecific trauma was the most frequently reported known cause of mortality in both sexes and all age classes by elk producers. Ranked on perceived economic importance, producers cited trauma, chronic wasting disease, calf scours, dystocia, pneumonia, winter tick, tuberculosis, and grain overload. One-year mortality rates for adults and yearlings were 2.6% and 2.7%, respectively. Mortality rates for male and female adult animals were 2.4% and 2.7%, respectively. In general, the major findings of the survey matched reported causes for mortality provided by elk producers.     

A case of chronic wasting disease in an elk imported to Korea from Canada

A seven-year-old male elk (Cervus elaphus nelsoni) was euthanized and necropsied after having a 3-week history of body weight loss, emaciation, excessive salivation, teeth grinding, fever, anorexia, and respiratory distress. The elk was imported into Korea from Canada on March 9, 1997. Gross pathologic findings were restricted to a diffuse fibrinous pneumonia. Microscopic lesions included mild neuronal vacuolation and spongiform change in the neuropil of selected brain stem nuclei and generalized astrocytosis. Immunohistochemistry for protease-resistant prion protein (PrP(res)) was positive in all brain sections but more pronounced in the section of the obex of the medulla. And the PrP(res) was also detected by western immunoblotting in the brain and spinal cord. All the remaining elk and deer that had been in contact with this elk were destroyed and negative for chronic wasting disease (CWD). To our knowledge, this is the first case of CWD occurring outside of the U.S.A. and Canada.     

Use of naloxone to reverse carfentanil citrate-induced hypoxemia and cardiopulmonary depression in Rocky Mountain wapiti (Cervus elaphus nelsoni).

With the use of a crossover study design, we investigated the respiratory and cardiovascular effects of naloxone administration in eight healthy Rocky Mountain wapiti (Cervus elaphus nelsoni) anesthetized with carfentanil (10 microg/kg i.m.) and xylazine (0.1 mg/kg). Anesthetized animals showed profound hypoxemia with mild hypercapnia, tachycardia, hypertension, and acidosis prior to naloxone administration. After monitoring equipment was placed, animals were administered either naloxone (2 microg/microg carfentanil i.v.) or an equivalent volume of normal saline. Mean values for PaO2, PaCO2, heart rate, and respiratory rate were significantly different between naloxone- and saline-treated groups, but mean blood pressure, hematocrit, and serum electrolyte concentrations were not. Mean PaO2 was 23.0 +/- 4.1 mm Hg prior to administration of naloxone or saline and increased to 50.2 +/- 7.3 mm Hg after naloxone administration. Mean PaO2 of saline-treated animals did not change significantly. Electrocardiograms of three saline-treated animals suggested myocardial hypoxia. Hypoxemia appeared to be caused by respiratory depression, hemodynamic alterations, and lateral recumbency. All but one animal remained anesthetized after naloxone administration. Anesthesia in all animals was reversed in < or = 4 min with naltrexone (100 mg/mg carfentanil i.v. s.c.) and yohimbine (0.1 mg/kg i.v.). One bolus of naloxone improved oxygenation in carfentanil-xylazine-anesthetized wapiti.     

Immunohistochemical diagnosis of chronic wasting disease in preclinically affected elk from a captive herd.

An immunohistochemical (IHC) method was used to test brain tissues from 17 elk in a captive herd in which chronic wasting disease (CWD) had previously occurred. The IHC technique detects the protease-resistant prion protein (PrP-res), which is considered a disease-specific marker for transmissible spongiform encephalopathies (TSE), regardless of the species affected. Of the 17 elk tested, 10 were positive by IHC. Only 2 of these 10 animals had shown clinical signs and histologic lesions of CWD, and an additional animal had histologic lesions only. The most consistently IHC-positive tissue was medulla oblongata, especially the obex. These results show that the PrP-res IHC test on brain tissue, specifically medulla oblongata at the obex, should be considered an essential component of any surveillance study intended to determine the incidence of CWD in captive or free-ranging cervids.     

Susceptibility of cattle to first-passage intracerebral inoculation with chronic wasting disease agent from white-tailed deer

Fourteen, 3-month-old calves were intracerebrally inoculated with the agent of chronic wasting disease (CWD) from white-tailed deer (CWDwtd) to compare the clinical signs and neuropathologic findings with those of certain other transmissible spongiform encephalopathies (TSE, prion diseases) that have been shown to be experimentally transmissible to cattle (sheep scrapie, CWD of mule deer [CWDmd], bovine spongiform encephalopathy [BSE], and transmissible mink encephalopathy). Two uninoculated calves served as controls. Within 26 months postinoculation (MPI), 12 inoculated calves had lost considerable weight and eventually became recumbent. Of the 12 inoculated calves, 11 (92%) developed clinical signs. Although spongiform encephalopathy (SE) was not observed, abnormal prion protein (PrPd) was detected by immunohistochemistry (IHC) and Western blot (WB) in central nervous system tissues. The absence of SE with presence of PrPd has also been observed when other TSE agents (scrapie and CWDmd) were similarly inoculated into cattle. The IHC and WB findings suggest that the diagnostic techniques currently used to confirm BSE would detect CWDwtd in cattle, should it occur naturally. Also, the absence of SE and a distinctive IHC pattern of CWDwtd and CWDmd in cattle suggests that it should be possible to distinguish these conditions from other TSEs that have been experimentally transmitted to cattle.     

Experimental transmission of malignant catarrhal fever to red deer (Cervus elaphus)

Abstract

Malignant catarrhal fever was transmitted from affected to recipient red deer (Cervus eluphus) using bloodor lymphoid suspension as inoculum. Incubation periods ranged from 11 to 26 days. The disease was also transmitted using lymphoid suspension stored overnight at 4°C or at ?70°C for 8 months.

The experimental disease was characterised by fever, depression, anorexia, diarrhoea and dysentry. The course of the disease was approximately 96 hours.

Major lesions consisted of acute mesenteric lymphadenitis and acute haemorrhagic typhlitis and colitis. Lesions in the caecum and colon started as multifocal mucosal haemorrhages and progressed rapidly to massive mucosal haemorrhage.     

Quantification of the transmission of bovine herpesvirus 1 among red deer (Cervus elaphus) under experimental conditions

Bovine herpesvirus 1 (BHV1) is endemically present in a cattle population that lives in a nature reserve in the Netherlands. Red deer (Cervus elaphus), living in the same nature reserve, can come into contact with the BHV1-infected cattle and could then become infected with BHV1. For the eradication of BHV1 in cattle, it is, therefore, important to know whether red deer alone can play a role in the transmission of BHV1. For that reason, we quantified the transmission of BHV1 among farmed red deer under experimental conditions. Two groups of ten animals were formed. In each group, five of these animals were inoculated with BHV1 and the other five served as contact animals. Three inoculated animals in each transmission experiment became infected and none of the contact animals became infected. The one-sided 95% confidence interval for R [0.0-0.94] showed that limited transmission might occur among red deer. Based on these results, we would expect only minor outbreaks of BHV1 to occur in red deer populations. We concluded that BHV1 will probably not survive longer than a few decades (several times the mean deer lifetime) in red deer populations. Consequently, it is not necessary for the eradication of BHV1 in cattle to eradicate BHV1 in red deer populations as well.     

Blood constituents of farmed red deer (Cervus elaphus): I. Haematological values

Approximately 100 farmed male and female red deer aged three months and over were blood-sampled and haematological parameters were measured. The deer were sampled by jugular venepuncture without tranquillization. Mean values were haemoglobin (16.0g/dl), packed cell volume (44.6%), mean corpuscular haemoglobin concentration (35.8g/dl), total plasma protein (66.0g/l). fibrinogen (4.8g/l) and white blood-cell numbers (5.80 x 10⁹/l). Differential white blood-cell counts were neutrophils (53.9%), eosinophils (4.8%), basophils (3.0%), lymphocytes (37.2%) and monocytes (1.1%). There were no significant differences in any parameter measured between sexes or between age groups three to eight months, nine to eighteen months, or older.     

A preliminary study of the genetic diversity of Xinjiang Tarim red deer (Cervus elaphus yarkandensis) using the microsatellite DNA method.

To evaluate the genetic diversity of the Xinjiang Tarim red deer (Cervus elaphus yarkandensis) population, we analyzed the frequencies of microsatellite alleles. Samples were collected from 3 isolated populations in Xaya, Lopnur and Qarqan of Xinjiang. Although 10 microsatellite loci were examined, alleles of 133 to 190 base-pairs were detected for only 3 loci: BM5004, BM4208 and BM888. The average observed multilocus heterozygosity was 0.08 +/- 0.04 for the Xaya, 0 for the Lopnur, and 0.17 +/- 0.08 for the Qarqan population. The average heterozygosity of all populations was 0.08 +/- 0.02. The observed heterozygosities were significantly lower than the expected values. The present results suggest that the bottleneck effect has occurred in the populations of the Xinjiang Tarim red deer.