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From: TSS ()
Subject: Experimental Second Passage of CWD (CWDmule deer) Agent to Cattle
Date: April 25, 2006 at 8:01 am PST

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.

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Received; April 4th; 2005

Accepted; July 23rd; 2005 

Chronic Wasting Disease in Cattle 69





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