Exse

MoCaa Secb Hoc Nod Fin

Veterinary Microbiology 162 (2013) 499–506

A R

Artic

Rece

Rece

Acce

Keyw

Con

Exp

Orf

Para

Poxv

Rein

Wild

*

Dep

of V

Tel.:

1

Facu

Wom

037

http

perimental parapoxvirus infection (contagious ecthyma) inmi-domesticated reindeer (Rangifer tarandus tarandus)

rten Tryland a,*, Jorn Klein b,1, Therese Berger a, Terje D. Josefsen c,rlos G. das Neves a, Antti Oksanen d, Kjetil Asbakk a

tion of Arctic Veterinary Medicine, Department of Food Safety and Infection Biology, The Norwegian School of Veterinary Science, NO-9010 Tromsø, Norway

st Microbe Interaction, Department of Medical Biology, University of Tromsø, NO-9037 Tromsø, Norway

rwegian Veterinary Institute, Stakkevollveien 23, NO-9010 Tromsø, Norway

nish Food Safety Authority Evira, Production Animal and Wildlife Health Research Unit, Elektroniikkatie 3, FI-90590 Oulu, Finland

1. Introduction

Contagious ecthyma (contagious pustular dermatitis,orf) is a disease that occurs world-wide in sheep and goatsand is caused by orf virus, a member of the genusParapoxvirus in the Poxviridae family. The disease is usuallynot fatal. Lambs and kids, however, can develop severelesions, with secondary bacterial infections, and may alsobe prevented from suckling since the udder of the mother

T I C L E I N F O

le history:

ived 18 June 2012

ived in revised form 23 September 2012

pted 5 October 2012

ords:

tagious ecthyma

erimental infection

poxvirus

irus

deer

life

A B S T R A C T

Contagious ecthyma (contagious pustular dermatitis, orf) occurs world-wide in sheep and

goats and is caused by orf virus (genus Parapoxvirus, family Poxviridae). Contagious

ecthyma outbreaks have been described in semi-domesticated reindeer (Rangifer tarandus

tarandus) in Sweden, Finland and Norway, occasionally with high mortality. Fourteen one-

year-old reindeer were corralled in mid-April. One week after arrival, two animals

received a commercial live orf virus vaccine for sheep (Scabivax1) on scarified skin of the

medial thigh. Four weeks later, the two vaccinated and six additional animals were

inoculated in scarified oral mucosa with parapoxvirus obtained from reindeer with clinical

contagious ecthyma. The remaining six reindeer were kept as sentinels, sharing feed and

water with the inoculated animals. A small whitish lesion appeared on the inoculation site

and the labial skin-mucosa junction of three animals five days post inoculation (p.i.).

Twelve days p.i., typical ecthyma lesions were visible on the inoculation site in six of eight

animals, including both vaccinees. Four inoculated animals (including both vaccinees) and

one sentinel seroconverted 12 days p.i., and five animals (including one sentinel)

seroconverted 20 days p.i. No contagious ecthyma-like lesions were detected in the

sentinels. All animals were euthanized at 26–29 days p.i. Histological examination of

lesions showed proliferative dermatitis with epidermal hyperplasia, hyperkeratosis, intra-

epithelial pustules and ulcers. Orf virus DNA was detected in mandibular lymph nodes,

tonsils and mucosal lesions of four animals, including one sentinel, which showed that

virus transmission took place. The commercial orf virus vaccine may be difficult to

administer due to the need for close-cropping and its zoonotic nature, and did not indicate

significant protection, although the latter has to be verified with a larger number of

animals.

� 2012 Elsevier B.V. All rights reserved.

Corresponding author at: Section of Arctic Veterinary Medicine,

artment of Food Safety and Infection Biology, The Norwegian School

eterinary Science, Stakkevollveien 23b, NO-9010 Tromsø, Norway.

+47 77665400; fax: +47 77694911.

E-mail address: morten.tryland@nvh.no (M. Tryland).

Current address: Norwegian University of Science and Technology,

lty of Medicine, Department for Laboratory Medicine, Children’s and

en’s Health, P.O. Box 8905, 7491 Trondheim, Norway.

Contents lists available at SciVerse ScienceDirect

Veterinary Microbiology

jo u rn al ho m epag e: ww w.els evier .c o m/lo cat e/vetmic

8-1135/$ – see front matter � 2012 Elsevier B.V. All rights reserved.

://dx.doi.org/10.1016/j.vetmic.2012.10.039

M. Tryland et al. / Veterinary Microbiology 162 (2013) 499–506500

can be infected and tender. Parapoxvirus has also beenisolated from contagious ecthyma-like lesions in otherdomestic ruminants, as well as in camelids and wildlifespecies, such as muskoxen (Ovibos moschatus), squirrels,and seals (Khalafalla et al., 1994; Nettleton et al., 1995;Sainsbury and Ward, 1996; Tryland et al., 2004; Vikørenet al., 2008). Parapoxviruses causing contagious ecthymaor similar lesions are zoonotic, causing cutaneous infec-tions in fingers and hands of people handling infectedanimals (Damon, 2007).

Contagious ecthyma has been reported in semi-domesticated reindeer (Rangifer tarandus tarandus) inSweden (Nordkvist, 1973), Norway (experimental ani-mals; Kummeneje and Krogsrud, 1979) and in Finland,starting during the winter 1992–1993 (Buttner et al.,1995). The first serious outbreak of contagious ecthymaunder regular reindeer herding conditions in Norwayappeared in Nordland County in April 2000, affecting about30 animals, of which eight died (Tryland et al., 2001). Basedon the experience in Finland and Norway, it seems evident,that contagious ecthyma outbreaks in reindeer can beassociated to corralling and supplementary feeding ofanimals (Tryland et al., 2001). Orf virus seems to be able tobe sub-clinically present in reindeer, since parapoxvirusDNA was detected in various tissues from six of 48 animalsfound dead on winter pastures (1999–2000) in FinnmarkCounty, Norway, where contagious ecthyma never hasbeen reported in reindeer (Tryland, 2002).

In Finland, earlier outbreaks were caused by orf virus,whereas more recent virus isolates (since 1999) havemostly been characterized as being pseudocowpoxvirus,another zoonotic parapoxvirus, causing pseudocowpox incows and milkers’ nodules in people (Tikkanen et al., 2004;Hautaniemi et al., 2010). In Norway, however, studies haveconsistently shown that parapoxvirus obtained fromsubclinically infected animals and from reindeer withclinical contagious ecthyma has been orf virus (Tryland,2002; Klein and Tryland, 2005), also being similar to a virusisolated from muskoxen (Vikøren et al., 2008). Thus, wehave no reason to believe that reindeer hosts its ownspecific parapoxvirus, as has been shown for red deer inNew Zealand (Robinson and Mercer, 1995). Orf virus ispresumably being transferred from sheep and possiblygoats through shared pastures via infected organic matter(scabs), in corrals used for both sheep and reindeer, intransport vehicles etc.

Vaccinating sheep against contagious ecthyma, eitherwith commercial vaccine or autovaccine produced from orfvirus obtained from the herd to be vaccinated, is notcompletely protective and the effect is usually short-lived.It has also proved to be challenging, since vaccine strainshave also caused regular outbreaks of orf in sheep (Gilrayet al., 1998). The humoral immune response has beencharacterized as relatively poor and short lived in sheepand may thus be of minor importance for the immunityagainst parapoxvirus infections (Damon, 2007). Further, itseems that passive administration of antibodies (hyper-immune serum) does not protect lambs from infection(Czerny et al., 1997; Haig and McInnes, 2002). Theimmunological response to parapoxvirus infections inreindeer is virtually unknown.

The aim of this study was to see whether semi-domesticated reindeer and sheep were susceptible toexperimental infections with parapoxvirus isolated fromreindeer with contagious ecthyma, to establish theincubation period for reindeer, and to look for indica-tions whether a commercial orf virus vaccine for sheepmay have the potential to prevent clinical disease inreindeer.

2. Materials and methods

2.1. Animals

Fourteen one-year-old semi-domesticated reindeer(seven males, seven females), were blood sampled(jugular vein), ear-tagged (R1–R14; Table 1) and corralledin mid-April. During a 5-week period, animals wereaccustomed to people and to commercial pelleted feed forreindeer (Reinsdyrfor, Felleskjøpet, Trondheim, Norway).In addition, they were fed lichen (Cladina spp.) and haythrough the experimental period. Four weeks after thearrival of reindeer, two one-year-old male sheep (S1, S2;Table 1) were introduced to a neighbouring pen, having nocontact with the reindeer. The sheep were fed hay andcommercial pelleted feed for sheep (Formel Favør 10,Felleskjøpet).

2.2. Vaccination

One week after arrival (Day – 28; 4 weeks prior toinoculation of virus), two reindeer (R1 and R4) werevaccinated with a commercial vaccine (Scabivax1, Scher-ing Plough Animal Health Corp., Oslo, Norway), based onlive orf virus (strain NZ2) originating from lambs withclinical contagious ecthyma, and expecting to induceimmunity in sheep within 3–4 weeks. The medial side ofthe right thigh was close-cropped with scissors and thevaccine was applied by a cross-scarification of the skinwith the vaccine-bottle device, introducing the live virusvaccine suspension in the skin lesion, as recommended forsheep.

2.3. Virus and inoculation

Live parapoxvirus, for inoculation of reindeer and asantigen in ELISA, was purified from scabs obtained fromreindeer with contagious ecthyma (Tryland et al., 2001) asdescribed previously (Klein and Tryland, 2005). Four weekspost vaccination (Day 0) the two vaccinated (R1 and R4)and three females (R8, R9, R10) and three males (R2, R12,R13) were inoculated with live parapoxvirus. An area(approximately 2 cm � 1 cm) of the mucosa of the lower lipwas scarified by sandpaper (grade 80), and the suspensionof live parapoxvirus was introduced to the scarifiedmucosa with a cotton stick. The remaining six reindeerreceived no treatment and were kept as sentinels, sharingfeed and water with the inoculated animals. The two sheepwere inoculated at the same time as the reindeer (Day 0) bythe same procedure. The experimental animal researchprotocol was approved by the Norwegian Animal ResearchAuthority.

Table 1

Clinical symptoms, pathology, serology and PCR results from experimental inoculation of semi-domesticated reindeer (Rangifer t. tarandus) (R1–R14) and domestic sheep (S1 and S2) with parapoxvirus originally

isolated from reindeer. d.p.i.: days post inoculation.

Animal Sex Status Lesions in oral mucosa, detected clinically (5 and 12 d.p.i.) or at necropsy (26-29 d.p.i.) Serologya +(d.p.i.)/� PCR positive (blood

and saliva, and

tissue at necropsy)

5 12 26–29 (euthanization/necropsy)

R1b F Vaccinatedc +

inoculated

Lesion (2 mm� 8 mm)

skin–mucosal junction

Lesion (5 mm)

lower lip

Multiple lesions lower lip (ulcerated),

gum and hard palate

+ (20) Oral lesion,

mandibular

lymph node

R4b M Vaccinatedc +

inoculated

Lesion (3 mm� 5 mm)

lower lip

Two lesions (appr. 6 mm� 10 mm)

lower lip

Multiple lesions lower lip and hard palate + (12)

R2 M Inoculated – – – + (20) Tonsil

R8b F Inoculated – Coalescing lesions covering most

of lower lip

Entire rostral part of lower lip covered

by hyperplastic and ulcerated epithelium

(Fig. 1C). Smaller lesions upper lip and

hard palate

+ (12) Oral lesion

R9 F Inoculated Lesion (3x6 mm)

lower lip

Lesion (5 mm� 8 mm) lower lip Ulcerated lesion lower lip + (20) Oral lesion

R10b F Inoculated – Lesion (20 mm� 40 mm) lower lip Ulcerated lesion lower lip and gum + (12)

R12 M Inoculated – Lesion (4 mm� 8 mm) lower lip Lesions inside (ulcerated) and outside

lower lip, and on dental pad

+ (20)

R13 M Inoculated – – – �R3 F Sentinel – – Lesion hard palate (2 mm� 4 mm) + (20) Oral lesion

R5 M Sentinel – – – �R6 M Sentinel – – – �R7 F Sentinel – – – + (20)

R11 F Sentinel – – – �R14 M Sentinel – – – �S1 F Inoculated – Small lesion on muzzle (skin) – � Skin lesion

S2 F Inoculated – Lesion (3 mm� 4 mm) on lip

mucosa

– + (12) Blood (Day 5 + 26 p.i.).

Lesiona The Day post inoculation from which the antibody level (ELISA percentage relative to controls) was increasing.b Full necropsy. For the other animals, samples were obtained after euthanization and only heads were subjected to a full necropsy.c Vaccinated (live virus; Scabivax1; Schering Plough) on cropped and scarified skin of the medial thigh 28 days prior to inoculation. Description of the local skin reaction on the vaccination site is given in the text

only.

M.

Try

lan

d et

al.

/ V

eterina

ry M

icrob

iolo

gy

16

2 (2

01

3)

49

9–

50

6

50

1

M. Tryland et al. / Veterinary Microbiology 162 (2013) 499–506502

2.4. Infection period

The animals were fed and inspected three times a dayduring the adaptation and the infection period, a total of 9weeks. The animals were inspected on the inoculation siteand in the oral cavity for clinical signs approximately onceevery week (Day 5, 12, 20 and 26–29 post inoculation, p.i.),and blood samples were obtained and serum prepared forserology and saliva was sampled for PCR using a cottonswab. At Day 26 and Day 28 p.i., animals were euthanizedusing a captive bolt stunning gun followed by bleeding ofthe jugular vein.

2.5. Pathology

After euthanization, four animals were fully necropsied,while the remaining animals were necropsied and sampledin the field. Heads from all animals were subjected to athorough pathological examination, and tissue fromlesions on lip, gum, dental pad and palate were fixed in4% buffered formaldehyde. Paraffin sections were preparedand stained with haematoxylin–eosin (HE). Tissue samplesfrom mucosal lesions, tonsils, mandibular lymph node,lung, mediastinal lymph node, spleen, liver, kidney,testicle, ovary, and the trigeminal ganglion were collectedand frozen at �80 8C until DNA extraction and PCR.

2.6. Serology

For preparation of antigen for ELISA, a primary culture ofcells from an adult reindeer from a herd with no knownhistory of contagious ecthyma was established. Mucosatissue (2 cm � 2 cm, depth 5 mm) from the lower lip wasplaced in Hanks’ balanced salt solution (Gibco 14065-049,Invitrogen; Auckland NZ) with 0.1 mg/ml of gentamicin(Gibco), 100 U/ml of penicillin, 0.1 mg/ml of streptomycineand 0.25 mg/ml of amphotericin B (Sigma A5955; Sigma–Aldrich, St. Louis, MO, USA). Specimens (3 mm � 3 mm) withthe mucosal side intact were incubated (37 8C, 5% CO2) in0.25% trypsin–EDTA solution (Sigma T4049). After 6 h,epidermis was peeled off, aggregates of cells were releasedin 5 ml Eagles Minimum Essential Medium (Sigma M4655)with antibiotic–antimycotic solution as described above,with the supplement of 4 mM L-glutamine (Sigma G7513),15% newborn calf serum and 10 ng/ml of epidermal growthfactor (EGF, Sigma E4127), transferred to 25 cm2 cell cultureflasks (Costar 3050, Costar Corp., Cambridge, USA; 5 ml perflask) and incubated at 37 8C in 5% CO2. After one passage bytrypsinization (0.25% Trypsin–EDTA solution, 5 min at 37 8C,5% CO2) and incubation until 30–40% confluency, mediumwas gradually changed to serum-free medium (OptiPro SFM,Gibco 12309) supplemented with antibiotic–antimycoticsolution and EGF as described above. After four days, the cellculture was inoculated with parapoxvirus (100 ml per75 cm2 flask of the virus suspension described above). After2 weeks of incubation, no typical cytopathic effect (CPE) wasobserved,but in contrast to the controlcells (uninfected), theinfected culture had a much higher growth rate, with cells inmanylayers,visiblewithoutamicroscope.Supernatantfrominoculated cultures was centrifuged (1500 � g, 10 min) toremove cells and debris, and stored at �20 8C.

Microtitre plates (96-well, PolySorp; Nunc, Roskilde,Denmark) were coated with culture supernatant (50 ml/10 ml 0.1 M sodium carbonate buffer pH 9.6, 100 ml per well,1 h at 37 8C). Free binding sites were blocked with washingbuffer (50 mM Tris–HCl, pH 7.4, with 150 mM NaCl, 5% non-fat dry milk powder and 1% Triton X-100). After washing(3�), duplicate wells were incubated (1 h, 37 8C) withreindeer or sheep test serum samples and controls, diluted1:50 in wash buffer, washed (3�) and incubated withhorseradish peroxidase conjugated Protein G (cat. no. 10-1223, Zymed Laboratories, Inc., San Francisco, CA, USA,1:1000 in washing buffer). After a final washing procedure,substrate (o-phenylenediamine; Dako A/S, Glostrup, Den-mark) was added. Colour development was stopped after40 min (1 M H2SO4) and optical density was read at 492 nm(OD492). If readings for duplicate wells differed by morethan 5%, the sample was re-assayed.

A positive and a negative control serum were run inevery plate. The positive control was obtained from aFinnish reindeer previously experimentally infected withorf virus. The negative control serum was obtained from aSvalbard reindeer (Rangifer tarandus platyrhynchus) in1995, since there are no reports on parapoxvirus infectionsin this subspecies of reindeer. The binding ability of proteinG to immunoglobulin of sheep and reindeer was verified inadvance by coating microtiter plate wells with sheep orreindeer sera diluted 1:1000. The ELISA-results wereexpressed as a percentage relative to the positive andthe negative controls (Guan et al., 2005).

2.7. Polymerase chain reaction

DNA was extracted from 25 mg of each tissue sampleobtained during necropsy of all animals and from salivaand the white blood cell fraction (‘‘buffy coat’’) fromsamples obtained on Day 5, 12, 20, 26 and 29 p.i. Tissueswere homogenized in phosphate buffered saline (PBS) witha pellet pestle device (Kontes, Fisher Thermo Scientific Inc.,Walthan, MA, USA). DNA was extracted with QIagen tissuekit (QIAamp DNA mini kit; QIAGEN, Oslo, Norway). Primersobtained from the B2L-gene (Inoshima et al., 2000) and theGIF-gene (Klein and Tryland, 2005) were used, givingexpected amplicon size of 594 and 408 base pairs (bp),respectively. DNA extracted from scabs from reindeer withcontagious ecthyma (Tryland et al., 2001) was used aspositive control, whereas water and DNA extracted from anorthopoxvirus lesion in a cat (Tryland et al., 1998) wereused as negative controls.

PCR amplicons were analyzed by horizontal gelelectrophoresis and sequencing, as described previously(Tryland et al., 2011). Raw sequence data were edited byChromas Pro software (Version 1.41; Technelysium PtyLtd., Tewantin, Qld., Australia) and BioEdit SequenceAlignment Editor (Version 7.0.4; Tom Hall, Departmentof Microbiology, North Carolina State University, NC, USA).

2.8. Statistical analyses

The means of the ELISA-results for the sample seriesfrom the different groups of reindeer (controls, inoculatedand vaccinated/inoculated) were compared by using

one(SA

3. R

3.1.

derthaRaiindvirubrosecinowhhaiandpostion

inoboraniandFigino

Fig.

inoc

cros

inoc

vacc

deve

5 da

mul

M. Tryland et al. / Veterinary Microbiology 162 (2013) 499–506 503

way Anova and the statistical software JMP ver. 8.0S Institute Inc., NC, USA).

esults

Clinical symptoms

The two vaccinated reindeer (R1 and R4) developed amal reaction along the lines of scarification of the skint were inoculated with the live vaccine (R4, Fig. 1A).sed labia developed on both sides of the scarification,icating proliferative tissue growth, characteristic of orfs infection. The reactive zone was about 1–1.5 cm

ad, with no open lesions, pus or other indications ofondary bacterial infections on the inoculation site. Theculation site with the reactive tissue remained hair free,ereas the neighbouring close-cropped skin regained ther growth. The reaction at the inoculation site was pink

reactive, but seemed to be less reactive after week 4t vaccination, leaving a hairless scar from the scarifica-

needle.On inspection of inoculated animals on Day 5 postculation (p.i.), a lesion was evident on the lip at theder between skin and mucosa of the two vaccinatedmals (R1, Fig. 1B, and R4), and one of the non-vaccinated

inoculated animals (R9). In one of the animals (R8,. 1C) the lesions had, by Day 26 p.i., spread from theculation site and were present as one large and

coalescing proliferative process, covering the inside ofthe lower lip (Fig. 1C). A small whitish lesion was evidenton the inoculation site in two of the other reindeer, R4 andR9 (Fig. 1D). On Day 12 p.i., typical ecthyma lesions werevisible on the inoculation site in 6 of the 8 inoculatedanimals (Fig. 1E), including the two vaccinated animals. OnDay 20 p.i., secondary lesions were seen on the lip, mucosaand gingiva (Fig. 1F). No contagious ecthyma-like lesionscould be detected clinically in any of the sentinels.

One of the sheep (S1) developed a small ecthyma-likelesion on the muzzle (not shown), whereas the other (S2)developed a small mucosal lesion at the inoculation site,which healed within a few days and disappeared.

3.2. Pathology

At euthanasia, all reindeer were in good body condition,as judged by visual inspection of muscles and fat depots.The presence of oral lesions at necropsy is summarized inTable 1. The lesions at the inoculation site typically spreadto other parts of the lower lip, and to other sites of the oralmucosa (Table 1).

Histological examination showed a proliferative der-matitis characterized by epidermal hyperplasia andformation of elongated rete ridges (Fig. 2B and C). In somelesions hyperkeratosis was prominent. Accumulation ofinflammatory cells, dominated by lymphocytes andmacrophages, was found at the dermal/epidermal junction

1. Clinical symptoms in semi-domesticated reindeer (Rangifer tarandus tarandus) after vaccination (skin; commercial vaccine) and experimental

ulation (oral mucosa; parapoxvirus isolated from reindeer). (A) Animal R4 was vaccinated in close-cropped skin of the medial side of the thigh, by a

s-scarification with the vaccination device, producing a local and proliferative reaction (6 weeks post vaccination, p.v.). (B) Animal R1 (vaccinated and

ulated) had a viral lesion on the skin–mucosa junction on the lower lip (arrow) 33 days p.v. and 5 days post inoculation (p.i.). (C) Animal R8 (non-

inated and inoculated) at Day 26 p.i. (necropsy). This animal had multiple proliferative and coalescing lesions in the oral mucosa at Day 12 p.i., which

loped more severely as compared to other affected animals, and showed no signs of healing at necropsy. Animal R9 (non-vaccinated and inoculated) at

ys p.i., (D) 12 days p.i. (E) and 20 days p.i. (F), showing a whitish lesion in the mucosa, a typical proliferative lesion, and a more developed lesion with

tiple secondary lesions (arrows), respectively.

M. Tryland et al. / Veterinary Microbiology 162 (2013) 499–506504

and in the dermal papillae. Intra-epithelial pustulesoccurred in some areas. Ulcerated areas had more heavyinflammatory infiltrates, often with purulent foci, andplant fragments could often be detected in dermis belowulcerated lesions.

One animal (R8) had more severe lesions than theothers. The whole rostral part of the lower lip was reddishbrown with small humps on the surface, resembling araspberry (Fig. 1C). Histologically, this animal had thesame type of lesions as the others, but the rete ridges wereextremely elongated (Fig. 2C). The epidermis showedsevere hyperkeratosis and there were frequently smallulcerations on the top of the dermal papillae.

Three of the sentinel reindeer had single (n = 2) ormultiple (n = 1) small white spots on the lower lip. One ofthe single spots was examined histologically, and showedno pathological changes, but a lack of melanin pigment inthe affected area. Histological examination of the animalwith multiple white spots showed multifocal areas of mildepidermal hyperplasia and hyperkeratosis, without anyinflammatory cells or other signs of inflammatory reaction.The aetiology of these changes is unknown, but it wasinterpreted as not related to orf virus infection. Onesentinel animal (R3) had a small lesion, 2 mm � 4 mm, inthe oral mucosa of the cranial part of the hard palate, whichmacroscopically could resemble an orf lesion. This lesionwas not sampled for histological evaluation, but a samplewas obtained for PCR.

3.3. Serology

The positive control serum gave an OD492 value of 0.7(ELISA percentage defined as 100%) and the negativecontrol serum an OD492 value of 0.06 (ELISA percentagedefined as 0%). The ELISA results are given in Table 1. Thetwo vaccinated and inoculated reindeer (R1 and R4)showed increased levels of antibodies at Day 20 p.i. (R1)and Day 12 p.i. (R4), respectively (Fig. 3A). The ELISAresults for the six reindeer that were inoculated but notvaccinated are depicted in Fig. 3B.

Of the six sentinels, antibody levels for four (R5, R6, R11,R14) remained at a level comparable to the negativecontrol serum sample throughout the experimental period.In one sentinel (R7), the ELISA percentage appearedmarkedly elevated by Day 29 p.i., whereas for R3, a slightelevation in ELISA percentage was observed from Day 12 toDay 20 p.i., but for this particular animal we had no bloodsample past this point (data not shown).

By Day 20 p.i. the ELISA-percentage for the animals thatwere both vaccinated and inoculated (mean = 36.1,SD = 6.3, n = 2) was significantly higher than for the groupthat was non-vaccinated and inoculated (mean = 11.8,SD = 4.0, n = 5) (oneway Anova, p = 0.01) and for the groupof sentinels (mean = 6.8, SD = 4.0, n = 5) (oneway Anova,p = 0.004). The difference between the inoculated non-vaccinated animals and the sentinel group was notsignificant (p = 0.4).

For sheep S1, the antibody level remained around thelevel of the negative control throughout the experimentalperiod, whereas for S2, the level increased sharply afterDay 12 p.i. (data not shown).

3.4. Polymerase chain reaction

Results from the PCR investigation of tissues, blood andsaliva (reindeer and sheep) are presented in Table 1.Amplicons were sequenced and verified to be identical toparapoxvirus used for the inoculation (Klein and Tryland,2005; data not shown). PCR amplicons of relevant size andsequence were generated from the live parapoxviruspreparation used for inoculation and from the supernatantof the inoculated primary cell culture used as antigen in theELISA.

4. Discussion

This is the first report of experimental infection ofreindeer with parapoxvirus, and also the first reporton vaccination of reindeer with a commercial orfvirus vaccine. This experiment revealed that even fit

Fig. 2. Histological sections from the lower lip of 3 different reindeer. (A) Normal lip epithelium (animal R2, inoculated, no clinical signs). (B) Lip epithelium

with rete ridge formation (animal R1, inoculated, with a healing lesion in lower lip). (C) Lip epithelium with extremely elongated rete ridges and

hyperkeratosis (animal R8). HE-stain, magnification (25�) identical in the three slides for comparison.

anifeeeiglesidemday

lesiaroprerein199to

mucomin tsensoumanosand

Fig.

(Ran

inoc

prio

rein

rela

M. Tryland et al. / Veterinary Microbiology 162 (2013) 499–506 505

mals in good condition and with ad libitum access tod were susceptible to parapoxvirus infection. Six ofht inoculated animals developed typical ecthymaons in the oral mucosa at the inoculation site,onstrating an incubation time of approximately five

s.The fact that most lesions, including secondaryons, appeared in the oral mucosa and not in the skinund the mouth, is comparable to the clinical pictureviously seen during contagious ecthyma outbreaks indeer under regular herding conditions (Buttner et al.,5; Tryland et al., 2001). This is, however, in contrast

contagious ecthyma outbreaks in sheep, goats andskoxen, where both mucosal and skin affections aremon (Vikøren et al., 2008). From the results obtained

his study, indicating virus transmission to one of thetinels, it may seem that saliva may be an importantrce of infection within a reindeer herd, where salivay be transferred between animals through directe-to-nose contact or licking, or by sharing troughs

water sources. This was, however, not supported by

the PCR, since parapoxvirus DNA could not be detected inany of the saliva samples obtained on Day 5, 12, 20, 26and 29 p.i.

Orf virus is an epiteliotrophic virus, infectingdamaged or scarified skin and mucosa, replicating inregenerating epidermal keratinocytes (McKeever et al.,1988). The general clinical progression is erythema,vesicle, pustule and then scab formation. Gross andhistopathological changes in the oral lesions of thereindeer in this experiment are consistent with orflesions as previously described (Ginn et al., 2007). Orflesions usually heal within 4–6 weeks from when theyfirst appear. Most reindeer in this experiment hadsmaller lesions, and at the time of autopsy the ulcera-tions were superficial with narrow spaces, roundededges and fibrosis, indicating that complete healingwithin few weeks was probable. One reindeer (R8) had amore chronic persisting and proliferative lesion (Fig. 1C).Individual animals with proliferative lesions persistingfor weeks and months are commonly seen in naturaloutbreaks of contagious ecthyma in sheep (Reid, 2000),and also seems to be common in muskoxen (Guo et al.,2004; Vikøren et al., 2008). In the present case theexperimental period was too short to state whether thisparticular reindeer would develop a severe chroniclesion, or if the lesion would regress with onlymoderately prolonged healing time, but for the otheranimals showing clinical symptoms, the lesions wereregressing at the time of euthanization.

Of the six non-vaccinated and inoculated reindeer,two animals (R8, R10) showed increased antibody levelsby Day 20 p.i., and a similar increase was observed forthree other animals (R2, R9, R12) at Day 26 p.i.(euthanization), indicating that the experimental periodpossibly ended too early to have the full overview ofantibody development. The humoral immune responsein the inoculated animals was similar to the response inindividuals that were both vaccinated and inoculated,although one of the two vaccinated reindeer had anearlier onset, evident by Day 12 p.i. For one of the non-vaccinated and inoculated animals (R13), no increase inantibody level could be observed, no clinical signs wereevident, and all tissue samples were negative forparapoxvirus DNA, suggesting that an infection wasnot established.

The two vaccinated animals demonstrated a markedrise in ELISA percentage at Day 12 p.i. (R4) and Day 20 p.i.(R1), showing a significantly stronger humoral immuneresponse than most of the animals that were inoculatedbut not vaccinated (Fig. 3). This immune response was notfully protective, however, since both animals developedsmall lesions in the lower lip at the inoculation site, similarto the lesions in other animals. The lesions on thevaccination site at the medial thigh were restricted tothe scarified skin area only, and had a proliferative nature(Fig. 1A), although not as strong as the processes caused bythe inoculated virus in the oral mucosa. There are noprevious reports on vaccination of reindeer againstcontagious ecthyma, and the restricted protectionrecorded from the vaccination in this study must beevaluated in light of the fact that very few animals were

3. Development of antibody response in semi-domesticated reindeer

gifer tarandus tarandus) following vaccination and experimental

ulation of parapoxvirus. (A) Reindeer vaccinated (Scabivax1) 28 days

r to inoculation with parapoxvirus (n = 2). (B) Non-vaccinated

deer inoculated with parapoxvirus (n = 6). Y-Axis: ELISA-percentage

tive to positive control.

M. Tryland et al. / Veterinary Microbiology 162 (2013) 499–506506

vaccinated and challenged. For a thorough evaluation ofthe efficacy of vaccination of reindeer against contagiousecthyma, a broader study with this as the main focusshould be conducted. Nevertheless, it is fair to say that wehave no indication from this study that the vaccineprovokes protection against orf virus in reindeer. We alsorecorded other aspects of the vaccination, which may pointat a restricted use in reindeer. First, the fact that thevaccination site needs to be close-cropped to ensure thatthe vaccine reaches the skin, which is laborious andchallenging, especially when dealing with many animals.Second, the vaccine in question is based on live orf viruswhich is also zoonotic, and it might be challenging to avoidinfection from the vaccine components when workingunder field conditions.

Although the six sentinel animals shared feed andwater with the inoculated ones, they did not show clinicalsymptoms 3 weeks p.i., which is a more than 2 week longerperiod than the recorded incubation time for the inocu-lated animals. However, parapoxvirus DNA was amplifiedfrom one small lesion in the oral mucosa of one of thesentinels (R3), suggesting that the virus was transferredfrom the inoculated animals to the sentinel, although thisparticular lesion did not resemble a typical contagiousecthyma lesion and the animal did not develop the typicaldisease symptoms during the study period.

Both sheep in this study developed small ecthyma-likelesions, but the lesions were very restricted and did notspread, as commonly seen during contagious ecthyma inlambs, and the production of antibodies was restricted. Itwas evident that the sheep were susceptible for thereindeer virus, but the data may also indicate a lowersusceptibility among the sheep for this particular virusisolate, as compared to reindeer. Another possibility is thatthe sheep were previously exposed to an antigenicallysimilar virus strain and thus more resistant to infection.From previous studies, it is evident that the virus used forthis inoculation indeed is orf virus, based on comparison ofnucleotide sequences from virus obtained from contagiousecthyma outbreaks in reindeer (Norway; 2000 and Fin-land; 1994) and from sheep and goat (Klein and Tryland,2005).

Contagious ecthyma seems to be a multi-factorialdisease in reindeer causing clinical symptoms and diseaseunder certain circumstances, such as bad nutritional statusof the animals, reduced food quality, and stress, oftenassociated to herding and handling of animals and keepingthem corralled for a period of time (Buttner et al., 1995;Tryland et al., 2001). Clinical outbreaks of contagiousecthyma in reindeer have not been reported in Norwaysince 2001, whereas disease outbreaks occur regularly inFinland. Further work is needed to determine the majorrisk factors associated to outbreaks of contagious ecthymain reindeer.

Acknowledgements

We greatly acknowledge assistance from Heidi R.Bendiksen, Siri Enoksen and Børre With during handlingof animals. This project was partly financed by the

References

Buttner, M., von Einem, C., McInnes, C., Oksanen, A., 1995. Klinik undDiagnostik einer schweren Parapocken-Epidemie beim Rentier inFinland. Tierarztl. Prax. 23, 614–618.

Czerny, C.P., Waldmann, R., Scheubeck, T., 1997. Identification of threedistinct antigenic sites in parapoxviruses. Arch. Virol. 142, 807–821.

Damon, I., 2007. Poxviruses. In: Knipe, D.M., Howley, P.M. (Eds.), FieldsVirology. 5th ed. Lippincott, Williams & Wilkins, New York, pp.2947–2975.

Gilray, J.A., Nettleton, P.F., Pow, I., Lewis, C.J., Stephens, S.A., Madeley, J.D.,Reid, H.W., 1998. Restriction endonuclease profiles of orf virus iso-lates from the British Isles. Vet. Rec. 143, 237–240.

Ginn, P.E., Mansell, J.E.K.L., Rakich, P.M., 2007. Skin and appendages. In:Grant Maxie, M. (Ed.), Jubb, Kennedy, and Palmers Pathology ofDomestic Animals, 5th ed., vol. 1Saunders Elsevier, London, pp. 553–781 (Chapter 5).

Guan, G., Luo, J., Ma, M., Yang, D., Wang, Y., Gao, J., Sun, H., Liu, Z., Liu, A.,Dang, Z., Boulard, C., Yin, H., 2005. Sero-epidemiological surveillanceof hypodermosis in yaks and cattle in north China by ELISA. Vet.Parasitol. 129, 133–137.

Guo, J., Rasmussen, J., Wunschmann, A., de la Concha-Beremjillo, A., 2004.Genetic characterization of orf viruses from various ruminant speciesof a zoo. Vet. Microbiol. 99, 81–92.

Haig, D.M., McInnes, C.J., 2002. Immunity and counter-immunity duringinfection with the parapoxvirus orf virus. Virus Res. 88, 3–16.

Hautaniemi, M., Ueda, N., Tuimala, J., Mercer, A.A., Lahdenpera, J.,McInnes, C.J., 2010. The genome of pseudocowpoxvirus: comparisonof a reindeer isolate and a reference strain. J. Gen. Virol. 91, 1560–1576.

Inoshima, Y., Morooka, A., Sentsui, H., 2000. Detection and diagnosis ofparapoxvirus by the polymerase chain reaction. J. Virol. Methods 84,201–208.

Khalafalla, A.I., Agab, H., Abbas, B., 1994. An outbreak of contagiousecthyma in camels (Camelus dromedarius) in eastern Sudan. Anim.Health Prod. 26, 253–254.

Klein, J., Tryland, M., 2005. Characterisation of parapoxviruses isolatedfrom Norwegian semi-domesticated reindeer (Rangifer tarandus tar-andus). Virol. J. 2, 79.

Kummeneje, K., Krogsrud, J., 1979. Contagious ecthyma (orf) in reindeer(Rangifer t. tarandus). Vet. Rec. 105, 60–61.

McKeever, D.J., McEwan Jenkinson, D., Hutchinson, G., Reid, H.W., 1988.Studies of the pathogenesis of orf virus infection in sheep. J. Comp.Pathol. 99, 317–328.

Nettleton, P.F., Munro, R., Pow, I., Gilray, J., Gray, E.W., Reid, H.W., 1995.Isolation of a parapoxvirus from a grey seal (Halichoerus grypus). Vet.Rec. 137, 562–564.

Nordkvist, M., 1973. Munvartsjuka – en ny rensjukdom? Rennaringsnytt8–9, 6–8.

Reid, H.W., 2000. Orf. In: Martin, W.B., Aitken, I.D. (Eds.), Diseases ofSheep. 3rd ed. Blackwell Science Ltd., Oxford, pp. 61–266.

Robinson, A.J., Mercer, A.A., 1995. Parapoxvirus of red deer: evidence forits inclusion as a new member in the genus parapoxvirus. Virology208, 812–815.

Sainsbury, T., Ward, L., 1996. Parapoxvirus infection in red squirrels. Vet.Rec. 138, 400.

Tikkanen, M.K., McInnes, C.J., Mercer, A.A., Buttner, M., Tuimala, J., Hir-vela-Koski, V., Neuvonen, E., Huovilainen, A., 2004. Recent isolates ofparapoxvirus of Finnish reindeer (Rangifer tarandus tarandus) areclosely related to bovine pseudocowpox virus. J. Gen. Virol. 85,1413–1418.

Tryland, M., 2002. Asymptomatic parapoxvirus infections in semi-domes-ticated reindeer (Rangifer tarandus tarandus). In: Proceedings of theXIVth International Poxvirus & Iridovirus Workshop, 20–25 Septem-ber, Lake Placid, New York, USA, P75, p. 169.

Tryland, M., Myrmel, H., Holtet, L., Haukenes, G., Traavik, T., 1998. Clinicalcowpox cases in Norway. Scand. J. Infect. Dis. 30, 301–303.

Tryland, M., Josefsen, T.D., Oksanen, A., Aschfalk, A., 2001. Contagiousecthyma in Norwegian semidomesticated reindeer (Rangifer tarandustarandus). Vet. Rec. 149, 394–395.

Tryland, M., Klein, J., Nordøy, E.S., Blix, A.S., 2004. Isolation and partialcharacterization of a parapoxvirus isolated from a Weddell seal. VirusRes. 108, 83–87.

Tryland, M., Okeke, M.I., Af Segerstad, C.H., Morner, T., Traavik, T., Ryser-Degiorgis, M.P., 2011. Orthopoxvirus DNA in Eurasian lynx, Sweden.Emerg. Infect. Dis. 17, 626–632.

Vikøren, T., Haugum, T., Schulze, J., Akerstedt, J., Lillehaug, A., Tryland, M.,2008. A severe outbreak of contagious ecthyma (orf) in a free-ranging

musk ox (Ovibos moschatus) population in Norway. Vet. Microbiol.127, 10–20. Norwegian Reindeer Husbandry Development Fund (RUF).