Broad-spectrum detection of papillomaviruses in bovine teat papillomas and healthy teat skin

Tomoko Ogawa1,2, Yoshimi Tomita1, Mineyuki Okada2, Kuniko Shinozaki2, Hiroko Kubonoya2, Ikuo Kaiho2 and Hiroshi Shirasawa1

1 Department of Molecular Virology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuou-ku, Chiba 260-8670, Japan
2 Division of Virology, Chiba Prefectural Institute of Public Health, 666-2 Nitona-cho, Chuou-ku, Chiba 260-8715, Japan

Correspondence
Yoshimi Tomita
tomita{at}faculty.chiba-u.jp


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To investigate the prevalence of bovine papillomavirus (BPV) in bovine papilloma and healthy skin, DNA extracted from teat papillomas and healthy teat skin swabs was analysed by PCR using the primer pairs FAP59/FAP64 and MY09/MY11. Papillomavirus (PV) DNA was detected in all 15 papilloma specimens using FAP59/FAP64 and in 8 of the 15 papilloma specimens using MY09/MY11. In swab samples, 21 and 8 of the 122 samples were PV DNA positive using FAP59/FAP64 and MY09/MY11, respectively. Four BPV types (BPV-1, -3, -5 and -6), two previously identified putative BPV types (BAA1 and -5) and 11 putative new PV types (designated BAPV1 to -10 and BAPV11MY) were found in the 39 PV DNA-positive samples. Amino acid sequence alignments of the putative new PV types with reported BPVs and phylogenetic analyses of the putative new PV types with human and animal PV types showed that BAPV1 to -10 and BAPV11MY are putative new BPV types. These results also showed the genomic diversity and extent of subclinical infection of BPV.

The GenBank accession numbers of the sequences reported in this paper are AY300817 (BAPV1), AY300818 (BAPV2), AY300819 (BAPV3), AY426550 (BAPV4), AY426551 (BAPV5), AY426552 (BAPV6), AY426553 (BAPV7), AY426554 (BAPV8), AY426555 (BAPV9), AY426556 (BAPV10), AY300820 (BAPV11MY) and AY555237 (BAPV6MY).


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To date, six bovine papillomavirus (BPV) types, BPV-1 to -6, have been isolated (Campo & Coggins, 1982; Campo et al., 1980, 1981; Chen et al., 1982; Jarrett et al., 1984; Pfister et al., 1979) and the complete genomes of BPV-1 to -5 and a partial genome of BPV-6 have been sequenced. It has been reported that infections with these BPVs cause skin warts, papillomatosis and cancer in cows (Campo, 2002). However, it has been reported that the DNA of human papillomaviruses (HPVs), originally found in skin lesions, can frequently be detected in healthy skin by PCR, demonstrating latent or subclinical infections of these HPVs (Antonsson et al., 2000; Astori et al., 1998; Boxman et al., 1997).

The PCR primer pair FAP59/FAP64 was designed from two relatively conserved regions found in the L1 gene of most HPV types to detect a broad spectrum of HPV types in cutaneous tumours and normal skin (Forslund et al., 1999). Using this primer pair, it was shown that HPV is a divergent group and is found ubiquitously and subclinically in human healthy skin, suggesting a commensal nature of these viruses (Antonsson et al., 2000, 2003a, b). The FAP59/FAP64 PCR has also been shown to amplify papillomavirus (PV) DNA in the healthy skin of many animal species including cows, in which five putative BPV types (BAA1 to -5) have been detected in forehead skin (Antonsson & Hansson, 2002). The PCR primer pair MY09/MY11 was originally designed to detect mucosal HPV types (Manos et al., 1989) and has been reported to amplify the L1 gene of most genital HPV types (Bernard et al., 1994; Chan et al., 1995; Kado et al., 2001), as well as some animal PV types, including BPV-1, -3, -5 and -6 (Chan et al., 1995).

In this study, we showed the prevalence of BPVs and putative BPVs in teat papillomas and healthy teat skin by PCR using the primer pairs FAP59/FAP64 and MY09/MY11, and also detected 11 putative new BPV types.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Papilloma and swab samples.
All samples were collected from dairy cows kept in five prefectures of Japan. Fifteen papilloma specimens of bovine teat were collected from 15 dairy cows in different farms. These papilloma specimens were taken by hand (wearing gloves, changed for each cow) and were suspended in 1 ml 0·9 % NaCl solution and stored at –80 °C. A swab sample was collected from each of 122 dairy cows kept in 95 farms using cotton-tipped swabs pre-wetted with 0·9 % NaCl solution, which were drawn back and forth five times over the teats and then suspended in 1 ml 0·9 % NaCl solution. All samples were stored at 4 °C for less than 24 h before being used for DNA extractions.

DNA extraction.
The frozen specimens were homogenized under liquid nitrogen. DNA was extracted from papilloma specimens and swab samples using the High Pure PCR template preparation kit (Roche Diagnostics). Purified DNAs were eluted from the column in a volume of 200 µl and 2·5 µl was used for each PCR.

PCR.
DNA samples were amplified using two PCR primer pairs: FAP59 (forward; 5'-TAACWGTIGGICAYCCWTATT-3') and FAP64 (reverse; 5'-CCWATATCWVHCATITCICCATC-3') (Forslund et al., 1999); and MY11 (forward; 5'-GCMCAGGGWCATAAYAATGG-3') (Manos et al., 1989) and MY09 (reverse; 5'-CGTCCMARRGGAWACTGATC-3'). PCR was carried out in a volume of 50 µl containing 2·5 µl DNA sample, 0·25 µM of each primer, 200 µM dNTP mixture, 5 µl 10x EX Taq buffer and 1·25 U EX Taq polymerase (Takara Shuzo). For the primer pair FAP59/FAP64, after an initial denaturation at 94 °C for 10 min, the PCR consisted of 45 cycles of 1·5 min at 94 °C, 1·5 min at 50 °C and 1·5 min at 72 °C, followed by extension at 72 °C for 5 min. For the primer pair MY09/MY11, after an initial denaturation at 95 °C for 5 min, the PCR consisted of 35 cycles of 30 s at 95 °C, 30 s at 55 °C and 1 min at 72 °C, followed by extension at 72 °C for 5 min. The mixed-primer PCR using FAP59 and MY09 was carried out under the same conditions used for the primer pair FAP59/FAP64. Five microlitres of the amplified material was analysed by electrophoresis on a 1·5 % agarose gel in TBE buffer (Sambrook & Russell, 2001), followed by staining with ethidium bromide for 30 min. PV DNA-specific bands were identified by size determination under UV light.

Sequence analysis.
The PCR products were purified using the High Pure PCR products purification kit (Roche Diagnostics) and direct sequencing was carried out using the ABI PRISM Big Dye Terminator cycle sequencing kit (Applied Biosystems) using an ABI Genetic Analyzer 310 (Applied Biosystems) with forward and reverse primers. Some PCR products were cloned using the TOPO TA Cloning kit (Invitrogen). Six to ten clones from each PV-positive sample were isolated and sequenced. The forward and reverse complementary sequences were aligned using SEQED computer software (version 1.0.3). The DNA sequences were compared with all sequences in GenBank through the BLAST server (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/blast/). The percentage similarity of the PV sequences determined in this work in comparison with previously determined PV sequences was estimated using GENETYX computer software (version 11). The partial sequences of the BPV-6 L1 gene were determined after PCR amplification using FAP59/FAP64 and MY09/MY11.

The guidelines from the Papillomavirus Nomenclature Committee 1995 (14th International Papillomavirus Conference, Quebec City, Quebec, Canada) were followed to identify putative new PV types. An isolate was defined as a new PV type if the sequence of its L1 gene displayed less than 90 % similarity with the L1 genes of all types already known. The isolated novel sequences were called putative new PV types instead of PV types, since the PCR products represent only part of the L1 gene (de Villiers, 2001; Antonsson & Hansson, 2002). In this study, the putative new PV types that were amplified using primers FAP59/FAP64 were designated BAPV1 to -10. The DNA sequences of the putative new PV types that were amplified using primers MY09/MY11 were designated BAPV6MY and BAPV11MY.

Alignment of amino acid sequences and phylogenetic analysis of DNA sequences.
Sequences of 96–98 aa from FAP59/FAP64 PCR-amplified DNA segments corresponding to nt 5528–5821 of BPV-3 and 96 aa sequences of MY09/MY11 PCR-amplified DNA segments corresponding to nt 6446–6733 of BPV-3 were selected and used for the alignment. The CLUSTAL X multiple alignment program (version 1.83) (Thompson et al., 1997) was used for these alignments. Phylogenetic analysis was based on the neighbour-joining method. The DNA sequences of the HPV and animal PV types were selected by similarity searches with the DNA sequence corresponding to nt 5484–5933 and nt 6293–6753 of BPV-3. The former region was used for phylogenetic analysis of BAA1 to -5 and BAPV1 to -10 and the latter region was used for phylogenetic analysis of BAPV6MY and BAPV11MY. The phylogenetic tree was constructed using TreeView version 1.6.6.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
PCR of papilloma specimens and swab samples
DNA was extracted from 15 papilloma specimens and 122 swab samples and analysed by PCR using the primer pairs FAP59/FAP64 and MY09/MY11 (Table 1). These results showed that the sensitivity of the FAP59/FAP64 PCR was higher than that of the MY09/MY11 PCR for DNA samples from both papilloma specimens and swab samples.


View this table:
[in this window]
[in a new window]
 
Table 1. Detection of PV DNA in papilloma specimens and swab samples by PCR using primers FAP59/FAP64 and MY09/MY11

 
Detection of BPV types, putative BPV types and putative new PV types
The PCR products were sequenced and BPV types, previously reported putative BPV types and putative new PV types were determined after comparison with previously published PV types and putative PV types. As shown in Table 2, BPV-1 and -3, BAA5 and seven putative new PV types were detected in DNA samples from papilloma specimens. In swab samples, BPV-1, -3, -5 and -6, BAA1 and -5, and ten putative new PV types were detected. Double or multiple types were frequently detected by FAP59/FAP64 PCR in one sample. The most prevalent type was BAPV6. To examine whether the MY09/MY11 PCR products in P3, P8, P9, S4, S7 and S21 were downstream sequences of BAA5, BAPV2, -5 and -6, PCR using the mixed primers FAP59 and MY09 was carried out on the DNA in these samples. This PCR amplified a 1·3 kb DNA fragment from P8, but not from the other samples (data not shown). The 5'-terminal region of this 1·3 kb DNA was identical to BAPV6 and the 3'-terminal region was identical to BAPV6MY, showing that BAPV6MY is a downstream sequence of BAPV6. These results also suggested that BAPV11MY is one of the putative new PV types, and not a downstream sequence of BAA5 or BAPV2 or -5.


View this table:
[in this window]
[in a new window]
 
Table 2. Detection of BPV types, putative BPV types and putative new PV types using FAP59/FAP64 PCR and MY09/MY11 PCR

P1–P15, DNA from papilloma specimens; S1–S24, DNA from swab samples. F, PCR positive using primers FAP59/FAP64; M, PCR positive using primers MY09/MY11.

 
The similarities of the nucleotide sequences were determined for the putative new PV types to those of the HPV types, animal PV types, putative HPV types, putative animal PV types and putative new PV types found in this study. As shown in Table 3, the percentage similarity of BAPV1 to -10 to BPV types or putative new PV types was 67–79 %. The percentage similarity of BAPV11MY to BPV types was relatively low compared with that of BAPV1 to -10.


View this table:
[in this window]
[in a new window]
 
Table 3. Nucleotide sequence similarity of the putative new PV types to their closest related PV, putative new PV and BPV types

 
Alignment of amino acid sequences
To verify that the DNA sequences of the putative new PV types encoded conservative amino acids of the BPV L1 genes, we performed amino acid sequence alignments of BPV-1 to -6 and the results were compared with those of BAPV1 to -10 (Fig. 1a) or BAPV6MY and BAPV11MY (Fig. 1b). These results showed that 31 of the 96–98 aa residues were strictly conserved in BPV-1 to -6 and BAPV1 to -10, and 17 of the 96 aa residues were strictly conserved in BPV-1 to -6, BAPV6MY and BAPV11MY, suggesting that BAPV1 to -10 and BAPV11MY found in this study are putative new BPV types.



View larger version (71K):
[in this window]
[in a new window]
 
Fig. 1. (a) Amino acid sequence alignments of L1 subsegments of BPV-1 to -6 and BAPV1 to -10 (labelled B1–B10). (b) Amino acid sequence alignments of L1 subsegments of BPV-1 to -6, BAPV6MY (B6MY) and BAPV11MY (B11MY). Asterisks indicate residues that are fully conserved among all the sequences analysed.

 
Phylogenetic analysis of DNA sequences
A phylogenetic tree of HPV types, animal PV types, putative BPV types and BAPV1 to -10 found in this study was obtained by the neighbour-joining method (Fig. 2a). The tree was divided into the supergroups A–E (Chan et al., 1995; Myers, 1997; Antonsson & Hansson, 2002) and at least two other clusters comprising BAA2 and -3 and BAPV5 to -7. One of these clusters has been reported previously (Antonsson & Hansson, 2002). BAPV1, -3, -8, -9 and -10 were included in supergroup D, also containing BPV-3, -4 and -6 and BAA1 and -5. BAPV2 and -4 were found in supergroup C, which also included BPV-1, -2 and -5 and other animal PV types. No putative new PV types were found in supergroups A, B or E. The BAPV6MY and BAPV11MY DNA sequences were also subjected to phylogenetic analysis; BAPV11MY could not be classified in supergroups A–E (Fig. 2b). These results also showed that BAPV1 to -10 and BAPV11MY are putative new BPV types, although BAPV5 to -7 and BAPV11MY are relatively close to HPV, which is classified as supergroup B.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2. (a) Phylogenetic analysis of HPV types, animal PV types, putative BPV types and putative new PV types. BAPV1 to -10 are indicated by B1 to B10. (b) Phylogenetic analysis of HPV types, animal PV types, BAPV6MY and BAPV11MY. BAPV6MY and BAPV11MY are indicated by B6MY and B11MY. HPV types are indicated by numbers only. OvPV, Ovine PV; EEPV, European elk PV; DPV, deer PV; COPV, canine oral PV; CRPV, cottontail rabbit PV; ROPV, rabbit oral PV.

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Currently, various generic or consensus PCR primers are used for the detection of HPV. We previously tested the relative sensitivities and specificities of these PCR primer sets for HPV detection using a combination of three primer sets for the L1 consensus sequences and two sets for the E6/E7 consensus sequence (Kado et al., 2001). As a result, we showed that a high rate of detection of HPVs was achievable using the five pairs of consensus PCR primers. In this study, two L1 consensus primer pairs, FAP59/FAP64 and MY09/MY11, were used to detect BPV in bovine papilloma and healthy teat skin. We detected 16 BPV types, putative BPV types and putative new BPV types in total using the primers FAP59/FAP64 and four BPV types and putative new BPV types in total using the primers MY09/MY11. These results also suggested that application of multiple primer sets for PCR could be a successful method to detect a broad range of PV DNA. In general, the sensitivity and specificity of the PCR may be affected by primary factors such as concentration and purity of the sample DNA. In this study, we extracted DNA from the cotton swab sample prior to PCR, which resulted in the successful detection of PV DNA in swab samples of bovine teat skin. Without DNA extraction, the PCR showed very low PV DNA detection rates (data not shown).

Recently, it has been reported that a large number of putative HPV types and putative animal PV types have been isolated by PCR from the healthy skin of humans and other animals (Antonsson & Hansson, 2002; Antonsson et al., 2000, 2003a, b; Astori et al., 1998; Boxman et al., 1997). These reports showed latent or subclinical infection of skin PV and a commensal nature of PV. BPV-1 to -6 were originally isolated from bovine papillomas and these have been thought to be a causal agent of papillomatosis (Campo, 2002; Jarrett et al., 1984). In this study, however, genome DNA of BPV-1, -3 and -6, BAA1 and -5 and most putative new BPV was detected in swab samples of healthy teat skin on which papilloma was not apparent, suggesting latent or subclinical infections of these BPV types. In addition, the potential to induce tumours caused by BAPV2 and -4, which are relatively close to BPV-5 in the phylogenetic analysis, remains to be elucidated.


   ACKNOWLEDGEMENTS
 
We thank Dr M. Saveria Campo for supplying the clone of BPV-6 and E. Shinohara and J. Nagai for helpful advice and collecting samples.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Antonsson, A. & Hansson, B. G. (2002). Healthy skin of many animal species harbors papillomaviruses which are closely related to their human counterparts. J Virol 76, 12537–12542.[Abstract/Free Full Text]

Antonsson, A., Forslund, O., Ekberg, H., Stemer, G. & Hansson, B. G. (2000). The ubiquity and impressive genomic diversity of human skin papillomavirus suggest a commensalic nature of these viruses. J Virol 74, 11636–11641.[Abstract/Free Full Text]

Antonsson, A., Erfurt, C., Hazard, K., Holmgren, V., Simon, M., Kataoka, A., Hossain, S., Håkangård, C. & Hansonn, B. G. (2003a). Prevalence and type spectrum of human papillomavirus in healthy skin samples collected in three continents. J Gen Virol 84, 1881–1886.[Abstract/Free Full Text]

Antonsson, A., Karanfilovska, S., Lindqvist, P. & Hansson, B. G. (2003b). General acquisition of human skin papillomavirus infections occurs in early infancy. J Clin Microbiol 41, 2509–2514.[Abstract/Free Full Text]

Astori, G., Lavergne, D., Benton, C., Höckmayr, B., Egawa, K., Garbe, C. & de Villiers, E.-M. (1998). Human papillomavirus are commonly found in normal skin of immunocompetent hosts. J Invest Dermatol 110, 752–755.[Abstract]

Bernard, H.-U., Chan, S.-Y., Manos, M. M., Ong, C.-K., Villa, L. L., Delius, H., Peyton, C. L., Bauer, H. M. & Wheeler, C. M. (1994). Identification and assessment of known and novel papillomaviruses by polymerase chain reaction amplification, restriction fragment length polymorphisms, nucleotide sequence, and phylogenetic algorithms. J Infect Dis 170, 1077–1085.[Medline]

Boxman, I. L., Berkhout, R. J., Mulder, L. H., Wolkers, M. C., Bouwes Bavinck, J. N., Vermeer, B. J. & ter Schegget, J. (1997). Detection of human papillomavirus DNA in plucked hairs from renal transplant recipients and healthy volunteers. J Invest Dermatol 108, 712–715.[Abstract]

Campo, M. S. (2002). Animal models of papillomavirus pathogenesis. Virus Res 89, 249–261.[CrossRef][Medline]

Campo, M. S. & Coggins, L. W. (1982). Molecular cloning of bovine papillomavirus genomes and comparison of their sequence homologies by heteroduplex mapping. J Gen Virol 63, 255–264.[Abstract]

Campo, M. S., Moar, M. H., Jarrett, W. F. H. & Laird, H. M. (1980). A new papillomavirus associated with alimentary cancer in cattle. Nature 286, 180–182.[Medline]

Campo, M. S., Moar, M. H., Laird, H. M. & Jarrett, W. F. H. (1981). Molecular heterogeneity and lesion site specificity of cutaneous bovine papillomaviruses. Virology 113, 323–335.[CrossRef][Medline]

Chan, S.-Y., Delius, H., Halpern, A. & Bernard, H.-U. (1995). Analysis of genomic sequences of 95 papillomavirus types: uniting typing phylogeny, and taxonomy. J Virol 69, 3074–3083.[Abstract]

Chen, E. Y., Howley, P. M., Levinson, A. D. & Seeburg, P. H. (1982). The primary structure and genetic organization of the bovine papillomavirus type 1 genome. Nature 299, 529–534.[Medline]

de Villiers, E. M. (2001). Taxonomic classification of papillomaviruses. Papillomavirus Rep 12, 57–63.

Forslund, O., Antonsson, A., Nordin, P., Stenquist, B. & Hansson, B. G. (1999). A broad range of human papillomavirus types detected with a general PCR method suitable for analysis of cutaneous tumours and normal skin. J Gen Virol 80, 2437–2443.[Abstract/Free Full Text]

Jarrett, W. F. H., Campo, M. S., O'Neil, B. W., Laird, H. M. & Coggins, L. W. (1984). A novel bovine papillomavirus (BPV-6) causing true epithelial papillomas of the mammary gland skin: a member of a proposed new BPV subgroup. Virology 136, 255–264.[CrossRef][Medline]

Kado, S., Kawamata, Y., Shino, Y. & 9 other authors (2001). Detection of human papillomaviruses in cervical neoplasias using multiple sets of generic polymerase chain reaction primers. Gynecol Oncol 81, 47–52.[CrossRef][Medline]

Manos, M. M., Ting, Y., Wright, D. K., Lewis, A. J., Broker, T. R. & Wolinsky, S. M. (1989). The use of polymerase chain reaction amplification for the detection of genital human papillomaviruses. Cancer Cell 7, 209–214.

Myers, G. (1997). Alignments, P.I-3. In Human Papillomaviruses 1997. HPV Sequence Database. Edited by G. Myers, C. Baker, K. Münger, F. Sverdrup, A. McBride & H. U. Bernard. Los Alamos, NM: Los Alamos National Laboratory.

Pfister, H., Linz, U., Gissmann, L., Huchthausen, B., Hoffman, D. & zur Hausen, H. (1979). Partial characterization of a new type of bovine papillomavirus. Virology 96, 1–8.[CrossRef][Medline]

Sambrook, J. & Russell, W. D. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.[Abstract/Free Full Text]

Received 4 March 2004; accepted 1 April 2004.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Ogawa, T.
Articles by Shirasawa, H.
Articles citing this Article
PubMed
PubMed Citation
Articles by Ogawa, T.
Articles by Shirasawa, H.
Agricola
Articles by Ogawa, T.
Articles by Shirasawa, H.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS