Published Ahead of Print 12 December 2012. 2013, 87(4):1947. DOI: 10.1128/JVI.02592-12. J. Virol. 

Hayes-Klug and Edward A. HooverSusan L. Kraft, Kevin Carnes, Kelly R. Anderson, Jeanette Candace K. Mathiason, Amy V. Nalls, Davis M. Seelig, Wasting DiseaseSusceptibility of Domestic Cats to Chronic

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Susceptibility of Domestic Cats to Chronic Wasting Disease

Candace K. Mathiason,a Amy V. Nalls,a Davis M. Seelig,a Susan L. Kraft,b Kevin Carnes,b Kelly R. Anderson,a Jeanette Hayes-Klug,a

Edward A. Hoovera

Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USAa; Department of Environmental and Radiological HealthSciences, Colorado State University, Fort Collins, Colorado, USAb

Domestic and nondomestic cats have been shown to be susceptible to feline spongiform encephalopathy (FSE), almost cer-tainly caused by consumption of bovine spongiform encephalopathy (BSE)-contaminated meat. Because domestic andfree-ranging nondomestic felids scavenge cervid carcasses, including those in areas affected by chronic wasting disease(CWD), we evaluated the susceptibility of the domestic cat (Felis catus) to CWD infection experimentally. Cohorts of 5cats each were inoculated intracerebrally (i.c.) or orally (p.o.) with CWD-infected deer brain. At 40 and 42 months postin-oculation, two i.c.-inoculated cats developed signs consistent with prion disease, including a stilted gait, weight loss, an-orexia, polydipsia, patterned motor behaviors, head and tail tremors, and ataxia, and the cats progressed to terminal dis-ease within 5 months. Brains from these two cats were pooled and inoculated into cohorts of cats by the i.c., p.o., andintraperitoneal and subcutaneous (i.p./s.c.) routes. Upon subpassage, feline CWD was transmitted to all i.c.-inoculatedcats with a decreased incubation period of 23 to 27 months. Feline-adapted CWD (FelCWD) was demonstrated in the brainsof all of the affected cats by Western blotting and immunohistochemical analysis. Magnetic resonance imaging revealedabnormalities in clinically ill cats, which included multifocal T2 fluid attenuated inversion recovery (FLAIR) signal hyper-intensities, ventricular size increases, prominent sulci, and white matter tract cavitation. Currently, 3 of 4 i.p./s.c.- and 2 of4 p.o. secondary passage-inoculated cats have developed abnormal behavior patterns consistent with the early stage of fe-line CWD. These results demonstrate that CWD can be transmitted and adapted to the domestic cat, thus raising the issueof potential cervid-to-feline transmission in nature.

Trans-species transmission of prion diseases is unpredictableand remains poorly understood, yet has obvious public health

implications. These facts drive continued appraisal of presentconcepts regarding both transmissible spongiform encephalopa-thy (TSE) spread and intervention strategies. It is clear that variantCreutzfeldt-Jakob disease (vCJD) (1) and feline spongiform en-cephalopathy (FSE) (2) can be attributed to the ingestion of meatand bone from bovine spongiform encephalopathy (BSE)-in-fected cattle. Transmissible mink encephalopathy (TME) in theUnited States is also attributed to the consumption of scrapie- orBSE-infected meat products (3). It has been hypothesized thatsheep scrapie may have crossed the species barrier from scrapie-infected sheep to cattle as BSE, and possibly from sheep to deer aschronic wasting disease (CWD) (4), yet this remains far from cer-tain, and spontaneous generation cannot be excluded. Thus, thereis accruing evidence for the trans-species transmission of prions,with potentially grave consequences for animals and humans.

FSE, the fatal TSE of domestic and nondomestic cats, was firstdescribed shortly after the BSE epidemic (5) and strain typed to bethe result of the consumption of BSE-contaminated beef (1). Thisprovided support for the adaptation and transmission of orallyconsumed prions, as well as evidence that felids are susceptible toprion infection. Chronic wasting disease has been shown to bereadily transmitted via multiple means, including direct contactwith a CWD-infected cervid (6–8), exposure to infectious CWDprions in the environment (9, 10), and ingestion of bodily fluidsand tissues from a CWD-infected cervid (11–15). Recent researchby Miller and colleagues (16) performed in an area of Coloradowhere CWD is endemic, where nearly one-fourth of the adultmule deer are prion infected, confirmed the assumption that af-flicted animals become prey to mountain lions more often than

healthy ones. In addition, experimental studies have demon-strated that prions infect and cause disease in several rodent scav-enger species (i.e., voles and deer mice) overlapping in distribu-tion with CWD and scrapie outbreaks (17, 18). Such scavengingrodents are known to cannibalize and are also an important foodsource for many larger predators and scavengers in nature, henceproviding a reservoir and a potential bridge for cross-speciestransmission of prions from the natural host to predator/scaven-ger species.

Thus, the facile transmission of CWD, coupled with its grow-ing geographical distribution (19) and propensity to persist in theenvironment (20–22), provide ample opportunity for the bio-availability of CWD prions to many species coexisting with CWDin nature, including felids.

We undertook this study to determine (i) whether felids aresusceptible to deer origin CWD prion infection and disease pro-gression and (ii) whether disease course and transmission effi-ciency are altered upon subsequent passage of feline-adaptedCWD in cats.

Received 21 September 2012 Accepted 4 December 2012

Published ahead of print 12 December 2012

Address correspondence to Candace K. Mathiason, candace.mathiason@colostate.edu.

C.K.M. and A.V.N. contributed equally to this article as first authors.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.02592-12

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MATERIALS AND METHODSDomestic cats, genotyping, and prion protein sequence comparison.Four-month-old domestic cats were provided by the Andrea D. Lau-erman specific-pathogen-free (SPF) colony, Department of Microbi-ology, Immunology and Pathology (DMIP), College of VeterinaryMedicine and Biological Sciences (CVMBS), Colorado State Univer-sity (CSU).

All animals were handled in strict accordance with good animalpractice, as defined by relevant national and/or local animal welfarebodies, and all animal work was approved by Colorado State Univer-sity Animal Care and Use Committee (ACUC approval no. 02-151A,08-175A, and 11-2622A). All cats were genotyped to determine theprion protein gene (PRNP) coding sequence in the laboratory of Wil-fred Goldmann, The Roslin Institute, University of Edinburgh, Scot-land, United Kingdom.

Prion protein amino acid sequences were recovered from GenBank forthe domestic cat (ACA50727.1) and 8 other mammalian species, includ-ing mountain lions (ACA50739.1), bobcats (ACA50731.1), Canadianlynx (ACA50730.1), white-tailed deer (AAP37447.1), mule deer(AAC33174.2), elk (AAC12860.2), cattle (CAA39368.1), and humans(BAG32276.1). The sequences were aligned and pairwise identities calcu-lated using default parameters for the MUSCLE algorithm (multiple se-quence comparison by log expectation) in the Geneious v.5.6 (Biomatters,Ltd.) software.

Biocontainment protocols. Protocols to preclude extraneous expo-sure and cross-contamination between cohorts of animals as previouslydescribed (12) included protective shower-in requirements, Tyvek cloth-ing, masks, head covers, and footwear, while maintaining stringent hus-bandry. Tissues were harvested with sample-specific instruments to min-imize possibility of cross-contamination. Solid and liquid waste from eachsuite was either incinerated or collected and denatured by alkaline diges-tion.

Inocula. (i) Primary passage DeerCWD. Deer origin CWD (DeerCWD)brain inoculum that was confirmed as CWD positive by immunohisto-chemistry (IHC) was from a free-ranging mule deer (989-09147) pro-vided by Terry Spraker at the Colorado State Veterinary Diagnostic Lab-oratory (CSVDL), which was utilized in previous bioassay studies (12).The CWD-negative brain inoculum for sham controls was sourced fromtwo white-tailed deer (UGA 1 and 2) born and raised in a CWD-free areaprovided by the Warnell School of Forestry and Natural Resources, Uni-versity of Georgia, Athens (UGA).

(ii) Secondary passage FelCWD. Inocula for feline-adapted CWD(FelCWD) secondary passage studies consisted of pooled brain sectionsfrom two primary passage cats (4152 and 4137) confirmed as CWD pos-itive by Western blotting and IHC or one primary passage CWD-negativesham-inoculated cat (4149).

Inoculation cohorts. Cohorts of 2 to 5 4-month-old SPF domesticcats were kept in separate group housing suites throughout the study.Suite-specific and dedicated protective clothing, utensils, and waste dis-posal were utilized to exclude cross-contamination by fomites, bedding,food, excretions, or contact.

Primary passage. Cohorts of 5 SPF domestic cats were inoculated withDeerCWD brain either intracerebrally (i.c.; 0.5 g into the left parietal cor-tex) or orally (p.o.; a total of 1 g administered by syringe over 2 consecu-tive days at 0.5 g/day). Age-matched sham controls were inoculated in thesame manner with brain from CWD-negative deer.

Secondary passage. FelCWD brain pool was inoculated by multipleroutes into cohorts of SPF domestic cats: i.c. (0.5 g into the left parietalcortex; n � 5), p.o. (a total of 1 g syringe administered over 2 consecutivedays at 0.5 g/day; n � 4), or intraperitoneally and subcutaneously (i.p./s.c.) (0.25 g each; n � 4). Two cats were cohoused with the i.c.-inoculatedcohort to monitor for horizontal/environmental shedding and transmis-sion of disease-causing prions. As lingual abrasions have been shown tofacilitate prion transmission following oral exposure (23, 24), two abra-sions (2 cm in length) were made on the dorsal and ventral aspects of the

tongue by lightly scratching the surfaces with a 20-gauge needle prior tosecond-passage p.o. inoculations.

Monitoring and sample collection. Following inoculation, cats weremonitored daily for behavioral changes and euthanized at or before onsetof late-stage clinical signs. At study termination, bodily secretions (bloodand saliva), excreta (urine and feces), and tissues from each cat wereharvested, fixed, and/or frozen for the detection of FelCWD.

Western blotting. Tissue homogenates were prepared from the obexregion of the medulla oblongata at 10% (wt/vol) in NP-40 buffer (10 mMTris-HCl buffer [pH 7.5], 0.5% NP-40, 0.5% sodium deoxycholate) in aFastPrep FP120 cell disrupter (Qbiogene) with one 45-s cycle at a speedsetting of 6.5 and then cooled on ice. Homogenates were mixed withproteinase K (PK) (Invitrogen) at a final concentration of 50 �g/ml andincubated at 37°C for 30 min with shaking. Samples were mixed with 10�reducing agent– 4� lithium dodecyl sulfate (LDS) sample buffer (Invit-rogen) at a final concentration of 1�, heated to 95°C for 5 min, and thenrun through a NuPAGE 10% Bis-Tris gel at 100 V for 2.5 h. Proteins weretransferred to a polyvinylidene fluoride (PVDF) membrane at 100 V for 1h in transfer buffer (0.025 M Trizma base, 0.2 M glycine, 20% methanol[pH 8.3]). The membrane was loaded into a prewetted SNAP i.d. blotholder (Millipore) and then placed in the SNAP i.d. system (Millipore).Blocking buffer (Blocker casein in Tris-buffered saline [TBS] [ThermoScientific] with 0.1% Tween 20) was added to the blot holder for 10 min,followed by vacuum removal. Primary antibody BAR224 (CaymanChemical) conjugated to horseradish peroxidase (HRP) was diluted inblocking buffer to 0.2 �g/ml, added to the blot holder, incubated for 12min, and then vacuumed through the membrane. The membrane waswashed three times with 30 ml of wash buffer (50% Blocker casein in TBS,50% 1� TBS, 0.1% Tween 20) with continuous vacuum and then devel-oped with ECL Plus enhanced chemiluminescence Western blotting de-tection reagents (GE) and viewed on a Luminescent image analyzer LAS-3000 (Fujifilm).

IHC. Tissues were fixed in 10% neutral buffered formalin or para-formaldehyde-lysine-periodate (PLP) for 5 days or 2 days, respec-tively, and stored in 60% ethanol. Tissues were treated for 1 h with 88%formic acid, rinsed with tap water, and embedded in paraffin, and6-�m sections were placed on positively charged slides. Deparaffinizedand rehydrated sections were PK digested (20 mg/ml) at 37°C in 1�TBS for 15 min, washed for 10 min in TNT buffer (0.1 M Tris-HCl [pH7.5], 0.15 M NaCl, 0.05% Tween 20), and subjected to heat-inducedepitope retrieval in the 2100-Retriever (Prestige Medical) in sodiumcitrate buffer (0.01 M sodium citrate, 0.05% Tween 20 [pH 6.0]). Atyramide signal amplification (TSA) detection kit (PerkinElmer) wasused to enhance the signal, as described in previous work (25), usingthe anti-prion protein antibody L42 (R-Biopharm) at 0.077 �g/ml andEnvision� anti-mouse HRP-labeled polymer (Dako). Following TSAtreatment, slides were incubated with 3-amino-9-ethylcarbazole(AEC) substrate-chromogen (Dako), counterstained with hematoxy-lin and bluing reagent (0.1% sodium bicarbonate), and coverslippedwith an aqueous mounting medium (Vector Laboratories).

RT-QuIC. Real-time quaking-induced conversion (RT-QuIC), as firstdescribed by Atarashi (26), Wilham (27), and Orru (28), was used toassess prion-converting activity in brain tissue from all terminated cats.Serial dilutions (10�2 to 10�9) of tissue homogenates or “seeds” wereprepared from the obex region of the medulla oblongata in seed buffer(0.1% SDS, 1� phosphate-buffered saline [PBS], 1� N2 medium supple-ment [Gibco]). Positive and negative assay controls included serial dilu-tions (10�2 to 10�9) of naïve and CWD-infected white-tailed deer and catobex. Reaction mixtures, set up in duplicate in 96-well black bottom opticplates (Nalgene Nunc), consisted of 2 �l seed in 98 �l real-time quaking-induced conversion (RT-QuIC) buffer (final concentrations of 1� PBS,300 mM NaCl, 1 mM EDTA, 10 �M thioflavin T [ThT], 0.1 mg/ml re-combinant hamster PrPC substrate [with glycerol stocks generously pro-vided by Byron Caughey, NIH]). Plates containing reaction mixtures wereplaced in a BMG Fluostar plate reader at 42°C with cycles of 1 min of

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shaking and 1 min at rest and with ThT fluorescence measurements takenat 15-min intervals for 50 h.

MRI. Anatomic brain magnetic resonance imaging (MRI) scans, in-cluding proton density, T2-weighted, fluid attenuated inversion recovery(FLAIR), and T1-weighted (pre- and postcontrast) scans were performedon 10 i.c.-inoculated cats: 6 clinically ill (1 primary passage, 5 secondarypassage), 2 asymptomatic (primary passage), and 2 age-matched shamcontrols, as well as 1 uninoculated age-matched control. MRI was donewith a Signa LX 1.5 T MR HiSpeed Plus system (General Electric), aspreviously described (29). Briefly, anesthetized cats were positioned ster-nally with the head centered within the quadrature head coil. The pituitaryfossa was centered in the imaging coil, and laser light localization was usedto standardize a consistent longitudinal orientation along the z-axis. Theanatomic brain MRI scan included transverse dual fast-spin echo protondensity/T2-weighted scan (repetition time [TR] � 4,000 ms, echo time[TE] � 17/115 ms, echo train length � 16, slice thickness � 3 mm, imagematrix � 256 � 256), T2 FLAIR (TR � 8,125 ms, TE � 120, inversiontime [TI] � 2,200 ms), and T1-weighted scans (TR � 550 ms, TE �minimal) pre- and postcontrast after intravenous diethylenetriaminepen-taacetic acid (gd-DTPA; Magnevist) at 0.5 mmol/kg (Bayer Pharmaceu-ticals). Diffusion-weighted imaging (DW-MRI) was performed with bfactor 2000 using TE � 90 to 110 ms, TR � 8,000 ms, field of view(FOV) � 24 mm, slice thickness � 5 mm, image matrix � 124 � 124, anda number of excitations (NEX) of 4. All MRI pulse sequences were re-viewed qualitatively for gross lesions.

RESULTSPRNP genotypes and sequence comparison. No single-amino-acid polymorphisms were detected in any of the 39 inoculatedcats. Four cats were heterozygous for the nonapeptide repeats inthe N terminus: 1 i.c. DeerCWD inoculate (clinical disease at 40months postinfection [p.i.]; cat 4152) and 2 sham controls fromthe primary passage cohort (cats 4154 and 4151), as well as 1s.c./i.p. FelCWD inoculate (no clinical disease at 40 months p.i.; cat4338) from the secondary passage cohort (Table 1).

The alignment and pairwise analysis of the PRNP aminoacid sequences of domestic cats, bobcats, Canadian lynx,white-tailed deer, mule deer, elk, cattle, and humans (Table 2;see Fig. 5) revealed that pairwise genetic identity for these spe-cies ranges from 84% (humans and mountain lions) to 100%(white-tailed deer and mule deer). The domestic cat PRNPshares 94.9 to 99.1% homology with nondomestic cats and 92.1to 92.5% homology with cervid species. While an octarepeatdeletion (amino acids [aa] 69 to 77) was identified in themountain lion sequence, the PRNP amino acid sequences fordomestic and nondomestic cats are highly homologous com-pared to those of the other 5 species analyzed. For comparison,we analyzed the PRNP amino acid sequence for species inwhich successful cross-species prion transmission has beendocumented— cattle to humans (87.9% homology) and cattleto domestic cats (87.7% homology).

Clinical signs of TSE. (i) Primary passage of DeerCWD intodomestic cats. Project-dedicated caretakers intimately familiarwith each animal observed the cats daily. Subtle clinical signs con-sistent with TSE, including a stilted gait, weight loss, anorexia,polydipsia, patterned motor behaviors, head and tail tremors, andataxia, were detected 40 to 42 months p.i. in 2/5 primary passagei.c.-inoculated cats, which ultimately mandated euthanasia 45 to47 months p.i. (Table 1). Of the 3 remaining i.c.-inoculated cats, 1was euthanized at 25 months p.i. for disease screening, while 2remained asymptomatic for TSE disease and were euthanized at86 months p.i. The p.o.-inoculated cohort did not develop signs of

TSE disease (0/5); 2/5 were euthanized at 19 and 25 months p.i.,and the remaining 3 were euthanized at 85 months p.i. to permitexamination for FelCWD. No cats receiving naïve deer brain devel-oped TSE disease.

(ii) Secondary passage of FelCWD into domestic cats. Five of 5secondary passage i.c.-inoculated cats developed slowly progress-ing signs of TSE sooner than the primary passage cats, at 20 to 24mo p.i., with some individuals displaying additional signs of ag-gressiveness and hyperreactivity. Symptomatic i.c.-inoculated catswere euthanized 23 to 27 months p.i. (Table 1). Currently (40months p.i.), 3/4 i.p./s.c.- and 2/4 p.o.-inoculated cats are showingearly TSE signs, including a stilted gait, head and tail tremors, andhyperreactivity. All remaining cats (i.p./s.c. or p.o. inoculated andcohoused) will continue to be monitored for further developmentof TSE disease.

Detection of FelCWD in tissues at necropsy. (i) Primary pas-sage of DeerCWD into domestic cats. At necropsy, 2/5 i.c.-inocu-lated symptomatic cats were prion positive, as indicated by West-ern blotting, IHC, and RT-QuIC detection of FelCWD in themedulla oblongata at the level of the obex. This PK-resistant ma-terial had a slightly lower molecular mass than CWD-infecteddeer brain homogenate (Fig. 1A). IHC revealed small amounts ofFelCWD immunostaining, primarily within neurons (Fig. 2A).Neuroanatomically, the FelCWD deposits were confined to nucleiof the caudal brain stem, including the dorsal motor nucleus of thevagus and the nucleus of the solitary tract. Morphologically, theFelCWD deposits were finely granular and intraneuronal. No evi-dence of FelCWD was found in animals that did not develop clinicaldisease.

(ii) Secondary passage of FelCWD into domestic cats. Westernblot analysis of the medulla oblongata at the level of the obexregion of all five i.c.-inoculated cats revealed PK-resistant materialwith a molecular mass signature similar to that of primary passagecats (Fig. 1A). While antibody BAR224 recognized both deer andcat origin PrP, monoclonal antibody (MAb) 3F4 recognizedonly cat origin PrPs (Fig. 1). Morphologically, in contrast to theinitial passage cats, in which mild, intraneuronal, and finely gran-ular FelCWD deposits dominated, the FelCWD deposits in the obexregions of the second passage cats were (i) more extensive, (ii)more heterogenous (finely granular, coarsely granular, andclumped), and (iii) identified in multiple cellular patterns (intra-neuronal, perineuronal, and linear) (Fig. 2).

Detection of prion-converting activity by RT-QuIC. PrPC

converting activity, analyzed by RT-QuIC of brain tissue, wasdetected in 7/7 i.c.-inoculated cats that developed prion disease(Fig. 3), providing 100% specificity and 92% sensitivity com-pared to Western blotting and IHC analysis. (A total of 13/14duplicates were positive in cats that were positive by Westernblotting and IHC.) RT-QuIC did not detect prions in CWD-inoculated cats that tested Western blot and IHC negative (3i.c.-inoculated and 5 p.o.-inoculated primary passage cats) orin sham-inoculated cats.

MRI detects abnormalities in clinically ill cats. Abnormalitieswere detected in 4/6 i.c.-inoculated clinically ill cats (1/1 primarypassage and 3/5 secondary passage) (Fig. 4). The abnormalities in1 DeerCWD i.c.-inoculated cat (4137) consisted of small sparse T2and T2 FLAIR signal hyperintensities associated with the whitematter of the internal capsule and minimal lateral ventricular en-largement. The multifocal T2 hyperintensities in this cat lackedmass effect or contrast enhancement. Two FelCWD i.c.-inoculated

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cats (4346 and 4357) had asymmetric lateral ventricle enlarge-ment, one (4346) presenting with a focal dilation of the ventricu-lar extension into the olfactory bulb. These changes are consistentwith ex vacuo ventricular enlargement and probable loss of brainvolume, since there was otherwise a lack of mass effect or signs ofventricular obstruction. One cat (4341) had a small linear cere-

brospinal fluid (CSF)-filled cavitation involving a portion of thecorona radiata of the parietal cerebrum, consistent with previousbrain degeneration or other form of insult. Three cats (4137, 4156,and 4341) had mild subjective ventricular and sulcal prominencesthat could accompany early brain atrophy. Scans of three age-matched controls were normal.

TABLE 1 Study cohorts, design, and outcome of cats inoculated with CWDa

CohortAnimalno.

Inoculationroute

Genotypecodon 57

MPI:

Qualitative MRIanalysis result

Age (mo)at MRI

To early/late stageof TSE disease At euthanasia

Primary passageDeerCWD brain 4137 i.c. TC 40/45 45 Abnormal 56

4142 TT � 25 ND ND4146 CC � 86 Normal 814152 CND 42/47 47 ND ND4156 TC � 86 Abnormal 804133 p.o. CC � 85 ND ND4143 TT � 25 ND ND4148 TC � 85 ND ND4150 TT � 85 ND ND4155 CC � 19 ND ND

Naïve deer brain 4136 i.c. TC � 85 ND ND4144 TC � 49 Normal 574149 TT � 26 ND ND4154 CND � 85 ND ND4157 TC � 45 ND ND4132 p.o. TC � 21 ND ND4141 CC � 85 ND ND4145 CC � 26 ND ND4147 CC � 85 ND ND4151 CND � 85 ND ND

Secondary passageFelCWD brain 4332 i.c. CC 19/23 23 Abnormal 32

4346 TT 21/27 27 Abnormal 354357 TT 19/25 25 Abnormal 294360 TT 23/25 25 Normalb 284341 CC 24/26 26 Abnormal 344329 p.o. CC ° CTM ND ND4337 CC 42/* CTM ND ND4352 TT ° CTM ND ND4344 CC 35/* CTM ND ND4361 i.p./s.c. TT 31/* CTM ND ND4338 CND ° CTM ND ND4345 CC 29/* CTM ND ND4348 CC 29/* CTM ND ND4340 Cohoused CC ° CTM ND ND4356 TT ° CTM ND ND

Primary passage-negativefeline brain

4334 i.c. TT ° CTM ND ND4359 CC � CTM Normal 31.54349 TT ° CTM ND ND4353 CC ° CTM ND ND

Uninoculated 4135 ND ND � 65 Normal 65a MPI, month postinfection; CND, cannot determine repeat number variation in heterozygous state; �, did not develop TSE clinical disease, nor was PrPCWD detected fromterminal brain tissue; *, no late-stage clinical signs at the time of this report (continuing to monitor [CTM]at 42 MPI); °, no clinical signs at the time of this report (continuing tomonitor at 42 MPI); ND, not done.b In this cat, MRI revealed a normal brain but mandibular lymph node asymmetry.

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DISCUSSIONTransmission of deer origin CWD to domestic cats. CWD, theonly prion disease in a wildlife population, exhibits high transmis-sion rates among cervids in their natural environment. Becauseof the facile transmission between cervids, questions arise regard-ing interspecies transmission and alternate host reservoirs in scav-enger/predator species sympatric with CWD in nature. In thisstudy, we have demonstrated transmission of deer origin CWD(DeerCWD) to one such scavenger species, the domestic cat, via theclassical i.c. route of inoculation.

Transmission of cat-adapted CWD to domestic cats. The suc-cessful transmission of prions to alternate hosts is determined bythe species barrier, the strength of which, for prions, is dependentupon interactions between host PrPC and the infecting prionstrain (30). Transmission across a species barrier requires adapta-tion by both the host and the infectious agent, as the original hostPrPRES templates its conformation onto the new host PrPC (31).The new host PrPRES thus formed takes on strain-specific proper-ties in the tertiary structure. Therefore, subsequent passage intothis new host requires less time for efficient templating, conver-sion, rogue prion deposition, and progression to terminal clinicaldisease (31, 32). The results of this study support this host adap-tation hypothesis, as upon secondary passage of cat-adapted CWD(FelCWD), 100% of FelCWD i.c.-inoculated cats developed clinicaldisease in about half the time required for DeerCWD-inoculatedcats. In conjunction with an appreciably shortened course of clin-ical disease, FelCWD deposition and distribution were more exten-

sive and diffuse (Fig. 2), suggesting a broadening of host cell tro-pism, i.e., the ability of feline-adapted CWD prions to infect andamplify in or on a greater variety of feline cell types.

Clinical disease in peripherally inoculated domestic cats. Aplausible route for interspecies transmission of prion diseases isvia breach of mucosal surfaces during ingestion of infectious pri-ons found in carcasses, their by-products, and in the environment.Precedence for prion trans-species transmission has been welldocumented with vCJD, FSE, and TME (4, 33). Studies have alsodemonstrated that prion diseases can be orally transmitted tomany species: i.e., CWD to voles (34), Peromyscus mice (34), andferrets (35), scrapie to squirrel monkeys (36) and hamsters (37–40), BSE to sheep (41–43), goats (41), cynomolgus macaques (44,45), and lemurs (46), and CJD and Kuru to squirrel monkeys (36),with some requiring prior in vivo or in vitro adaptation.

In our studies, p.o. inoculation of DeerCWD was not sufficientto induce TSE. Western blotting, IHC, and RT-QuIC failed toshow the presence of FelCWD in these animals, demonstrating theexistence of a considerable species barrier. However, early signs ofdisease are currently observed in second passage p.o.- and i.p./s.cFelCWD-inoculated cats, suggesting that the ingestion of and/oropen wound exposure to CWD-contaminated material couldcause feline TSE disease. Naïve cats cohoused with the i.c.-inocu-lated FelCWD-positive cohort continue to serve as horizontal/en-vironmental shedding test subjects and are currently housed withthe i.p./s.c.-inoculated cohort. These remaining cats will continueto be monitored for further development of clinical disease, and

TABLE 2 Pairwise genetic distances between domestic cats and 7 other mammalian species sharing a home range in North America

Species

Genetic distance (%) froma:

Domestic cats Mountain lions Bobcats Canadian lynx White-tailed deer Mule deer Elk Humans Cattle

Domestic cats 100 94.9 98.6 99.1 92.1 92.1 92.5 89.7 87.7Mountain lions — 100 96.2 96.5 88 88 88.4 84 84.6Bobcats — — 100 99.6 90.4 90.4 90.8 86.4 86.9Canadian lynx — — — 100 90.8 90.8 91.2 86.8 87.3White-tailed deer — — — — 100 100 99.6 90.2 94.7Mule deer — — — — — 100 99.6 90.2 94.7Elk — — — — — — 100 89.8 94.3Humans — — — — — — — 100 87.9Cattle 100a —, repeat comparison.

FIG 1 Immunoblot demonstration of FelCWD in brain (medulla) of cats. (A) Lanes 2 and 4 demonstrate complete proteinase K (PK) digestion of PrPC inCWD-negative inoculated cat and deer. Lanes 6 and 8 demonstrate FelCWD detection after PK digestion in 2 cats i.c. inoculated with deer origin CWD(primary passage DeerCWD), while lane 10 demonstrates FelCWD detection in 1 representative cat i.c. inoculated with cat-adapted CWD (secondarypassage FelCWD). Lane 11 shows the molecular mass shift upon partial PK digestion of DeerCWD in a CWD� deer. The MAb used was BAR224. (B) Lanes1 to 4 demonstrate the absence of detection of PrPC and DeerCWD in deer. Lanes 5 to 8 show detection of PrPC and FelCWD in cats inoculated with DeerCWD

or FelCWD. The MAb used was 3F4.

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terminally harvested tissues will be analyzed for the presence ofFelCWD.

Detection of MRI abnormalities in clinically ill cats. CerebralMRI has been used to detect specific abnormalities betweensporadic CJD (sCJD), vCJD, and nonprion rapidly progressivedementias (47–52). In sCJD patients, T2 hyperintensities areobserved in the cortex and deep gray matter, in particular thestriatum, whereas in vCJD, T2 hyperintensities are most com-

monly seen in posterior pulvinar and medial thalamus (47,52–56). The histological basis of the T2 hyperintensities is notcertain, but for at least certain subtypes of CJD, it may berelated to vacuolar fluid accumulation (57). Cerebral atrophycan also become evident on MRI in CJD patients (58). DW-MRI is physiologically based and is a highly sensitive and spe-cific marker of restricted brain diffusion in CJD patients (59,60) and has improved the imaging diagnosis over conventional

FIG 2 FelCWD detection by immunohistochemistry in brain (medulla oblongata at obex) of cats. (A and B) Detection (red deposits) demonstrated in catsinoculated with deer origin DeerCWD (A) and cat-adapted FelCWD (B). Panel C demonstrates the lack of FelCWD detection in cats inoculated with age-matchedcat-adapted CWD-negative deer obex. Scale bars, 10 �m. The MAb used was L42. Panels D and E represent DeerCWD detection and the lack thereof inCWD-infected and naïve deer brain, respectively.

FIG 3 RT-QuIC detection of PrPC converting activity in the brain (medulla) of DeerCWD- and FelCWD-inoculated cats. Reaction mixtures were seeded, induplicate, with cat or deer brain homogenate at a dilution of 10�4. Each data point represents the average ThT fluorescence of each duplicate reaction set. (A)Detection of PrPC converting activity in 2 of 5 primary passage DeerCWD i.c.-inoculated cats (cats 1 and 4). (B) Detection of PrPC converting activity in 5 of 5secondary passage FelCWD i.c.-inoculated cats. Panels A and B demonstrate detection of PrPC converting activity in CWD� deer (red dotted lines with X) and lackof converting activity in CWD-negative deer (black circles) and cat (black diamonds) controls.

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MRI. Restricted diffusion and DW-MRI signal hyperintensityhave been found in the basal ganglia, thalamus, and cortex ofCJD patients (60). Again, the proposed explanations for re-stricted diffusion vary with the CJD subtype but may be relatedto intraneuronal microvacuolation (57), prion protein deposi-tion, and spongiform change (61). The combination of DW-MRI with FLAIR imaging has sensitivity and specificity re-ported at �91 to 98% (59, 62). Those sequences are importantfor early detection to inform appropriate diagnostic testing(61) and to enable timely intervention as new therapies aredeveloped (59). Although only a small number of studies haveused MRI to investigate animal models of prion disease, it hasbeen shown that focal blood-brain barrier disruption occurs ina hamster scrapie model (63), that there are increases in T2measurements in the septum, hippocampus, thalamus, andcortex of mice inoculated with 139A scrapie (64), and that thediffusion of tissue water was significantly reduced in the latepreclinical period in mice inoculated with ME7 scrapie (65).Furthermore, a recent MRI study in sheep naturally exposed toscrapie demonstrates a generalized cerebral atrophy (66).

In this study, MRIs were performed on cats inoculated withdeer or cat origin CWD. Abnormalities resembling those found inaforementioned MRI studies in CJD patients (47, 52–56, 58), in-cluding nonspecific T2 and T2 FLAIR hyperintense signal changesand ventricular enlargement consistent with cerebral atrophy,were present in the symptomatic cat inoculated with DeerCWD,whereas only ventricular enlargement was found in symptomaticcats inoculated with FelCWD. Based on the limited MRI data inother prion animal models, it appears that FelCWD most closelyresembles the atrophy demonstrated in sheep scrapie. How orwhether these signal abnormalities correlate with histopathologi-cal data is currently being assessed.

Comparison of CWD and BSE transmission to domestic cats.Striking similarities are seen in the clinical presentation and dura-tion of TSEs (4, 55). In cats, both BSE and CWD are marked by

changes in behavior and gait, hyperaesthesia, ataxia, and tremors,and both progress to terminal-phase disease 3 weeks to 5 monthspostobservation of initial TSE signs (67–69).

BSE appears to be more efficiently transmitted to domestic catsby oral exposure than CWD. In this study, while we have shownthat cats are susceptible to CWD infection by the classical intra-cranial route, oral transmission was seen only after subpassageinto a second cohort of cats that had received lingual abrasionsknown to enhance transmission potential (23, 24). While the pres-ence or absence of oral lesions was not reported in FSE cases ob-served by Wyatt (68, 69) and Legget (67), it is hypothesized thatthe affected cats consumed BSE-contaminated meat and bone incommercial cat foods.

Implications associated with trans-species transmission ofCWD prions. It has been determined that, once a prion strain hasbeen adapted to a new host species, the prions from this new hostspecies propagate more efficiently in a third host. Specifically, thepassage of cow BSE prions in sheep or goats markedly increasedthe transmission efficiency into human transgenic mice (70). Al-though a substantial species barrier appears to exist between deerand cats, barring an invasive route of inoculation, we must con-sider the epidemiologic and ecologic implications associated withCWD transmission to felids, which could potentially result in thegeneration of prion strains adapted for natural (mucosal) routesof transmission to felids and/or other noncervid species. The highgenetic identity homology observed between domestic and non-domestic cats compared to 5 other mammalian species sharing ahome range with CWD-infected cervids, while warranting furtheranalysis of protein structure and protein-protein interactions,suggests that nondomestic cats may be a susceptible reservoir spe-cies in nature (Fig. 5). If CWD has the ability to infect and establishalternate host reservoirs in nature or to initiate the adaptationsnecessary for trans-species transmission, this will impact not onlywildlife, but also domestic species, which can lead to serious con-sequences for human health. Here, we have demonstrated that

FIG 4 Abnormalities recorded in magnetic resonance imaging brain scans of cats i.c. inoculated with CWD. (A to D) CWD� inoculated cats; (E) CWD-negativeinoculated cat. White arrows indicate abnormalities. (A) Inflammatory lesions; (B) focal dilation of the olfactory extension/horn; (C) distended lateral ventricle;(D) focal cavitation. Panel E demonstrates the lack of abnormalities in an age-matched cat i.c. inoculated with CWD-naïve deer obex.

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one scavenger/predator species (the domestic cat) that cohabitateswith the natural hosts of CWD could become an intermediate hostfor this prion disease in nature.

ACKNOWLEDGMENTS

We thank Sallie Dahmes and David Osborn for providing the naïve white-tailed deer used in our studies, Terry Spraker for CWD-positive mule deerinoculum, Gary Mason and Nathaniel Denkers for contributions to inoc-ulations, Nicholas Haley, Clare Hoover, Craig Miller, and Martha Mac-Millan for necropsy expertise, Kelly Walton, Tracy Webb, CristinaWeiner, Matthew Rosenbaum, Winona Burgess, and Elizabeth Magdenfor animal evaluations, Alan Elder and Amber Mayfield for laboratorysupport, Anca Selariu for editing, Wilfred Goldmann and Paula Stewartfor generously genotyping the cats, and Justin Lee for sequence analysis.

This work was supported by contract N01-AI25491 and grant R01-NS061902 from the National Institutes of Health (NIH) and by the Col-lege Research Counsel (CRC) at Colorado State University, College ofVeterinary Medicine and Biological Sciences (CSU-CVMBS), and the De-partment of Microbiology, Immunology and Pathology (DMIP).

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