Colorado Division of Wildlife, Wildlife Research Center, 317 W. Prospect Road, Fort Collins, CO 80526, USA1
Colorado State Veterinary Diagnostic Laboratory, Colorado State University, Fort Collins, CO 80523, USA2
Department of Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA3
Animal Disease Research Unit, Agricultural Research Service, US Department of Agriculture, 337 Bustad Hall, Washington State University, Pullman, WA 99164, USA4
Author for correspondence: Margaret A. Wild.Current address: National Park Service, Biological Resource Management Division, 1201 Oak Ridge Dr., Suite 200, Fort Collins, CO 80525, USA. Fax +1 970 225 3585. e-mail margaret_wild{at}nps.gov
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Abstract |
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Introduction |
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In addition to application to post-mortem samples, IHC staining of lymphoid tissue has been used as an ante-mortem test to detect preclinical scrapie in sheep (Schreuder et al., 1998 ; ORourke et al., 2000
) and to diagnose variant CJD in humans (Hill et al., 1999
). Such a preclinical test for CWD could greatly enhance epidemiological studies and potentially aid in management activities where maintenance of live animals is indicated. The objective of this study was to determine the feasibility of using tonsillar biopsies obtained from captive mule deer and white-tailed deer (O. virginianus) as a preclinical test for CWD. Serial tonsillar biopsies collected from mule deer and white-tailed deer confirmed that PrPCWD accumulations occur prior to onset of clinical CWD and can precede CWD-related death by at least 20 months.
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Methods |
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Sample collection.
Serial tonsillar biopsies were collected from mule deer at 6 to 9 month intervals between July 1996 and January 1999 and from white-tailed deer in April 1998. Deer were anaesthetized by intramuscular administration of 5 to 12·5 mg/kg ketamine.HCl (Ketaset, Fort Dodge Animal Health) combined with 1 to 2·5 mg/kg xylazine (Cervizine, Wildlife Laboratories or Rompun, Bayer Corporation). Alternatively, deer received 4 mg/kg Telazol (Fort Dodge Animal Health) combined with 2 mg/kg xylazine. When anaesthetized, deer were blindfolded and placed in sternal recumbency for sampling. The mouth was held open with a metal mouth gag and the palatine tonsil visualized using a laryngoscope with a 30 cm blade (Jorgensen Laboratories). The biopsy was collected with a 30 cm Jackson endoscopic forceps with 4 mm cup (Sontec Instruments, Inc.). The cup of the forceps was placed in the tonsillar crypt and pressed against the ventral-medial wall to obtain one to several tissue samples. Biopsy specimens were placed in 10% buffered neutral formalin. Deer were given prophylactic antibiotics and analgesic. The mouth gag and blade of the laryngoscope were wiped with 20% bleach solution and rinsed in water. The Jackson forceps was cleaned, soaked in 20% bleach solution for 30 min, and then rinsed in water.
Deer were euthanized after showing clinical signs of CWD or, in some cases, died naturally with or without clinical signs of CWD. Brain, and in most cases tonsil, was collected and formalin fixed to determine CWD status by histology and IHC.
Immunohistochemical staining.
Biopsy specimens were processed and embedded in paraffin blocks within 10 days of collection. Tissue sections were mounted onto positively charged glass slides, deparaffinized and hydrated in preparation for IHC. Tissue treatment performed prior to IHC consisted of slide immersion in 88% formic acid solution for 5 min followed by a rinse in water. Tissue sections were then autoclaved for 12 min at 121 °C in a buffer solution (DAKO target antigen retrieval) and cooled for 30 min.
The IHC protocol employed an automated immunostainer (Ventana Medical Systems) and PrP monoclonal antibody (mAb) F99/97.6.1, a biotinylated secondary antibody, an alkaline phosphatasestreptavidin conjugate, a substrate chromagen (fast red A), and a haematoxylin and bluing counterstain (Ventana Medical Systems). mAb F99/97.6.1 binds residues 220225 of the cervid PrP protein (QYQRES) (ORourke et al., 2000 ) and has been validated for IHC staining of brain and tonsil in mule deer (Spraker et al., 2002a
). Positive and negative control deer brain and tonsil sections were included in each run. CWD-negative deer were harvested from the CWD non-endemic area (non-endemic area established by Miller et al., 2000
) and were confirmed as CWD negative by the absence of histological brain lesions and negative IHC staining for PrPCWD in brain and tonsil.
Number of follicles staining positive (Spraker et al., 2002a ) and total number of intact tonsillar follicles were determined for each section. Results were categorized into one of three interpretations based on Schreuder et al. (1998): positive (PrPCWD in any number of follicles), negative (no detectable immunostaining in at least three follicles) or inconclusive (less than three follicles in the sample with no detectable immunostaining). We refer to biopsies where positive or negative interpretation was possible as adequate samples.
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Results |
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Immunohistochemical staining
Positive staining was characterized by coarse granular, bright red material in lymphoid follicles (Fig. 1). With one exception (Table 1
, animal M92), all follicles within an individual biopsy stained uniformly either positive or negative. No staining was seen in negative control samples from deer harvested outside the CWD-endemic area.
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CWD was confirmed by post-mortem examination of brain in all mule deer (n=30) and white-tailed deer (n=3) with clinical signs indicative of CWD. All deer with confirmed CWD for which tonsil samples were available (n=28) showed positive immunostaining in tonsil tissue collected post-mortem. Two mule deer and one white-tailed deer that died or were euthanized without clinical signs of CWD were IHC positive on post-mortem examination of brain. The remaining two mule deer and one white-tailed deer were IHC negative on tonsillar biopsy and on post-mortem examination of brain.
Live animal testing
Ten mule deer had positive tonsillar IHC when first sampled (Table 1, group a) at ages ranging from 10 to 64 months. Those ten deer died or were euthanized with clinical signs of CWD 2 to 20 months after sampling (Fig. 2
, group a). CWD was confirmed post-mortem by examination of brain and tonsillar tissue in all ten of these deer.
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Six mule deer with negative biopsy samples (Table 1, group c) died or were euthanized 12 to 40 months after the last biopsy collection at ages ranging from 31 to 80 months. Death occurred 27 to 55 months after the first biopsy sampling. Two of these deer (Table 1
, C92 and J93) died due to causes other than CWD; however, CWD was confirmed in all six deer by post-mortem IHC analysis of the brain. Tonsil samples collected post-mortem from each deer were also IHC positive.
Two mule deer remained negative throughout (Table 1, group d). These deer died or were euthanized due to causes other than CWD 7 and 13 months after the first biopsy was obtained. Deer were age 11 and 98 months at death.
Of the 34 mule deer sampled, ten were members of a cohort born in 1997. These deer were evaluated by sequential sampling at 3, 10 and 19 months of age, although not all samplings yielded adequate biopsy samples. None of the six deer from which adequate tonsillar biopsies were collected at 3 months of age were IHC positive. Seven of the ten deer had at least one positive tonsillar biopsy by 19 months of age: 2/7 deer successfully sampled at 10 months and an additional 5/5 deer successfully sampled at 19 months of age were tonsillar IHC positive. The remaining three deer were test negative at 3 and 10 months and no adequate sample was collected at 19 months. One of these deer died at 11 months of age from non-CWD related causes while the remaining nine all died from CWD at ages 26 to 42 months.
White-tailed deer were 58 months of age at sampling. Four of five white-tailed deer had IHC-positive tonsillar biopsy. CWD was confirmed in all four deer at death 2 to 18 months after sampling. One of these deer had not exhibited clinical signs of CWD when it was euthanized for non-CWD related causes 18 months following a positive tonsillar biopsy. The deer that was negative on tonsillar biopsy was CWD-negative on post-mortem examination of brain when it was euthanized for non-CWD related causes 18 months after sampling.
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Discussion |
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Although biopsy collection was primarily from adult deer, one cohort of young mule deer born at this CWD-endemic facility was followed and provided insight into early exposure to CWD. All biopsies (n=6) collected at 3 months of age were negative. The youngest deer to test IHC positive were two 10-month-old mule deer. Five other mule deer tested IHC positive at 19 months of age.
PrPCWD accumulation in tonsillar biopsies was documented up to 20 months prior to death and 14 months prior to onset of clinical signs of CWD (Table 1). However, the period of preclinical lymphoid accumulation of PrPCWD in our study was likely underestimated in some cases due to the time lapse between sample collections and because we sampled primarily adult deer. Many adult deer may have had disease exposure and progression prior to initiation of biopsy sampling. In fact, 5/8 mule deer with IHC-positive tonsillar biopsies
16 months prior to death (Table 1
) were from the 1997 cohort, the only deer that we began sampling as juveniles. Therefore, reported preclinical periods should be considered minimum estimates in these captive deer.
Although use of the 4 mm Jackson endoscopic forceps was adequate for collection of biopsies in many cases, a modified technique that more reliably obtains numerous tonsillar follicles in the biopsy should be developed for highest efficiency. Availability of an affordable disposable biopsy instrument also would be useful to minimize the risk of unintentional PrPCWD transmission. Although we believed that our inactivation technique for surgical instruments was adequate, recent research underscores the difficulty in fully inactivating PrPres (Taylor, 2000 ), particularly on stainless steel surgical instruments (Zobeley et al., 1999
). Although we cannot exclude the possibility that iatrogenic transmission occurred, high rates of CWD in deer used in our study (94% in mule deer and 80% in white-tailed deer) should not be regarded as evidence that cross-contamination from the biopsy technique occurred. From 1970 to 1981, 90% of mule deer that were resident at this same facility for 2 years or longer developed CWD (Williams & Young, 1992
). In our study, the mean interval from first biopsy collection to death from CWD in deer with negative initial samples was 36 months (SD=7 months) for deer ranging in age from 3 to 128 months at sampling. In a group of non-biopsied mule deer (n=8) held at this facility concurrently, mean time from arrival at the facility to death from CWD was also 36 months (M. W. Miller, unpublished data).
Tonsillar biopsy was successful in identifying CWD-affected live deer, in most cases well before the onset of clinical signs of the disease. Although few white-tailed deer were available for biopsy, findings were consistent with those in mule deer and support similarity in lymphoid accumulation of PrPCWD between the species that has been observed post-mortem. However, because PrPCWD does not appear to accumulate in lymphoid tissue to the same degree in elk as deer (T. R. Spraker, unpublished data), the technique is not currently applicable to elk. However, where intensive management actions are possible, use of this technique will aid in the clinical diagnosis of CWD in individual captive or free-ranging deer and in epidemiological investigations of CWD in populations of mule deer and white-tailed deer.
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Acknowledgments |
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References |
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Received 26 April 2002;
accepted 3 July 2002.