- Email: [email protected]
Molecular Detection of Equus caballus Papillomavirus Type 2 in Genital Swabs From Healthy Horses in the Republic of Korea
Accepted Manuscript Molecular detection of Equus caballus papillomavirus type 2 in genital swabs from healthy horses in the Republic of Korea Sang-Kyu Lee, Jung Keun Lee, Inhyung Lee PII:
S0737-0806(18)30515-X
DOI:
https://doi.org/10.1016/j.jevs.2018.10.015
Reference:
YJEVS 2609
To appear in:
Journal of Equine Veterinary Science
Received Date: 21 July 2018 Revised Date:
7 October 2018
Accepted Date: 8 October 2018
Please cite this article as: Lee SK, Lee JK, Lee I, Molecular detection of Equus caballus papillomavirus type 2 in genital swabs from healthy horses in the Republic of Korea, Journal of Equine Veterinary Science (2018), doi: https://doi.org/10.1016/j.jevs.2018.10.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Molecular detection of Equus caballus papillomavirus type 2 in genital swabs from
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healthy horses in the Republic of Korea
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Sang-Kyu Leea,b, Jung Keun Leec and Inhyung Leea,*
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College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea
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Veterinary center, Korea Racing Authority, Gwacheon 13822, Republic of Korea
IDEXX Laboratories, Westbrook 04092, United States of America
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*Corresponding author
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Inhyung Lee, DVM, MS, PhD
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College of Veterinary Medicine, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul
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08826, Republic of Korea
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Tel: +82-2-880-1252
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Email: [email protected]
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Molecular detection of Equus caballus papillomavirus type 2 in genital swabs from
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healthy horses in the Republic of Korea
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Abstract
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Equus caballus papillomavirus type 2 (EcPV-2) is implicated in genital neoplasms in horses,
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including equine penile papillomas, penile intraepithelial neoplasia, and squamous cell
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carcinoma (SCC). This virus seldom regresses spontaneously and can result in the
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development of SCC, which may result in significant clinical damage and economic cost.
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However, the prevalence of this virus is unknown in clinically unaffected horses. The aim of
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this study was to determine the prevalence of EcPV-2 DNA in genital swabs from healthy
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horses in the Republic of Korea and to investigate genetic variability within EcPV-2. A total
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of 797 genital swab samples obtained from Thoroughbred horses (35 stallions and 762 mares)
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were tested for the presence of the EcPV-2 gene E1 using a polymerase chain reaction (PCR)
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assay. Positive results were confirmed by repeat PCR to detect the presence of E6 and
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amplicon sequencing. EcPV-2 DNA was detected in 1% (8/762) of mare swabs but in none of
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stallion swabs (0/35). All EcPV-2 positive mares had been imported from the USA. EcPV-2
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E6 gene sequences were used for phylogenetic analysis and revealed three subdivisions relate
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to the European strains, and three isolates were located on two separate branches. This study
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documents the molecular prevalence and phylogenetic analysis of EcPV-2 DNA in the
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Republic of Korea.
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Keywords: papillomavirus, horse, Equus caballus papillomavirus type 2, phylogenetic
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analysis
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Papillomaviruses are small, non-enveloped, double-stranded DNA viruses that are associated
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with benign and malignant tumors of the skin and mucous membranes in humans and animals
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[1]. They are classified on the basis of their species of origin, and, to date, seven types of
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EcPVs have been reported in horses [1, 2]. EcPV-1 is associated with cutaneous papillomas
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on the legs and muzzle while EcPV-3 is related to aural plaques [2, 3]. Among EcPV types,
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EcPV-2 is considered the most clinically important due to its mucosotropic nature and is
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considered to be a causal factor for the development of squamous cell carcinoma (SCC) [8, 9]
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and papillomas affecting the genital area of horses [2]. Genital plaques, papillomas, and
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carcinoma in situ have been recognized as genital SCC precursors and can be induced by
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EcPV-2 infection [9, 12, 16].
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SCC is the second most common neoplasia found in horses and is the most common on
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the external genitalia of horses [4, 5]. SCC can metastasize and may recur after treatment,
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therefore long-term prognosis may be poor [5, 6]. SCC can also seriously affect the welfare
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of affected horses, causing substantial financial losses to their owners [7]. EcPV-2 DNA is
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most commonly detected in samples from SCC and papillomas; however, low levels have
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been recently reported in genital swabs from clinically healthy horses where the prevalence
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of EcPV-2 is not known [1, 9].
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This study was performed to determine the prevalence and to genetically characterize EcPV-2 in genital swabs from healthy horses in the Republic of Korea.
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2. Material and Methods
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2.1. Sample material Seven hundred ninety-seven genital swabs were obtained from healthy Thoroughbred
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breeding horses in the Republic of Korea, with ages ranging from 4 to 25 years. A routine
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clinical examination was performed on all horses and no evidence of genital tumors were
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observed. Penile swabs (n = 35) were taken from the surface of the penis, the fossa glandis
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and the urethral sinus of stallions that had been sedated for Contagious Equine Metritis
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sampling. Vulvovaginal swabs (n = 762) were taken from the clitoral fossa and sinuses of
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mares for the same sampling. All swabs were taken by using sterile flocked swabs (Noblebio,
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Republic of Korea). Immediately after sample collection, the samples were stored in sterile
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tubes at –20°C until use.
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2.2. Sample processing and PCR
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DNA was extracted from genital swab samples using a cador Pathogen 96 QIAcube HT
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Kit (Qiagen, Germany) with the QIAcube HT system (Qiagen, Germany) according to the
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manufacturer’s instructions and stored at –20°C. Successful DNA purification was confirmed
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by equine beta-actin PCR, using a previously described method [10]. For the EcPV-2 DNA
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screening test, the following EcPV-NB primer set was used: EcPV2-NB forward (5′-
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GCGGACTGCGCGTCACAAGAGGGGC-3′)
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ACGCAAGCACCACCCACTGCTTGGCA-3′), which amplified a 679-base pair (bp)
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fragment of the E1 gene [4]. PCR was performed in a 20 µL reaction mixture, containing 1
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unit HotstarTaq Plus DNA polymerase (Qiagen, Germany), 200 µM of each dNTP, a reaction
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buffer with 1.5 mM MgCl2, and 2.5 µL of template DNA. The concentration of each primer
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was 0.5 µM. The reaction profile consisted of 5 minutes of denaturation at 95°C followed by
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35 cycles of 30 seconds at 94°C, 40 seconds at 68°C, and 1 minute at 72°C, followed by a
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and
reverse
primer
(5′-
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final incubation at 72°C for 10 minutes. The PCR was performed in a SimpliAmp thermal
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cycler (Applied Biosystems, USA). Amplification products were visualized on 1.5% TBE (Tris-borate-EDTA) agarose gels
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by Safe Shine Green (Biosesang, Republic of Korea) staining under UV light. Confirmed
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EcPV-2 DNA from a stored sample of a horse with a penile SCC served as a positive control
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[11], and nuclease-free water (Qiagen, Germany) functioned as a negative control. PCR
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products were sent to a commercial sequencing laboratory (Macrogen, Republic of Korea)
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where amplicons were purified using the QIAquick PCR Purification Kit (Qiagen, Germany)
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and sequenced using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction
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Kit V.3.1 and with an ABI3730 Genetic Analyzer (Applied Biosystems Inc, USA). The results
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were
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(http://www.ncbi.nlm.nih.gov/Genbank/) using the basic local alignment search tool (BLAST;
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http://blast.ncbi.nlm.nih.gov/Blast.cgi). A second round of PCR was performed on the
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positive samples identified using the EcPV-2 NB primer sets. The second PCR assay
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amplified a 1643-bp fragment of the E6 region using an EcPV2_URRE6E7 forward and
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reverse primers [4]. PCR was carried out in a 50-µL volume containing 2 µL of extracted
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DNA and Hotstar Taq Plus Master Mix Kit (Qiagen, Germany), and 2.5 µM of each primer.
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PCR conditions were as follows: 95°C for 5 minutes; 30 cycles at 94°C for 30 seconds, 60°C
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for 30 seconds, and 72°C for 2 minutes; and a final extension step at 72°C for 10 minutes.
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Electrophoresis, purification, and sequencing were performed as described above. After
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sequencing the amplicons with the same primers, the results were merged and homology of
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the deduced nucleotide sequences for EcPV-2 was analyzed via the GenBank database using
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BLAST.
known
sequences
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GenBank
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with
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2.3. Phylogenetic analysis
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genetic relationship with those reported sequences from other countries. Eighteen additional
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sequences were added to the GenBank database (EU503122, HM153757, HM153758,
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HM153759, HM153760, HM153762, HM461973, JN664032, JN664040, JN664042,
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KX349717, KX349718, KX349719, KX349721, KX349722, KX349724, KY827401, and
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KY827403). All PCR-positive samples for E6 gene sequences of 127 to 400 bp were
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evaluated. Sequences of the EcPV-2 E6 gene identified in this study were aligned using
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CLUSTAL X v.2.0 and Bioedit v.7.2.6 software. Phylogenetic analysis was performed using
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the neighbor-joining method with 1,000 bootstrap values using MEGA 7 software.
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3. Results
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3.1. Molecular prevalence of EcPV-2 in apparently healthy horses
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The initial PCR method targeting the E1 gene revealed 8 of 797 (1%) genital samples positive for EcPV-2; 1/115 samples collected from the inland region and 7/682 samples
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collected from Jeju island in the Republic of Korea (Table 1). All positive samples obtained
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were from vulvovaginal swabs in mares (Table 2) with an age range of 6 to 20 years old, and
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all were imported from the USA (Table 3).
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3.2. Phylogenetic analysis
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Clustal X algorithm in MEGA 7 (Fig. 1). The alignment of the E6 gene of 8 positive
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sequences in this study along with 10 EcPV-2 sequences retrieved from GenBank consisted
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of 274 positions, with nucleotide identities ranging from 97.8 to 100%. In this reduced
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fragment, 10 different variants could be defined. Two genetic strains in this study were a
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match to the retrieved sequences. Three samples (V2, V7, V8) isolated from vulvovaginal
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swabs were slightly distant from the other sequences. Four sequences showing a 3-bp
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deletion in the E6 gene clustered along with sequences without this deletion.
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EcPV-2 is associated with genital papillomas and squamous cell carcinomas in horses [8] and
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is considered to be a causal agent of equine genital SCC [6-8]. EcPV-2 has frequently been
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detected in equine SCC’s, whereas there is a low prevalence rate in apparently healthy horse
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mucosae [7, 8, 12]. However, few studies focusing on the prevalence of EcPV-2 in clinically
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healthy horses have been reported. Four of 94 (4.3%) horses were found to be positive in a
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report by Sykora et al. [7], three of 72 (4.2%) were isolated in a Knight et al. report [12], and
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six of 59 (6.8%) were detected in normal genital swab samples in a report by Bogaert et al.
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using molecular diagnosis [4]. In this study, apparently healthy horses from the Republic of
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Korea showed a lower prevalence rate (1%) of EcPV-2 DNA, as determined by testing of
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genital swabs when compared to the previous studies. Interestingly, in our study, EcPV-2
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DNA was only detected in vulvovaginal samples, in contrast with previous studies where a
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higher incidence was detected in penile swab samples [4, 7, 12]. EcPV-2 spreads via sexual
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or close-contact transmission in horses [8]. Also of interest was the fact that stallions that had
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mated with the EcPV-2-positive mares over the previous year were found to be negative for
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EcPV-2 DNA. Due to their relatively high economic value, Thoroughbred horses are often
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looked after more carefully by their owners, with stallions typically being treated with even
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greater care. It was therefore speculated that the increased attention giving to Thoroughbred
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stallions, including thorough hygienic care of the genital regions may explain why the
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prevalence rate in stallions was lower in this study when compare to previous reports that had
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sampled other horse breeds including Warmbloods, Arabians, Icelandic horses and Trotters
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amongst others [4, 7]. Additionally, strict hygiene procedure of Thoroughbred mares prior to
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mating may also reduce EcPV-2 transmission between mares and stallions. The irritant and
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carcinogenic properties of equine smegma has been proposed as a promoter of penile SCC
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development [5, 6, 13]. EcPV-2 DNA has been detected in smegma samples and smegma can
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ACCEPTED MANUSCRIPT act as an EcPV-2 reservoir [4, 9, 10]. Stallions have a tendency to gather less smegma than
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geldings [6, 13] Male genitalia samples were from stallions only without geldings in this
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study. Therefore it may be another factor of lower EcPV-2 DNA prevalence of penile swab
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samples, in contrast with the previous studies [4, 7, 12]. The horses sampled in this study
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originated from 13 different countries, with the majority originating from
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and the Republic of Korea (35.1%) (Table 4). All EcPV-2-positive horses found in this study
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had been imported from the USA. Seven of 8 (87.5%) EcPV-2-positive horses were over 10
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years of age (range 6-20 years). The reported average age of horses with genital papilloma
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and SCC are 16 to 18 years and 13 to 28 years, respectively [13, 14] and given that EcPV-2
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infection is associated with equine genital neoplasia, there may be an association between
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EcPV-2 infection and age that requires further investigation.
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the USA (53.9%)
Phylogenetic analysis based on the E6 gene and other sequences showed that various
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EcPV-2-related variants circulate in horses. Three EcPV-2 isolates (V2, V7 and V8) found in
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this study were quite different from all previously reported strains and were located on two
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separate branches, with V7 and V8 in the same subgroup and V2 on a separate branch. Three
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EcPV-2 isolates (V1, V5 and V6) were clustered in the same subgroup as isolates from
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Austria, Belgium, and Turkey. The other EcPV-2 isolates (V3 and V4) were closely located
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with strains from the United Kingdom and Austria. Results of this study show that EcPV-2
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isolates from the Republic of Korea are genetically diverse. In 2012, Bogaert et al. reported a
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phylogenetic tree for the E6 gene based on 7 sequences retrieved from GenBank and 10 other
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isolates [4]. However, one of seven retrieved E6 sequences from Genebank was actually an
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E1 gene (HM153764) sequence, which was isolated from Aron penile SCC. In this study, the
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E6 gene (HM153757) sequence, which was isolated from Aron penile SCC as the E6 gene,
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was added to the phylogenetic analysis instead of HM153764.
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Following infection with a papilloma virus has infected to horses, the virus becomes
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environmental factors which may lead to papilloma development [15]. Classical viral
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papillomas typically regress within 4 months but may last up to 9 months [15]. Genital
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papillomas associated with EcPV-2, have not been reported to spontaneously regress and may
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continue to develop into in situ or invasive SCC [15]. Although EcPV-2 DNA has been
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detected more commonly in penile rather than vulval swabs [7, 8, 12], the prevalence of
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EcPV-2 was higher in mares in this study. Therefore, early detection by regular examination
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of the external genitalia and regular hygienic care for both stallions and mares is
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recommended to avoid development and progression of genital tumors associated with EcPV-
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ACCEPTED MANUSCRIPT 5. Conclusion
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This is the first report of EcPV-2 infection surveillance in genital mucosa from a large
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number of apparently healthy Thoroughbred horses in the Republic of Korea. The prevalence
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of EcPV-2 in this study was lower in penile swab samples than previously reported [7, 8, 12].
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Three EcPV-2 isolates in this study were unique and 5 isolates showing links to previously
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reported sequences (Fig. 1) .
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Acknowledgement
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This study was supported by veterinary center, Korea Racing Authority.
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Conflict of Interest
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The authors declare that they have no conflicts of interest.
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Animal welfare/ethical statement
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Our study didn't conduct any animal experiments. Therefore, we didn't violate animal welfare
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in this study.
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ACCEPTED MANUSCRIPT References.
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[1] Fischer NM, Favrot C, Birkmann K, Jackson M, Schwarzwald CC, Muller M, et al.
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Serum antibodies and DNA indicate a high prevalence of equine papillomavirus 2 (EcPV2)
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among horses in Switzerland. Vet Dermatol. 2014;25:210-e54.
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[2] Sears KP, Sellon DC. Papillomavirus Infections. In: Sellon DC, Long MT, editors. Equine
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Infectious Diseases. 2nd ed. St. Louis, Missouri: Saunders/Elsevier; 2013. p. 244-5.
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[3] Quinn PJ, Markey BK, Leonard FC, Hartigan P, Fanning S, Fitzpatrick ES. Veterinary
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Microbiology and Microbial Disease: Wiley; 2011. p. 583-7.
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[4] Bogaert L, Willemsen A, Vanderstraeten E, Bracho MA, De Baere C, Bravo IG, et al.
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EcPV2 DNA in equine genital squamous cell carcinomas and normal genital mucosa. Vet
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Microbiol. 2012;158:33-41.
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[5] Gerald VDT. Squamous cell carcinoma of the penis and prepuce. In: Sprayberry KA,
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Robinson NE, editors. Robinson's Current Therapy in Equine Medicine. 7th ed:
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Elsevier/Saunders; 2014. p. 418.
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[6] Knight CG, Munday JS, Peters J, Dunowska M. Equine penile squamous cell carcinomas
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are associated with the presence of equine papillomavirus type 2 DNA sequences. Vet Pathol.
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2011;48:1190-4.
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[7] Sykora S, Samek L, Schonthaler K, Palm F, Borzacchiello G, Aurich C, et al. EcPV-2 is
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transcriptionally active in equine SCC but only rarely detectable in swabs and semen from
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healthy horses. Vet Microbiol. 2012;158:194-8.
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[8] Scase T, Brandt S, Kainzbauer C, Sykora S, Bijmholt S, Hughes K, et al. Equus caballus
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papillomavirus-2 (EcPV-2): an infectious cause for equine genital cancer? Equine Vet J.
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2010;42:738-45.
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[9] Sykora S, Jindra C, Hofer M, Steinborn R, Brandt S. Equine papillomavirus type 2: An
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equine equivalent to human papillomavirus 16? Vet J. 2017;225:3-8.
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ACCEPTED MANUSCRIPT [10] Brandt S, Haralambus R, Schoster A, Kirnbauer R, Stanek C. Peripheral blood
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mononuclear cells represent a reservoir of bovine papillomavirus DNA in sarcoid-affected
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equines. J Gen Virol. 2008;89:1390-5.
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[11] Lee S-K, Lee JK, Lee I. Penile squamous cell carcinoma associated with Equus caballus
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papilloma virus 2 in a miniature Appaloosa horse. In:Proceedings of the 67th congress of
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Korean Society of veterinary clinics, Seoul, Republic of Korea; 2017. p. 19.
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[12] Knight CG, Dunowska M, Munday JS, Peters-Kennedy J, Rosa BV. Comparison of the
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levels of Equus caballus papillomavirus type 2 (EcPV-2) DNA in equine squamous cell
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carcinomas and non-cancerous tissues using quantitative PCR. Vet Microbiol. 2013;166:257-
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62.
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[13] Scott DW, Miller WH. Equine Dermatology. 2nd ed: Elsevier/Saunders; 2010. p. 468-76.
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[14] van den Top JG, de Heer N, Klein WR, Ensink JM. Penile and preputial tumours in the
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horse: a retrospective study of 114 affected horses. Equine Vet J. 2008;40:528-32.
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[15] Torres SM, Koch SN. Papillomavirus-associated diseases. Vet Clin North Am Equine
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Pract. 2013;29:643-55.
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[16] Sykora S, Brandt S. Papillomavirus infection and squamous cell carcinoma in horses.
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Vet J. 2017;233:48-54.
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Table 1. Summary of the EcPV-2 molecular prevalence of genital swab samples by region in
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the Republic of Korea. Sample
Stallions
Mares
collection Pos (%)
No.
Inland area
7
0 (0%)
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Jeju island
28
0 (0%)
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1 (1%)
7 (1%)
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Abbreviations: No, number of examined samples; Pos (%); number (percentage) of positive samples for EcPV2 PCR.
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Pos (%)
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Table 2. Summary of the EcPV-2 molecular prevalence in genital swab samples from healthy
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horses. Penile swabs
Vulvovaginal swabs
group
No.
Pos (%)
Stallions
35
0 (0%)
Mares
No.
Pos (%)
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Sample
762
8 (1%)
Abbreviations: No, number of examined samples; Pos (%), number (percentage) of positive
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samples for EcPV-2 PCR.
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No.
Country of foaling
6 years
1
USA
10 years
1
USA
11 years
1
12 years
1
14 years
1
16 years
2
20 years
1
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Age
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Abbreviations: No., number of EcPV-2 PCR positive horses; USA, United States of America.
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Table 3. The age and origin of EcPV-2 positive horses.
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USA
KOR
AUS
JPN
CAN
UK
IRL
NZL
Other countries
430
280
30
27
12
5
4
4
5
54.0% 17
35.1% 12
3.8% 1
3.4% 4
1.5% -
0.6% -
0.5% 1
0.5% -
0.6% -
413
268
29
23
12
5
3
4
5
8
-
-
-
-
-
-
-
-
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Abbreviations: KOR, Republic of Korea; AUS, Australia; JPN, Japan; CAN, Canada; UK,
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United Kingdom; IRL, Ireland; NZL, New Zealand; Pos, positive samples.
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Fig. 1. Neighbor-joining phylogenetic tree for partial E6 gene based on 8 sequences obtained
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in this study along with 18 sequences retrieved from GenBank (11 from genital SCC, 2 from
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nasal cavity SCC, 1 from vulvovaginal swab, 1 from skin, and 1 from normal penile mucosa).
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GenBank accession numbers are shown for retrieved sequences. Bootstrap percentage values
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are shown on the branches.
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Abbreviations: SCC, squamous cell carcinoma; V, vulvovaginal swab.
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ACCEPTED MANUSCRIPT Highlights · The prevalence of Equus caballus papillomavirus type 2 (EcPV-2) in genital swabs from
· Molecular prevalence of EcPV-2 was lower than earlier reports.
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healthy horses in the Republic of Korea was investigated using PCR.
. The Korean EcPV-2 isolates also showed genetic diversity comparable to previously
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reported sequences.
ACCEPTED MANUSCRIPT Animal welfare/ethical statement This study didn't conduct any animal experiments. Therefore, this study didn't violate animal
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welfare guidlines.
ACCEPTED MANUSCRIPT CRediT author statement
Sang-Kyu Lee: Conceptualization, Methodology, Investigation, Software, Writing – Original
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Writing-Reviewing and Editing, Supervision.
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Draft, Visualization, Data curation. Jung Keun Lee: Investigation. Inhyung Lee: Validation,
S0737-0806(18)30515-X
DOI:
https://doi.org/10.1016/j.jevs.2018.10.015
Reference:
YJEVS 2609
To appear in:
Journal of Equine Veterinary Science
Received Date: 21 July 2018 Revised Date:
7 October 2018
Accepted Date: 8 October 2018
Please cite this article as: Lee SK, Lee JK, Lee I, Molecular detection of Equus caballus papillomavirus type 2 in genital swabs from healthy horses in the Republic of Korea, Journal of Equine Veterinary Science (2018), doi: https://doi.org/10.1016/j.jevs.2018.10.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Molecular detection of Equus caballus papillomavirus type 2 in genital swabs from
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healthy horses in the Republic of Korea
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Sang-Kyu Leea,b, Jung Keun Leec and Inhyung Leea,*
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a
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b
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College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea
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Veterinary center, Korea Racing Authority, Gwacheon 13822, Republic of Korea
IDEXX Laboratories, Westbrook 04092, United States of America
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*Corresponding author
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Inhyung Lee, DVM, MS, PhD
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College of Veterinary Medicine, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul
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08826, Republic of Korea
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Tel: +82-2-880-1252
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Email: [email protected]
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Molecular detection of Equus caballus papillomavirus type 2 in genital swabs from
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healthy horses in the Republic of Korea
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Abstract
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Equus caballus papillomavirus type 2 (EcPV-2) is implicated in genital neoplasms in horses,
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including equine penile papillomas, penile intraepithelial neoplasia, and squamous cell
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carcinoma (SCC). This virus seldom regresses spontaneously and can result in the
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development of SCC, which may result in significant clinical damage and economic cost.
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However, the prevalence of this virus is unknown in clinically unaffected horses. The aim of
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this study was to determine the prevalence of EcPV-2 DNA in genital swabs from healthy
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horses in the Republic of Korea and to investigate genetic variability within EcPV-2. A total
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of 797 genital swab samples obtained from Thoroughbred horses (35 stallions and 762 mares)
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were tested for the presence of the EcPV-2 gene E1 using a polymerase chain reaction (PCR)
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assay. Positive results were confirmed by repeat PCR to detect the presence of E6 and
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amplicon sequencing. EcPV-2 DNA was detected in 1% (8/762) of mare swabs but in none of
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stallion swabs (0/35). All EcPV-2 positive mares had been imported from the USA. EcPV-2
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E6 gene sequences were used for phylogenetic analysis and revealed three subdivisions relate
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to the European strains, and three isolates were located on two separate branches. This study
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documents the molecular prevalence and phylogenetic analysis of EcPV-2 DNA in the
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Republic of Korea.
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Keywords: papillomavirus, horse, Equus caballus papillomavirus type 2, phylogenetic
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analysis
ACCEPTED MANUSCRIPT 1. Introduction
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Papillomaviruses are small, non-enveloped, double-stranded DNA viruses that are associated
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with benign and malignant tumors of the skin and mucous membranes in humans and animals
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[1]. They are classified on the basis of their species of origin, and, to date, seven types of
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EcPVs have been reported in horses [1, 2]. EcPV-1 is associated with cutaneous papillomas
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on the legs and muzzle while EcPV-3 is related to aural plaques [2, 3]. Among EcPV types,
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EcPV-2 is considered the most clinically important due to its mucosotropic nature and is
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considered to be a causal factor for the development of squamous cell carcinoma (SCC) [8, 9]
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and papillomas affecting the genital area of horses [2]. Genital plaques, papillomas, and
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carcinoma in situ have been recognized as genital SCC precursors and can be induced by
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EcPV-2 infection [9, 12, 16].
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SCC is the second most common neoplasia found in horses and is the most common on
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the external genitalia of horses [4, 5]. SCC can metastasize and may recur after treatment,
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therefore long-term prognosis may be poor [5, 6]. SCC can also seriously affect the welfare
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of affected horses, causing substantial financial losses to their owners [7]. EcPV-2 DNA is
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most commonly detected in samples from SCC and papillomas; however, low levels have
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been recently reported in genital swabs from clinically healthy horses where the prevalence
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of EcPV-2 is not known [1, 9].
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This study was performed to determine the prevalence and to genetically characterize EcPV-2 in genital swabs from healthy horses in the Republic of Korea.
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2. Material and Methods
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2.1. Sample material Seven hundred ninety-seven genital swabs were obtained from healthy Thoroughbred
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breeding horses in the Republic of Korea, with ages ranging from 4 to 25 years. A routine
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clinical examination was performed on all horses and no evidence of genital tumors were
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observed. Penile swabs (n = 35) were taken from the surface of the penis, the fossa glandis
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and the urethral sinus of stallions that had been sedated for Contagious Equine Metritis
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sampling. Vulvovaginal swabs (n = 762) were taken from the clitoral fossa and sinuses of
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mares for the same sampling. All swabs were taken by using sterile flocked swabs (Noblebio,
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Republic of Korea). Immediately after sample collection, the samples were stored in sterile
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tubes at –20°C until use.
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2.2. Sample processing and PCR
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DNA was extracted from genital swab samples using a cador Pathogen 96 QIAcube HT
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Kit (Qiagen, Germany) with the QIAcube HT system (Qiagen, Germany) according to the
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manufacturer’s instructions and stored at –20°C. Successful DNA purification was confirmed
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by equine beta-actin PCR, using a previously described method [10]. For the EcPV-2 DNA
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screening test, the following EcPV-NB primer set was used: EcPV2-NB forward (5′-
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GCGGACTGCGCGTCACAAGAGGGGC-3′)
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ACGCAAGCACCACCCACTGCTTGGCA-3′), which amplified a 679-base pair (bp)
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fragment of the E1 gene [4]. PCR was performed in a 20 µL reaction mixture, containing 1
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unit HotstarTaq Plus DNA polymerase (Qiagen, Germany), 200 µM of each dNTP, a reaction
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buffer with 1.5 mM MgCl2, and 2.5 µL of template DNA. The concentration of each primer
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was 0.5 µM. The reaction profile consisted of 5 minutes of denaturation at 95°C followed by
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35 cycles of 30 seconds at 94°C, 40 seconds at 68°C, and 1 minute at 72°C, followed by a
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and
reverse
primer
(5′-
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final incubation at 72°C for 10 minutes. The PCR was performed in a SimpliAmp thermal
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cycler (Applied Biosystems, USA). Amplification products were visualized on 1.5% TBE (Tris-borate-EDTA) agarose gels
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by Safe Shine Green (Biosesang, Republic of Korea) staining under UV light. Confirmed
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EcPV-2 DNA from a stored sample of a horse with a penile SCC served as a positive control
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[11], and nuclease-free water (Qiagen, Germany) functioned as a negative control. PCR
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products were sent to a commercial sequencing laboratory (Macrogen, Republic of Korea)
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where amplicons were purified using the QIAquick PCR Purification Kit (Qiagen, Germany)
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and sequenced using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction
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Kit V.3.1 and with an ABI3730 Genetic Analyzer (Applied Biosystems Inc, USA). The results
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were
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(http://www.ncbi.nlm.nih.gov/Genbank/) using the basic local alignment search tool (BLAST;
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http://blast.ncbi.nlm.nih.gov/Blast.cgi). A second round of PCR was performed on the
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positive samples identified using the EcPV-2 NB primer sets. The second PCR assay
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amplified a 1643-bp fragment of the E6 region using an EcPV2_URRE6E7 forward and
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reverse primers [4]. PCR was carried out in a 50-µL volume containing 2 µL of extracted
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DNA and Hotstar Taq Plus Master Mix Kit (Qiagen, Germany), and 2.5 µM of each primer.
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PCR conditions were as follows: 95°C for 5 minutes; 30 cycles at 94°C for 30 seconds, 60°C
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for 30 seconds, and 72°C for 2 minutes; and a final extension step at 72°C for 10 minutes.
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Electrophoresis, purification, and sequencing were performed as described above. After
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sequencing the amplicons with the same primers, the results were merged and homology of
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the deduced nucleotide sequences for EcPV-2 was analyzed via the GenBank database using
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BLAST.
known
sequences
from
GenBank
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with
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2.3. Phylogenetic analysis
ACCEPTED MANUSCRIPT Phylogenetic analyses of the isolated EcPV-2 sequences was performed to evaluate their
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genetic relationship with those reported sequences from other countries. Eighteen additional
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sequences were added to the GenBank database (EU503122, HM153757, HM153758,
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HM153759, HM153760, HM153762, HM461973, JN664032, JN664040, JN664042,
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KX349717, KX349718, KX349719, KX349721, KX349722, KX349724, KY827401, and
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KY827403). All PCR-positive samples for E6 gene sequences of 127 to 400 bp were
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evaluated. Sequences of the EcPV-2 E6 gene identified in this study were aligned using
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CLUSTAL X v.2.0 and Bioedit v.7.2.6 software. Phylogenetic analysis was performed using
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the neighbor-joining method with 1,000 bootstrap values using MEGA 7 software.
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3. Results
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3.1. Molecular prevalence of EcPV-2 in apparently healthy horses
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The initial PCR method targeting the E1 gene revealed 8 of 797 (1%) genital samples positive for EcPV-2; 1/115 samples collected from the inland region and 7/682 samples
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collected from Jeju island in the Republic of Korea (Table 1). All positive samples obtained
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were from vulvovaginal swabs in mares (Table 2) with an age range of 6 to 20 years old, and
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all were imported from the USA (Table 3).
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3.2. Phylogenetic analysis
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A phylogenetic tree was generated using the neighbor-joining method, as implemented in
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Clustal X algorithm in MEGA 7 (Fig. 1). The alignment of the E6 gene of 8 positive
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sequences in this study along with 10 EcPV-2 sequences retrieved from GenBank consisted
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of 274 positions, with nucleotide identities ranging from 97.8 to 100%. In this reduced
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fragment, 10 different variants could be defined. Two genetic strains in this study were a
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match to the retrieved sequences. Three samples (V2, V7, V8) isolated from vulvovaginal
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swabs were slightly distant from the other sequences. Four sequences showing a 3-bp
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deletion in the E6 gene clustered along with sequences without this deletion.
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ACCEPTED MANUSCRIPT 4. Discussion
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EcPV-2 is associated with genital papillomas and squamous cell carcinomas in horses [8] and
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is considered to be a causal agent of equine genital SCC [6-8]. EcPV-2 has frequently been
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detected in equine SCC’s, whereas there is a low prevalence rate in apparently healthy horse
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mucosae [7, 8, 12]. However, few studies focusing on the prevalence of EcPV-2 in clinically
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healthy horses have been reported. Four of 94 (4.3%) horses were found to be positive in a
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report by Sykora et al. [7], three of 72 (4.2%) were isolated in a Knight et al. report [12], and
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six of 59 (6.8%) were detected in normal genital swab samples in a report by Bogaert et al.
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using molecular diagnosis [4]. In this study, apparently healthy horses from the Republic of
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Korea showed a lower prevalence rate (1%) of EcPV-2 DNA, as determined by testing of
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genital swabs when compared to the previous studies. Interestingly, in our study, EcPV-2
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DNA was only detected in vulvovaginal samples, in contrast with previous studies where a
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higher incidence was detected in penile swab samples [4, 7, 12]. EcPV-2 spreads via sexual
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or close-contact transmission in horses [8]. Also of interest was the fact that stallions that had
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mated with the EcPV-2-positive mares over the previous year were found to be negative for
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EcPV-2 DNA. Due to their relatively high economic value, Thoroughbred horses are often
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looked after more carefully by their owners, with stallions typically being treated with even
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greater care. It was therefore speculated that the increased attention giving to Thoroughbred
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stallions, including thorough hygienic care of the genital regions may explain why the
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prevalence rate in stallions was lower in this study when compare to previous reports that had
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sampled other horse breeds including Warmbloods, Arabians, Icelandic horses and Trotters
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amongst others [4, 7]. Additionally, strict hygiene procedure of Thoroughbred mares prior to
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mating may also reduce EcPV-2 transmission between mares and stallions. The irritant and
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carcinogenic properties of equine smegma has been proposed as a promoter of penile SCC
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development [5, 6, 13]. EcPV-2 DNA has been detected in smegma samples and smegma can
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ACCEPTED MANUSCRIPT act as an EcPV-2 reservoir [4, 9, 10]. Stallions have a tendency to gather less smegma than
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geldings [6, 13] Male genitalia samples were from stallions only without geldings in this
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study. Therefore it may be another factor of lower EcPV-2 DNA prevalence of penile swab
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samples, in contrast with the previous studies [4, 7, 12]. The horses sampled in this study
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originated from 13 different countries, with the majority originating from
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and the Republic of Korea (35.1%) (Table 4). All EcPV-2-positive horses found in this study
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had been imported from the USA. Seven of 8 (87.5%) EcPV-2-positive horses were over 10
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years of age (range 6-20 years). The reported average age of horses with genital papilloma
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and SCC are 16 to 18 years and 13 to 28 years, respectively [13, 14] and given that EcPV-2
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infection is associated with equine genital neoplasia, there may be an association between
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EcPV-2 infection and age that requires further investigation.
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the USA (53.9%)
Phylogenetic analysis based on the E6 gene and other sequences showed that various
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EcPV-2-related variants circulate in horses. Three EcPV-2 isolates (V2, V7 and V8) found in
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this study were quite different from all previously reported strains and were located on two
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separate branches, with V7 and V8 in the same subgroup and V2 on a separate branch. Three
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EcPV-2 isolates (V1, V5 and V6) were clustered in the same subgroup as isolates from
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Austria, Belgium, and Turkey. The other EcPV-2 isolates (V3 and V4) were closely located
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with strains from the United Kingdom and Austria. Results of this study show that EcPV-2
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isolates from the Republic of Korea are genetically diverse. In 2012, Bogaert et al. reported a
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phylogenetic tree for the E6 gene based on 7 sequences retrieved from GenBank and 10 other
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isolates [4]. However, one of seven retrieved E6 sequences from Genebank was actually an
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E1 gene (HM153764) sequence, which was isolated from Aron penile SCC. In this study, the
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E6 gene (HM153757) sequence, which was isolated from Aron penile SCC as the E6 gene,
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was added to the phylogenetic analysis instead of HM153764.
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Following infection with a papilloma virus has infected to horses, the virus becomes
ACCEPTED MANUSCRIPT latent and may recrudesce following immunosuppression as well as genetic and
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environmental factors which may lead to papilloma development [15]. Classical viral
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papillomas typically regress within 4 months but may last up to 9 months [15]. Genital
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papillomas associated with EcPV-2, have not been reported to spontaneously regress and may
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continue to develop into in situ or invasive SCC [15]. Although EcPV-2 DNA has been
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detected more commonly in penile rather than vulval swabs [7, 8, 12], the prevalence of
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EcPV-2 was higher in mares in this study. Therefore, early detection by regular examination
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of the external genitalia and regular hygienic care for both stallions and mares is
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recommended to avoid development and progression of genital tumors associated with EcPV-
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2.
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ACCEPTED MANUSCRIPT 5. Conclusion
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This is the first report of EcPV-2 infection surveillance in genital mucosa from a large
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number of apparently healthy Thoroughbred horses in the Republic of Korea. The prevalence
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of EcPV-2 in this study was lower in penile swab samples than previously reported [7, 8, 12].
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Three EcPV-2 isolates in this study were unique and 5 isolates showing links to previously
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reported sequences (Fig. 1) .
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Acknowledgement
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This study was supported by veterinary center, Korea Racing Authority.
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Conflict of Interest
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The authors declare that they have no conflicts of interest.
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Animal welfare/ethical statement
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Our study didn't conduct any animal experiments. Therefore, we didn't violate animal welfare
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in this study.
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ACCEPTED MANUSCRIPT References.
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[1] Fischer NM, Favrot C, Birkmann K, Jackson M, Schwarzwald CC, Muller M, et al.
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Serum antibodies and DNA indicate a high prevalence of equine papillomavirus 2 (EcPV2)
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among horses in Switzerland. Vet Dermatol. 2014;25:210-e54.
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[2] Sears KP, Sellon DC. Papillomavirus Infections. In: Sellon DC, Long MT, editors. Equine
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Infectious Diseases. 2nd ed. St. Louis, Missouri: Saunders/Elsevier; 2013. p. 244-5.
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[3] Quinn PJ, Markey BK, Leonard FC, Hartigan P, Fanning S, Fitzpatrick ES. Veterinary
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Microbiology and Microbial Disease: Wiley; 2011. p. 583-7.
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[4] Bogaert L, Willemsen A, Vanderstraeten E, Bracho MA, De Baere C, Bravo IG, et al.
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EcPV2 DNA in equine genital squamous cell carcinomas and normal genital mucosa. Vet
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Microbiol. 2012;158:33-41.
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[5] Gerald VDT. Squamous cell carcinoma of the penis and prepuce. In: Sprayberry KA,
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Robinson NE, editors. Robinson's Current Therapy in Equine Medicine. 7th ed:
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Elsevier/Saunders; 2014. p. 418.
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[6] Knight CG, Munday JS, Peters J, Dunowska M. Equine penile squamous cell carcinomas
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are associated with the presence of equine papillomavirus type 2 DNA sequences. Vet Pathol.
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2011;48:1190-4.
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[7] Sykora S, Samek L, Schonthaler K, Palm F, Borzacchiello G, Aurich C, et al. EcPV-2 is
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transcriptionally active in equine SCC but only rarely detectable in swabs and semen from
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healthy horses. Vet Microbiol. 2012;158:194-8.
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[8] Scase T, Brandt S, Kainzbauer C, Sykora S, Bijmholt S, Hughes K, et al. Equus caballus
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papillomavirus-2 (EcPV-2): an infectious cause for equine genital cancer? Equine Vet J.
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2010;42:738-45.
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[9] Sykora S, Jindra C, Hofer M, Steinborn R, Brandt S. Equine papillomavirus type 2: An
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ACCEPTED MANUSCRIPT [10] Brandt S, Haralambus R, Schoster A, Kirnbauer R, Stanek C. Peripheral blood
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mononuclear cells represent a reservoir of bovine papillomavirus DNA in sarcoid-affected
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equines. J Gen Virol. 2008;89:1390-5.
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[11] Lee S-K, Lee JK, Lee I. Penile squamous cell carcinoma associated with Equus caballus
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papilloma virus 2 in a miniature Appaloosa horse. In:Proceedings of the 67th congress of
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Korean Society of veterinary clinics, Seoul, Republic of Korea; 2017. p. 19.
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[12] Knight CG, Dunowska M, Munday JS, Peters-Kennedy J, Rosa BV. Comparison of the
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levels of Equus caballus papillomavirus type 2 (EcPV-2) DNA in equine squamous cell
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carcinomas and non-cancerous tissues using quantitative PCR. Vet Microbiol. 2013;166:257-
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62.
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[13] Scott DW, Miller WH. Equine Dermatology. 2nd ed: Elsevier/Saunders; 2010. p. 468-76.
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[14] van den Top JG, de Heer N, Klein WR, Ensink JM. Penile and preputial tumours in the
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horse: a retrospective study of 114 affected horses. Equine Vet J. 2008;40:528-32.
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[15] Torres SM, Koch SN. Papillomavirus-associated diseases. Vet Clin North Am Equine
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[16] Sykora S, Brandt S. Papillomavirus infection and squamous cell carcinoma in horses.
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Vet J. 2017;233:48-54.
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Table 1. Summary of the EcPV-2 molecular prevalence of genital swab samples by region in
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the Republic of Korea. Sample
Stallions
Mares
collection Pos (%)
No.
Inland area
7
0 (0%)
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Jeju island
28
0 (0%)
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1 (1%)
7 (1%)
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Abbreviations: No, number of examined samples; Pos (%); number (percentage) of positive samples for EcPV2 PCR.
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Pos (%)
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No. region
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Table 2. Summary of the EcPV-2 molecular prevalence in genital swab samples from healthy
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horses. Penile swabs
Vulvovaginal swabs
group
No.
Pos (%)
Stallions
35
0 (0%)
Mares
No.
Pos (%)
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Sample
762
8 (1%)
Abbreviations: No, number of examined samples; Pos (%), number (percentage) of positive
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samples for EcPV-2 PCR.
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No.
Country of foaling
6 years
1
USA
10 years
1
USA
11 years
1
12 years
1
14 years
1
16 years
2
20 years
1
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Abbreviations: No., number of EcPV-2 PCR positive horses; USA, United States of America.
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Table 3. The age and origin of EcPV-2 positive horses.
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USA
KOR
AUS
JPN
CAN
UK
IRL
NZL
Other countries
430
280
30
27
12
5
4
4
5
54.0% 17
35.1% 12
3.8% 1
3.4% 4
1.5% -
0.6% -
0.5% 1
0.5% -
0.6% -
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268
29
23
12
5
3
4
5
8
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-
-
-
-
-
-
-
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Abbreviations: KOR, Republic of Korea; AUS, Australia; JPN, Japan; CAN, Canada; UK,
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United Kingdom; IRL, Ireland; NZL, New Zealand; Pos, positive samples.
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Fig. 1. Neighbor-joining phylogenetic tree for partial E6 gene based on 8 sequences obtained
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in this study along with 18 sequences retrieved from GenBank (11 from genital SCC, 2 from
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nasal cavity SCC, 1 from vulvovaginal swab, 1 from skin, and 1 from normal penile mucosa).
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GenBank accession numbers are shown for retrieved sequences. Bootstrap percentage values
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are shown on the branches.
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Abbreviations: SCC, squamous cell carcinoma; V, vulvovaginal swab.
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ACCEPTED MANUSCRIPT Highlights · The prevalence of Equus caballus papillomavirus type 2 (EcPV-2) in genital swabs from
· Molecular prevalence of EcPV-2 was lower than earlier reports.
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healthy horses in the Republic of Korea was investigated using PCR.
. The Korean EcPV-2 isolates also showed genetic diversity comparable to previously
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reported sequences.
ACCEPTED MANUSCRIPT Animal welfare/ethical statement This study didn't conduct any animal experiments. Therefore, this study didn't violate animal
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welfare guidlines.
ACCEPTED MANUSCRIPT CRediT author statement
Sang-Kyu Lee: Conceptualization, Methodology, Investigation, Software, Writing – Original
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Writing-Reviewing and Editing, Supervision.
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Draft, Visualization, Data curation. Jung Keun Lee: Investigation. Inhyung Lee: Validation,