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Short Communication |
1 Colorado Division of Wildlife, Wildlife Research Center, 317 West Prospect Road, Fort Collins, CO 80526-2097, USA
2 Colorado State University Veterinary Diagnostic Laboratory, Colorado State University, Fort Collins, CO 80523, USA
3 Veterinary Laboratories Agency – Lasswade, Pentlands Science Park, Penicuik EH26 0PZ, UK
4 Moredun Research Institute, Pentlands Science Park, Penicuik EH26 0PZ, UK
5 Department of Molecular Biology, University of Wyoming, 1000 E. University Avenue, Laramie, WY 82071, USA
6 Department of Veterinary Sciences, University of Wyoming, 1174 Snowy Range Road, Laramie, WY 82070, USA
Correspondence
Lisa L. Wolfe
lisa.wolfe{at}state.co.us
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ABSTRACT |
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), a contagious prion disease of free-ranging and captive cervids, occurs in foci scattered across North America (Williams, 2005). More extensive surveillance appears essential to develop a more complete understanding of CWD distribution and occurrence, and to control its spread. Accumulation of abnormal, disease-associated prion protein (PrP) has been used as a diagnostic marker for prion disease in a variety of susceptible species, including CWD in mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), wapiti (Cervus elaphus nelsoni) and moose (Alces alces) (Spraker et al., 1997; Miller et al., 2000; Miller & Williams, 2002; Hibler et al., 2003; Williams, 2005; Baeten et al., 2007). In deer, pre-clinical CWD can be diagnosed by demonstrating the accumulation of disease-associated PrP (PrPCWD) in lymphatic or nervous tissues (Miller & Williams, 2002; Wild et al., 2002; Wolfe et al., 2002). Tonsil and retropharyngeal lymph node are favoured for use in surveillance (Miller & Williams, 2002; Hibler et al., 2003), and thus established procedures for CWD surveillance rely primarily on sampling lymphatic tissues from the heads of hunted or slaughtered deer. However, because the requisite handling and disposing of submitted heads present significant logistical and financial challenges for large-scale programmes, alternatives for simpler or more efficient sampling are needed if long-term commitments to CWD surveillance are to be sustained.
Recent studies have revealed that scrapie-associated PrP (PrPSc) is deposited in the rectoanal mucosa-associated lymphatic tissue (RMALT) of infected sheep relatively early in the disease course and that samples from this site can be used to diagnose pre-clinical scrapie (Espenes et al., 2003, 2006; González et al., 2005, 2006). Given the extensive similarities between scrapie pathogenesis in sheep and CWD pathogenesis in deer (Williams, 2005; Fox et al., 2006), we surmised that sampling of this site might also be useful in pre-clinical CWD diagnosis in deer. Here, we describe PrPCWD deposition in RMALT samples from CWD-infected mule and white-tailed deer.
To assess the utility of RMALT sampling for diagnosis of CWD infections in deer, we used 19 captive mule deer and 21 captive white-tailed deer that had been experimentally infected at about 6 months of age by oral inoculation with 1 g of conspecific, undiluted, infectious brain tissue pool (based on previous analyses, inoculum pools contained about 3 or 6 µg PrPCWD g–1; Sigurdson et al., 1999; Raymond et al., 2000); these inoculated deer were part of an ongoing study of agent shedding patterns (Colorado Division of Wildlife Animal Care and Use file 7–2004). To facilitate sampling, we anaesthetized deer with xylaine (50–100 mg) and ketamine (40–100 mg) and applied a topical analgesic cream (2.5 % lidocaine and prilocaine, Fougara Cream; E. Fougara and Co.) to the distal rectal mucosa. Sampling methods were adapted from those used by L. González and M. P. Dagleish in sheep (González et al., 2005; and unpublished data). About 10 min after applying the local anaesthetic cream, we exposed the rectal mucosal border by manually exteriorizing the anal mucosa or isolating it using a home-made speculum (M. P. Dagleish, unpublished data). Using Brown–Adson forceps, the mucosa was lifted from a depression between the rectal columns in the 0.8–1 cm immediately rostral to the transition between the anal orifice and the mucosa (muco-cutaneous junction). A small piece (5–6 mm diameter) of the elevated mucosa was cut with fine-point scissors or rectal biopsy forceps. Bleeding was controlled with direct pressure and Gel Foam (Pharmacia & Upjohn Company) applied as needed. Additional topical analgesic cream was applied directly to the biopsy sites. We collected rectal biopsies at 253 days post-inoculation (p.i.) from all deer and at 299, 342, 381, 477, 552, 661 and 751 days p.i. when previous results were negative or samples were inadequate. For comparison, we also collected tonsil biopsies at 253 days p.i. using methods described previously (Wolfe et al., 2002) and again at 342 and 477 days p.i. in individuals with negative biopsies at the preceding sampling. Tissue samples were placed in 10 % neutral buffered formalin and submitted for evaluation for PrPCWD by immunohistochemistry using methods established for tonsil biopsy (Miller & Williams, 2002; Wolfe et al., 2002). We also sampled 45 naturally exposed and 20 unexposed adult (
1.5 years old) mule deer using the methods described above. In addition to RMALT sampling, we collected blood or tissue, extracted the DNA and determined the PrP genotype using established methods (O'Rourke et al., 2004; Jewell et al., 2005).
All 19 experimentally infected mule deer had PrPCWD deposits in tonsil biopsies (termed ‘tonsil positive’) when sampled at 253 days p.i. Concurrently, we observed PrPCWD deposits in rectal mucosal biopsies (termed ‘RMALT positive’) from 15 out of 17 mule deer (88 %) where adequate samples were collected (Fig. 1); two samples were insufficient. By 381 days p.i., all 19 mule deer were RMALT positive.
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Of the 21 captive, experimentally infected white-tailed deer, 16 were tonsil positive at 253 days p.i. and nine (56 %) of those were also RMALT positive. However, the PrP genotype [encoding combinations of glycine (G) and serine (S) at codon 96, denoted 96GG, 96GS or 96SS; O'Rourke et al., 2004] appeared to influence the patterns of PrPCWD deposition in tonsil and RMALT of white-tailed deer (Table 1). Nine out of ten 96GG white-tailed deer were tonsil positive when sampled at 253 days p.i. and eight (89 %) of the nine tonsil-positive deer were also RMALT positive; all ten were tonsil positive by 342 days p.i. and nine (90 %) of these were also RMALT positive. In contrast, seven out of eight 96GS white-tailed deer were tonsil positive when sampled at 253 days p.i., but only one (14 %) of the seven tonsil-positive deer was RMALT positive; all eight were tonsil positive by 342 days p.i., but only four (50 %) of these were RMALT positive by 381 days p.i. By 751 days p.i., five of the six 96GS white-tailed deer (83 %) with useable samples were RMALT positive. The three 96SS white-tailed deer were tonsil and RMALT negative at 253 and 342 days p.i. (Table 1). At 477 days p.i., all three 96SS white-tailed deer became weakly tonsil positive, but they all remained RMALT negative up to 751 days p.i. As all mule deer in our inoculation study were homozygous for S at codon 225, we did not have an opportunity to explore whether PrP genotype [encoding S or phenylalanine (F)] might have any influence on the patterns of PrPCWD deposition in this species; however, two of the four naturally exposed deer that were tonsil positive but RMALT negative were SF at codon 225. In light of the apparent effects of PrP genotype on other aspects of CWD pathogenesis (Jewell et al., 2005; Fox et al., 2006), influences similar to those seen here in white-tailed deer would not be surprising.
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), sample quality should be considered when interpreting data from RMALT biopsies to diagnose CWD in individual deer. Overall, 138 out of 161 RMALT biopsies (86 %) had observable lymphoid follicles; based on experiences with tonsil biopsy (Wolfe et al., 2002), we anticipate that sampling consistency will probably improve with experience and minor technique improvements. Overall sampling success was similar for the two deer species: 58 out of 69 rectal biopsies (84 %) from white-tailed deer and 80 out of 92 rectal biopsies (87 %) from mule deer contained follicles. Individual variation may have contributed to problems with sample quality: six of the 15 ‘insufficient’ mule deer biopsies came from three animals. Although not studied here, age could further influence the abundance, location or appearance of true lymphoid follicles in deer: in the white-tailed deer that were serially sampled, eight out of 11 insufficient samples were from the later sampling periods, suggesting that age or repeated sampling may affect RMALT biopsy quality.
Potential variation in sample quality should be less of a consideration with post-mortem sampling, because the distal rectum can be opened for sampling and larger amounts of tissue can be collected for analysis (Espenes et al., 2003, 2006; González et al., 2005, 2006). In addition to examining biopsies from live deer, we also collected and examined rectal mucosa samples post-mortem from 48 naturally exposed mule deer to examine the correlation between PrPCWD deposits in medial retropharyngeal lymph nodes and lymphoid follicles in the rectal mucosa; 26 of these deer had PrPCWD deposits in retropharyngeal lymph node tissue whilst 22 did not. We saw agreement between immunohistochemistry of rectal mucosa and retropharyngeal lymph node in 47 of the 48 cases (
=0.96; 95 % CI 0.88–0.99); no PrPCWD was detected in the rectal mucosa of one infected deer. Moreover, when we screened independent rectal mucosa samples from 28 of these deer using an ELISA previously validated for use in CWD surveillance (Hibler et al., 2003), PrPCWD was detected in 12 of the 14 infected deer (absorbance values 0.201). Based on observations from microscopic examination of immunohistochemistry samples, failure to detect two of the 14 infected deer by ELISA seemed to be more likely to be due to sample inadequacy than to failure of the ELISA to detect PrPCWD in these samples.
Our findings support the further evaluation of RMALT sampling as an alternative to sampling lymphatic tissues of the head and neck in CWD surveillance programmes. For screening of either free-ranging or captive deer populations to detect the presence of CWD, our data show that RMALT sampling should detect a high proportion of infected individuals, particularly those in the later stages of infection; technical improvements with increased experience should also lead to better results in early infection. However, the use of RMALT sampling in CWD surveillance may be limited by the ability to acquire a sample with adequate follicles for evaluation. Additional data from naturally infected mule deer and white-tailed deer would be useful for better estimation of the sensitivity of RMALT sampling compared with cranial lymph node sampling; given such data, sample sizes or prevalence estimates could be adjusted to accommodate the somewhat lower sensitivity anticipated from our data. When the total cost of sampling and disposal from the head and neck are considered, rectal mucosal sampling testing is likely to be more economical and efficient than the existing testing system. Even taking into account the possible need to use greater numbers of rectal mucosal tests to achieve the same sensitivity as testing tissues of the head, the greater ease of sampling, cost of disposal and its suitability for rapid test analysis (L. González et al., unpublished data) suggest that rectal mucosal testing offers a more economical and efficient approach for large-scale surveillance schemes.
As a tool for assessing individual infection status for movement or management purposes, RMALT biopsy may not be as reliable as tonsil biopsy in deer, as PrPCWD appears to be deposited somewhat later in lymphoid tissues of the rectal mucosa compared with tonsil follicles; based on our data from white-tailed deer, relevant PrP genotypes probably need to be considered for both deer species in interpreting negative results. Rectal mucosal biopsy could be useful as a screening tool in captive herds or free-ranging conditions where anaesthetizing individuals for handling is either undesirable or unnecessary. However, our data show that tonsil biopsy or tonsil biopsy in addition to rectal biopsy is still preferred for reliable detection of CWD infection in individual deer.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Espenes, A., Press, C. M., Gunnes, G., Aleksandersen, M., Ulvund, M. J. & Landsverk, T. (2003). PrPSc in rectal lymphoid tissue of sheep experimentally infected with scrapie. Proceedings of the Conference on Methods for Control of Scrapie, Oslo, Norway, 15–16 May 2003, p. 47.
Espenes, A., Press, C. M., Landsverk, T., Tranulis, M. A., Aleksandersen, M., Gunnes, G., Benestad, S. L., Fuglestveit, R. & Ulvund, M. J. (2006). Detection of PrPSc in rectal biopsy and necropsy samples from sheep with experimental scrapie. J Comp Pathol 134, 115–125.[CrossRef][Medline]
Fox, K. A., Jewell, J. E., Williams, E. S. & Miller, M. W. (2006). Patterns of PrPCWD accumulation during the course of chronic wasting disease infection in orally inoculated mule deer (Odocoileus hemionus). J Gen Virol 87, 3451–3461.
González, L., Jeffrey, M., Sisó, S., Martin, S., Bellworthy, S. J., Stack, M. J., Chaplin, M. J., Davis, L. A., Dagleish, M. P. & Reid, H. W. (2005). Diagnosis of preclinical scrapie in samples of rectal mucosa. Vet Rec 156, 846–847.
González, L., Dagleish, M. P., Bellworthy, S. J., Sisó, S., Stack, M. J., Chaplin, M. J., Davis, L. A., Hawkins, S. A. C., Hughes, J. & Jeffrey, M. (2006). Postmortem diagnosis of preclinical and clinical scrapie in sheep by the detection of disease-associated PrP in their rectal mucosa. Vet Rec 158, 325–331.
Hibler, C. P., Wilson, K. L., Spraker, T. R., Miller, M. W., Zink, R. R., DeBuse, L. L., Andersen, E., Schweitzer, D., Kennedy, J. A. & other authors (2003). 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). J Vet Diagn Invest 15, 311–319.
Jewell, J. E., Conner, M. M., Wolfe, L. L. & Williams, E. S. (2005). Low frequency of PrP genotype 225SF among free-ranging mule deer (Odocoileus hemionus) with chronic wasting disease. J Gen Virol 86, 2127–2134.
Miller, M. W. & Williams, E. S. (2002). Detection of PrPCWD in mule deer by immunohistochemistry of lymphoid tissues. Vet Rec 151, 610–612.
Miller, M. W., Williams, E. S., McCarty, C. W., Spraker, T. R., Kreeger, T. J., Larsen, C. T. & Thorne, E. T. (2000). Epizootiology of chronic wasting disease in free-ranging cervids in Colorado and Wyoming. J Wildl Dis 36, 676–690.[Abstract]
O'Rourke, K. I., Baszler, T. V., Besser, T. E., Miller, J. M., Cutlip, R. C., Wells, G. A. H., Ryder, S. J., Parish, S. M., Hamir, A. N. & other authors (2000). Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue. J Clin Microbiol 38, 3254–3259.
O'Rourke, K. I., Spraker, T. R., Hamburg, L. K., Besser, T. E., Brayton, K. A. & Knowles, D. P. (2004). Polymorphisms in the prion precursor functional gene but not the pseudogene are associated with susceptibility to chronic wasting disease in white-tailed deer. J Gen Virol 85, 1339–1346.
Raymond, G. J., Bossers, A., Raymond, L. D., O'Rourke, K. I., McHolland, L. E., Bryant, P. K., III, Miller, M. W., Williams, E. S., Smits, M. & Caughey, B. (2000). Evidence of a molecular barrier limiting susceptibility of humans, cattle, and sheep to chronic wasting disease. EMBO J 19, 4425–4430.[CrossRef][Medline]
Sigurdson, C. J., Williams, E. S., Miller, M. W., Spraker, T. R., O'Rourke, K. I. & Hoover, E. A. (1999). Oral transmission and early lymphoid tropism of chronic wasting disease PrPres in mule deer fawns (Odocoileus hemionus). J Gen Virol 80, 2757–2764.
Spraker, T. R., Miller, M. W., Williams, E. S., Getzy, D. M., Adrian, W. J., Schoonveld, G. G., Spowart, R. A., O'Rourke, K. I., Miller, J. M. & Merz, P. A. (1997). Spongiform encephalopathy in free-ranging mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus nelsoni) in northcentral Colorado. J Wildl Dis 33, 1–6.[Abstract]
Wild, M. A., Spraker, T. R., Sigurdson, C. J., O'Rourke, K. I. & Miller, M. W. (2002). Preclinical diagnosis of chronic wasting disease in captive mule deer (Odocoileus hemionus) and white-tailed deer (Odocoileus virginianus) using tonsillar biopsy. J Gen Virol 83, 2629–2634.
Williams, E. S. (2005). Chronic wasting disease. Vet Pathol 42, 530–549.
Williams, E. S. & Young, S. (1980). Chronic wasting disease of captive mule deer: a spongiform encephalopathy. J Wildl Dis 16, 89–98.[Abstract]
Wolfe, L. L., Conner, M. M., Baker, T. H., Dreitz, V. J., Burnham, K. P., Williams, E. S., Hobbs, N. T. & Miller, M. W. (2002). Evaluation of antemortem sampling to estimate chronic wasting disease prevalence in free-ranging mule deer. J Wildl Manage 66, 564–573.[CrossRef]
Received 30 June 2006;
accepted 6 March 2007.
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