Clinicopathological characteristics and papillomavirus types in cutaneous warts in bovine

Abstract

Thirty-one bovine cutaneous warts were submitted to macroscopic and histological analyses and to molecular analyses to partial amplification and sequencing of the L1 gene of bovine papillomavirus (BPV). Viral types detected were BPV1 (52%), BPV2 (29%), BPV6 (16%) and BPV10 (3%). BPV2 had lower frequency in papilloma in comparison to that in fibropapilloma (p = 0.002).

Electronic supplementary material

The online version of this article (10.1007/s42770-019-00121-2) contains supplementary material, which is available to authorized users.

Introduction

Bovine cutaneous papillomas or warts are benign exophytic proliferative lesions usually associated with bovine papillomavirus (BPV). These lesions may cause significant economic losses for cattle industry, depending on the lesion extension and anatomic localization [6].

Papillomaviruses have double-stranded circular DNA genomes of approximately 8 kb [2], and are currently classified in Papillomaviridae family [33]. According to Papillomavirus episteme, BPVs have been classified in five genera based on L1 nucleotide sequences and biological properties, named Deltapapillomavirus 4 (BPV 1, BPV 2, BPV 13, and BPV 14), Xipapillomavirus 1 (BPV 3, BPV 4, BPV 6, BPV 9, BPV 10, BPV 11, BPV 12, and BPV 15) and Xipapillomavirus 2 (BPV 12), Xipopapillomavirus which does not have the classified species (BPV17, BPV20, BPV 23, BPV 24, and 26), Epsilonpapillomavirus 1 (BPV5, BPV 8, and BPV 25), Dyoxipapillomavirus 1 (BPV7), and Dyokappappillomavirus genus (BPV16, BPV18, and BPV 22). Other three BPV types (BPV19, BPV21, and BPV 27) described correspond to unclassified genus ([32]; https://pave.niaid.nih.gov/). In general, Deltapapillomavirus and Epsilonpapillomavirus genera are linked to both epithelial papillomas and cutaneous fibropapillomas, which affects both epithelial and dermal components [22, 30]. Xipapillomavirus are responsible for the occurrence of squamous papillomas, strictly epitheliotropic [17].

BPVs have been previously detected in bovine cutaneous warts from different Brazilian regions [1, 3, 7–9, 12]; however, its diversity is still low when compared to human papillomaviruses, for which almost 200 types have been described (https://pave.niaid.nih.gov/). Thus, reporting BPV types is an important tool to enlarge the knowledge of their distribution and eventually enabling the discovery of novel BPVs.

Moreover, there is no clear information about BPV types and the warts’ clinicopathological characteristics. Therefore, the aim of the present study was to describe the BPV types present in cutaneous warts of cattle raised in Rio Grande do Sul state, southern Brazil, and to evaluate possible association between BPV types and warts features within this geographic area.

Bovine cutaneous warts (n = 31) received at Instituto de Pesquisas Veterinárias Desidério Finamor between April 2012 and November 2013 were used in this research. Veterinarians who received previous instructions on the collection in order to avoid possible cross-contamination surgically extracted the samples. Samples were collected individually and all procedures were performed using exclusive scalpel blades and gloves. Information regarding lesions and clinical features were obtained, and the local Ethics Committee (Number 07/2012) approved the procedures. Only samples extracted of bovines from Rio Grande do Sul were included.

In the laboratory, the specimens were sectioned into two fragments, one of them was placed in 10% buffered formalin for 24 h for subsequent histological analysis, and the other was frozen at − 20 °C for DNA extraction. The lesions were classified according to the anatomical localization and were sorted by gross morphologic characteristics into three groups (cauliflower-like, flat and round, or filamentous). For histological evaluation, samples were embedded in paraffin by standard procedures. Consecutive sections of 4 μm were obtained from each block, stained and hematoxylin eosin (HE) and evaluated under light microscope [25]. The lesions were classified according to the type of proliferation in papillomas and fibropapillomas [15].

DNA was extracted with DNAzol® (Invitrogen) according to the manufacturer’s instructions and quantified by spectrophotometry (L-quant, Loccus Biotechnology, Brazil). The presence of inhibitors in DNA was verified by partial amplification of bovine glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) [20]. Amplification of a segment of the BPV L1 gene was performed by PCR using primers FAP59 and FAP64 [14] and MY09 and MY11 [24], which target DNA fragments of 478 bp and 450 bp, respectively. The PCR was performed in a total volume of 25 μL containing approximately 100 ng of DNA, 1 × PCR buffer, 1.5 mM magnesium chloride, 1 unit of Taq DNA polymerase, 0.1 mM of each dNTP and 10 μM of each primer. PCR conditions were as follows: initial denaturation for 5 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min, and a final extension at 72 °C for 7 min.

Amplicons were submitted to electrophoresis in an ethidium bromide stained 1.5% (w/v) agarose gel and visualized under ultraviolet light. Amplicons were purified by PureLink™ Quick Gel Extraction and PCR Purification Combo Kit (Life technologies) following the manufacturer’s protocol and subjected to direct nucleotide sequencing using a ABI 3100 using BigDye v3.1 (Applied Biosystems, USA). Sequences were initially evaluated by BLAST and subsequently analyzed with Geneious software (version 8.1.8) [18]. Multiple sequence alignments were performed with MUSCLE. Phylogenetic trees were constructed by the maximum likelihood (ML) method inferred on the basis of the best fit nucleotide substitution model (GTR+G+I) for BPV L1 gene implemented in MEGA 6 package (version 6.06) [23, 29]. The reliability of the ML trees was evaluated by analyzing 1,000 bootstrap replicates and bootstrap values of > 60% were considered significant.

Possible associations between BPV types (1 or 2) and samples characteristics (anatomical site, macroscopy, microscopy and lesion size) were analyzed by Fisher’s Exact Test using the GraphPad® software. Significant associations were considered when p < 0.05.

The warts were collected from dairy (n = 12), beef (n = 9) and mixed (2) bovine herds from 13 cattle herds. Data on the clinical aspects and BPV types are summarized in Supplementary table. Macroscopically, papillomas were pedunculated, irregular and had cornified surface (cauliflower-like; Supplementary Fig. S2-A) in 71.1% (22/31) of the cases and in 22.6% (7/31) were rounded, flattened and had smooth surface (flat and round) and only 6% two were filamentous 6.5 (2/31; Supplementary Fig. S2-B). Microscopically, 45% (14/31) of the lesions were characterized by moderate to marked epidermal hyperplasia sustained by dermal stroma. Epithelial cells were well differentiated and had moderate to marked acanthosis, hypergranulosis, koilocytosis and basal layer hyperplasia. Hyperkeratosis was also observed a few cases with areas of parakeratosis. These lesions were classified as papillomas (Supplementary Fig.S2-C). In 54% (17/31) of the cases, the lesions consisted of mild to moderate hyperkeratosis and acanthosis and moderate to intense proliferation of dermal fibroblasts, sometimes forming tightly curled and fascicles of fibroblasts and collagen (fibropapillomas; Supplementary Fig. S2-D).

BPV genomes were detected in all examined warts. Twenty-two out of 31 samples were amplified using FAP primers; six specimens required the use of the MY primers and three samples amplified both primers (FAP and MY). Deltapapillomavirus was identified in 25 lesions (81%) and Xipapillomavirus in 6 (19%) (Figs.1a and 1b). BPV1 was the main viral type identified (16 samples; 51%), followed by BPV2 (9 samples; 29%), BPV6 (5 samples; 16%) and BPV10 (1 sample; 3%) (Table 1).

Fig. 1.

a Phylogenetic analysis of the partial BPV L1 gene. Maximum likelihood phylogenetic tree of twenty-five nucleotide sequences obtained by FAP59/FAP64 amplification (black diamond shape) and reference sequences for each BPV type was constructed using GTR+G+I as nucleotide substitution model and bootstrap of 1,000 replicates. Bootstrap values greater than 60% are shown. b Phylogenetic analysis of the partial BPV L1 gene. Phylogenetic tree based on nucleotide sequences obtained by MY09/MY11 amplification (black diamond shape) and reference sequences for each BPV type was constructed using maximum likelihood (ML) method, GTR+G+I as nucleotide substitution model and bootstrap of 1,000 replicates. Bootstrap values greater than 60% are shown

Table 1.

Clinical and morphological characteristics of evaluated samples and BPV types (n = 31)

	BPV1			BPV2			BPV6			BPV 10
	Positive (%)	Negative (%)	p*	Positive (%)	Negative (%)	p*	Positive (%)	Negative (%)	p **	Positive (%)	Negative (%)	p**
Anatomic site
Head, neck or dewlap	7 (22.6)	8 (25.8)	1.00	5 (16.1)	10 (32.2)	0.85	3 (9.7)	12 (38.7)	–	0 (0.0)	0 (0.0)	–
Abdomen, thorax or groin	7 (22.6)	6 (19.3)	3 (9.8)	2 (6.4)	3 (9.7)	10 (32.3)	1(100)	0 (0.0)
Udder, teats or prepuce	2 (6.4)	1 (3.2)	1 (3.2)	2 (6.4)	0 (0.0)	3 (9.7)	0 (0.0)	0 (0.0)
Macroscopy
Cauliflower	10 (32.2)	14 (45.1)	0.08	8 (25.8)	16 (51.6)	0.63	6 (19.3)	18 (58.6)	–	1 (100.0)	0 (0.0)	–
Flat, round, or filamentous	6 (19.3)	1 (3.2)	1 (3.2)	6 (19.3)	0	7 (22.5)	0 (0.0)	0 (0.0)
Microscopy
Papilloma	7 (22.6)	7 (22.6)	1.00	1 (3.2)	13 (41.9)	0.02	6 (19.3)	8 (25.8)	–	1 (100.0)	0 (0.0)	–
Fibropapilloma	9 (29.0)	8 (25.8)	8 (25.8)	9 (29.0)	0 (0.0)	17(54.8)	0 (0.0)	0 (0.0)
Tumor size
≤ 1.0 cm	6 (22..2)	4 (14.8)	0.44	1 (3.7)	9 (33.3)	0.19	3 (11.1)	7 (25.9)	–	1 (100.0)	0 (0.0)	–
>1.0 cm	7 (25.9)	10 (37.0)	7 (25.9)	10 (37.0)	3 (11.1)	14 (51.8)	0 (0.0)	0 (0.0)

*p value calculated using Fischer’s exact test

**Unrealized analysis due to the small number of cases

BPV1 and 6 were detected in warts from different anatomic localizations in the same animal (A1). Moreover, different BPV types were recovered from animals in the same herd (herds 1, 2, and 6) (Online Resource 1). BPV2 had lower frequency in papilloma (p = 0.002) in comparison to that in fibropapilloma. There was no significant association between the BPV1 or BPV2 with the anatomical site of occurrence (head, neck, or dewlap × abdomen, thorax, or groin × udder, teats or prepuce) or the macroscopic pattern (cauliflower × flat, round, and filamentous) (Table 1).

Obtained sequences were subjected to phylogenetic analysis and two trees were generated using BPV reference sequences available in GenBank (http://www.ncbi.nlm.nih.gov/genbank/). The L1 nucleotide sequences shared high degree of identity with BPV genomes available at GenBank, ranging from 97.4 to 99.8%. BPV1 clustered along with reference strains reported in USA (accession no. NC 001522), United Kington (accession no. JX678969), and Japan (accession no. AB626705) (Fig. 1a and b). BPV2 clustered along with BPV identified in lesions in China (accession no. KC878306_and KM 455051) (Figs. 1a and 1b). The 6 sequences of BPV6 clustered along with BPV detected in Japan (accession no. AB845589) and Germany (accession no. AJ620208BPV6) (Fig. 1a), whereas BPV10 clustered with BPV identified in a bovine lingual papilloma in Japan (accession no. KF017607) (Fig. 1a).

Partial sequence of nucleotides corresponding to the L1 gene of BPV obtained in the present study were submitted to the GenBank database under accession numbers KU728462, KU728463, KU728469, KU728470, KU865637, KU728455, KU728456, KU743470, KU728474, KU865634, KU865638, KU865635, KU728467, KU736826, KU7284743, KU728465, KU728464, KU728457, KU865636, KU865633, KU728475,KU728466, KU728476, KU728477, KU728459, KU728458, KU728478, KU728460, KU728461, KU865640, and KU728471.

Papillomaviruses are part of skin microbiota; however, they can cause infections with variable clinical presentations as cutaneous and mucosal warts, and neoplasia in bovines and other animals [4]. Bovine cutaneous warts due to papillomaviruses infections are prone to begin after micro-injuries, which expose the epithelium basal layer to the virus [21]. Accordantly, most of the cutaneous lesions retrieved in the present study were located in anatomical sites more susceptible to physical attrition.

The most frequent genotype BPVs identified here were BPV1, BPV2, and BPV6. In Brazil, these BPVs were already detected in Rio Grande do Sul [28] and in other Southern states [8–10, 19, 31]. They have also been detected in North-East [3, 7], Amazon [12, 13, 28], and Southeast [19] Brazilian regions. The fact that these types have been detected in different herds and in different years indicates that they are frequent types in the bovine population.

Regarding the phylogenetic analysis, this study shows overall high genetic identity with other BPV around the world, suggesting low selection pressure. Recently, new BPV types were suggested to occur in Brazil [11], which nucleotide identities with other genomes were lower, around 90%. It is possible that the primers used in the present study select those types more conserved, precluding the identification of new types; thus, likewise for elucidation of BPV present in normal skin, metagenomics approach is a valuable tool to gain insight on lesions’ etiology and also to better understand the viral genetic diversity based on viral complete genome rather than just one gene.

BPV-1 has been linked to teat fibropapillomas and penile fibropapillomas [4]; however in sampled animals, it was detected in papillomas and fibropapillomas in the same proportion. Thus, it was difficult to establish a link between the BPV1 and the lesions’ etiology. BPV2 was observed in a lower frequency in papillomas indicating tropism of this viral type by the fibropapillomas of the skin as already reported [22, 31].

In addition, although BPV6 has been previously associated with squamous papillomas on teats and udder [27, 31], here it was rescued from squamous papillomas of head, neck, and abdomen, confirming that the viral type cannot be assigned to specific lesions as previously thought [7, 8].

BPV10 was detected in one case in a papilloma located in the groin. This BPV type was previously detected in squamous papillomas of the teats and udder [16, 26]. In Brazil, BPV10 had already been detected in cattle herds in Southern [19, 31], Southeast [19], and North-East [3, 7] and the present finding reveals that they are also present in Rio Grande do Sul. Apparently, there is no restrict anatomical site in cattle to its occurrence, as BPV10 genomes were detected in fibropapillomas located in different anatomical sites including shoulder, around the eye, neck, muzzle, dewlap, head [3], and in teats [19, 31].

No relationship between BPV types and the macroscopic aspect or anatomical site of the lesion could be established. This lack of association can be attributed to co-infections, which has been reported to occur [3, 7, 10, 13]; recently, seven different BPV types were detected in the same lesion [13]. In the present study, it was not possible to detect more than one BPV type in the same lesion; however, different BPV types were detected in animals from three herds and in different anatomic sites from one animal. It has been suggested that the occurrence of mixed infections can contribute to persistent infections due to immunosuppression [3, 7]. Finally, another possibility is that the detected viruses are not responsible for the infection. A study using a metagenomics approach described 396 different human papillomaviruses in healthy skin, of which 229 putative types were previously unknown [5]. To evaluate this hypothesis, the health skin of these animals should have been analyzed.

Although the sample size has been restricted to a period of time and sampling has been performed for convenience, it is important to report the BPV types circulating in evaluated regions. Thus, in the present study, four BPV types were detected in bovine papillomas, besides the occurrence of mixed infections involving the BPVs 1, 2, and 6. No association could be detected between the BPV type or genus and the lesions’ anatomical region or macroscopic characteristics. The present study provides information on the BPV types circulating in cattle from southern Brazil.

Funding information

This work was financially supported by Financiadora de Estudos e Projetos (FINEP/SANIMARS - Grant number 01100783-00) and by Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), for financing the project and concession of scholarship on scientific initiation.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in the study involving animals were in accordance with the ethical standards of the institution.