Searching bovine papillomavirus presence in lesions seen on teats of cows

The samples were taken from 106 cows with various-looking lesions on their teats and ranged in age from 2 to 8 years. Enzyme-linked immunosorbent assay (ELISA) antigen (Ag) positive for the bovine papillomavirus (BPV) was found in 59 (55.7%) blood serum samples. PCR using FAP59/64 primers was positive for 24 (22.6%) samples. BPV-2 (40, 37.7%), BPV-6 (28, 26.4%), BPV-8 (30, 28.3%), BPV-9 (36, 34%), BPV-10 (32, 30.3%), and BPV-12 (22, 20.8%) were found in a PCR type-specific analysis of single and mixed type teat warts. The highest positivity was observed in BPV-2, BPV-9 and BPV-10 in flat and round forms, BPV-6, BPV-10, BPV-12, and mixed types in rice grain-cauliflower forms, BPV-9 and mixed types in filiform in the distribution of types based on the macroscopic appearance of teat lesions. As for the distribution of BPV types according to age, the most BPV-2 types were found in the age group of two years, the most BPV-10 types in the age group of three years, the most BPV-9 types in the age group of four years, the most BPV-8+BPV-12 types in the age group of five years, and the most mixed types between the ages of six and eight years. The existence of the virus was then checked using electron microscopy on the chosen samples (at least one investigation was conducted), and it was positively identified using BPV type-specific primers. The authors concluded that BPV detection using an ELISA (Ag) test from blood serum samples was shown to be less sensitive than BPV type-specific PCR from wart samples.

Equine papillomavirus detection in aural plaques by qPCR

Papillomatosis occupy a prominent position both in human and veterinary medicine, since it is a viral skin disease with potential to develop malignancy. Equus caballus papillomavirus (EcPV) are associated with several diseases in horses, i.e. classical papillomatosis associated with EcPV 1; squamous cell carcinoma associated with EcPV 2; tumors in mucous membranes on the genital area (EcPV 2 and EcPV 7); aural plaque associated with EcPV 1, 3, 4, 5 and 6; and equine sarcoid, associated with bovine papillomavirus (BPV 1 and 2). The aural plaque is characterized by small papules (1-2 cm), hypochromic and keratinized on the internal surface of the pinnae and can evolve and coalesce into larger lesions. To obtain a specific diagnostic test, both sensitive and fast to identify these viruses, a quantitative real time PCR (qPCR) was standardized for EcPV 3, 4, 5 and 6. Applying the qPCR technique in the 103 equine aural plaque samples resulted in 90.29% of at least one viral type prevalence, which was distributed as following: EcPV3, 36.89%; EcPV4, 82.52%; EcPV5, 0.97%; and EcPV6, 10.68%. This study represents an evolution in the area related to aural plaque and equine papillomatosis and raises new questions for future research.

Equine papillomavirus detection in aural plaques by qPCR

Papillomatosis occupy a prominent position both in human and veterinary medicine, since it is a viral skin disease with potential to develop malignancy. Equus caballus papillomavirus (EcPV) are associated with several diseases in horses, i.e. classical papillomatosis associated with EcPV 1; squamous cell carcinoma associated with EcPV 2; tumors in mucous membranes on the genital area (EcPV 2 and EcPV 7); aural plaque associated with EcPV 1, 3, 4, 5 and 6; and equine sarcoid, associated with bovine papillomavirus (BPV 1 and 2). The aural plaque is characterized by small papules (1-2 cm), hypochromic and keratinized on the internal surface of the pinnae and can evolve and coalesce into larger lesions. To obtain a specific diagnostic test, both sensitive and fast to identify these viruses, a quantitative real time PCR (qPCR) was standardized for EcPV 3, 4, 5 and 6. Applying the qPCR technique in the 103 equine aural plaque samples resulted in 90.29% of at least one viral type prevalence, which was distributed as following: EcPV3, 36.89%; EcPV4, 82.52%; EcPV5, 0.97%; and EcPV6, 10.68%. This study represents an evolution in the area related to aural plaque and equine papillomatosis and raises new questions for future research.(AU)

Sarcoid associated with infection by Habronema spp. in equines in Brazil / Sarcoide associado à infecção por Habronema spp. em equinos no Brasil

Background: Equine sarcoid, supposed to be caused by infection with bovine papillomavirus type 1 or 2, is usually observed in previously traumatized skin areas, including lesions of habronemosis due to inoculation of third stage larvae in ulcerated wounds by Musca domestica or Stomoxys calcitrans. Little is known about the occurrence of diseases associated with equine sarcoid, mainly because limitations on clinical diagnosis, due to the different skin diseases that have to be considered as differential diagnoses. This report aimed to describe three cases of equine sarcoid associated with habronemosis in horses in the semiarid region of Northeastern Brazil. Cases: Three cases of sarcoid associated with habronemosis in equines were diagnosed at the Animal Pathology Laboratory of the Veterinary Hospital of the Federal University of Campina Grande, Patos, Paraíba. Case 1. A 5-year-old female showed in the ventral branch of the mandible a nodule of 3 cm in diameter, partially covered with skin and hair intercepted by areas of irregular surface with yellow-red ulcerations. The cut surface was formed by whitish and firm tissue. Case 2. It was a biopsy from a 4-year-old mare, who was not informed of the macroscopic characteristics of the lesion. Case 3. A 5-year-old horse presenting a nodular mass in the region of the tarsal-metatarsal joint, measuring 8.0x5.0x3.0 cm with an irregular [...]

Sarcoid associated with infection by Habronema spp. in equines in Brazil / Sarcoide associado à infecção por Habronema spp. em equinos no Brasil

Background: Equine sarcoid, supposed to be caused by infection with bovine papillomavirus type 1 or 2, is usually observed in previously traumatized skin areas, including lesions of habronemosis due to inoculation of third stage larvae in ulcerated wounds by Musca domestica or Stomoxys calcitrans. Little is known about the occurrence of diseases associated with equine sarcoid, mainly because limitations on clinical diagnosis, due to the different skin diseases that have to be considered as differential diagnoses. This report aimed to describe three cases of equine sarcoid associated with habronemosis in horses in the semiarid region of Northeastern Brazil. Cases: Three cases of sarcoid associated with habronemosis in equines were diagnosed at the Animal Pathology Laboratory of the Veterinary Hospital of the Federal University of Campina Grande, Patos, Paraíba. Case 1. A 5-year-old female showed in the ventral branch of the mandible a nodule of 3 cm in diameter, partially covered with skin and hair intercepted by areas of irregular surface with yellow-red ulcerations. The cut surface was formed by whitish and firm tissue. Case 2. It was a biopsy from a 4-year-old mare, who was not informed of the macroscopic characteristics of the lesion. Case 3. A 5-year-old horse presenting a nodular mass in the region of the tarsal-metatarsal joint, measuring 8.0x5.0x3.0 cm with an irregular [...](AU)

Caracterização molecular de DNA de Delta papillomavirus bovino (BPV1, 2 e 13) em sarcoides equinos / Molecular characterization of bovine Deltapapillomavirus (BPV1, 2, and 13) DNA in equine sarcoids

Sarcoides são tumores fibroblásticos, considerados os tumores de pele mais comuns em pele de equinos e que raramente apresentam regressão espontânea. Papilomavírus bovino (BPV) tipos 1 e 2 são relacionados com a patogenia do sarcoide e, provavelmente, o BPV tipo 13 (BPV13), recentemente descrito, também pode estar associado com a formação dessa lesão. Neste estudo, 20 amostras de lesões cutâneas, sendo 12 constituídas por tecidos frescos e 8 amostras de tecido fixado em formalina e embebido em parafina, provenientes de 15 cavalos foram utilizadas para a identificação do DNA de BPV. A análise histopatológica (HE) confirmou todas as lesões como sarcoide. Para a amplificação do DNA de papilomavírus (PV) foram realizadas três reações de PCR. Como triagem, os primers IFNR2/IDNT2 foram utilizados para amplificar um fragmento da ORF L1 do PV. O segundo par de primersutilizado é complementar a sequência dos genes E5 e L2 de BPVs 1, 2 e 13. O terceiro par de primers(FAP59/FAP64) utilizado tem o gene L1 como alvo. A primeira e a segunda PCRs permitiram amplificar produtos em todas as amostras avaliadas. Entretanto, na terceira reação, na qual foram utilizados os primers FAP, foi possível amplificar produtos com tamanho molecular esperado somente nas amostras constituídas por tecidos frescos. O sequenciamento de nucleotídeos e as análises filogenéticas realizadas nos fragmentos E5L2 resultaram na identificação de BPV1, 2 e 13 em 14 (70%), 2 (10%) e em 4 (20%) amostras de sarcoides, respectivamente. As amostras de sarcoides de um dos animais continha somente o DNA de BPV1. Entretanto, nas amostras provenientes do segundo cavalo foi possível identificar o DNA de três tipos de Deltapapillomavirus bovino (BPV1, 2 e 13) em lesões distintas. Este estudo ratifica a presença do DNA de BPV1, 2 e 13 em lesões de sarcoides em equinos, além de identificar três tipos de BPVs em um mesmo animal e descrever pela primeira vez no Brasil a presença de BPV1 e 2 nesse tipo de lesão.(AU)

Sarcoids are fibroblastic lesions, which are considered as the most common skin tumors of horses; spontaneous regression rarely occurs. The bovine papillomavirus (BPV) types 1 and 2 may be involved in the pathogenesis of sarcoids, and probably the recently described BPV type (BPV13) might be associated with the pathogenesis of this lesion. This study characterized the DNA of BPVs in sarcoids from 15 horses from Brazil by analyzing 20 cutaneous lesions (12 recently collected; 8 from formalin-fixed paraffin-embedded (FFPE) tissues). Histopathology confirmed the proliferative lesions as sarcoids. Three PCRs were performed to amplify papillomavirus (PV) DNA. For screening, the primers IFNR2/IDNT2 were used to amplify a fragment of the PV L1 ORF. The second primer set was complementary to a common sequence of the E5L2 genomic region of BPV1, 2, and 13. The third primer pair (FAP59/FAP64) targeted a fragment of the PVs L1 ORF. The screening and E5L2 PCRs yielded amplicons in all samples evaluated. The FAP amplicons identified BPV1, 2, and 13 only from fresh tissue samples. The phylogenetic analyses of E5L2 resulted in the identification of BPV1, 2, and 13 in 14 (70%), 2 (10%), and 4 (20%) sarcoids, respectively. Two horses demonstrated multiple lesions: the sarcoids of one of these contained only BPV1 DNA and those of the other contained three types of bovine Deltapapillomavirus (BPV1, 2, and 13). This study confirmed the presence of BPV1, 2, and 13 DNA in equine sarcoids. Moreover, these findings represent the first description of three types of BPV diagnosed in the same horse, as well as the first confirmation of BPV1 and 2 in horses from Brazil.(AU)

PCR Amplification of DNA Sequence of Bovine Papillomavirus Type 2 in Urinary Bladder of Cattle with Enzootic Hematuria in Espirito Santo, Brazil

The goal of this study was to investigate the presence of bovine papillomavirus type 2 (BPV-2) genomic DNA using polymerase chain reaction (PCR) assays, in the urinary bladder of cattle with bovine enzootic hematuria (BEH), in the neoplastic and non-neoplastic lesions in Espírito Santo, Brazil. Thus, 46 bladders with gross BEH lesions were collected from adult cattle at slaughterhouses. The samples were divided into two portions: one for use in histopathological analysis and the other one for PCR. Microscopic analysis showed that 56.52% of the bladders presented neoplastic lesions, of which 50% were benign, 23.08% were malignant and 26.92% presented both types of neoplasms. The remaining 43.48% of the bladders did not show any neoplasms. Presence of BPV-2 was confirmed in 43.48% of the bladders: 65% of these were found in neoplastic lesions and 35% in non-neoplastic lesions. Among the neoplastic lesions, detection of BPV-2 was more frequent in benign lesions (53.84%), followed by malignant lesions (23.08%) and by those presenting both benign and malignant lesions (23.08%). These results confirm the presence of BPV-2 in both neoplastic and non-neoplastic conditions in urinary bladders of cattle with enzootic hematuria from the studied geographic area.

PCR Amplification of DNA Sequence of Bovine Papillomavirus Type 2 in Urinary Bladder of Cattle with Enzootic Hematuria in Espirito Santo, Brazil

The goal of this study was to investigate the presence of bovine papillomavirus type 2 (BPV-2) genomic DNA using polymerase chain reaction (PCR) assays, in the urinary bladder of cattle with bovine enzootic hematuria (BEH), in the neoplastic and non-neoplastic lesions in Espírito Santo, Brazil. Thus, 46 bladders with gross BEH lesions were collected from adult cattle at slaughterhouses. The samples were divided into two portions: one for use in histopathological analysis and the other one for PCR. Microscopic analysis showed that 56.52% of the bladders presented neoplastic lesions, of which 50% were benign, 23.08% were malignant and 26.92% presented both types of neoplasms. The remaining 43.48% of the bladders did not show any neoplasms. Presence of BPV-2 was confirmed in 43.48% of the bladders: 65% of these were found in neoplastic lesions and 35% in non-neoplastic lesions. Among the neoplastic lesions, detection of BPV-2 was more frequent in benign lesions (53.84%), followed by malignant lesions (23.08%) and by those presenting both benign and malignant lesions (23.08%). These results confirm the presence of BPV-2 in both neoplastic and non-neoplastic conditions in urinary bladders of cattle with enzootic hematuria from the studied geographic area.(AU)

Carcinomas de células escamosas do trato alimentar superior de bovinos associados à intoxicação crônica por samambaia (Pteridium arachnoideum)

Os neoplasmas do trato alimentar superior (TAS) são raros em bovinos, porém em algumas regiões, como no Sul do Brasil, os carcinomas de células escamosas (CCEs) do TAS são relativamente comuns e estão associados à ocorrência concomitante de papilomas e ao consumo de samambaia. Embora haja uma teoria de patogênese que considera o papilomavírus bovino tipo 4 (BPV-4) como um co-fator no desenvolvimento desses CCEs, ainda há aspectos da etiopatogênese dessa condição a serem investigados, pois a detecção do BPV-4 em outras regiões geográficas, além das poucas previamente publicadas, é escassa. Além disso, no Brasil, os CCEs esofágicos são observados com uma apresentação macroscópica incomum, como um espessamento anelar estenosante da parede esofágica, que é diferente das formas comuns de apresentação deste tumor que são massas exofíticas ou endofíticas ulceradas ou infiltrativas. Portanto, os objetivos desta tese foram (1) analisar os aspectos epidemiológicos, clínicos e patológicos de 100 casos de CCEs do TAS em bovinos que pastejavam em áreas contaminadas por samambaia (Pteridium arachnoideum), bem como investigar as associações entre esses parâmetros; (2) investigar a presença de papilomavírus nos papilomas do TAS por reação em cadeia da polimerase (PCR) e por imuno-histoquímica; (3) e detalhar os aspectos clínicos e patológicos de 13 casos de CCEs esofágicos anelares estenosantes. No primeiro estudo, sobre os 100 casos de CCEs no TAS, associações estatisticamente significativas entre os sinais clínicos e a localização do tumor no TAS, entre o grau de diferenciação histológica e a localização do tumor e uma tendência a associação significativa entre o grau de diferenciação histológica e a presença de metástases foram observadas. A média de idade dos bovinos com CCEs orofaríngeos foi 7,39 anos, com diferença significativa quando comparado com bovinos com neoplasmas esofágicos (8,6 anos). DNA ou antígeno de papilomavírus não foram detectados nos papilomas por PCR e imuno-histoquímica, respectivamente. Desta forma, os resultados mantem aberta a possibilidade de que papilomas de TAS podem não estar associados ao papilomavírus. Assim, a infecção por BPV-4 pode não ser um co-fator no desenvolvimento dos CCEs em bovinos da região estudada. No segundo estudo, sobre os CCEs esofágicos anelares estenosantes, massas endofíticas, circunferenciais (anelares) no esôfago, com estreitamento luminal (estenose) foram observadas. Os ceratinócitos neoplásicos eram circundados por tecido conjuntivo fibroso moderado a abundante (reação desmoplástica). A coloração de Picrosírius sob luz polarizada demonstrou fibras de colágeno tipo I abundantes, que contribuiram para as características estenosantes deste tumor. Esses CCEs esofágicos devem ser incluídos no diagnóstico diferencial de estenose esofágica.

Upper digestive tract (UDT) neoplasms are rare in cattle, but in some regions, such as Southern Brazil, UDT squamous cell carcinomas (SCCs) are relatively common and have been associated with the concomitant occurrence of papillomas and the consumption of bracken fern. Although there is a theory of pathogenesis that considers bovine papillomavirus type 4 (BPV-4) as a co-factor in the development of these SCCs, some aspects of the etiopathogenesis of this condition need to be investigated, since the detection of BPV-4 in other geographic regions, in addition to the few ones previously published, is scarce. Additionally, esophageal SCCs in Brazil are observed with an uncommon gross presentation, such as an annular stenotic thickening of the esophageal wall, which is different from the usual forms of presentation of this tumor, which are exophytic or ulcerative or infiltrative endophytic neoplasms. Therefore, the aims of this thesis were (1) to analyze the epidemiological, clinical and pathological aspects of 100 cases of SCCs in the UDT of cattle grazing on bracken fern (Pteridium arachnoideum) highly contaminated areas, as well as to investigate associations between these parameters; (2) to investigate the presence of papillomavirus in the UDT papillomas by polymerase chain reaction (PCR) and by immunohistochemistry; (3) and to elucidate the clinical and pathological aspects of annular stenotic esophageal SCCs. In the first paper, about 100 cases of SCCs of the UDT, there were statistically significant associations between clinical signs and tumor localization in the UDT, between histological grade of differentiation and tumor localization, and a trend towards significant association between histological grade of differentiation and presence of metastases. The average age of cattle with oropharyngeal SCCs was 7.39 years old, with significant difference compared to cattle with esophageal SCCs (8.6 years). No papillomaviral DNA or antigen were detected in papillomas by PCR and immunohistochemistry, respectively. Therefore, these results keep open the possibility that papillomas of the UDT may not be associated with papillomavirus. Thus, the BPV-4 infection may not be a co-factor to the development the SCCs in cattle from the region studied. In the second paper, about annular stenotic esophageal SCCs, endophytic masses, circumferential (annular) within the esophageal with luminal narrowing (stenosis) were observed. The neoplastic keratinocytes were surrounded by moderate to abundant fibrous connective tissue (desmoplastic reaction). Picrosirius red-stained sections under polarized light showed abundant collagen type I fibers, which contributed to the stenosing characteristics of this tumor. These esophageal SCCs should be included in the differential diagnosis of esophageal stenosis.

Penectomia parcial em equino com sarcoide de glande / Partial equine penectomy withglans sarcoid / Penectomía parcial en equino con sarcoide de glande

O sarcoide é a neoplasia cutânea mais frequente em equinos, independentemente da raça e coloração da pelagem. Há evidências consideráveis que o fator mais importante da oncogênese do sarcoide equino é a infecção pelo papilomavírus bovino (BPV) tipo 1 e 2, sendo o contato direto de equinos com bovinos a rota mais comum de transmissão. É uma neoplasia localmente invasiva e não metastática, clinicamente parecida com outras doenças, como carcinoma de células escamosas e habronemose, sendo o diagnóstico definitivo realizado através da biópsia e exame histopatológico. O prognóstico depende do lugar e do tipo de tratamentoe, em muitos casos, a excisão cirúrgica é a única opção viável para o tratamento do sarcoide. Um equino, macho, não castrado, da raça Crioula, de cinco anos de idade e 410 kg de peso, foi atendido no Setor de Grandes Animais do Hospital de Clínicas Veterinárias da UFRGS, apresentando aumento de volume na região da glande, com aspecto nodular e presença de miíase. O diagnóstico do sarcoide foi realizado através do exame histopatológico e o animal foi submetido a cirurgia de penectomia parcial, onde a neoplasia pôde ser retirada completamente. Descreveu-se a técnica cirúrgica utilizada e acompanhou-se o pós-cirúrgico do paciente, o qual não apresentou complicações e pôde retornar à suas atividades em um curto período de tempo.

The sarcoid is the most common cutaneous neoplasm in horses, no matter the breed and coat coloring. There are considerable evidence that the most important facto r in equine sarcoid oncogenesis is the bovine papillomavirus infection (BPI). type 1 and 2, in which the direct contact of horses with cattle the most common route of transmission. It is a locally invasive and non-metastatic neoplasm, clinically similar with other diseases, such as squamous cell carcinoma and habronema. with the definitive diagnosis being made through the biopsy and histopathological examination. The prognosis depends on the place and the kind of treatment and, in many cases, a surgical excision is the only viable option for the treatment of sarcoid. A horse, male, non-castrated, Criollo breed, five years old and weighting 410 kg, was attended at the Large Animais Sector of the Hospital of Veterinary Clinics of UFRGS, presenting an increase in volume in the glans region, with nodular appearance and presence of myiasis. The diagnosis of the sarcoid was made through histopathological examination, and the animal underwent partial penectomy surgery, where the neoplasm could be completely removed. The surgical technique was described and the patient's postoperative period was followed, which presented no complications and was able to return to its activities in a short period of time.

El sarcoide es la neoplasia cutánea más frecuente en los equinos, independientemente de laraza y coloración del pelaje. Hay evidencias considerables que el factor más importante de la oncogénesis del sarcoide equino es la infección por el papilomavirus bovino (BPV) del tipo 1 y el tipo 2, siendo el contacto directo de equinos con bovinos la ruta más común de transmisión. Es una neoplasia localmente invasiva y no metastásica, clínicamente parecida a otras enfermedades, como carcinoma de células escamosas y habronemose, siendo el diagnóstico definitivo realizado a través de la biopsia y el examen histopatológico. El pronóstico depende del lugar y del tipo de tratamiento y, en muchos casos, la excisión quirúrgica es la única opción viable para el tratamiento del sarcoide. Un equino, macho, no castrado, de la raza Criolla, de cinco anos de edad y 41 O kg de peso, fue atendido en el Sector de Grandes Animales del Hospital de Clínicas Veterinarias de la UFRGS, presentando un aumento de volumen em la región dei glande, con aspecto nodular y presencia de miasis. El diagnóstico del sarcoide fue realizado a través del examen histopatológico y el animal fue sometido a cirugía de penectomía parcial, donde la neoplasia pudo ser retirada completamente. Se describió la técnica quirúrgica utilizada y se acompanó al postquirúrgico del paciente, el cual no presentó complicaciones y pudo regresar a sus actividades en un corto período de tiempo.

Penectomia parcial em equino com sarcoide de glande / Partial equine penectomy withglans sarcoid / Penectomía parcial en equino con sarcoide de glande

O sarcoide é a neoplasia cutânea mais frequente em equinos, independentemente da raça e coloração da pelagem. Há evidências consideráveis que o fator mais importante da oncogênese do sarcoide equino é a infecção pelo papilomavírus bovino (BPV) tipo 1 e 2, sendo o contato direto de equinos com bovinos a rota mais comum de transmissão. É uma neoplasia localmente invasiva e não metastática, clinicamente parecida com outras doenças, como carcinoma de células escamosas e habronemose, sendo o diagnóstico definitivo realizado através da biópsia e exame histopatológico. O prognóstico depende do lugar e do tipo de tratamentoe, em muitos casos, a excisão cirúrgica é a única opção viável para o tratamento do sarcoide. Um equino, macho, não castrado, da raça Crioula, de cinco anos de idade e 410 kg de peso, foi atendido no Setor de Grandes Animais do Hospital de Clínicas Veterinárias da UFRGS, apresentando aumento de volume na região da glande, com aspecto nodular e presença de miíase. O diagnóstico do sarcoide foi realizado através do exame histopatológico e o animal foi submetido a cirurgia de penectomia parcial, onde a neoplasia pôde ser retirada completamente. Descreveu-se a técnica cirúrgica utilizada e acompanhou-se o pós-cirúrgico do paciente, o qual não apresentou complicações e pôde retornar à suas atividades em um curto período de tempo.(AU)

The sarcoid is the most common cutaneous neoplasm in horses, no matter the breed and coat coloring. There are considerable evidence that the most important facto r in equine sarcoid oncogenesis is the bovine papillomavirus infection (BPI). type 1 and 2, in which the direct contact of horses with cattle the most common route of transmission. It is a locally invasive and non-metastatic neoplasm, clinically similar with other diseases, such as squamous cell carcinoma and habronema. with the definitive diagnosis being made through the biopsy and histopathological examination. The prognosis depends on the place and the kind of treatment and, in many cases, a surgical excision is the only viable option for the treatment of sarcoid. A horse, male, non-castrated, Criollo breed, five years old and weighting 410 kg, was attended at the Large Animais Sector of the Hospital of Veterinary Clinics of UFRGS, presenting an increase in volume in the glans region, with nodular appearance and presence of myiasis. The diagnosis of the sarcoid was made through histopathological examination, and the animal underwent partial penectomy surgery, where the neoplasm could be completely removed. The surgical technique was described and the patient's postoperative period was followed, which presented no complications and was able to return to its activities in a short period of time.(AU)

El sarcoide es la neoplasia cutánea más frecuente en los equinos, independientemente de laraza y coloración del pelaje. Hay evidencias considerables que el factor más importante de la oncogénesis del sarcoide equino es la infección por el papilomavirus bovino (BPV) del tipo 1 y el tipo 2, siendo el contacto directo de equinos con bovinos la ruta más común de transmisión. Es una neoplasia localmente invasiva y no metastásica, clínicamente parecida a otras enfermedades, como carcinoma de células escamosas y habronemose, siendo el diagnóstico definitivo realizado a través de la biopsia y el examen histopatológico. El pronóstico depende del lugar y del tipo de tratamiento y, en muchos casos, la excisión quirúrgica es la única opción viable para el tratamiento del sarcoide. Un equino, macho, no castrado, de la raza Criolla, de cinco anos de edad y 41 O kg de peso, fue atendido en el Sector de Grandes Animales del Hospital de Clínicas Veterinarias de la UFRGS, presentando un aumento de volumen em la región dei glande, con aspecto nodular y presencia de miasis. El diagnóstico del sarcoide fue realizado a través del examen histopatológico y el animal fue sometido a cirugía de penectomía parcial, donde la neoplasia pudo ser retirada completamente. Se describió la técnica quirúrgica utilizada y se acompanó al postquirúrgico del paciente, el cual no presentó complicaciones y pudo regresar a sus actividades en un corto período de tiempo.(AU)

Análise genética de papilomavírus bovino da região Norte do Brasil

Os papilomavírus (PV) são vírus epiteliotrópicos e constituem um grande grupo geneticamente diverso da família Papillomaviridae que infecta diferentes espécies de mamíferos, répteis e aves. Estes vírus caracterizam-se por sua propriedade oncogênica, sendo responsáveis pela indução de tumores benignos e malignos nos epitélios cutâneo e mucoso de suas espécies hospedeiras. Nos bovinos, a papilomatose causa importantes prejuízos econômicos à pecuária de corte e leiteira, dentre as quais destacam-se as infecções secundárias, a dificuldade de amamentação do bezerro, a depreciação do couro e o descarte precoce de animais e, ainda, no caso de rebanhos de aptidão leiteira, a papilomatose mamária ocasiona dificuldade de ordenha, retenção de leite e predispõe à mastite. Independente do nível de tecnificação da exploração na pecuária, estes agentes podem ser encontrados em quase todos os países. No entanto, poucos tipos de papilomavírus bovinos (BPV) foram caracterizados até o presente momento. Desta forma, este trabalho teve como objetivo realizar um estudo de caracterização genética e patológica das infecções causadas pelo BPV na região norte do Brasil. No Capítulo 1, buscou-se avaliar a diversidade dos BPVs em lesões cutâneas de bovinos na região Amazônica brasileira utilizando o par de primers degenerados FAP (FAP59 e FAP64). Foram encontrados os BPVs tipo 1, 2, 11, e 13 além de quatro prováveis novos tipos virais que apresentaram baixa identidade com os BPVs 7, 8, 11 e 12. No Capítulo 2, o genoma de um novo tipo de papilomavírus bovino pertencente ao gênero Xipapillomavirus, espécie Xipapillomavirus 1, foi caracterizado aplicando a técnica de amplificação por círculo rolante seguida de sequenciamento de alta eficiência utilizando a plataforma Illumina. Este novo tipo apresentou maior similaridade com o BPV3 (79% de identidade a nível genômico) e preenche os requisitos para ser considerado um novo tipo de BPV. Histopatologicamente, a lesão causada por esse novo BPV foi caracterizada como papiloma exofítico, apresentando células bem diferenciadas, marcada acantose e paraqueratose. No Capítulo 3, foi realizado o sequenciamento de um genoma completo de BPV5 utilizando o mesmo protocolo do Capítulo 2. Este foi o primeiro genoma de BPV5 da América do Sul completamente sequenciado e caracterizado. Assim, os resultados obtidos nesta tese contribuem para o melhor conhecimento da variabilidade genética do BPV e mostra a importância da utilização de ferramentas moleculares na sua caracterização, principalmente em regiões carentes de estudos e que podem abrigar tipos desconhecidos.

Papillomaviruses (PV) are epitheliotropic viruses that constitute a large genetically diverse group of the Papillomaviridae family which can infect different species of mammals, reptiles and birds. These viruses are characterized by their oncogenic properties and are responsible for the induction of benign and malignant tumors in cutaneous and mucosal epithelia. In cattle, papillomatosis causes significant economic losses to beef and dairy cattle characterized by secondary infections, calf breastfeeding difficulties, leather depreciation, early animal discarding, mammary papillomatosis, retention of milk and predisposes to mastitis. Regardless of the level of technification of livestock farming, these agents can be found in almost all countries. However, few types of bovine papillomavirus (BPV) have been characterized to date. Thereby, this work had as objective to carry out a study of genetic and pathological characterization of the infections caused by the BPV from Northern Brazil. In Chapter 1, we evaluated the genetic diversity of BPVs in bovine cutaneous lesions from Brazilian Amazon using the degenerate FAP pairs of primers (FAP59 and FAP64). BPV1, 2, 11, and 13 were found in addition to four probable new viral types that had low identity with BPVs 7, 8, 11 and 12. In Chapter 2, the whole genome of a new BPV type belonging to genus Xipapillomavirus, species Xipapillomavirus 1, was characterized applying the technique of rolling circle amplification followed by high-throughput sequencing using the Illumina platform. This new type showed greater similarity with BPV3 (79% identity at the genomic level) and fulfills the requirements to be considered a new BPV type. Histopathology was characterized by exophytic papilloma, presenting well differentiated cells, marked acanthosis and parakeratosis. In Chapter 3, a complete BPV5 genome was sequenced using the same protocol used in Chapter 2. This was the first fully sequenced and characterized BPV5 genome from South America. Thus, the results obtained in this thesis contribute to a better knowledge of the genetic variability of BPV and shows the importance of the use of molecular tools in their characterization, especially in regions that are lacking in studies and which may harbor unknown types.

DETECÇÃO DE PAPILOMAVÍRUS EQUINO EM PLACA AURAL EQUINA POR QPCR

As papilomatoses ocupam uma posição de destaque na medicina humana e veterinária uma vez que são enfermidades virais dermatológicas com potencial para desenvolver malignidade tumoral. Nos equinos os papilomas estão associados às seguintes enfermidades: papilomatose clássica, associada ao EcPV1; carcinoma de células escamosas associado ao EcPV2; Massas tumorais em mucosas genitais (EcPV2 e EcPV7); placa aural, associado ao EcPV3, 4, 5 e 6; sarcóide equino, associado ao papilomavírus bovino 1 e 2 (BPV1 e 2). Dessas, a que está em estudo nesse trabalho é a placa aural, caracterizada por lesões puntiformes de poucos milímetros, despigmentadas e queratinizadas na face interior do pavilhão auricular, podendo evoluir e coalescer em lesões maiores e por vezes se estendendo por todo pavilhão auricular. Visando obter um teste de diagnóstico específico, sensível e rápido para identificação destes vírus, foi padronizada a técnica de PCR em tempo real (qPCR) para o EcPV3, 4, 5 e 6. E com a finalidade de avaliar a incidência dessa enfermidade no rebanho nas cinco regiões do país e estabelecer a prevalência de cada tipo viral a partir da técnica padronizada, foram coletadas 104 amostras das regiões Nordeste, Norte, Sul, Sudeste e Centro-Oeste. Como resultado obteve-se a padronização da técnica. Na análise das amostras a prevalência de pelo menos um tipo viral na lesão foi 90,39%, com as seguintes prevalências por tipo: EcPV3 36,54%, EcPV4 81,73%, EcPV5 0,96% e EcPV6 10,58%, apresentando variação entre as diferentes regiões. O presente estudo traz uma evolução no que se refere às pesquisas relacionadas a Placa Aural e também levanta novos questionamentos para estudos futuros.

Papillomatosis occupy a prominent position both in human and veterinary medicine, since they are viral skin diseases with potential to develop malignancy. In horses papillomas are associated with the following diseases: classical papillomatosis associated with EcPV1; squamous cell carcinoma associated with EcPV2; tumors in mucous membranes on the genital area (EcPV2 and EcPV7); aural plaque associated with EcPV3, 4, 5 and 6; equine sarcoid, associated with bovine papillomavirus (BPV1 and 2). Of all equine papillomatosis, aural plaque is the one analyzed in this work, which is characterized by small papules, with a few millimeters, hypochromic and keratinized on internal surface of the pinnae and can evolve and coalesce into larger lesions. To obtain a specific diagnostic test, also sensitive and quick to identify these viruses, a Real Time PCR was standardized for EcPV3, 4, 5 and 6. In order to evaluate the incidence of this disease in equine herds distributed in the five regions of Brazil and establish the prevalence of each viral type using the standardized technique, 109 samples were collected in the Northeast, North, South, Southeast and Midwest. The main result was the technique standardization of the technique. Applying the qPCR technique in the samples resulted in 90,39% of at least on viral type prevalence, the prevalence for each type was: EcPV3 36,54%, EcPV4 81,73%, EcPV5 0,96% e EcPV6 10,58%, with variation between different regions. This study represents an evolution in the area related to aural plaque and equine papillomatosis and raises new questions for future research.

Preliminary binary ethylenimine (BEI) inactivated bovine papillomavirus (BPV) vaccine trial against cutaneous warts in bull calves: a pathological assessment

A preliminary prophylactic and therapeutic vaccine trial was successfully conducted in bull calves with binary ethylenimine (BEI) inactivated saponized crude cutaneous warts bovine papillomavirus 1 and 2 (CW BPV-1 & -2) vaccine. In Prophylactic Group of vaccinated animals no temperature, untoward reaction or nodule formation was seen at vaccine inoculation site. After challenge with BPV inoculum, cutaneous warts (CWs) failed to develop in this group indicating that vaccine was effective. In Therapeutic Group, before vaccination in two animals CWs were experimentally induced. In one animal, luxuriant cauliflower-like and in other linear slow growth was observed. After vaccination, CWs in both animals became dried and growth was arrested. It appeared that tumours were regressing which was confirmed by further clinical observations as very little tumour-scar was left at termination of experiment.Histopathologically, both cases were diagnosed as regressing occult/ early fibroblastic type papilloma. Mononuclear cellular infiltration/ aggregates around hair follicles and in tumour stroma were noted. As compared to first case (cauliflower- like/ fast growing) in second case (linear growth/ fast regressing), in addition to other findings mononuclear cellular inflammatory reaction were more pronounced. The clinical regression results are in accordance with histopathological findings of degree of infiltration of lymphocytes and this trial showed that both type use of BEI inactivated saponized crude CW BPV-1&-2 vaccine gave encouraging results with fast regression/ prevention of CWs.

Preliminary binary ethylenimine (BEI) inactivated bovine papillomavirus (BPV) vaccine trial against cutaneous warts in bull calves: a pathological assessment

A preliminary prophylactic and therapeutic vaccine trial was successfully conducted in bull calves with binary ethylenimine (BEI) inactivated saponized crude cutaneous warts bovine papillomavirus 1 and 2 (CW BPV-1 & -2) vaccine. In Prophylactic Group of vaccinated animals no temperature, untoward reaction or nodule formation was seen at vaccine inoculation site. After challenge with BPV inoculum, cutaneous warts (CWs) failed to develop in this group indicating that vaccine was effective. In Therapeutic Group, before vaccination in two animals CWs were experimentally induced. In one animal, luxuriant cauliflower-like and in other linear slow growth was observed. After vaccination, CWs in both animals became dried and growth was arrested. It appeared that tumours were regressing which was confirmed by further clinical observations as very little tumour-scar was left at termination of experiment.Histopathologically, both cases were diagnosed as regressing occult/ early fibroblastic type papilloma. Mononuclear cellular infiltration/ aggregates around hair follicles and in tumour stroma were noted. As compared to first case (cauliflower- like/ fast growing) in second case (linear growth/ fast regressing), in addition to other findings mononuclear cellular inflammatory reaction were more pronounced. The clinical regression results are in accordance with histopathological findings of degree of infiltration of lymphocytes and this trial showed that both type use of BEI inactivated saponized crude CW BPV-1&-2 vaccine gave encouraging results with fast regression/ prevention of CWs.(AU)

Clonagem e expressão do gene E6 do Papilomavírus Bovino do tipo 1 / Cloning and expression of Bovine Papillomavirus type 1 E6 gene

O Papilomavírus Bovino do tipo 1 (BPV-1) causa fibropapilomas em bovinos e sarcóide em equinos, associados à expressão de oncoproteínas virais, principalmente E6 e E7. A purificação de oncoproteínas a partir de sistema recombinante possibilita seu estudo. Os objetivos foram expressar o gene E6 do BPV-1, purificar e analisar in silico a proteína recombinante obtida. O amplificado foi clonado em E. coli e os plasmídeos foram sequenciados, confirmando a matriz correta de leitura. Após indução da expressão, a proteína recombinante foi identificada e purificada. A microscopia eletrônica mostrou a formação de corpúsculos de inclusão. As análises in silico apontaram as mutações das sequências gênica e proteica de E6-1 em relação às depositadas. Foi realizada a predição tridimensional da estrutura da proteína recombinante e identificadas as regiões conservadas, antigênicas e capazes de realizar ligações cátion-p. Logo, a proteína recombinante E6 do BPV-1 foi obtida e purificada com sucesso, possibilitando o início de novos experimentos e estudos mais detalhados.

Bovine Papillomavirus type 1 (BPV-1) causes fibropapillomas in cattle and sarcoid in equines, associated with the expression of viral oncoproteins, E6 and E7 particularly. Purification of oncoproteins from recombinant system allows their study. The aim of this study was cloning and expressing BPV-1 E6 gene, purify and analyze in silico recombinant protein. Amplicon was cloned into E. coli and the plasmids were sequenced to confirm the correct reading frame. After induction of expression, the recombinant protein was identified and purified. Electron microscopy showed the inclusion bodies formation. In silico analysis showed mutations in E6-1 gene and protein sequences when there were compared to sequences available in current database. Three-dimensional structure prediction of the recombinant protein was performed and conserved, antigenic and cation-p interaction regions were identified. Therefore, BPV-1 E6 recombinant protein was obtained and successfully purified enabling new experiments and more detailed studies.

Clonagem e expressão do gene E6 do Papilomavírus Bovino do tipo 1 / Cloning and expression of Bovine Papillomavirus type 1 E6 gene

O Papilomavírus Bovino do tipo 1 (BPV-1) causa fibropapilomas em bovinos e sarcóide em equinos, associados à expressão de oncoproteínas virais, principalmente E6 e E7. A purificação de oncoproteínas a partir de sistema recombinante possibilita seu estudo. Os objetivos foram expressar o gene E6 do BPV-1, purificar e analisar in silico a proteína recombinante obtida. O amplificado foi clonado em E. coli e os plasmídeos foram sequenciados, confirmando a matriz correta de leitura. Após indução da expressão, a proteína recombinante foi identificada e purificada. A microscopia eletrônica mostrou a formação de corpúsculos de inclusão. As análises in silico apontaram as mutações das sequências gênica e proteica de E6-1 em relação às depositadas. Foi realizada a predição tridimensional da estrutura da proteína recombinante e identificadas as regiões conservadas, antigênicas e capazes de realizar ligações cátion-p. Logo, a proteína recombinante E6 do BPV-1 foi obtida e purificada com sucesso, possibilitando o início de novos experimentos e estudos mais detalhados. (AU)

Análise filogenética de papilomavírus bovino associado com lesões cutâneas em rebanhos do Estado do Paraná / Phylogenetic analysis of bovine papillomavirus associated with skin warts in cattle herds from the state of Paraná

A infecção pelo papilomavírus bovino (BPV) causa lesões hiperplásicas no epitélio cutâneo dos animais. De acordo com a localização e as características morfológicas das lesões, os seis tipos de BPV são classificados em dois sub-grupos. O objetivo desse trabalho foi identificar os tipos de BPV presentes em lesões cutâneas em bovinos de rebanhos do Estado do Paraná. Os primers degenerados FAP59 e FAP64 foram utilizados para a amplificação de um fragmento com 478 pb do gene L1 do BPV bovino em nove amostras de papilomas cutâneos obtidos de seis animais provenientes de quatro rebanhos bovinos do Estado. Em todas as amostras foi possível a amplificação de um produto com a massa molecular esperada. Por meio da análise filogenética das seqüências dos amplicons foi possível identificar o BPV-2 em três amostras, o BPV-1 em uma e o BPV-6 em cinco amostras de papilomas. O BPV-6 foi encontrado tanto em papilomas localizados no teto quanto em outras partes do corpo. Em um dos animais, do qual foram colhidas mais de uma amostra, foi detectada infecção concomitante do BPV-1 com o BPV-2. As cinco amostras positivas para o BPV-6 apresentaram 100 por cento de identidade de nucleotídeos com a amostra padrão disponível no GenBank. No entanto, foram identificadas diferenças entre as amostras do BPV-2 e BPV-1 e aquelas depositadas neste banco de dados. Esse estudo demonstrou a diversidade de tipos do BPV circulantes em rebanhos do Estado do Paraná.(AU)

Bovine papillomavirus (BPV) infection causes hyperplastic lesions in the cutaneous epithelium of cattle. Six types of BPV were classified in two sub-groups, being correlated to the anatomical regions of the infection and morphologic characteristics of the lesions. The present study was carried out to identify the types of BPV present in skin warts of cattle from the state of Paraná, Brazil. The generic primers FAP59 and FAP64 were used for amplification of a 478 bp fragment of BPV L1 gene in nine cutaneous papilloma samples obtained from six animals in four herds. In all papillomas examined, a product with the expected molecular size was amplified. Phylogenetic analysis of the PCR products identified BPV-2 in three samples, BPV-1 in one, and BPV-6 in five papillomas. BPV-6 was detected in cutaneous papillomas of the teat and in other body parts as well. In one animal, from which more than one sample was collected, a concomitant infection by BPV-1 and BPV-2 was identified. The five positive BPV-6 samples showed a nucleotide identity of 100 percent with the sequence of the reference strain available in GenBank. However, differences among BPV-2 and BPV-1 Brazilian samples and the respective reference sequences deposited in GenBank were observed. Molecular comparison of the two BPV-2 strains identified showed the involvement of two viral variants. This study revealed the diversity of BPV types circulating in the state of Paraná.(AU)

Análise filogenética de papilomavírus bovino associado com lesões cutâneas em rebanhos do Estado do Paraná / Phylogenetic analysis of bovine papillomavirus associated with skin warts in cattle herds from the state of Paraná

A infecção pelo papilomavírus bovino (BPV) causa lesões hiperplásicas no epitélio cutâneo dos animais. De acordo com a localização e as características morfológicas das lesões, os seis tipos de BPV são classificados em dois sub-grupos. O objetivo desse trabalho foi identificar os tipos de BPV presentes em lesões cutâneas em bovinos de rebanhos do Estado do Paraná. Os primers degenerados FAP59 e FAP64 foram utilizados para a amplificação de um fragmento com 478 pb do gene L1 do BPV bovino em nove amostras de papilomas cutâneos obtidos de seis animais provenientes de quatro rebanhos bovinos do Estado. Em todas as amostras foi possível a amplificação de um produto com a massa molecular esperada. Por meio da análise filogenética das seqüências dos amplicons foi possível identificar o BPV-2 em três amostras, o BPV-1 em uma e o BPV-6 em cinco amostras de papilomas. O BPV-6 foi encontrado tanto em papilomas localizados no teto quanto em outras partes do corpo. Em um dos animais, do qual foram colhidas mais de uma amostra, foi detectada infecção concomitante do BPV-1 com o BPV-2. As cinco amostras positivas para o BPV-6 apresentaram 100 por cento de identidade de nucleotídeos com a amostra padrão disponível no GenBank. No entanto, foram identificadas diferenças entre as amostras do BPV-2 e BPV-1 e aquelas depositadas neste banco de dados. Esse estudo demonstrou a diversidade de tipos do BPV circulantes em rebanhos do Estado do Paraná.

Bovine papillomavirus (BPV) infection causes hyperplastic lesions in the cutaneous epithelium of cattle. Six types of BPV were classified in two sub-groups, being correlated to the anatomical regions of the infection and morphologic characteristics of the lesions. The present study was carried out to identify the types of BPV present in skin warts of cattle from the state of Paraná, Brazil. The generic primers FAP59 and FAP64 were used for amplification of a 478 bp fragment of BPV L1 gene in nine cutaneous papilloma samples obtained from six animals in four herds. In all papillomas examined, a product with the expected molecular size was amplified. Phylogenetic analysis of the PCR products identified BPV-2 in three samples, BPV-1 in one, and BPV-6 in five papillomas. BPV-6 was detected in cutaneous papillomas of the teat and in other body parts as well. In one animal, from which more than one sample was collected, a concomitant infection by BPV-1 and BPV-2 was identified. The five positive BPV-6 samples showed a nucleotide identity of 100 percent with the sequence of the reference strain available in GenBank. However, differences among BPV-2 and BPV-1 Brazilian samples and the respective reference sequences deposited in GenBank were observed. Molecular comparison of the two BPV-2 strains identified showed the involvement of two viral variants. This study revealed the diversity of BPV types circulating in the state of Paraná.

Expressão e purificação da proteína recombinante L2 do Papilomavírus bovino tipo-2 em sistema bacteriano / Expression and purification of recombinant protein L2 of Bovine papillomavirus type-2 in bacterial system

A papilomatose bovina é uma doença infectocontagiosa de ocorrência mundial, que assola o rebanho brasileiro, sem qualquer atitude efetiva de controle, e que tem como enfermidades associadas a tumores de bexiga hematúria enzoótica e tumores de trato digestório superior caraguatá, responsáveis por sensíveis perdas para a pecuária. Várias tentativas vacinais têm sido empreendidas com finalidades profiláticas ou terapêuticas, porém sem resultados eficazes. Esta situação se deve a aspectos relacionados à estrutura viral que dificultam uma manipulação eficiente para produção de produtos vacinais. Para que tais dados possam ser obtidos, faz-se necessário uma melhor compreensão da ação das proteínas recombinantes. A clonagem em vetores bacterianos para a expressão e purificação dessas proteínas serve a diferentes propósitos. Entre eles, a produção de insumos imunológicos, como testes diagnósticos ou mesmo vacinas. O presente projeto visou à expressão e purificação da proteína de capsídeo recombinante L2 de BPV-2. A proteína foi expressa em bactéria e tentou-se a purificação por coluna de afinidade; entretanto, problemas na purificação não possibilitaram a conclusão deste objetivo. Todas as abordagens e protocolos utilizados nesse trabalho são discutidos para contribuição ao conhecimento do processo

The bovine papillomatosis is an infectious disease of worldwide occurrence, plaguing the Brazilian herd, without any effective attitude control, and whose illnesses associated with bladder tumors /"enzootic hematuria/" and upper digestive tract tumors /"caraguatá/" sensitive responsible for losses to livestock. Several attempts have been undertaken vaccine with prophylactic or therapeutic purposes, but without effective results. This is due to issues related to viral structure that hinder efficient manipulation for production of vaccine products. In order to obtain such information, it is necessary better understanding of the action of recombinant proteins. The bacterial cloning vectors for the expression and purification of such proteins serve different purposes. Among them, the production of immune inputs, such as diagnostic tests or vaccines. This project aimed the expression and purification of recombinant L2 capsid protein of BPV-2. The protein was expressed in bacteria and purification was carried out by affinity column. However, difficulties in the purification process, impaired the full completion of this objective. All the attempted approaches and protocols were discussed and potential solutions proposed