Abstract
Teat papillomatosis is caused by different bovine papillomavirus (BPV) types and is especially important for dairy cows, because it results in severe damage to the health and structure of the mammary gland. This work describes the molecular and pathological aspects of teat papillomatosis in dairy cows in southern Brazil. Samples of teat papillomas were collect from 73 slaughtered dairy cows. Fragments of the lesions were collected in individual pools per animal and subjected to PCR using the FAP primer pair and sequencing of the amplification products. Teats with the remaining lesions were fixed in 10% neutral buffered formalin, routinely processed for histopathology, and stained with hematoxylin and eosin (H&E). Papillomatous lesions were characterized by three macroscopic patterns, namely exophytic (5 [6.9%]), flat (29 [39.7%]), and mixed (39 [53.4%]). Histologically, all samples were identified as squamous papillomas. Partial sequencing of the L1 gene resulted in the detection of 8 classical BPV types (BPVs 4, 6, 7, 8, 9, 10, 11, and 12) in 27 samples, 6 previously reported putative BPV types in 17 samples, and 10 putative new BPV types in 15 samples. Four sequences could not be classified, and 10 were negative in the PCR. There was no correlation between the gross pattern and the BPV type identified, and all the samples were characterized by squamous papillomas under histological examination. However, 24 different BPV types were identified, demonstrating high genetic diversity among BPVs associated with teat papillomatosis in dairy cows in southern Brazil.
Keywords: Dairy cattle, Papillomatosis, BPV, Viral disease, Veterinary pathology, PCR
Introduction
Papillomaviruses (PVs) are non-enveloped, double-stranded circular DNA viruses belonging to the Papillomaviridae family [12, 23, 25]. In cattle, they cause benign cutaneous papillomatous lesions, which may involve the teats. In addition, they are also associated with malignant tumors in the bladder and upper digestive tract [7, 16, 18].
Teat papillomatosis has been widely described in dairy cattle and may result in damage to the health and structure of the mammary gland. Papillomas may be large enough to interfere with the milking process and milk flow, especially when they are located near the sphincter of the teat, predisposing the cattle to mastitis. In addition, ulceration and rupture of the lesions may result in bleeding and distortion of the lactiferous ducts [5, 6, 8, 15, 19, 25].
According to the papillomavirus episteme (PaVE) [22], there are currently 24 fully characterized bovine papillomavirus (BPV) types that are classified into five genera, namely: the Deltapapillomavirus genus with one species (Deltapapillomavirus 4) and four types (BPVs 1, 2, 13, and 14); Epsilonpapillomavirus genus with one species (Epsilonpapillomavirus 1) and two types (BPVs 5 and 8); Dyoxipapillomavirus genus with one species (Dyoxipapillomavirus 1) and one type (BPV7); Dyokappapilomavirus genus with three types (BPVs 16, 18, and 22); and Xipapillomavirus genus, composed of the species Xipapillomavirus 1, which encompasses BPVs 3, 4, 6, 9, 10, 11, and 15; and Xipapillomavirus 2, consisting of BPV12. Additionally, there are BPVs 17, 20, 23, and 24 that also belong to the genus Xipapillomavirus but do not present species demarcation, as well as an unclassified genus that includes BPVs 19 and 21.
Although 24 BPV types have been identified and most of these have recently been characterized, this number still contrasts with the more than 200 human papillomavirus (HPV) types described [22]. Therefore, the aim of this study was to describe the molecular and pathological aspects of teat papillomatosis in dairy cows in southern Brazil, as well as to report the identification of 10 putative new BPV types.
Materials and methods
Sampling and histopathology
The dairy cows were slaughtered in two slaughterhouses located in the state of Rio Grande do Sul, southern Brazil, from August 2016 to March 2017. The mammary glands were inspected and, were selected. The teats, along with a fragment of the skin of the udder base, were collected from 73 cows presenting with papillomatous lesions in the teats, and occasionally also in the udder. Small fragments of the papillomas of each cow were pooled, consisting of one sample per cow, and stored frozen at − 20 °C for subsequent molecular analysis. The remaining material was fixed in 10% neutral buffered formalin, routinely processed for histopathology, and stained with hematoxylin and eosin (H&E).
DNA isolation
Papilloma specimens were ground with sterile sand in 10 mL of phosphate buffered saline (PBS) (pH 7.4), centrifuged at 720×g for 10 min, and 1000 μL of the supernatant was stored at − 20 °C for molecular analysis. DNA was isolated of 100 μL using phenol-chloroform following usual procedures [26] and eluted in 50 μL of ultrapure water. The quality and quantity of the DNA were assessed through spectrophotometry and fluorometry, using a NanoDrop™ (Thermo Fisher Scientific) and Qubit™ (Thermo Fisher Scientific) respectively.
PCR and Sanger sequencing
Partial amplification of the L1 gene was performed with the forward primer FAP59 (5′-TAA CWG TIG GIC AYC CWT ATT-3′) (Position in BPV strain X02346: 5712-5752) and the reverse primer FAP64 (5′-CCW ATA TCW VHC ATI TCI CCA TC-3′) (Position in BPV strain X02346: 6206-6185) [14]. Briefly, 100 ng of extracted DNA was mixed with [1×] PCR buffer, 20 pmol of each primer, 2 mM MgCl2, 200 μM dNTPs, and 1 U GoTaq® DNA Polymerase (Promega, Madison, WI, USA) in a total volume of 25 μL adjusted with ultrapure water. After an initial incubation at 95 °C for 5 min, the reaction conditions consisted of 40 cycles of denaturation at 95 °C for 1 min, annealing at 50 °C for 1 min, and extension at 75 °C for 1 min. The PCR products were purified using a PureLink™ Quick PCR Purification Kit (Invitrogen, Carlsbad, CA, USA). Aliquots from the reactions were analyzed by electrophoresis in 1% agarose gels stained with GelRed Loading Buffer (Biotium Inc., Hayward, CA, USA) and examined under UV light. To confirm the PCR results, both strands were sequenced with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using a BigDye Terminator v.3.1 cycle sequencing kit (Applied Biosystems).
Sequence analysis
To determine sequence identity, the sequences were compared with all the GenBank sequences using the Basic Local Alignment Search Tool (BLAST) [27]. Representative ruminant PV sequences were retrieved from GenBank. Nucleotide alignment was performed using MUSCLE software [13]. The percentage similarity between the PV sequences determined in this work, and those previously determined was estimated using MEGA6 computer software (version 6.0) [24]. The similarity between the sequences of the putative new BPV types detected in this study was determined by constructing an identity matrix using GENEIOUS software (version 9.0). This matrix aimed to identify homologous sequences with more than 90% of identity which would represent a same putative new type.
The taxonomy criteria of the BPV samples were based on the L1 gene [4]. To be considered a new type, the entire L1 gene sequence would have to be more than 10% different compared with the closest know type. Putative new BPV types were defined as those displaying less than 90% nucleotide identity within the L1 gene fragment of all PV types already classified [12, 21].
Results
Gross and histopathological findings
The four teats were affected by papillomatous lesions in most of the 73 cows (Fig. 1a). Grossly, the papillomas were characterized according to three patterns. Pattern 1 was identified in 5 cows (6.9%) and consisted of brown to blackish, exophytic projections of varying sizes and vegetative, digitiform, or filiform aspect (Fig. 1b). Pattern 2 was observed in 29 cows (39.7%) and was characterized by slightly elevated whitish projections with a flat to round surface (Fig. 1c). The third and most frequent pattern (pattern 3 or mixed) was observed in 39 cows (53.4%) and presented a mixed morphology. Papillomatous lesions similar to those from both patterns 1 and 2 were observed in the same teat or in the same cow (Fig. 1d).
Fig. 1.
Gross findings of teat papillomatosis in dairy cows. a All four teats are affected by papillomas. b Brown to blackish and exophytic projections with a vegetative aspect are observed on the teat surface (pattern 1). c Flat, slightly elevated, and whitish projections are observed on the teat surface (pattern 2). d Papillomatous lesions similar to those described in b and c are observed in the same teat (pattern 3)
Histologically, patterns 1 and 2 showed very similar morphological characteristics, differing only in surface appearance, which was vegetating to digitiform in pattern 1 (Fig. 2a) and flat to undulating in pattern 2 (Fig. 2b). All the lesions were identified as squamous papillomas and were characterized by marked epidermal hyperplasia, ortho or parakeratotic hyperkeratosis, and an increase in the amount of keratohyaline granules. Swollen keratinocytes with a lightly staining eosinophilic cytoplasm and a pyknotic nucleus surrounded by a clear halo (koilocytes) were observed in the spinous and granular epidermal layers in 28 of the 73 cows (Fig. 2c). In addition, intranuclear amphophilic inclusion bodies in keratinocytes were observed only in one cow (Fig. 2d).
Fig. 2.
Histological findings of teat papillomatosis in dairy cows. a Histological aspect of gross pattern 1, characterized by marked epidermal hyperplasia and orthokeratotic hyperkeratosis with a vegetating to digitiform surface. Hematoxylin and eosin (H&E), 4×. b Histological aspect of gross pattern 2, characterized by marked epidermal hyperplasia and orthokeratotic hyperkeratosis with a flat surface. H&E, 4×. c There are numerous swollen keratinocytes, with a lightly eosinophilic cytoplasm and a pyknotic nucleus surrounded by a clear halo (koilocytes) in epidermal spinous and granular layers. H&E, 40×. d A keratinocyte with an intranuclear amphophilic inclusion body is visible (arrow) in the center of the figure. Note the marked increase in the amount of keratohyaline granules. H&E, 40×
PCR and Sanger sequencing
Conventional PCR using the FAP59/FAP64 primer pair generated the expected 480 bp L1 gene fragment in 63 of the 73 papilloma samples [14]. All the fragments were submitted to Sanger sequencing to confirm the PCR results and determine the BPV type involved in the lesion. The sequences were categorized according to previously determined (classical) BPV types, previously reported putative BPV types, and putative new BPV types after comparison of the nucleotide similarity of the L1 gene fragment. Sequences with less than 90% identity with classical BPV types were divided into previously reported putative BPV types and putative new BPV types. Previously reported putative BPV types were those displaying more than 90% identity with other reported BPV types, while putative new BPV types were those exhibiting less than 90% nucleotide similarity, with any sequence retrieved from the genetic database.
Classical BPV types were detected in 27 samples, the most frequent of which was BPV8 (12/27), followed by BPVs 6 and 7, both of which were found in four samples (Table 1). There were 17 sequences associated with previously reported putative BPV types, the most frequent of which was BAPV8 (6/17) (Table 2). Fifteen sequences were associated with putative new BPV types and presented nucleotide similarities ranging from 74.3 to 89.1% with the PV sequences available in the GenBank database (Table 3). As shown in the identity matrix (Fig. 3), there were 10 putative new BPV types, since five sequences (AP3878-16, AP3881-16, AP4169-16, AP4829-16, and AP781-17) shared more than 90% nucleotide similarity, and the same was recorded with two other sequences (AP4144-16 and AP4174-16). Four sequences could not be classified because the fragments were shorter than expected.
Table 1.
Nucleotide sequence similarity of the detected classical BPV types and gross pattern of the identified papillomas
| BPV type | GenBank accession number | Sample | Similarity (%) | Gross pattern |
|---|---|---|---|---|
| BPV4 | X05817 | AP3891-16 | 98.0 | Flat |
| BPV6 | AJ620208 | AP4147-16 | 98.7 | Flat |
| AP4155-16 | 99.5 | Exophytic | ||
| AP4165-16 | 99.5 | Flat | ||
| N772-17 | 100.0 | Flat | ||
| BPV7 | DQ217793 | AP4151-16 | 99.1 | Exophytic |
| AP4159-16 | 97.3 | Flat | ||
| AP4160-16 | 99.1 | Flat | ||
| AP4163-16 | 98.0 | Mixed | ||
| BPV8 | DQ098913 DQ098917 | AP3880-16 | 99.7 | Mixed |
| AP4150-16 | 99.2 | Mixed | ||
| AP4152-16 | 97.6 | Flat | ||
| AP4171-16 | 99.5 | Flat | ||
| AP4834-16 | 98.8 | Flat | ||
| AP4837-16 | 99.5 | Flat | ||
| AP768-17 | 99.6 | Flat | ||
| AP773-17 | 98.1 | Flat | ||
| AP782-17 | 89.7 | Flat | ||
| AP3670-16 | 98.5 | Mixed | ||
| AP3682-16 | 99.5 | Mixed | ||
| AP3697-16 | 99.5 | Mixed | ||
| BPV9 | AB331650 | AP3870-16 | 97.1 | Mixed |
| BPV10 | AB331651 KF017607 | AP772-17 | 98.5 | Mixed |
| N770-17 | 98.9 | Flat | ||
| N771-17 | 98.7 | Mixed | ||
| BPV11 | AB543507 | AP3898-16 | 99.1 | Mixed |
| BPV12 | JF834524 | AP764-17 | 90.7 | Mixed |
Table 2.
Nucleotide sequence similarities between the sequences obtained in this study, their closest related putative BPV type already reported, and gross patterns of the papillomas identified
| BPV type | GenBank accession number | Sample | Similarity (%) | Gross pattern |
|---|---|---|---|---|
| BAPV4 | AY426550 | AP3694-16 | 99.3 | Flat |
| BAPV8 | AY426554 | AP3689-16 | 93.1 | Mixed |
| AP3696-16 | 92.5 | Mixed | ||
| AP3895-16 | 92.6 | Mixed | ||
| AP4176-16 | 93.7 | Flat | ||
| AP4181-16 | 92.7 | Mixed | ||
| AP775-17 | 92.0 | Mixed | ||
| BAPV9 | AY426555 | AP3884-16 | 99.5 | Mixed |
| AP3896-16 | 99.5 | Mixed | ||
| AP4828-16 | 98.1 | Flat | ||
| AP778-17 | 98.8 | Mixed | ||
| BPV/BR-UEL2 | EU293538 | AP3707-16 | 91.1 | Flat |
| AP3888-16 | 91.1 | Mixed | ||
| BPV/BR-UEL5 | EU293541 | AP4182-16 | 100.0 | Flat |
| BPV/CHI-SW2 | KF751803 | AP3690-16 | 97.8 | Mixed |
| AP3703-16 | 97.9 | Mixed | ||
| AP4826-16 | 97.9 | Flat |
Table 3.
Nucleotide sequence similarities between the putative new BPV types, their closest related putative BPV type, and BPV type and gross patterns of the papillomas identified
| Sample | BPV type or putative BPV type closest related | BPV type closest related | Gross pattern | ||||
|---|---|---|---|---|---|---|---|
| Best Blastn Hit | GenBank accession number | Similarity (%) | Best Blastn Hit | GenBank accession number | Similarity | ||
| AP3878-16 | BPV/UFPE03BR | JQ897974 | 79.6 | BPV24 | MG602223 | 73.4% | Mixed |
| AP3881-16 | BPV/UFPE03BR | JQ897974 | 80.8 | Unclassified | nd | nd | Mixed |
| AP3885-16 | BPV12 | JF834524 | 78.2 | BPV12 | JF834524 | 78.2% | Flat |
| AP3892-16 | BAPV8 | AY426554 | 89.1 | BPV12 | JF834524 | 76.3% | Mixed |
| AP3899-16 | BPV12 | JF834524 | 87.8 | BPV12 | JF834524 | 87.8% | Exophytic |
| AP4144-16 | BPV/BR-UEL3 | EU293539 | 74.3 | BPV9 | AB331650 | 76.3% | Mixed |
| AP4157-16 | BPV/UFPE03BR | JQ897974 | 80.9 | BPV24 | MG602223 | 75.3% | Flat |
| AP4167-16 | BAA1 | AF485375 | 76.6 | BPV12 | JF834524 | 77.9% | Exophytic |
| AP4169-16 | BPV/UFPE03BR | JQ897974 | 80.5 | BPV24 | MG602223 | 74.6% | Mixed |
| AP4174-16 | IZ1214/02SP/BR/2009 | HQ612180 | 76.6 | Aks-02 | KM983393 | 80.3% | Mixed |
| AP4178-16 | Aks-02 | KM983393 | 74.6 | Aks-02 | KM983393 | 74.6% | Flat |
| AP4829-16 | BPV/UFPE03BR | JQ897974 | 80.9 | BPV24 | MG602223 | 75.3% | Flat |
| AP4835-16 | BAPV9 | AY426555 | 83.5 | Unclassified | nd | nd | Mixed |
| AP771-17 | BPV/BR-UEL6 | KP892554 | 76.8 | Unclassified | nd | nd | Mixed |
| AP781-17 | BPV/UFPE03BR | JQ897974 | 80.1 | BPV24 | MG602223 | 75.5% | Flat |
Fig. 3.
Matrix of nucleotide sequence similarity among the putative new BPV types detected in this study
Discussion
In this study, diagnosis of teat papillomatosis in dairy cows was based on gross and microscopic findings, and the associated BPV types were determined through molecular analysis, allowing the identification of a wide variety of BPVs.
The four teats were affected in most of the cows. This is a frequent feature of teat papillomatosis described in dairy cows [19] and may be associated with the infection process. To infect an animal, BPVs require microabrasions or cutaneous wounds. In addition, they are highly contagious and may affect the entire herd, since transmission occurs easily either by direct contact, or indirectly through fomites, insects, and pastures [6, 19, 20]. For dairy cattle, the milking process also plays an important role in transmission, namely through milking equipment and hand milking [15].
Different patterns of teat papillomatosis were observed in the macroscopic evaluation. However, the histological changes were similar among the patterns and were characterized by squamous papillomas as only the epidermis was affected. The BPVs of the genus Deltapapillomavirus (1, 2, 14, and 15) normally induce fibropapilloma formation since they infect the epidermis and the dermis. However, these four types, as well as BPV5 that is known to cause both fibropapillomas and squamous papillomas, were not observed in this study [12, 16–18]. BPV8 can also induce both types of papillomas [20]. However, despite being identified in 12 cows, all the lesions associated with BPV8 were squamous papillomas.
BPV DNA from 59 of the 73 teat papilloma samples collected was amplified and sequenced, leading to the identification of 8 classical BPV types (n = 27), 6 previously reported putative BPV types (n = 17), and 10 putative new BPV types (n = 15). The identification of different BPV types reflects the wide genetic diversity of the strains related to teat papillomatosis in dairy cows in the analyzed region. However, despite the wide variety of BPVs identified in this study, the same papilloma-associated histological changes were observed in all the samples, and there was no correlation between the gross pattern and the BPV type identified. Although not considered pathognomonic of BPV infection, the presence of koilocytes observed in the histological analysis likely indicates the cytopathic effect of the papillomavirus in the tissues [2, 5].
PCR assays using the FAP59/FAP64 degenerate primers to amplify partial fragments of the BPV L1 gene, followed by sequencing of the amplified product, is an important tool that is widely used to determine the presence of different BPV types in cattle herds from different geographical regions [1, 10, 18, 21, 23, 25]. The 10 negative results were likely due to inefficient base pairing in the 3 region of both primers [11], although is not possible to discard the presence of PCR inhibitors in the purified DNA samples nor that there was no BPV DNA in the analyzed samples [23].
The entire genome must be sequenced for considering new PV types and the L1 open reading frame (ORF) have to differ by more than 10% in comparison with the closest PV type [12]. Considering this criteria, eight classical BPV types (BPVs 4, 6, 7, 8, 9, 10, 11, and 12) were identified in this study. Among these, BPVs 6, 7, 9, and 10 have been widely detected in papillomas from cattle teats or udders [17, 19–21, 25]. However, BPV8 is associated primarily with papillomas from other regions of the skin [3, 10, 20] and is rarely found associated with teat papillomatosis [21]. In this work, BPV8 was identified in 12 cows, demonstrating that it has a large capacity for infecting different anatomical sites.
The wide diversity of BPV types identified in our study was similar to that found in studies conducted in different regions of Brazil that focused on cutaneous and teat papillomatosis of cattle [2, 3, 9, 10, 23, 25]. However, our results reinforce the importance of using genotyping studies to better determine BPV genetic diversity, since the immune response against the papillomavirus is type-specific [25] and fundamental for the control of the disease.
Teat papillomatous lesions were characterized according to three gross patterns (exophytic, flat, and mixed), and the mixed pattern was identified in more than 50% of the cows examined. Histologically, all the lesions were characterized by squamous papillomas. Eight classical BPV types, 6 previously reported putative BPV types, and 10 putative new BPV types were identified in the molecular analysis, indicating large variation in viral types and emphasizing the importance of teat papillomatosis in dairy cows.
Funding information
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and PROPESQ/UFRGS.
Compliance with ethical standards
Conflict of interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Footnotes
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