First chronic wasting disease (CWD) surveillance of roe deer (Capreolus capreolus) in the northern part of Belgium

Cases of chronic wasting disease (CWD) in wild cervids have yet not been reported in Europe, whereas the disease is considered enzootic in free-ranging mule deer, Rocky mountain elk and white-tailed deer in the area of Colorado, Wyoming, and Nebraska. New foci of CWD continue to be detected in other parts of the United States. However, no large-scale active epidemiosurveillance of European wild cervids is yet installed in Europe. In accordance with the opinion of the European Scientific Steering Committee, a preliminary (active) surveillance scheme was installed, in order to improve the knowledge of the CWD status of wild cervids (roe deer) in the Northern part of Belgium. Spleen samples (n=206) and brain samples (n=222) of roe deer collected in the Northern part of Belgium, were examined for CWD using the antigen-capture enzyme-linked immunoassay (EIA) of IDEXX. Afterwards, the EIA was systematically confirmed by immunohistochemistry using three antibodies, namely R524, 2G11 and 12F10. There were no indications on the occurrence of TSE in any of the samples. A Bayesian framework was used for the estimation of the true prevalence of CWD in the Northern part of Belgium that was estimated to have a median value of zero with a 95th percentile value of 0.0049 and 0.0045 for spleen and brain samples respectively.     

Abundant PrP(CWD) in tonsil from mule deer with preclinical chronic wasting disease.

A monoclonal antibody dot-blot assay was used to evaluate detergent lysates of tonsil tissue from mule deer to detect PrP(CWD), the marker for the cervid transmissible spongiform encephalopathy chronic wasting disease (CWD). Samples of formalin-fixed brain and tonsil tissues from mule deer were examined for PrP(CWD) using immunohistochemistry (IHC) with Mab F99/97.6.1, the gold standard for diagnosis of preclinical CWD. The contralateral tonsil from each of the 143 deer was prepared for confirmatory IHC and as a 10% (wt/vol) detergent lysate without purification or enrichment steps for monoclonal antibody dot-blot assay. PrP(CWD) was detected by dot-blot assay in 49 of 50 samples considered positive by IHC. Forty-eight of the positive samples were evaluated with a quantitative dot-blot assay calibrated with recombinant PrP. Tonsillar PrP(CWD) concentrations ranged from 34 to 1,188 ng per 0.5 mg starting wet weight of tissue. The abundant PrP(CWD) in mule deer tonsil will facilitate development and validation of high-throughput screening tests for CWD in large populations of free-ranging deer.     

Development of monoclonal antibodies against the abnormal prion protein isoform (PrPres) associated with chronic wasting disease (CWD)

Monoclonal antibodies (mAbs) specific for the abnormal prion protein isoform (PrP^res) are indispensable for diagnosing chronic wasting disease (CWD). In this study, eight mAbs were developed by immunizing PrP knockout mice with recombinant elk PrP and an immunogenic PrP peptide. The reactivity of the mAbs to recombinant PrP and the PrP peptide was measured, and their isotypes were subsequently determined. Among them, four mAbs (B85-05, B85-08, B85-12, and B77-75) were shown by Western blotting to recognize proteinase K-treated brain homogenate derived from an elk suffering from CWD.     

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.     

Experimental transmission of chronic wasting disease (CWD) from elk and white-tailed deer to fallow deer by intracerebral route: final report

Final observations on experimental transmission of chronic wasting disease (CWD) from elk (Cervus elaphus nelsoni) and white-tailed deer (Odocoileus virginianus) to fallow deer (Dama dama) are reported herein. During the 5-year study, 13 fawns were inoculated intracerebrally with CWD-infected brain material from white-tailed deer (n = 7; Group A) or elk (n = 6; Group B), and 3 other fawns were kept as uninoculated controls (Group C). As described previously, 3 CWD-inoculated deer were euthanized at 7.6 mo post-inoculation (MPI). None revealed presence of abnormal prion protein (PrP(d)) in their tissues. At 24 (Group A) and 26 (Group B) MPI, 2 deer were necropsied. Both animals had a small focal accumulation of PrP(d) in their midbrains. Between 29 and 37 MPI, 3 other deer (all from Group A) were euthanized. The 5 remaining deer became sick and were euthanized between 51 and 60 MPI (1 from Group A and 4 from Group B). Microscopic lesions of spongiform encephalopathy (SE) were observed in only these 5 animals; however, PrP(d) was detected in tissues of the central nervous system by immunohistochemistry, Western blot, and by commercial rapid test in all animals that survived beyond 24 MPI. This study demonstrates that intracerebrally inoculated fallow deer not only amplify CWD prions, but also develop lesions of spongiform encephalopathy.     

Variable patterns of distribution of PrP(CWD) in the obex and cranial lymphoid tissues of Rocky Mountain elk (Cervus elaphus nelsoni) with subclinical chronic wasting disease

Sections of medulla oblongata, taken at the level of the obex, palatine tonsil and medial retropharyngeal lymph node from 10,269 captive Rocky Mountain elk (Cervus elaphus nelsoni), were examined by immunohistochemical staining with monoclonal antibody for the prion protein associated with the transmissible spongiform encephalopathy of cervids, chronic wasting disease (PrP(CWD)). The protein was detected in 226 of them. On the basis of the anatomical location of the deposits in the brainstem of 183 elk, four distinct patterns of distribution of PrP(CWD) within the parasympathetic region of the dorsal motor nucleus of the vagus nerve and the adjacent nuclei were observed. Mild gross lesions of chronic wasting disease (serous atrophy of fat) were observed in only three elk, all with spongiform degeneration; the other elk were considered to be in the preclinical stage of the disease. In contrast with the relatively predictable distribution of prion protein (PrP) in the brain and cranial nodes of sheep and mule deer, the distribution of PrP(CWD) in the brain and nodes of the elk was more variable and unrelated to their PrP genotype. One hundred and fifty-five of the 226 positive elk had deposits of PrP(CWD) in the brainstem and lymphoid tissues, 43 had deposits only in the lymphoid tissue and 28 had deposits only in the brainstem.     

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.     

A prion disease of cervids: chronic wasting disease

Chronic wasting disease (CWD) is a prion disease of deer, elk, and moose, initially recognized in Colorado mule deer. The discovery of CWD beyond the borders of Colorado and Wyoming, in Canada and as far east as New York, has led to its emergence as a prion disease of international importance. Epidemiological studies indicate that CWD is horizontally transmitted among free-ranging animals, potentially indirectly by prion-containing secreta or excreta contaminating the environment. Experimental CWD transmission attempts to other wild and domestic mammals and to transgenic mice expressing the prion protein of cattle, sheep, and humans have shed light on CWD species barriers. Transgenic mice expressing the cervid prion protein have proven useful for assessing the genetic influences of Prnp polymorphisms on CWD susceptibility. Accumulating evidence of CWD pathogenesis indicates that the misfolded prion protein or prion infectivity seems to be widely disseminated in many nonneural organs and in blood. This review highlights contemporary research findings in this prion disease of free-ranging wildlife.     

Age-related lesions in laboratory-confined raccoons (Procyon lotor) inoculated with the agent of chronic wasting disease of mule deer.

This communication documents age-associated pathologic changes and final observations on experimental transmission of chronic wasting disease (CWD) by the intracerebral route to raccoons (Procyon lotor). Four kits were inoculated intracerebrally with a brain suspension from mule deer with CWD. Two uninoculated kits served as controls. One CWD-inoculated raccoon was humanely killed at 38 months after inoculation, and 1 control animal died at 68 months after inoculation. Both animals had lesions that were unrelated to transmissible spongiform encephalopathy. Six years after inoculation, none of the 3 remaining CWD-inoculated raccoons had shown clinical signs of neurologic disorder, and the experiment was terminated. Spongiform encephalopathy was not observed by light microscopy, and the presence of abnormal prion protein (PrP(d)) was not detected by either immunohistochemistry or Western blot techniques. Age-related lesions observed in these raccoons included islet-cell pancreatic amyloidosis (5/6), cystic endometrial hyperplasia (3/4), cerebrovascular mineralization (5/6), neuroaxonal degeneration (3/6), transitional-cell adenoma of the urinary bladder (1/6), and myocardial inclusions (4/6). The latter 2 pathologic conditions were not previously reported in raccoons.     

Distribution of protease-resistant prion protein and spongiform encephalopathy in free-ranging mule deer (Odocoileus hemionus) with chronic wasting disease

Serial sections of brain and palatine tonsil were examined by immunohistochemical staining (IHC) using monoclonal antibody F89/160.1.5 for detecting protease-resistant prion protein (PrP(res)) in 35 hunter-killed mule deer (Odocoileus hemionus) with chronic wasting disease. Serial sections of brain were stained with hematoxylin and eosin and examined for spongiform encephalopathy (SE). Clinical signs of disease were not observed in any of these deer. On the basis of the location and abundance of IHC and the location and severity of SE, deer were placed into four categories. Category 1 (n = 8) was characterized by IHC in the palatine tonsil with no evidence of IHC or SE in the brain. Category 2 (n = 13) was characterized by IHC in the palatine tonsil and IHC with or without SE in the dorsal motor nucleus of the vagus nerve (DMNV). Category 3 (n = 2) was characterized by IHC in the palatine tonsil, IHC with SE in the myelencephalon, and IHC without SE in the hypothalamus. Category 4 (n = 12) was characterized by IHC in the palatine tonsil and IHC with SE throughout the brain. Category I may represent early lymphoid tissue localization of PrP(res). The DMNV appears to be the most consistent single neuroanatomic site of detectable PrP(res). Categories 2-4 may represent a progression of spread of PrP(res) and SE throughout the brain. IHC in tonsil and brain and SE in brain were not detected in 208 control deer.     

Monitoring of chronic wasting disease using real-time quaking-induced conversion assay in Japan

There has been no report on Chronic wasting disease (CWD) cases in Japan to date; however, there is concern about the geographic spread of CWD. To clarify the CWD status in Japan, we conducted CWD monitoring using real-time quaking-induced conversion (RT-QuIC) assay which can detect the low level of CWD prions. A total of 690 obex samples collected from sika deer and Reeves’s muntjac in Hokkaido and Honshu was tested for CWD prions. No CWD-positive cases were found, suggesting that CWD is nonexistent in Japan. Our results also indicate that RT-QuIC assay is useful for continuous monitoring of CWD. Furthermore, nucleotide sequence analysis of the PrP gene revealed sika deer in Japan harbor CWD susceptible allele.     

First isolation of Trichinella britovi from a wild boar (Sus scrofa) in Belgium

Since 1992, when the European Union Council Directive requires that wild boars (Sus scrofa) hunted in EU for commercial purpose should be examined for Trichinella, the infection has not been detected in wild boars from Belgium, despite serological evidence of the presence of anti-Trichinella antibodies in wildlife and previous reports of Trichinella larvae in this host species. In November 2004, Trichinella larvae were detected in a wild boar hunted near Mettet, Namur province (Southern Belgium). Larvae were identified as Trichinella britovi by polymerase chain reaction methods. This is the first report of the identification of Trichinella larvae from Belgium at the species level. The detection of T. britovi in wildlife in Belgium is consistent with findings of this parasite in other European countries and confirms the need to test game meat for Trichinella to prevent its transmission to humans.