Teat Papillomatosis Associated with Bovine Papillomavirus Types 6, 7, 9, and 10 in Dairy Cattle from Brazil
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Brazilian Journal of Microbiology 44, 3, 905-909 (2013) Copyright © 2013, Sociedade Brasileira de Microbiologia ISSN 1678-4405 www.sbmicrobiologia.org.br Short Communication Teat papillomatosis associated with bovine papillomavirus types 6, 7, 9, and 10 in dairy cattle from Brazil 1,* 1,*,# 1 1 Claudia C. Tozato , Michele Lunardi , Alice F. Alfieri , Rodrigo A.A. Otonel , Giovana W. Di Santis2, Brígida K. de Alcântara1, Selwyn A. Headley2, Amauri A. Alfieri1 1Laboratório de Virologia Animal, Departamento de Medicina Veterinária Preventiva, Universidade Estadual de Londrina, Londrina, PR, Brazil. 2Laboratório de Patologia, Departamento de Medicina Veterinária Preventiva, Universidade Estadual de Londrina, Londrina, PR, Brazil. Submitted: February 7, 2012; Approved: September 10, 2012. Abstract This study describes the clinical, histopathological, and virological characterization of teat papillo- matosis from Brazilian dairy cattle herds. Four types of bovine papillomavirus were identified (BPV6, 7, 9, and 10); one of these (BPV7) is being detected for the first time in Brazilian cattle. Key words: papilloma, BPV, genotyping. Papillomaviruses (PVs) are small (52-55 nm), non- (BPV5 and 8), and a yet unnamed PV genus (BPV7) (Ber- enveloped, double-stranded DNA oncoviruses that repli- nard et al., 2010; Hatama et al., 2011; Zhu et al., 2012; cate in the nucleus of squamous epithelial cells and can in- Lunardi et al., 2013). Moreover, the occurrence of numer- duce warts in the skin and mucosal epithelia of most higher ous additional viral types has been proposed based on par- vertebrate species. Some specific viral types have the po- tial nucleotide sequence analysis of the major capsid pro- tential to cause malignant progression in papillomatous le- tein L1, obtained from both benign cutaneous lesions and sions of animals and humans (Antonsson et al., 2002; de swab samples of healthy skin from cattle (Antonsson and Villiers et al., 2004). In cattle, bovine papillomavirus Hansson 2002; Maeda et al., 2007; Ogawa et al., 2007; (BPV) is the etiological agent of cutaneous and teat papil- Claus et al., 2008; Claus et al., 2009c). lomatosis, cancers of the upper gastrointestinal tract and Teat papillomatosis has been reported in dairy herds urinary bladder (Campo 2002; Borzacchiello et al., 2003; worldwide as a cattle health problem resulting in economic Wosiacki et al., 2006). losses (Campo 2003). While milking can become difficult BPV produces characteristic gross lesions that are ei- in markedly affected individuals, ulceration and rupture of ther exophytic (proliferating outwards) or endophytic (in- established cutaneous lesions might predispose dairy cattle verted) and are composed of a hyperplastic epithelium to mastitis and distortion of the milk ducts. Additionally, supported by a discrete dermal tissue containing dilated the maintenance of affected cows with alteration in mam- capillaries (Ginn et al., 2007; Hargis et al., 2012). mary gland shape and/or even of herds with a high number Currently, the genomes of thirteen BPVs (BPV1 to of affected animals may prevent economic profits in the 13) have been fully characterized. They are classified into dairy industry (Campo 2002; Borzacchiello et al., 2008). four genera based on their genome relatedness and biologi- Although papilloma lesions found in teats and udders cal properties (de Villiers et al., 2004). These genera are can be caused by diverse BPV types, the BPV6 has often Deltapapillomavirus (BPV1, 2, and 13), Xipapillomavirus been identified in this anatomical location. Therefore, the (BPV3, 4, 6, 9, 10, 11, and 12), Epsilonpapillomavirus association of BPV1, 3, 5, 7, 8, 9, 10, 11, and other types Send correspondence to A.A. Alfieri. Laboratories of Animal Virology, Department of Preventive Veterinary Medicine, Universidade Estadual de Londrina, P.O. Box 10001, 860571-970, Londrina, Parana, Brazil. E-mail: [email protected]. *Both authors contributed equally to the development of this paper. #Current Adress: Laboratório de Microbiologia Veterinária, Hospital Veterinário Escola, Universidade de Cuiabá, Cuiabá, MT, Brazil. 906 Tozato et al. still to be characterized, have been confirmed in mammary PHRED software for quality analysis of chromatogram glands of cattle (Campo et al., 1981; Jarrett et al., 1984; readings. The sequences were accepted if the base quality Ogawa et al., 2004; Claus et al., 2007; Maeda et al., 2007; was equal to or higher than 20. Consensus sequences were Claus et al., 2008). determined by using the CAP3 software, and the sequence Despite the high number of cattle herds affected by identity was verified with all sequences deposited in the cutaneous papillomatosis in several geographical regions GenBank by using the BLAST software. The guidelines of of Brazil, studies investigating BPV diversity associated the Papillomavirus Nomenclature Committee 1995 (14th with different clinical outcomes are sporadic (Stocco dos International Papillomavirus Conference, Quebec City, Santos et al., 1998; Freitas et al., 2003; Wosiacki et al., Quebec, Canada) were followed to identify PV types (de 2005; Wosiacki et al., 2006; Claus et al., 2008; Claus et al., Villiers et al., 2004). 2009b; Claus et al., 2009c; Lunardi et al., 2010). Grossly, all cutaneous lesions were exophytic and The aim of this report is to describe the identification were histologically classified as benign squamous cell neo- of BPVs 6, 7, 9, and 10 in association with cases of teat plasms (cutaneous papilloma) irrespective of the type of vi- papillomatosis from two dairy cattle herds of Brazil. This rus isolated. Microscopically, all tumorous growths dem- study represents the first detection of the BPV7 in Brazilian onstrated similar histological features; being characterized cattle. by varying degrees of hyperkeratosis or parakeratosis with Seven exophytic teat papillomas were individually elongated digital-like proliferation of the squamous epithe- collected from three dairy cows belonging to two different lium (Figure 1A). In all tumors fragments evaluated, most cattle herds from the Northern region of Parana state in keratinocytes within the stratum spinosum demonstrated Southern Brazil. These lesions were observed on different clear perinuclear halo, some having pyknotic nucleus parts of the teat and coded for identification. A portion of (characterized as koilocytes) others revealed discrete bal- each lesion was fixed in 10% buffered formalin solution looning degeneration; in some areas, two or more adjacent and routinely processed for histopathological evaluation. degenerated cells fused to produce microvesciles (Figu- Fragments from each papilloma specimen were grounded re 1B-D). Further, there was acanthosis, reduced mitotic in phosphate-buffered saline solution (PBSpH 7.2) and sus- index (1-2 per 40x Obj.), foci of apoptosis of squamous epi- pensions (10-20% w/v) were centrifuged for 15 min at thelium, and severe accumulations of irregular kera- 3,000 x g at 4 °C. Aliquots (250 mL) from supernatant were tohyalin granules within cells of the stratum granulosum. treated with lysis buffer (10 mM Tris; 1 mM EDTA; 0.5% However, characteristic basophilic intranuclear viral inclu- Nonidet P40; 1% SDS; and 0.2 mg/mL proteinase K) sion bodies were not observed. (Invitrogen, Life Technologies, Carlsbad, CA, USA). After Amplicons of the expected length, approximately homogenization, the samples were incubated at 56 °C for 480 bp, were obtained from each of the seven teat papil- 30 min. For DNA extraction, a combination of the phe- loma DNA samples, via the FAP PCR assay. Negative con- nol/chloroform/isoamyl alcohol and silica/guanidine iso- trols were not amplified. thiocyanate methods was performed as previously de- Through direct sequencing of the FAP products, scribed (Alfieri et al., 2006). DNA was eluted in 50 mLof BPV6, 7, 9, and 10 were identified in the examined papil- ultrapure sterile water and kept at -20 °C until used. loma specimens. Two cows with more than one teat papil- Aliquots of ultrapure sterile water were included as nega- lomas evaluated were coinfected by different BPV types tive controls in the DNA extraction procedures. (BPV7 and 10, and BPV6 and 9, respectively). The BPV The PCR assay was carried out by using the primer strains identified were designated as BPV6/BR-UEL, pair FAP59 (forward: 5’-TAACWGTIGGICAYCCWTA BPV7/BR-UEL, BPV9/BR-UEL, and BPV10/BR-UEL TT-3’) and FAP64 (reverse: 5’-CCWATATCWVHCAT (GenBank accession numbers: HM245430, HM245431, ITCICCATC-3’) (Forslund et al., 1999), with modifica- HM245433, and HM245432, respectively). tions (Claus et al., 2007). Aliquots from the PCR products were analyzed by electrophoresis in a 2% agarose gel in The gross characteristics of the cutaneous lesions as TBE buffer, pH 8.4 (89 mM Tris; 89 mM boric acid; 2 mM well as the PV type classification and GenBank accession EDTA) at constant voltage (90 V) for approximately numbers of the obtained sequences are indicated in Table 1. 45 min, stained with ethidium bromide (0.5 mg/mL), and vi- However, an association between the macroscopic charac- sualized under UV light. teristics and the viral type present in the cutaneous teat le- PCR bands suggestive of PV amplification were ex- sions was not observed in our analysis. cised from the agarose gel and purified by using illustra BPV types 7, 9, and 10, in addition to BPV type 6, GFX PCR DNA and Gel Band Purification kit (GE were identified as causative agents of warts in dairy cow Healthcare, Little Chalfont, UK). Direct sequencing