|Home | About | Journals | Submit | Contact Us | Français|
Papillomas occur more frequently in cattle than other domestic animals. The causal agent of bovine papillomatosis is a virus that belongs to the family Papillomaviridae. In Tamaulipas, Mexico, the virus is considered a serious problem and has impeded the export of cattle to the United States, resulting in serious economic losses. Owing to the lack of information regarding the subtypes of papillomaviruses that infect cattle in Mexico, the aim of this study was to determine the subtypes in Tamaulipas. Fifty-two warts were analyzed with the use of polymerase chain reaction (PCR) involving primers that amplify the E7 gene of bovine papillomavirus (BPV). The PCR products were sequenced to differentiate the BPV-1 and BPV-2 subtypes. The sequencing quality was determined with the use of MEGA 6.0 software. Comparison of the Tamaulipas sequences with those of known BPV types by means of the MUSCLE algorithm showed that 53% of the former were BPV-1 and 47% were BPV-2. The distribution of the 2 subtypes in the cattle was homogeneous. This study demonstrated the presence of BPV-1 and BPV-2 in cattle from Tamaulipas and constitutes the first molecular characterization of papillomas in Mexico.
Les papillomes sont rencontrés plus fréquemment chez les bovins que chez n’importe quelle autre espèce domestiques. L’agent causal de la papillomatose bovine est un virus appartenant à la famille Papillomaviridae. Dans l’état mexicain de Tamaulipas le virus est considéré comme un problème sérieux et a empêché l’exportation de bovin vers les États-Unis d’Amérique, causant ainsi des pertes économiques importantes. Étant donné le manque d’information concernant les sous-types de papillomavirus qui infectent les bovins au Mexique, l’objectif de l’étude était de déterminer les sous-types présents dans l’état de Tamaulipas. Cinquante-deux verrues ont été analysées par réaction d’amplification en chaine par la polymérase (ACP) à l’aide d’amorces amplifiant le gène E7 du papillomavirus bovin (PVB). Les produits de l’ACP ont été séquencés afin de différencier les sous-types PVB-1 et PVB-2. La qualité du séquençage fut déterminée à l’aide du logiciel MEGA 6.0. La comparaison des séquences obtenues pour l’état de Tamaulipas avec celles des types connus de PVB par l’algorithme MUSCLE a permis de démontrer que 53 % étaient des PVB-1 et 47 % de PVB-2. La distribution des deux sous-types chez les bovins était homogène. La présente étude démontre la présence de PVB-1 et PVB-2 chez les bovins de Tamaulipas et constitue le premier rapport sur la caractérisation moléculaire des papillomes au Mexique.
(Traduit par Docteur Serge Messier)
Bovine papillomavirus (BPV) is a nonenveloped, icosahedral virus with a double-stranded circular DNA genome of approximately 8000 base pairs (bp) containing 5 or 6 open reading frames (ORFs) that are expressed early during infection and 2 ORFs that are expressed late during infection. Because BPV causes tumor-like lesions in the skin and mucosa (1), it is the carcinogenic virus most used as a study model in research pertaining to cattle. These viruses are divided into 29 genera according to the L1 gene sequence (2) and 12 subtypes, which include the genera Deltapapillomavirus, Xipapillomavirus, and Epsilonpapillomavirus, the last being a nonclassified BPV-7 (2–4). This classification is based on the lesion (i.e., warts and/or tumors in the skin and mucosa) or a tumor association (5). Subtypes 1 and 2 have been more widely studied than the other types, and only a few studies have reported the prevalence of the other virus types. Most molecular epidemiologic studies have demonstrated coinfection by 2 or more BPV types in the same sample (6,7).
Papillomaviruses are classically described as epitheliotropic. The lesions are mainly mucocutaneous warts and are in large numbers as a result of highly productive infection. These warts are often visible in the skin of the scalp, tongue, teats, penis, oral cavity, and upper digestive tract (8). Furthermore, the virus has been detected in different bodily fluids, such as semen, urine, blood, and milk (8). The presence of the virus has also been reported in the bladder, placenta, and lymphocytes, which may play roles as viral reservoirs (9–11).
To date, there are no reports of a predilection of any viral subtype for animals of a specific gender or age. Therefore, it is possible to find different types of lesions in a single animal. Although it is generally thought that papillomaviruses are species-specific, BPV has recently been detected in various livestock and bison (12), water buffaloes (13), giraffes, zebras, and antelopes (14), yaks (15), horses and donkeys (16,17), tapirs (18), and other species. Papillomas can disappear or progress to malignant tumors, and their progression is associated with some specific viral subtypes as well as expression of the early (E) genes of the virus (19). Interaction of the E5, E6, and E7 oncoproteins with the host DNA is associated with the progression of a viral infection to a tumor. The subtypes BPV-1, BPV-2, and BPV-4, which express these genes, are associated with malignant tumors (20). The L1 gene target is widely used for the molecular detection of BPV (21) because it is conserved and present in all papillomaviruses. Because the E7 gene encodes an oncoprotein, this gene was used in this study as an indicator of the viral subtypes causing tumor-associated papillomas and to identify animals in which the infection might progress to a malignant neoplasm.
Bovine papillomatosis is widely distributed throughout the world. No study conducted in Mexico has indicated which papillomavirus subtypes are present in the country’s herds, even though the presence of the disease is known. Veterinarians have observed virus-associated lesions in cattle in the major agricultural regions, including Tamaulipas, which ranks third in terms of the national production of cattle, accounting for 16.38% (22). In fact, this state is one of the most important localities for cattle exports to the United States, and in recent years this virus has become an animal health problem that has caused considerable economic losses. However, there are no epidemiologic data for bovine papillomatosis in Tamaulipas, and the only information known is that the incidence of this disease has increased considerably in Tamaulipas over the last 7 y. Thus, there is a strong need to identify which BPV subtypes are circulating within this geographic area. The purpose of this study was therefore to determine the subtypes of BPV that are present in cattle in Tamaulipas and to analyze them phylogenetically.
This study constituted a descriptive and cross-sectional investigation with respect to the prevalence of BPV infection in the population or subgroups of animals sampled within the population at a given time. Nonprobabilistic convenience sampling was done (23), and the data were analyzed with use of the SPSS descriptive statistics package, version 17.0 (SPSS, Chicago, Illinois, USA).
Biopsy samples of skin warts approximately 0.5 to 1 cm in diameter were collected from different areas of the bodies of 52 female and male cattle chronically affected with cutaneous papillomatosis in 2 regions of Tamaulipas: in the north, the municipalities of Gustavo Díaz Ordaz and Reynosa; and in the southeast, Soto La Marina and Aldama (Figure 1). Some samples were fixed in formalin and embedded in paraffin by routine methods. Sections from 3 different types of lesions were stained with hematoxylin and eosin.
The study protocols were approved by the Institutional Animal Care and Use Committee of the Centro Nacional de Investigación Disciplinaria en Microbiología Animal (approval 002-2014). All efforts were made to minimize animal suffering and distress according to the guidelines of Mexican Regulation NOM-033-ZOO-1995 for the euthanasia of domestic and wild animals.
A mechanical homogenizer (Qiagen, Hilden, Germany) was used to extract DNA, which was purified with the QIAamp DNA Mini Kit (Qiagen) extraction kit according to the manufacturer’s recommended protocol with some modifications. Briefly, 100 mg of each wart sample in 200 μL of lysis buffer ATL and 20 μL of proteinase K was homogenized by vortexing. The mixture was incubated at 56°C for approximately 2 h. After the samples were centrifuged, 200 μL of buffer AL was added, and the samples were incubated at 70°C for 10 min. Ethanol (200 μL) was added, and the mixture was placed in a mini QIAamp spin column and centrifuged at 6000 × g for 1 min. The column was washed with buffer AW1 and centrifuged as described previously. Finally, the DNA was eluted with 200 μL of elution buffer AE. The quantity and quality of the obtained DNA were determined spectrophotometrically. The DNA was then stored at −20°C until used.
The E7 BPV gene (383 bp) was cloned as a positive control for polymerase chain reaction (PCR). For detection of this gene the following primers were designed in house by amplifying a 203-bp fragment of the gene: E7BPV+, TAG GAA GCG AGG CWC AKA TA; and E7BPV−, CYC GMG GAC AAC ACA GG. The FastStart PCR High-Fidelity PCR System (Roche, Mannheim, Germany) was used with the following: 5 μL of 10× PCR buffer with magnesium, 10 mM of each primer, 2.5 U of High-Fidelity Enzyme Blend, 200 μM of deoxynucleotide triphosphates, 2 μL of DNA, and water of molecular-biology grade. The samples were amplified in an iCycler (BioRad, Hercules, California, USA) with the following thermocycling protocol: 95°C for 5 min; 35 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 30 s; and 72°C for 3 min. The amplification products were purified on 1.5% agarose gels with use of the QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer’s instructions. The purified products were quantified and visualized on a 1.5% agarose gel to determine their quality and the purified DNA was then sent for sequencing to the Instituto de Biotecnología, Universidad Nacional Autónoma de México and the quality of sequences was evaluated with MEGA 6.0 software (24).
Comparison and phylogenetic analyses were done with a 203-bp fragment of the E7 gene. The nucleotide sequences of all BPV subtypes of different species and geographic origins obtained from GenBank (National Center for Biotechnology Information, Bethesda, Maryland, USA) were included in an alignment done with the algorithm MUSCLE (multiple-sequence comparison by log expectation), which involved calculating the distance between pairs of nucleotides (25). From the nucleotide sequences in this dataset, a phylogenetic tree was constructed by means of the maximum likelihood method with the JC + G + I model. A consensus tree was obtained from 1000 bootstrap replicates.
The case data were analyzed by nonparametric tests, with frequencies used to determine the prevalence per herd and municipality, and a Chi-square test was conducted to determine statistical associations.
The prevalence of bovine papillomatosis in the cattle of Tamaulipas (Table I) ranged from 2% to only 5% in the northeastern region, averaging 3% in Reynosa and 5% in Díaz Ordaz, whereas it ranged from 2% to 70% in the southeastern region, averaging 9% in Soto la Marina and 14% in Aldama. The virus had no predilection for the gender or age of the animals, and the BPV-1 and BPV-2 subtypes were distributed homogeneously in the studied herds (Table II).
The animals presented a high degree of infection, showing several warts over the entire body. A cauliflower-like lesion was the most frequently found on different parts of the head, neck, flank, and foot. Histologic study of the cauliflower-like and flat warts showed alterations compatible with papillomavirus infection, such as hyperplasia of the epidermis, hyperkeratosis, and acanthosis (Figure 2).
The primers designed in-house to amplify the E7 gene were able to detect BPV subtypes 1 and 2 in the samples from 19 (36%) of the 52 animals: BPV-1 was detected in samples from 10 (53%) of the 19 and BPV-2 in 9 (47%). Hyperplasia was present primarily in the animals infected with the BPV-2 subtype. The dendrogram obtained (Figure 3) indicated 2 different clades, corresponding to subtypes 1 and 2. It is noteworthy that the Mexican sequences classified as subtype 1, which originated from the samples collected in Soto La Marina and Aldama, were grouped in a clade different from that containing the sequences classified as subtype 1 that were published in GenBank, which originated in Japan, the United States, Iraq, and Austria. No differences were found in the geographic distribution of the sequences grouped as subtype 2, which fell into 3 different subclades; these sequences corresponded to those of the samples obtained from Aldama, Soto La Marina, and Reynosa. The presence of both viral subtypes on the same farm was also demonstrated. The distance matrix showed little difference among the Mexican sequences but significant differences between the sequences of subtypes 1 and 2.
Before this study, there was no published information regarding the subtypes of BPV in any state of Mexico, despite the economic importance of bovine papillomatosis. We used PCR to assess the presence of BPV-1 and BPV-2 in samples of hyperplastic and macroscopic warts collected from chronically infected adult cattle in different herds in Tamaulipas, Mexico.
Of the 52 cattle evaluated by wart sampling, 19 (36%) were positive for 1 of 2 subtypes of BPV. Campo et al (26) detected BPV-2 DNA in natural tumors of immunosuppressed animals and demonstrated an association between BPV-2 and bovine bladder tumors. Borzacchiello et al (27) also demonstrated an association between BPV-2 and bovine bladder tumors and detected BPV-2 in samples with and without neoplastic change. Histopathological analysis of lesions is important for identifying intraepithelial tumors associated with oncogenic viruses such as BPV (28), and this analysis shows whether the virus has a predilection for anatomic areas related to a specific viral type. The histopathological findings reported in the literature for BPV include acanthosis, hyperkeratosis, parakeratosis, papillomatosis, and koilocytosis (29). We observed similar features in this study (Figure 2) and found that hyperplasia was present primarily in the animals infected with the BPV-2 subtype.
Both BPV-1 and BPV-2 have been reported to be associated with skin warts and fibropapillomas in cattle and sarcoids in horses. Owing to the presence of oncoproteins, BPV is genetically unstable. The E5, E6, and E7 genes, which are related to the transformation of cells, are found in BPV-1. The function of each viral oncogene depends on the viral subtype. The E5 gene is highly conserved among the group of BPVs, is the main transforming gene (26), and induces cell transformation, proliferation, and survival (30). Not all oncogenes are present in all BPV subtypes, some subtypes being deficient in E6, regardless of how the infection occurs (31). In contrast, E7 is a zinc-binding protein, and E5 and E7 coexpression increases the transforming capacity of BPV (32). The oncogene E7 leads to cellular transformation, degradation of the pRb tumor suppressor, deregulation of the cell cycle, and tumorigenesis (29,33–35). We were interested to determine the presence of the E7 gene in the sampled warts because of this gene’s association with cellular transformation. The E7 gene is important in oncogenesis, and independent of deletions or mutations its expression is related in some degree to tumor formation. Therefore, we selected this gene as the target in our molecular characterization study. It is more conserved and likely cannot be used to discriminate between some subtypes, whereas the E6 gene is not present in all subtypes.
The phylogenetic tree showed that the sequences of the detected BPV subtypes 1 and 2 were closest to those of North American strain type 1. Thus, these strains likely circulate between Mexico and the United States because of trade relations and may even infect wildlife species that can move freely across the border and have not been studied. The presence of both subtypes in different regions of Tamaulipas could indicate that the animals had been moved indiscriminately across the state. With respect to BPV-2, the tree showed that isolates from China and Mexico have a common ancestor (accession no. M02219), but its origin is not specified in GenBank. This finding was corroborated by the distance matrix, which revealed a value less than 0.02 for the aforementioned isolate.
The main finding of this study is identification of the BPV subtypes in beef cattle with papillomatosis in Tamaulipas, Mexico. This identification, the first molecular characterization of BPV-1 and 2 in papillomas done in Mexico, is of epidemiologic importance because of the economic value related to the export of cattle from Tamaulipas to the United States: in 2014 alone, 138 094 cattle were exported (33). Biosecurity measures should be implemented in herd management to prevent or reduce the overall transmission of these subtypes among the animals.
This research was supported by the Fundación Produce-Tamaulipas Project (grant 28-20120050) and Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias (grant SIGI-94039336749).