Article — Artikel

Detection and characterisation of papillomavirus in skin lesions ofgiraffe and sable antelope in South Africa

E van Dyka*, A-M Bosmanb, E van Wilpec, J H Williamsd, R G Bengise, J van Heerdenf and E H Venterb

INTRODUCTIONPapillomaviruses are classified in the

family Papillomaviridae6. This large familyof animal and human viruses generallyinfects epithelial cells causing hyperpro-liferative lesions. Papillomavirus also hasoncogenic potential18, infecting cutaneousand mucous epithelia in a variety of hoststhrough cuts or abrasions and inducesthe formation of papillomas or warts bytargeting the keratinocyte, the viralgrowth accompanying the steady matu-ration of the cell to the surface31. These

tumours are generally benign and self-limiting and spontaneously regress withthe animal recovering completely, butoccasionally benign tumours may persistand become malignant by progressing tosquamous cell carcinomas10.

Bovine papillomavirus induces exophy-tic papillomas of cutaneous or mucosalepithelia in cattle. Papillomas are benigntumours which generally regress un-eventfully; however, they do occasionallypersist and provide the focus for malig-nant transformation to squamous cellcarcinomas, particularly in the presence ofenvironmental co-factors. This has beenexperimentally demonstrated in cancer ofthe urinary bladder and upper alimen-tary tract in cattle feeding on bracken fern(Pteridium aquilinum)39. In equines BPVcauses a locally invasive, fibroblastic skintumour of horses, donkeys, mules23 andzebras37. These tumours appear as differ-ent clinical entities and can be classifiedinto 6 clinical types16. This cross-speciesinfection of Equidae by BPV 1 and 2 is theonly record until recently of a papilloma-virus crossing the species barrier, theresulting tumour being known as a sar-coid22. Bovine papillomavirus has beendemonstrated in lesions in the water

buffalo (Bubalis bubalis)28 and bison (Bisonbonasus)21.

Earlier it was thought that apart fromBPV 1 and 2, papillomaviruses are strictlyspecies-specific and only the natural hostis infected. Even under experimental con-ditions papillomaviruses did not appearto infect any host other than the naturalone. Lesions usually attributed to papil-lomavirus infection have been mostextensively studied in cattle2,9,14 andhorses3,7,11,24,25, but at least 50 mammalianspecies have been confirmed as beinginfected by species-specific papilloma-viruses33. Papillomaviruses appear to bewidespread and have been found in alarge number of vertebrate species andare assumed to have co-evolved withtheir hosts4,5. Virtually all mammalianspecies are hosts for one or more papil-lomaviruses33.

CASE HISTORIES

Giraffes 1 and 2An adult female giraffe (Giraffa camelo-

pardalis) (Giraffe 1) with extensive lesionsin the skin of the dorsal neck and back wasobserved near Shingwedzi rest camp inthe northern part of the Kruger NationalPark (KNP), South Africa (Fig. 1). She wasin a group of 6 animals, 1 of which was abull. The other animals in the groupshowed no lesions. This area is a naturalhabitat for giraffe as they occur in a varietyof dry savannas ranging from scrub towoodland29. Approximately 1 year latera 2nd affected giraffe (Giraffe 2), wasobserved in the vicinity of Skukuza restcamp and the Kruger Gate in the south-ern part of the KNP (Fig. 2). This wasalso an adult female and part of a groupof 5 of mixed sex and age. She was theonly animal in the group exhibitingwart-like lesions that were especiallyprominent in the skin of her head andneck, but had also spread to the rest of herbody.

SableA group of 30 sable antelope (Hippotragus

niger) was kept as a breeding herd on agame farm in the Kimberley district,Northern Cape Province, South Africa.

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ABSTRACTPapillomavirus was detected electron microscopically in cutaneous fibropapillomas of agiraffe (Giraffa camelopardalis) and a sable antelope (Hippotragus niger). The virus particlesmeasured 45 nm in diameter. Histopathologically, the lesions showed histopathologicalfeatures similar to those of equine sarcoid as well as positive immunoperoxidase-staining oftissue sections for papillomavirus antigen. Polymerase chain reaction (PCR) detectedbovine papillomavirus (BPV) DNA. Bovine papillomavirus-1 was characterised byreal-time PCR in the sable and giraffe, and cloning and sequencing of the PCR productrevealed a similarity to BPV-1. As in the 1st giraffe, the lesions from a 2nd giraffe revealedlocally malignant pleomorphism, possibly indicating the lesional end-point of papillomainfection. Neither virus particles nor positively staining papillomavirus antigen could bedemonstrated in the 2nd giraffe but papillomavirus DNA was detected by real-time PCRwhich corresponded with BPV-1 and BPV-2.

Keywords: cutaneous fibropapillomas, Giraffa camelopardalis, giraffe, Hippotragus niger,malignant pleomorphism, papillomavirus, sable antelope.

van Dyk E, Bosman A-M, van Wilpe E, Williams J H, Bengis R G, van Heerden J, Venter E HDetection and characterisation of papillomavirus in skin lesions of giraffe and sableantelope in South Africa. Journal of the South African Veterinary Association (2011) 82(2): 80–85(En.). Department of Companion Animal Clinical Studies, Faculty of Veterinary Science,University of Pretoria, Private Bag X04, Onderstepoort, 0110 South Africa.

aDepartment of Companion Animal Clinical Studies,Faculty of Veterinary Science, University of Pretoria,Private Bag X04, Onderstepoort, 0110 South Africa.

bDepartment of Veterinary Tropical Diseases, Faculty ofVeterinary Science, University of Pretoria, Private BagX04, Onderstepoort, 0110 South Africa.

cSection of Electron Microscopy, Department of Anatomyand Physiology, Faculty of Veterinary Science, Universityof Pretoria, Private Bag X04, Onderstepoort, 0110 SouthAfrica.

dSection of Pathology, Department of ParaclinicalSciences, Faculty of Veterinary Science, University ofPretoria, Private Bag X04, Onderstepoort, 0110 SouthAfrica.

eNational Department of Agriculture, PO Box 12,Skukuza, Kruger National Park, 1350 South Africa.

fKimberley Veterinary Clinic, 16 Dalham Road, Kimberley,8301 South Africa.

*Author for correspondence.E-mail: enettevd@gmail.com

Received: February 2011. Accepted: April 2011.

This arid area of South Africa is not thenatural habitat of sable as they are asavanna woodland species, but suchgame species are sometimes translocatedto private land and reserves outside theirnormal range. One cow was lame in theright hind leg and a wart-like lesion of60 × 60 mm was present in the skin of thelateral aspect of the distal part of the 2ndphalanx proximal to the right hind hoof.After surgical removal, growth recurredat the original site and other well-definedwart-like lesions were also noticed in theskin of the right shoulder and lip.

The purpose of this study was to deter-mine electron microscopically if BPV waspresent in the lesions of these animalsand, if so, to detect and distinguishbetween BPV-1 and BPV-2 DNA usingreal-time PCR37.

MATERIALS AND METHODS

Sample collectionGiraffe 1 was shot in May 2007 in the

vicinity of the Shingwedzi rest camp(23°97’42”S, 31°42’58”E). Samples of the

lesions were collected for PCR by excisingpieces of tissue 10 × 3 × 3 mm in size.These were stored in sterile tubes at 4 °Cuntil analysis. Lesion samples for histo-pathological examination were stored induplicate in 10 % neutral buffered forma-lin. The 2nd giraffe (Giraffe 2) was shotnear Skukuza rest camp (24°59’16”S,31°34’32”E) in October 2008 and sampleswere collected as described above. Thesable antelope was anaesthetised and thelesion on the pastern surgically removedin an effort to relieve the lameness. Sam-ples of the lesion were taken as describedabove.

Electron microscopyFormalin-fixed skin samples from both

giraffes were prepared for transmissionelectron microscopy (TEM) according tostandard procedures. Ultra-thin sectionswere stained with uranyl acetate and leadcitrate, and examined in a Philips CM 10transmission electron microscope oper-ated at 80 kV.

An unfixed skin sample of the sable cowwas prepared for TEM by grinding the

tissue in a mortar with a pestle in a smallvolume of sterile water and the mixturewas centrifuged at 13 000 rpm for 45 min.The resultant pellet was re-suspended inwater, stained with 3 % phosphotungsticacid and a drop of the suspension placedonto a formvar- and carbon-coated gridfor examination. A relevant area was alsoretrieved from the histological wax block(see below) of the skin sample from thepastern and was treated with 1 % OsO4 inxylene and embedded in an epoxy resin.

HistopathologyThe formalin-fixed samples from both

giraffes and the sable were dehydratedand paraffin wax-embedded for routinehistological processing. All tumour sectionswere stained with haematoxylin andeosin for light microscopy, as well asimmunohistochemically for evaluation ofpapillomavirus, using a polyclonal rabbitanti-papillomavirus antibody againstchemically disrupted BPV-1, by theavidin-biotin technique13. A section of abovine fibropapilloma was used as apositive control.

DNA extractionDNA was extracted from 25 mg of lesion

tissues from each animal using theQIAamp® DNA extraction kit (SouthernCross Biotechnologies) according to themanufacturer ’s instructions. ExtractedDNA was eluted in 100 elution bufferand stored at 4 °C until further analysis.

Real-time PCRThe hybridisation probe real-time PCR

assay, as described by van Dyk et al.,(2009)37 was used for the detection anddifferentiation of BPV-1 and BPV-2 DNAin the giraffe (1 & 2) and sable lesions. Theprimers (forward primer: 5’-CAA AGGCAA GAC TTT CTG AAA CAT-3’, andreverse primer: 5’-AGA CCT GTA CAGGAG CAC TCA A-3’) amplified a 244 and247 bp region of the E5 ORF of BPV-1 andBPV-2 respectively. For the specific detec-tion of BPV-1 amplicons, hybridisationprobes BPV-1 anchor (5’-ACT GG TGTACT ATG CCA AAT CTA TGG TTT CTATTG-Fluor-3’) and BPV-1 sensor (5’-LC-640-CTT GGG ACT AGT TGC TGC AATGCA ACT-Pho-3’) were used, and for thedetection of BPV-2 amplicons, hybridisa-tion probes BPV-2 anchor (5’-TTT AATCAC TGC CAT TTG TTT TTT TCA TATCTC GT-Fluor-3’) and BPV-2 sensor(5’-LC705-AGG CAT ACT ATG CCG AATCTA TGG TTT CTA TTG TT-Pho-3’) wereused.

All primers and fluorescent-labelledhybridisation probes were manufacturedby Metabion International AG (Martins-ried, Germany).

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Fig. 1: Multiple papillomas in the skin of Giraffe 1 euthanased in the vicinity of Shingwedzirest camp. (Photograph reproduced with kind permission of D S van Dyk, SANPARKS.)

Fig. 2: Giraffe 2 euthanased in the vicinity of Skukuza rest camp. Note the presence ofmultiple nodular fibropapilloma lesions in the skin of her neck, head and ears.

The PCR amplification mixture andreaction conditions were as previouslydescribed37. Plasmids obtained fromGiraffe 1 and the sable were used astemplate DNA (2 µl) and genomic DNAfrom Giraffe 2 was used. Fluorescencewas measured at 640 and 705 nm and theresults were analysed with the RocheLightCycler® Software v4.0. Specimens,from positive zebra sarcoid (BPV-1 andBPV-2)37 were included as positive con-trols.

Cloning and sequence analysisConventional PCR was used for the

amplification of a 637 bp DNA fragmentof a region of the E5 ORF of both BPV-1and BPV-2. The primers (F_3610: 5’-GCTAAC CAG GTA AAG TGC TAT C-3’;R_4247: 5’-TGC TTG CAT GTC CTG TACAGG T-3’), PCR amplification mixtureand reaction conditions were as previ-ously described37. However, a nested PCRstep was included using 1 µ of the 1stround amplicons as PCR template in Gi-raffe 1 and the sable. The amplicons ob-tained were purified, cloned into thepGEM®-T easy vector (Promega pGEM-TEasy Vector System, Promega, Madison,USA), and transformed into competentJM 109 E. coli cells (Promega, Madison,USA). Recombinant plasmid DNA wasisolated, directly sequenced using the ABIBigDye™ Terminator Cycle SequencingReady Reaction kit (PE AppliedBiosystems) and analysed on an ABI 3100sequencer at Inqaba Biotec (Pretoria,South Africa). Sequencing data were as-sembled and edited with theGAP4 programme of the Staden package(version 1.6.0 for Windows)30 and alignedwith published sequences of relatedgenera using ClustalX (version 1.81 forWindows). A BLAST search was per-formed using the Blastn algorithm.Phylogenetic trees were constructedusing the maximum parsimony method(Mega 3.0 software package) 17 in combi-nation with the bootstrap method12 (1000replicates/tree for distance methods and100 replicates/tree for parsimony methods).Human papillomavirus type 16 (K02718)was used as an outgroup.

RESULTS

Macroscopic lesionsThere were extensive skin lesions on the

head, neck and back of the 2 adult femalegiraffes (Giraffe 1 and 2) in the KrugerNational Park (Figs 1, 2).

Electron microscopyNegatively-stained spherical papil-

lomavirus particles with a diameter of52 nm and distinct capsomeres werepresent in the unfixed skin sample of the

sable cow (Fig. 3). The nuclei of the stratumgranulosum of both the sable cow andGiraffe 1 contained numerous randomlyscattered papillomavirus particles measur-ing 45 nm in diameter (Figs 4, 5). Thenuclei of infected cells displayed irregu-lar clumps of condensed chromatin. Novirus was demonstrated in samples ob-tained from Giraffe 2.

HistopathologyThe skin lesions of Giraffe 1 were

fibropapillomatous in nature and those ofthe sable resembled equine verrucosesarcoid. Small nuclei in the stratumgranulosum of both animals stainedimmunohistochemically positive forpapillomavirus antigen. The lesions ofGiraffe 2 were more sarcoid-like, expansileand infiltrative in the dermis. Lesions ofboth giraffes showed evidence of malig-nancy. Scattered, large fibroblasts with

bizarre large nuclei, some of whichappeared multinucleated, occurredthroughout the dermal tumour tissuewith those of Giraffe 2 being moreprevalent. Immunohistochemical stain-ing of sections from Giraffe 2 did notdemonstrate papillomavirus antigen. Thedetailed histopathology is described andillustrated elsewhere38.

Real-time PCRThe primers used in the real-time PCR

assay37 amplified either a 244 bp or 247 bpregion of the E5 ORF of BPV-1 and BPV-2,respectively. Two separate hybridisationprobes sets were used in a multiplexformat for the specific detection ofBPV-1 and BPV-2 DNA. For the detectionof BPV-1 DNA, an increase in fluores-cence is expected at 640 nm as well as 2BPV-1-specific melting peaks at 62.90 ±1.24 °C and 68.17 ± 0.71 °C. Similarly, for

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Fig. 3: Negatively-stained intranuclear papillomavirus particles in an unfixed sample fromthe sable cow.

Fig. 4: Intranuclear papillomavirus (encircled) particles stained positively with uranyl-acetate in ultra-thin sections of wax-retrieved epoxy sections of the sable cow skin.

the detection of BPV-2 DNA, an increasein fluorescence at 705 nm is expected aswell as 2 BPV-2-specific peaks at 58.86 ±0.60 °C and 64.06 ± 0.59 °C.

Bovine PV-1-positive material from sar-coids of Cape mountain zebras induced agraph with melting peaks showing a simi-lar configuration to those of material fromGiraffe 1 and the sable. Giraffe 2 demon-strated fluorescence at 58 °C as well as68 °C at 640 nm (Fig. 6) indicating bothBPV-1 and BPV-2.

Cloning and sequencingFragments, 636 bp in size, of the E5 ORF

of the sable and Giraffe 1 samples wereamplified, cloned and sequenced toconfirm that the amplicons obtained wereindeed related to BPV. Sequences wereedited and truncated to a length of 478 bp.Six [K10 (HM446170); K1 (HM446171); K3(HM446172); K7 (HM446173); K5(HM446174); K1-1 (HM446174)] of the 9Giraffe 1 clones had identical sequencesover the 478 bp region and a BLASTsearch revealed that these sequenceswere identical to those of the BPV -1 E2and E5 protein encoding genes previ-ously reported37 to occur in Cape moun-tain zebras (FJ648519 to FJ648528). Theother 3 clones [K4 (HM446168); K27(HM446169); K2 (HM446176)] were notidentical to each other, but the BLASTsearch showed the highest similarity(~99 %) with the BPV type 1 E2 and E5protein-encoding genes found in Capemountain zebras (FJ648519 to FJ648528).Only 2 sequences were obtained fromsable clones, Sable 4 (HM 446166) andSable 2 (HM 44167) and they showed 2nucleotide differences within the 478 bpfragment. Again, the BLAST searchshowed the highest similarity (~99 %)with the BPV- E2 and E5 protein encodinggenes found in Cape mountain zebras. Allsequences obtained from Giraffe 1 and

sable samples were closely related(~97 %) to the BPV-1 subtype IV E5 pro-tein-encoding gene (accession numberAY232260) detected in equine sarcoid11.A phylogenetic tree was constructed andthe results were concurrent with theBLAST results obtained (Fig. 7). In thecase of Giraffe 2, no good-quality se-quence data could be obtained and theDNA could only be detected by real-timePCR.

DISCUSSIONNumerous descriptions of papillo-

matous epithelial growths in the skin ofmany species of mammals have beenreported15,21,26–28,32,33,36. Epithelial growthswere reported in giraffes in the KrugerNational Park in 197835. They are charac-terised variably by a conspicuous amountof fibrous connective tissue underlyinghyperplastic epithelium and are com-monly associated with infection with apapillomavirus. This study documentsthe presence of BPV and/or DNA in suchgrowths in the skin of the 2 giraffes and asable antelope.

Papillomatosis is a naturally-occurring,generally species-specific infectious dis-ease. The lesions are regarded as hyper-plastic or a form of benign neoplasiaas they do not metastasise internally20.Although bovine-associated papilloma-virus DNA is consistently found in thesarcoid lesions of the horse1,19 and zebra37,papillomavirus particles have not beendemonstrated and the disease is thereforeconsidered to be a non-productive infec-tion in which the viral DNA exists epi-somally in these species. Most fibro-papillomas (sarcoids) in horses containidentifiable BPV DNA of either type 1 or23,7,11,24,25,34,40. The E1 gene of the papilloma-virus identified in sarcoids of donkeys isvery similar to BPV-1 and it has been pro-posed that it is a subtype of BPV-125. In the

present study the papillomavirus parti-cles that were demonstrated in the wart-like lesions of the sable antelope andGiraffe 1 are considered to be closely re-lated to BPV-I.

Ultrastructurally, virus particles havealso been demonstrated in skin papillomasin an impala and a giraffe in Kenya15 butsequencing was not done. In the waterbuffalo28 and the bison21 virus has alsobeen demonstrated electron microscopi-cally, and following its sequencing it wasreported to be homologous to the LCR ofBPV-1 (prototype sequence, accessionnumber X02346) in the water buffalo28

while the DNA sequence of 413 bp ampli-con derived from the European bisoncompared with the consensus sequenceof BPV-2 (GenBank Accession numberAY300818)21. The virus was also demon-strated in both species of animals by elec-tron microscopy21,28.

The real-time PCR method37 demon-strated the presence of BPV-1 in the sableand Giraffe 1 and was confirmed bycloning and sequencing. In the 2ndgiraffe, no good-quality sequences couldbe obtained, although, real-time assaydemonstrated the presence of both BPV-1and BPV-2. This can be attributed to thehigh sensitivity of the real-time PCR assayused, which is capable of demonstratingthe virus at less than 1.5 gene copies37.Another possible reason why the viruscould not be demonstrated was that thepapilloma had undergone a degree ofcellular malignant transformation, withmore pleomorphic fibroblasts beingpresent in the lesions than in Giraffe 1.The structural integrity of the virus ap-peared to be lost and the virus or viral an-tigen was no longer present20.

Although the geographical areas arewidely separated, the Giraffe 1 and sableclones clustered together with high simi-larity to BPV-1 sequences, and withsome of the zebra sequences reportedpreviously37. None of the sable or Giraffe 1sequences reported here were of theBPV-2 group, and only some of the previ-ously reported zebra samples37 clusteredin BPV-2. The human papillomavirustype 16 outgroup clustered completelydifferently (Fig. 7).

Clinical manifestation of latent infec-tion in the 3 animals described here mayhave been provoked by stress. In the caseof the sable antelope, not being in its typi-cal habitat may have induced immuno-suppression. In the case of the giraffes itcan only be speculated that drought con-ditions and the resultant high tannincontent of the Acacia trees as a result ofover-browsing acted as a co-factor inBPV-associated carcinogenesis as seen incattle following bracken fern ingestion8.

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Fig. 5: Numerous intranuclear papillomavirus particles in sections of formalin-fixed skinof Giraffe 1. Note nuclear chromatin clumping.

ACKNOWLEDGEMENTSThis work was supported by grants

from the South African Veterinary Foun-dation, the Research and DevelopmentFund of the University of Pretoria, andthe Department of Veterinary TropicalDiseases, Faculty of Veterinary Science,University of Pretoria. The authors thankProf. Roy Tustin for his critical commentson the manuscript.

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