The Journal of Veterinary Medical Science

Advance Publication

Accepted Date: 19 April 2021 J-STAGE Advance Published Date: 28 April 2021

1 PATHOLOGY: NOTE

2 Equine sarcoid of the glans penis with bovine papillomavirus type 1 in a miniature horse

3 (Falabella)

4 Running head: EQUINE SARCOID OF THE GLANS PENIS

5 Kikumi OGIHARA1, Akikazu ISHIHARA2, Makoto NAGAI3, Kazutaka YAMADA4,

6 Testuya MIZUTANI6, Mei HARAFUJI7, Hisanari NISHIO5, Hiroo MADARAME5

8 1Laboratory of Pathology, School of Life and Environmental Science, Azabu University, Kanagawa,

9 252-5201, Japan

10 2Laboratory of Farm Animal Internal Medicine, School of Veterinary Medicine, Azabu University,

11 Kanagawa, 252-5201, Japan

12 3Laboratory of Infectious Diseases, School of Veterinary Medicine, Azabu University, Kanagawa, 252-

13 5201, Japan

14 4Laboratory of Clinical Diagnosis, School of Veterinary Medicine, Azabu University, Kanagawa, 252-

15 5201, Japan

16 5Laboratory of Small Animal Clinics, Veterinary Teaching Hospital, Azabu University, Kanagawa,

17 252-5201, Japan

18 6Research and Education Center for Prevention of Global Infectious Diseases of Animal, Tokyo

19 University of Agriculture and Technology, Tokyo, 183-0054, Japan

20 7Nasu Animal Kingdom, Tochigi, 329-3223, Japan

22 Drs. Ogihara and Ishihara contributed equally to this manuscript.

24 Corresponding author: Hiroo Madarame

25 Laboratory of Small Animal Clinics, Veterinary Teaching Hospital, Azabu University

26 1-17-71, Fuchinobe, Chuo, Sagamihara, Kanagawa, 252-5201, Japan

27 Tel: +81-42-754-7111

28 E-mail: [email protected]

30 ABSTRACT

31 A 23-year-old Falabella gelding kept in Tochigi, Japan, for more than 20 years presented with a

32 recurrent mass of the glans penis that was first noticed about a year earlier. Partial phallectomy was

33 performed with no adjunctive therapy for local regrowth of the mass. The horse was euthanized 3

34 months after surgery for urinary retention due to suspected regrowth. The resected mass affected the

35 genital and urethral mucosa of the glans penis, and was diagnosed as equine sarcoid by histopathology

36 and identification of bovine papillomavirus (BPV) DNA. Phylogenetic analysis of the BPV genome

37 of the sarcoid showed high sequence homology to BPV type 1 (BPV-1) from Hokkaido, Japan,

38 suggesting a geographical relationship for BPV-1 in Japan.

40 Key words: bovine papillomavirus, equine sarcoid, glans penis, mucosal sarcoid, phylogenetic

41 analysis

56 Equine sarcoid is the most important and common papillomavirus (PV)-induced skin tumor in horses

57 worldwide [14, 16, 21], and genomic studies of causative bovine papillomavirus (BPV) have been

58 reported in many countries, including in Japan [29]. Although PVs are known to have a dual tropism

59 for the skin and mucosa [1, 29], there are few reported cases of mucosal sarcoid. Equine sarcoid of the

60 oral and orbital mucosa has not been reported, although two cases involving the penile mucosa of the

61 glans penis have been reported from Iran [10, 20]. This report provides a detailed morphological

62 description of equine sarcoid involving the genital and urethral mucosa of the glans penis and the

63 urethral process as well as a complete genomic sequence and phylogenetic analysis of BPV type 1

64 (BPV-1) in Japan.

65 A 23-year-old Falabella gelding that had been kept in the zoo of Tochigi prefecture for more than 20

66 years was referred to Azabu University for investigation and treatment of a mass of the glans penis,

67 which had recurred twice in the previous year. Clinically, the first mass of the glans penis had grown

68 over approximately 6 months after bleeding from the penile root, at which time the first resection was

69 performed. The resected mass was histopathologically diagnosed as suppurative inflammation and

70 degeneration/necrosis with formation of granulation tissue. Approximately 6 months later, the mass

71 regrew and a second resection was performed. A third resection of the mass of the glans penis was

72 performed at the Large Animal Veterinary Educational Center of Azabu University after a further 6

73 months. On physical examination, the mass of the glans penis was reddish, proud flesh-like, and partly

74 covered with yellowish-white pus (Fig. 1). There were no abnormalities of the penile shaft, no

75 preputial abnormalities, and no regional lymphadenopathy. Partial phallectomy was performed using

76 William’s technique, and no adjunctive therapy was administered to prevent local tumor regrowth. The

77 horse was euthanized 3 months after surgery for urinary retention due to suspected regrowth of the

78 mass. Necropsy was not performed.

79 Fresh tissue samples were fixed in 10% neutral buffered formalin, trimmed, embedded in paraffin

80 wax, and processed routinely for histopathological examination. Four-micrometer-thick sections were

81 stained with hematoxylin and eosin, and selected sections were stained with Azan trichrome.

82 Additional sections were subjected to immunohistochemical analyses. The primary antibodies used are

83 listed in Table 1. Labeled antigens were detected using a Histofine Simple Stain MAX PO (MULTI) kit

84 (Nichirei Biosciences, Tokyo, Japan). Each antibody was visualized using 3-3'- diaminobenzidine

85 (Nichirei Biosciences). The slides were counterstained with Mayer’s hematoxylin.

86 For electron microscopy, tissue samples were cut into 1-mm3 cubes, fixed in 2.5% glutaraldehyde, and

87 post-fixed in 1% osmium tetroxide (Nisshin EM, Tokyo, Japan). The fixed specimens were then

88 dehydrated with graded ethanol and embedded in epoxy resin (Quetol-812 set; Nisshin EM). Ultrathin

89 sections were cut using an ultramicrotome (EM UC7; Leica Microsystems, Wetzlar, Germany),

90 double-stained with uranyl acetate and lead citrate, and examined with a JEOL 1210 transmission

91 electron microscope (JEOL, Tokyo, Japan) at 80 kV.

92 For detection of BPV, frozen tissue samples were minced with scissors, diluted 1:10 in phosphate-

93 buffered saline, and homogenized for 20 s at 3,200 rpm in the presence of three stainless steel beads

94 (φ4 mm) using a μT-12 bead crusher (Taitec, Saitama, Japan), and centrifuged at 12,000 g for 5 min.

95 Viral DNA was extracted from the supernatant using a QIAamp DNA Mini Kit (Qiagen, Hilden,

96 Germany). Polymerase chain reaction (PCR) for detection of BPV DNA was performed based on

97 three-step cycle conditions using GoTaq® G2 Hot Start Taq Polymerase (Promega, Madison, WI,

98 USA) with the MY09-MY11 primer pair [13]. The PCR products were electrophoresed on 2% agarose

99 gel, stained with Midori Green Advance (Nippon Genetics, Tokyo, Japan), and visualized using a UV

100 transilluminator.

101 To obtain the complete genome sequence of BPV from the equine tissue samples, long PCR was

102 performed using PrimeSTAR GXL DNA polymerase (Takara Bio, Kusatsu, Japan) using three primer

103 pairs: BPV1_wholw_1, BPV1_whole_2, and BPV1_whole_3 [23]. A fragmented library was

104 constructed from the PCR products generated using the Nextera XT kit (Illumina, San Diego, CA,

105 USA). Samples were bar-coded using the Nextera XT index kit v2 (Illumina), and sequencing was

106 performed on a MiSeq sequencer. FASTQ files created by the MiSeq Reporter (Illumina) were

107 imported into the CLC Genomics Workbench 7.5.5 (CLC bio, Aarhus, Denmark). Contigs were 5

108 created from trimmed reads using de novo assembly with default parameters in the CLC Genomics

109 Workbench.

110 The complete genome sequence was deposited in the DDBJ/EMBL/GenBank database under the

111 accession number LC549663. Phylogenetic analysis was performed based on the complete genome nt

112 sequence using the neighbor-joining method in MEGA7 [12]. The trees were evaluated by bootstrap

113 analysis with 1000 replicates [6]. Pairwise sequence identities were calculated using the CLC

114 Genomics Workbench 7.5.5.

115 The resected specimen consisted of a large mass of the glans penis (approximately 14 × 12 × 10 cm).

116 On the cut surfaces, the mass had a whitish fibrotic appearance. The urethral process was partly buried

117 in the mass, and the external urethral orifice was shifted to the left side of the mass (Fig. 2).

118 Histologically, the overlying stratified squamous (mucosal) epithelium of the glans penis with the

119 urethral process was partly hyperkeratotic and irregularly hyperplastic with long, slender, and

120 anastomosing epithelium (so-called rete pegs or ridges) (Fig. 3). “Picket fence” formation, consists of

121 rows of fibroblasts with perpendicular orientation to the epidermal basement membrane, was absent

122 and no viral inclusions were identified. The remaining overlying epithelium was ulcerated and covered

123 with granulation tissue and suppurative inflammation. The submucosa was expanded by variable

124 amounts of collagen fibers and proliferation of mildly anisocytotic and anisokaryotic fibroblasts

125 arranged in a random, whorled, or crisscross pattern (Fig. 4). The mitotic index of the proliferating

126 fibroblasts was less than one per 10 high-power fields.

127 Immunohistochemically, there was strong, diffuse cytoplasmic positivity for vimentin, moderate

128 scattered cytoplasmic and nuclear positivity for S-100 (Fig. 5a), and moderate scattered cytoplasmic

129 positivity for claudin 1 in tumors (Fig. 5b). Cytokeratin AE1/AE3, laminin, desmin, alpha-smooth

130 muscle actin, glial fibrillary acid protein, CD34, calponin, and ionized calcium-binding adapter

131 molecule 1 (Iba-1) were negative. Immunolabeling for Iba-1 was observed in macrophages throughout

132 the submucosal tumor. The mucosal epithelium and submucosal tumor cells were negative for the PV

133 antigen and p16; p53 revealed weak, scattered, nuclear positive immunoreactivity in the submucosal

134 tumor cells. The immunohistochemical results are shown in Table 1.

135 Ultrastructurally, the tumor cells were characterized by various amounts of collagen fibrils and non-

136 cohesive spindle cells. The spindle cells had smooth cell membranes, a single round to deeply indented

137 nucleus, prominent rough endoplasmic reticulum, varying amounts of intermediate filaments, and no

138 myofilaments. They also had thin cytoplasmic processes, and the cell processes were in close contact

139 with those of adjacent cells with gap junctions (Fig. 6). These features are consistent with those

140 described for fibroblasts [11, 17].

141 PCR was conducted to detect the partial genome of the PV using the MY09-MY11 primer pair. The

142 amplicon was detected in the tissue samples from the glans penis. To amplify the complete genome of

143 the virus, an additional PCR assay was performed using three primer pairs [23]. PCR products were

144 successfully obtained and subjected to next-generation sequencing on the MiSeq platform. The 7945-

145 nucleotide complete genome sequence of the mucosal sarcoid LC549663/Equus caballus/JPN

146 (LC549663) obtained was aligned with sequences of BPV from equine sarcoid and bovine papilloma.

147 Phylogenetic analysis showed that LC549663 branched with Japanese bovine and equine strains and

148 was identified as BPV-1 (Fig. 7). LC549663 was most closely related to Japanese BPV from cutaneous

149 myopericytoma of the abdominal skin of a calf (DDBJ accession no. AB 626705) in Hokkaido,

150 exhibiting 99.96% nucleotide identity. LC549663 also shared high identity (99.87%) with Japanese

151 BPV from equine sarcoid (fibropapilloma) (DDBJ accession no. LC510377) on the foreskin of a

152 thoroughbred-cross gelding in Hokkaido [29].

153 PVs are ubiquitous but highly host species-specific [15]. Affinity of PV for the skin and mucosal

154 epithelium has been considered to be mutually exclusive [4], although recent studies show specific

155 subgroups of PVs could retain a dual tropism [1]. Equine sarcoid is caused by cross-species infection

156 with BPV-1, -2, or -13 and BPV ‘BsR-UEL-4’ [14, 15, 19] and is the most important and common

157 PV-induced skin tumor affecting horses worldwide, accounting for 35.3%–90% of all skin tumors in

158 the surveys conducted to date [2, 14, 21]. 7

159 Equine sarcoid can develop at any skin site on the body but has typical anatomical predilection sites

160 [14, 15, 19, 21], including the head, limbs, ventral abdomen, and paragenital region [2, 21]. Most cases

161 of equine sarcoid on the head develop at the mucocutaneous junction, including the eyelids and

162 commissure of the lips [2, 21], and sarcoid also affect the prepuce with the mucocutaneous junction

163 [22, 26]. To the best of our knowledge, equine sarcoid of the oral and orbital mucosa has not been

164 reported. Only two cases of sarcoid on the penis engulfing the glans penis have been reported in Iran

165 in recent years [10, 20], and fibropapilloma of the glans penis without BPV-1 or BPV-2 DNA has been

166 reported in the US [7]. Equine sarcoid is a cutaneous and mucosal tumor, although the scarcity of

167 published case reports on mucosal sarcoid may reflect its genuine rarity.

168 Histologically, equine sarcoid was originally considered to be a sarcoma-like mixed (fibroepithelial)

169 tumor [2, 9] but is now categorized as a tumor of soft tissue caused by proliferation of atypical

170 fibroblasts [8, 16, 19]. According to the third edition of the international histologic classification of

171 tumors of soft tissues of domestic animals, a diagnosis of equine sarcoid-affected mucosa of the glans

172 penis and urethral process is confirmed by a combination of histopathology and identification of BPV

173 DNA [19]. There are six clinical forms in equine sarcoid: flat/occult, verrucous, fibroblastic, nodular,

174 mixed, and malignant/“malevolent” [16, 19, 21]. According to clinical categorization by gross

175 appearance [14, 19, 21], the mucosal sarcoid in our case was fibroblastic (“proud flesh-like”) sarcoid,

176 as has already been reported to occur in the glans penis [10, 20].

177 Histopathology is required for a definitive diagnosis of equine sarcoid [14, 21] but may be challenging

178 due to the lack of clear diagnostic criteria [28]. Differentiation of equine sarcoid from other spindle

179 cell tumors or exuberant granulation tissue of the skin (proud flesh) can be difficult [15, 19, 21].

180 Furthermore, identification of BPV DNA by PCR is thought to be diagnostic for equine sarcoid [3, 8],

181 although BPV DNA can be demonstrated in 70% of equine sarcoids and in equine spindle cell tumors

182 in the skin other than equine sarcoid by BPV PCR in paraffin-embedded tissues [5]. The mere

183 presence of BPV DNA is of limited value in the diagnosis of equine sarcoid [16, 28].

184 Immunohistochemistry for equine sarcoid often requires additional diagnostic tests and differentiation

185 from other spindle cell tumors of the skin but may not be conclusive. Equine sarcoid cells are

186 vimentin-positive and may be laminin-positive, SMA-positive [3] and S100 positive [5]. The mucosal

187 sarcoid in our case showed positive immunoreactivity for vimentin and S-100 and negative

188 immunoreactivity for laminin and SMA. There was scattered cytoplasmic positivity for claudin 1.

189 Claudin 1 is a tight junction-related protein [18] and is not a marker of perineural cells in equine tissue

190 [27]. In the human medical literature, claudin-1 is reportedly expressed in perineurioma-like low-grade

191 fibromyxoid sarcoma [25]. Claudin-1 also induces epithelial-mesenchymal transition [24]. In our case

192 of equine sarcoid, PV and p16 immunohistochemistry studies revealed no immunoreactivity in the

193 mucous epithelium or submucosal tumor cells. Immunohistochemical detection of the presence of

194 BPV virus late proteins failed, and the BPV production in horses remains questionable in equine

195 sarcoid [16].

196 Clinically, equine sarcoid is a progressive disease that invades and destroys the surrounding tissue and

197 recurs frequently following surgical excision [19, 21]. Equine sarcoid is not lethal, but functional

198 impairment may necessitate euthanasia or slaughter [2]. Equine sarcoid becomes a dangerous problem

199 when the glans penis is involved and causes dysuria [10], as in our case.

200 Genomic studies of BPV in equine sarcoid have been reported worldwide, including in Japan [29].

201 Phylogenetic analysis of LC549663 revealed high sequence homology to BPVs derived from

202 cutaneous myopericytoma of a calf and from equine sarcoid of a horse kept in Hokkaido, Japan,

203 suggesting a geographical relationship of BPV-1 in Japan [29]. In this case, the old Falabella gelding

204 may have been infected with BPV-1 local to Japan. Domestic transport of horses and cattle may

205 explain the relationship between BPV-1, horses, and cattle in Japan.

206 In conclusion, equine sarcoid of the glans penis is a genital and urethral mucosal disease associated

207 with cross-species BPV-1 infection and may cause functional impairment. Phylogenetic analysis of

208 the BPV-1 genome in our case suggested a geographical relationship between BPV-1 in horses and

209 cattle in Japan. This report will contribute to further understanding of the pathology and geographical 9

210 epidemiology of mucosal sarcoid in horses.

211

212 CONFLICT OF INTEREST

213 The authors declare no conflicts of interest regarding the publication of this manuscript.

214

215 ACKNOWLEDGMENTS

216 The authors thank Ms. Kanako Satoh and Ms. Megumi Mori for their expert technical assistance. 217

218

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289

290 FIGURE LEGENDS

291 Fig. 1. Mass of the glans penis. Left lateral view showing the urethral process.

292 Fig. 2. Coronal section of the mass of the glans penis. The urethral process was partly buried in the

293 mass. External urethral orifice（arrow）. Scale bar = 1 cm.

294 Fig. 3. Coronal section of the urethral process. External urethral orifice. Urinary tract between the wall of

295 the urethral process. Genital and urethral mucosa covered with partly hyperkeratotic and irregularly

296 hyperplastic with long, slender, and anastomosing epithelium and granulation tissue. Hematoxylin and

297 eosin (HE) staining. Scale bar = 50 μm.

298 Fig. 4. Urethral submucosa. Mildly anisocytotic and anisokaryotic fibroblasts arranged in a whorled or

299 crisscross pattern. Hematoxylin and eosin (HE) staining. Scale bar = 50 μm.

300 Fig. 5. Immunohistochemistry. Urethral submucosa. a) Moderate, scattered cytoplasmic and nuclear

301 positivity for S-100 in tumor cells. Scale bar = 50 μm. b) Moderate, scattered cytoplasmic positivity

302 for claudin 1 in tumor cells. Scale bar = 50 μm.

303 Fig. 6. Transmission electron micrograph. Glans penis submucosa. Collagen fibrils and non-cohesive

304 spindle cells with prominent rough endoplasmic reticulum. Cell processes in close contact with those

305 of adjacent cells with gap junctions. Features of spindle cells consistent with fibroblasts. Scale bar = 1

306 μm.

307 Fig. 7. A phylogenetic tree was constructed using Neighbor-Joining in MEGA7 with 1000 bootstrap

308 replicates. The scale bar indicates the corrected genetic distance. LC549663 is indicated in bold red

309 text.

310

Fig. 1.

Fig. 2

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

MF384275.1MF384275/Equus Deltapapillomavirus asinus 4 isolate 01/GBR asi UK complete genome MF384285.1MF384285/Equus Deltapapillomavirus caballus 4 isolate 23 equ/CHE CH complete genome MF384290.1MF384290/Bos Deltapapillomavirus taurus 4 isolate/CHE 32 bos CH complete genome MF384291/BosMF384291.1 Deltapapillomavirus taurus 4 isolate/CHE 34 bos CH complete genome MF384287.1MF384287/Equus Deltapapillomavirus caballus 4 isolate 26 equ/CHE CH complete genome MF384279.1 Deltapapillomavirus 4 isolate 17 equ CH complete genome 95 MF384279/Equus caballus/CHE MF384294/BosMF384294.1 Deltapapillomavirus taurus 4 isolate/CHE 40 bos CH complete genome MF384284/EquusMF384284.1 Deltapapillomavirus caballus 4 isolate 22 equ/CHE CH complete genome MF384282/EquusMF384282.1 Deltapapillomavirus caballus 4 isolate 20 equ/CHE CH complete genome 61 94 MF384278/EquusMF384278.1 Deltapapillomavirus caballus 4 isolate 16 asi/CHE CH complete genome MF384283.1MF384283/Equus Deltapapillomavirus caballus 4 isolate 21 equ/CHE CH complete genome MF384277.1 Deltapapillomavirus 4 isolate 14 asi CH complete genome 100 MF384277/Equus asinus/CHE 65 MF384277/EquusMF384276.1 Deltapapillomavirus asinus 4 isolate 13/CHE asi CH complete genome MG977494.1MG977494/Bos Deltapapillomavirus taurus 4 isolate/ITA RD55948 complete genome 100 MF384289/EquusMF384289.1 Deltapapillomavirus asinus 4 isolate 09/GBR asi UK complete genome 99 MF384286/EquusMF384286.1 Deltapapillomavirus caballus 4 isolate 25 equ/CHE CH complete genome Bovine MF384280.1MF384280/Equus Deltapapillomavirus asinus 4 isolate 18/CHE equ CH complete genome 100 JX678969.1JX678969/Equus Bovine papillomavirus caballus type 1 isolate/GBR EqSarc1 complete genomePapillomavirus 100 MF384288/EquusMF384288.1 Deltapapillomavirus asinus 4 isolate 04/GBR asi UK complete genome

78 LC333380.1LC333380/Bos Deltapapillomavirus taurus 4 1347768929/JPN DNA complete genome 1 100 64 LC510385.1LC510385/Equus Deltapapillomavirus caballus 4 E190115 DNA/JPN complete genome 99 LC510379.1LC510379/Equus Deltapapillomavirus caballus 4 E171016 DNA/JPN complete genome KY886226/EquusKY886226.1 Deltapapillomavirus ferus 4 isolatecaballus Missouri complete/USA genome 96 94 X02346.1X02346/XXX/XXX Bovine papillomavirus - 1 complete genome 99 MF045489.1MF045489/XXX/XXX Bovine papillomavirus type 1 isolate GZLZ complete genome 99 MF435918/BosMF435918.1 Deltapapillomavirus taurus 4 strain/CHN BPV-GX-LA150909 complete genome KY746722.1KY746722/Bos Bovine papillomavirus taurus type/MAR 1 isolate PP002 A Inner complete genome MH197482.1 Deltapapillomavirus 4 complete genome 99 MH197482/Bos taurus/TUR Equine sarcoid 100 LC549663/Equus caballus/JPN 99 AB626705.1AB626705/Bos Bovine papillomavirus taurus type/JPN 1 DNA complete genome country: Japan:Hokkaido 8592 LC510377.1LC510377/ DeltapapillomavirusEquus caballus 4 E170428-2 DNA/JPN complete genome LC510378.1LC510378/ DeltapapillomavirusEquus caballus 4 E170801 DNA/JPN complete genome 100 MK347523.1MK347523/ DeltapapillomavirusBos taurus 4 strain/CHN BPV-GX-JX180408 complete genome MF435916.1 Deltapapillomavirus 4 strain BPV-GX-BS160810 complete genome 100 MF435916/Bos taurus/CHN MF435917.1 Deltapapillomavirus 4 strain BPV-GX-HX160815 complete genome 69 MF435917/Bos taurus/CHN KX907623.1KX907623/ Bovine Bospapillomavirus taurus type/CHN 1 isolate SY-12 complete genome 79 MG263871.1MG263871/ DeltapapillomavirusBos taurus 4 isolate/CHN GX01 complete genome MT459820.1MT459820/ DeltapapillomavirusEquus caballus4 strain DPV4/equine/Egypt/EGY complete genome 94 KU519390.1KU519390/ Bos taurusBos papillomavirus taurus/BRA 13 strain 14RO12 complete genome 77 MF741676.1MF741676/ Bovine Bospapillomavirus taurus type/BRA 13 isolate BPV13 BR/14/RO-13Bovine complete genome 100 JQ798171.1JQ798171/ Bovine Bospapillomavirus taurus type/BRA 13 complete genome MF327274.1MF327274/ Bovine XXXpapillomavirus/XXX type 13 clone BPV13-GZSBPapillomavirus complete genome 87 KM258443/KM258443.2 Bovine Bospapillomavirus taurus type/CHN 13 strain Hainan complete genome 93 MG818475.1MG818475/ Bovine papillomavirusBos taurus type/CHN 13 strain BPV-HN-170725 complete13 genome 100 KU674833.1KU674833/ Bovine Bospapillomavirus taurus type/CHN 2 strain BRA/09RO12 complete genome MH187961.1MH187961/ Bovine Bospapillomavirus taurus type/BRA 2 strain BPV2 BR/02AC12 complete genome

100 M20219/M20219.1 BovineBos papillomavirus taurus type/XXX 2 complete genome KM455051.1 Bovine papillomavirus type 2 isolate Aks-01 complete genomeBovine 86 KM455051/Bos taurus/CHN MF045490.1 Bovine papillomavirus type 2 isolate GZZN complete genome 100 MF045490/Bos taurus/XXX KC878306.1 Bovine papillomavirus type 2 isolate SW completePapillomavirus genome 100 KC878306/Bos taurus/CHN LC510383.1 Bos taurus papillomavirus 2 E180925-2 DNA complete genome 60 LC510383/Equus caballus/JPN 2 36 LC510384/LC510384.1 Bos taurusEquus papillomavirus caballus 2 E181025/JPN DNA complete genome 99 LC510376/LC510376.1 Bos taurusEquus papillomavirus caballus 2 E170428-1/JPN DNA complete genome

0.0100 Table 1. Immunohistochemistry and characterization of equine sarcoid of the glans penis Antibody Clone Source Dilution Tumor cells HPV BPV-1/1 H8 + CAMVIR Abcam (Cambridge, UK) 1:80 N Cytokeratin AE1/AE3 AE1, AE3 Nichirei (Tokyo, Japan) Prediluted N Vimentin Vim 3B4 Dako (Glostrup, Denmark) 1:100 S S-100 rabbit polyclonal Nichirei (Tokyo, Japan) 1:50 M Claudin 1 rabbit polyclonal Abcam (Cambridge, UK) 1:200 M Laminin rabbit polyclonal Abcam (Cambridge, UK) 1:200 N Desmin ZC18 Nichirei (Tokyo, Japan) 1:50 N αSMA 1A4 Nichirei (Tokyo, Japan) Prediluted N GFAP GA5 Nichirei (Tokyo, Japan) Prediluted N CD34 QBEnd 10 Dako (Glostrup, Denmark) Prediluted N Calponin CALP Dako (Glostrup, Denmark) 1:50 N p16 rabbit polyclonal Abcam (Cambridge, UK) 1:50 N p53 DO-7 Nichirei (Tokyo, Japan) Prediluted W Iba-1 rabbit polyclonal Wako (Osaka, Japan) 1:1000 N Ki-67 MIB-1 Dako (Glostrup, Denmark) Prediluted S (<10%) HPV, human papillomavirus; BPV, bovine papillomavirus; αSMA, alpha smooth muscle actin; GFAP, glial fibrillary acid protein; Iba-1, ionized calcium-binding adapter molecule 1. Abbreviations for staining intensity: N, negative; S, strong; M, moderate; W, weak.