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From: TSS ()
Experimental Second Passage of Chronic Wasting Disease (CWDmule deer) Agent to Cattle A. N. Hamir, R. A. Kunkle, J. M. Miller, J. J. Greenlee and J. A. Richt Agricultural Research Service, United States Department of Agriculture, National Animal Disease Center, 2300 Dayton Avenue, P.O. Box 70, Ames, IA 50010, USA Summary To compare clinicopathological findings in first and second passage chronic wasting disease (CWDmule deer) in cattle, six calves were inoculated intracerebrally with brain tissue derived froma first-passageCWD-affected calf in an earlier experiment. Two uninoculated calves served as controls. The inoculated animals began to lose both appetite and weight 10–12 months later, and five subsequently developed clinical signs of central nervous system (CNS) abnormality. By 16.5 months, all cattle had been subjected to euthanasia because of poor prognosis. None of the animals showed microscopical lesions of spongiform encephalopathy (SE) but PrPres was detected in their CNS tissues by immunohistochemistry (IHC) and rapid Western blot (WB) techniques. Thus, intracerebrally inoculated cattle not only amplified CWD PrPres from mule deer but also developed clinicalCNSsigns in the absence of SElesions.This situation has also been shown to occur in cattle inoculated with the scrapie agent. The study confirmed that the diagnostic techniques currently used for diagnosis of bovine spongiformencephalopathy (BSE) in theUS would detect CWDin cattle, should it occur naturally. Furthermore, it raised the possibility of distinguishing CWDfromBSE in cattle, due to the absence of neuropathological lesions and to a distinctive multifocal distribution of PrPres, as demonstrated by IHC which, in this study, appeared to be more sensitive than the WB technique. Published by Elsevier Ltd. Keywords: cattle; chronic wasting disease (CWD); deer; transmissible spongiform encephalopathy (TSE) Introduction Chronic wasting disease (CWD) is a fatal neurodegenerative transmissible spongiform encephalopathy (TSE), which has been identified in captive and free-ranging cervids (Williams and Young, 1992). Affected animals show accumulation of an abnormal form of prion protein (PrPres) in tissues of the central nervous system (CNS) and lymphatic system. Detection of PrPres in these tissues and characteristic histopathological changes in the brain are the basis of currently available diagnostic methods for TSEs (Hamir et al., 2001b). CWD has been experimentally transmitted by intracerebral inoculation of affected brain from mule deer into a variety of animal species, including a goat (Williams and Young, 1992). Recently, we reported results from a similar experiment in cattle (Hamir et al., 2001a, 2005a). Only 38% of the inoculated animals showed evidence of PrPres amplification, the incubation periods ranging from 2 to 5 years. Clinical signs were minimal, and the typical TSE lesions in brain were absent. Since cross-species transmission experiments provide valuable information about the biological characteristics of known TSE agents in a given species, we describe here a second passage of CWD in cattle. The primary objective of this study was to confirm and extend clinicopathological observations from the primary transmission experiment. Materials and Methods Cattle and Experimental Procedures Eight 3-month-old Jersey bull calves were obtained and assigned to inoculated (nZ6) and J. Comp. Path. 2006, Vol. 134, 63–69 www.elsevier.com/locate/jcpa 0021-9975/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.jcpa.2005.07.001 control (nZ2) groups. Inoculated calves were housed in a Biosafety Level 2 isolation barn at the National Animal Disease Center (NADC), Ames, Iowa. Husbandry of these animals has been described previously (Hamir et al., 2001b). Personnel wore protective clothing while in the isolation facility and showered before leaving. Material for inoculation was prepared from the brain of a first-passage CWD-affected animal (no. 1768) from a previous experiment in cattle (Hamir et al., 2001a). The brain material was positive for scrapie associated fibrils (SAF) by negative stain electron microscopy and for PrPres by immunohistochemical labelling (IHC) and Western blotting (Hamir et al., 2001b). Calves were inoculated intracerebrally with 1 ml of a 10% CWD brain inoculum as described by Cutlip et al. (1997). The two control calves were not inoculated. Sample Collection and Tests for PrP Animals were killed with pentobarbital and subjected to a complete necropsy. Representative samples of lung, liver, kidney, spleen, salivary gland, thyroid gland, reticulum, rumen, omasum, abomasum, intestines (ileum, colon), adrenal gland, pancreas, urinary bladder, lymph nodes (retropharyngeal, prescapular, mesenteric, popliteal), tonsil, striated muscle (heart, tongue, masseter, diaphragm), eye, sciatic nerve, trigeminal ganglion, pituitary gland and spinal cord (cervical, thoracic, lumbar) were immersion-fixed in 10% neutral buffered formalin. The brain was cut longitudinally, one-half being fixed in formalin and the remainder frozen (K20 8C). The formalin- fixed brain was cut into coronal sections, 2–4 mm wide. A minimum of 5 brain areas per animal were selected for examination by light microscopy. These were of rostral cerebrum (frontal lobe), hippocampus, midbrain (at the level of superior colliculus), cerebellum, and medulla (at the level of obex). Two sections of spinal cord at each location (cervical, thoracic, and lumbar) were selected. The tissues were processed for routine histopathology, embedded in paraffin wax, and sectioned at 5 mm. The sections were stained with haematoxylin and eosin (HE), or examined immunohistochemically for PrPres, as described previously (Hamir et al., 2004b), with a monoclonal antibody, F99/97.6.1 (O’Rourke et al., 2000). This antibody recognizes PrP sequences conserved in most mammalian species in which natural TSEs have been reported. It should be noted that the immunohistochemical (IHC) procedure used in this study, as in previous studies at this laboratory, did not incorporate a proteinase K (or any other proteinase) digestion step; however, for the purpose of simplicity of nomenclature, the term “PrPres” is used here to describe the abnormal prion within tissue sections. For immunodetection of PrPres, a rapid Western blot (WB) method described previously was used on frozen brain (midbrain and brain stem) tissue (Schaller et al., 1999). The antibody used in this technique, namely 6H4 was obtained from Prionics (Prionics-Check, CH-8952, Schlieren, Switzerland). Results Clinical Signs and Microscopical Findings Between 10 and 12 months after inoculation, cattle gradually lost appetite and started to lose weight. By the time of euthanasia (14.3–16.5 months after inoculation) they had lost at least 20% of bodyweight and became obviously weak. All but one of the inoculated animals (599) showed abnormal neurological signs, commencing 1 to 5 months after the onset of anorexia. The neurological signs, which were intermittent, included circling (usually to the left) in three animals, grinding of teeth in two, head pressing in one, depression and nonresponsiveness to external stimuli in two. The cattle often appeared depressed or non-responsive when undisturbed, but reacted to loud noises either by exaggerated posturing or by collapsing to lateral recumbency. Ultimately, all of the animals became recumbent and unable to rise without assistance. Within 16.5 months of intracerebral inoculation, all inoculated animals were killed to avoid unnecessary deterioration. The two control animals were killed at 10 and 11 days after euthanasia of the last inoculated animal. Microscopical examination of HE-stained sections of brain and spinal cord (cervical, thoracic and lumbar) failed to reveal lesions of spongiform encephalopathy in any of the experimental animals. A few isolated neurons with single, clear vacuoles of variable size were seen in the red nucleus of four inoculated animals (593, 595, 590 and 589). Also, the central canal in the caudal medulla of animal 593 showed a focal area of protrusion of neuropil with some glial cell infiltrations into the canal (Fig. 1). Neither increased gliosis nor degenerate neurons were seen in central nervous system (CNS) tissues. Significant lesions, apart from a few sarcocysts in striated muscles, were not observed in any of the animals. A.N. Hamir et al. 64 IHC and WB Findings The distribution and amount of PrPres IHC labelling within neuronal tissues is summarized in Table 1. There was a consistent pattern of multifocal labelling in brains of all six inoculated cattle (Figs. 2,3). In some tissues, particularly the midbrain, the foci were so numerous and large that they formed large confluent areas (Fig. 3). The labelled foci were located primarily in grey matter but sometimes also appeared in white matter, especially in the rostral cerebrum. Within a focus, the labelled particles occurred individually, in clumps, and occasionally in larger aggregates. They often appeared to be associated with glial cells, either in the cytoplasm or at the cell surface (Fig. 4). The most severely affected tissue was midbrain (Table 1), but the medulla typically showed many labelled foci. Immunoreactivity was less apparent in the hippocampus and rostral cerebrum. Labelling was apparently absent in the cerebellum of four cattle and in the other two inoculated animals was confined to a few small foci in white matter. IHC labelling in spinal cord sections was minimal and, when present, was usually restricted to the cervical cord. No non- CNS tissue, including lymphoid tissues, retina, Gasserian ganglion and pituitary gland showed any immunolabelling (Table 1). Immunoreactivity was absent within neuronal cytoplasm and at perineuronal, and perivascular locations. WB analysis was performed on samples from the brainstem and midbrain. In brainstem, only three samples (animals 593, 596, 599) were positive by this method whereas samples from the midbrain area of CWD-inoculated animals invariably gave a positive result (Fig. 5), albeit in one animal (590) weak. Samples from the midbrain area of control animals, however, remained negative (Fig. 5). Positive samples showed the typical profile of three bands of proteinase K-resistant isoforms of PrPres, representing the di-glycosylated, monoglycosylated and unglycosylated polypeptides. Comparison of these results from the second cattle passage with those from the first cattle passage revealed no obvious differences in the molecular weight between the three isoforms of PrPres (data not shown). It should be noted, however, that the unglycosylated isoform of PrPres has a similar staining intensity to that of the mono-glycosylated isoform. This is in contrast to typical bovine spongiform encephalopathy (BSE), in which the unglycosylated isoform shows the weakest staining Fig. 1. Brain (caudal medulla) of animal 593. The central canal is partly blocked and distorted. The blockage consists of chronic fibrous inflammation, with a multinucleated giant cell (arrow). HE. !250. Table 1 Immunohistochemical demonstration of PrPres in tissues of second-passage CWD-affected cattle Ear tag no. Immonolabelling of PrPres in brainstem cerebellum midbrain hippocampus cerebrum cervical sc thoracic sc lumbar sc 590 C – CC – – C – – 593 CC – CC CC C – – – 599 CC C CCC CC C C C C 596 C – CC C – – – – 589 CC – CCC CCC CC C C C 595 C C CCC C C C C C 695 – – – – – – – – 694 – – – – – – – – C, Minimal; CC, moderate; CCC, extensive. sc, Spinal cord. Retina, trigeminal ganglion and pituitary gland were invariably negative. Chronic Wasting Disease in Cattle 65 intensity and the di-glycosylated polypeptide is the most prominent of all three isoforms (Collinge et al., 1996). Discussion CWD, like all other TSEs, is characterized by a long incubation period, which in deer is seldom less than 18 months (Williams and Young, 1992). In an experimental study of cattle inoculated intracerebrally with CWD from mule deer (first passage), amplification of PrPres was demonstrated in only five of 13 (38%) cattle, after incubation periods that ranged from 23 to 63 months (Hamir et al., 2001a, 2005a). In contrast, all inoculated cattle in the present study were positive for PrPres within 16.5 months. This increased attack rate with shorter incubation periods probably indicates adaptation of the CWDmule deer agent to a new host. It could also be argued that the inoculum used for the primary passage (Hamir et al., 2001a, 2005a) had a lower infectivity titre than that used for the second passage. However, the former successfully transmitted CWD to each of five white tailed deer within two years of intracerebral inoculation (Kunkle et al., Unpublished). In cervids, clinical CWD is characterized by emaciation, changes in behaviour, and excessive salivation (Williams and Young, 1992). Although the latter was not observed in the CWD inoculated cattle, all animals showed anorexia and considerable weight loss. Five cattle also showed intermittent neurological signs. Although none of these animals showed histopathological changes in the brain, all were shown to be positive for PrPres by the IHC and WB methods. The presence of isolated vacuoles in the red nucleus is regarded as an incidental finding in cattle (McGill and Wells, 1993). The uniform susceptibility, relatively short incubation, and absence of microscopical lesions in cattle given CWD brain material passaged once through cattle resembled findings in cattle inoculated intracerebrally with the scrapie agent (Cutlip et al., 1997). In that experiment, 100% of cattle died 14–18 months after inoculation with material from the first cattle-passage of a US strain of the scrapie agent; none showed microscopical lesions and all were positive for PrPres. In the present experiment, the possibility that the PrPres seen in tissue sections represented residual CWD material from the inoculum was ruled out because of the multifocal distribution of Fig. 2. Brain (rostral cerebral cortex) of animal 589. There are four foci (three large and one small) of PrPres. IHC labelling. !80. Fig. 3. Brain (midbrain) of animal 589. There are numerous foci of PrPres. IHC labelling. !80. A.N. Hamir et al. 66 PrPres throughout the brain (excluding cerebellar folia) and cervical spinal cord of most of the affected animals. Had the PrPres represented residual inoculum, it would probably have been confined to the sites of deposition in the midbrain or cerebrum. Moreover, in studies on sheep scrapie, Hamir et al. (2002) showed that intracerebrally inoculated brain material containing PrPres was present for only a few days in sufficient quantity to be detectable immunohistochemically. The present work confirms previous observations that PrPres IHC labelling in cattle inoculated with the mule deer CWD agent is multifocal and glial cell-associated. This unusual pattern was first reported in descriptions of the primary CWD transmission to cattle (Hamir et al., 2001a, 2005a), and the study described here showed that it was maintained through the second passage in cattle. Further studies now in progress will determine whether this feature also characterizes CWD transmission to cattle fromother cervid species other than mule deer, namely, white tailed deer and elk. In this and an earlier study of CWD in cattle (Hamir et al., 2001a), IHC labelling differed from that seen in cattle with BSE or experimental transmissible mink encephalopathy (TME), both of which are associated with widespread diffuse labelling of grey matter neuropil, with labelled particles that are not obviously cell-associated except occasionally at neuronal cell membranes (Wells and Willsmith, 1995; Hamir et al., 2005a). The IHC pattern in bovine CWD also contrasts markedly with that seen in scrapie-inoculated cattle, in which intracytoplasmic labelling of neurons is a prominent feature (Cutlip et al., 1994, 1997). When brainstems of CWD-infected cattle were analysed by WB for the presence of PrPres, only three of six samples were found to be positive (Table 1). In contrast, all samples from the midbrain area were positive by this technique (Table 1; Fig. 5). It was noteworthy, however, that both brainstem and midbrain sections of all animals infected with CWD gave positive IHC results (Table 1) and a positive WB was associated with strong IHC labelling. This may indicate that the IHC procedure is more sensitive than the WB method for cattle-passaged CWD. However, given the multifocal nature of PrPres distribution in the CNS of CWD-infected cattle, this result is not surprising. WB analysis requires a small sample of brain tissue (e.g. 0.2 g, as in the present study) to produce a 10% homogenate; approximately 10 ml (1 mg brain tissue equivalent) of this homogenate are loaded on to an SDS-PAGE gel for further Fig. 4. Brain (midbrain) of animal 589. Higher magnification of one focus of labelled PrPres from Fig. 3. Note the absence of PrPres within neurons (arrows). IHC labelling. !160. Fig. 5.Western blot analysis with monoclonal antibody 6H4 showing distinct profile of PrPres in the five clearly positive animals (589, 593, 595, 596 and 599). Animal 590 is weakly positive. No specific signal is seen in animals 694 and 695, both non-inoculated control animals and classified as WB-negative. Molecular weight markers in kDa are indicated on the right side of the blot. Chronic Wasting Disease in Cattle 67 analysis. Bearing in mind the multifocal pattern of PrPres distribution, the brain tissue used for the preparation of WB homogenate, unlike the large amount examined in the IHC procedure, might well contain few if any foci of PrPres deposition, whereas the larger piece of tissue section used for IHC may contain detectable PrPres. In this respect, therefore, the IHC method would seem preferable to the WB procedure and to other procedures (e.g. ELISA-based tests) in which only small amounts of tissue are used for analysis. In comparison with experimental TME in cattle (Hamir et al., 2005b), the experimental bovine CWD in this study was associated with less extensive IHC labelling in non-CNS (i.e. other than brain and spinal cord) neural tissues. Whereas the retina was positive in all cattle inoculated with TME, none of the CWD-infected cattle in this experiment had any retinal labelling. Similarly, in the present study there was no labelling in the pituitary gland, a tissue sometimes positive in TME-infected cattle. Because the incubation time for second passage CWD transmission (mean of 468 days) was only slightly longer than for TME (mean of 430 days), it seems likely that these different tissue affinities reflect a biological difference between these two TSE agents. PrPres IHC labelling was not observed in striated muscles (heart, tongue, masseter, diaphragm) of the experimental animals. This observation accorded with our previous findings (Hamir et al., 2004a) in which striated muscle tissues from 20 animals (cattle, sheep, elk and raccoons) were examined for PrPres. In these animals, all of which had developed a TSE after experimental inoculation, PrPres was found by IHC examination in the brains, but not in muscle tissues. However, recent investigations with an enriched WB technique (Mulcahy et al., 2004) have enabled us to detect PrPres in the tongues of some sheep and elk experimentally inoculated with scrapie and CWD, respectively. This technique failed, however, to detect PrPres in cattle inoculated with CWD or TME (Bessen et al., unpublished). This study is still in progress, and the tongues of TSE-infected animals are currently being tested after careful removal from the carcasses to ensure non-contamination with infected brain material. The present study and a previous experiment (Hamir et al., 2005a) have established the biological characteristics of the CWDmule deer agent in cattle. However, isolates of CWD from other cervids (e.g. CWDwhite-tailed and CWDelk) may differ. Transmission experiments with different CWD isolates are therefore needed to examine the possibility of variation in the CWD agent in wild cervids. Such experiments have recently been initiated at the National Animal Disease Center (NADC). Acknowledgments We thank Dr Katherine I. O’Rourke for providing the antibody for the IHC procedure. Martha Church, Kevin Hassall, Dennis Orcutt, Jean Donald, Sharla Van Roekel, and animal handlers at the NADC provided expert technical assistance. This study was carried out under the guidelines of the institutional Animal Care and Use committee at NADC. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture. References Collinge, J., Sidle, K. C. L., Meads, J., Ironside, J. and Hill, A. F. (1996). Molecular analysis of prion strain variation and the aetiology of ‘new variant’ CJD. Nature, 383, 685–690. Cutlip, R. C., Miller, J. M., Race, R. E., Jenny, A. L., Katz, J. B., Lehmkuhl, H. D., DeBey, B. M. and Robinson, M. M. (1994). 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