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.

Synchronous detection of BPV and BVDV with duplex Taqman qPCR method

Background: Bovine parvovirus (BPV) and bovine viral diarrhea virus (BVDV) are commonly etiologies causing diarrheain dairy herds. BPV is a member of Bocaparvovirus genus with a non-enveloped capsid. BVDV, belonging to Pestivirusgenus in Flaviviridae, possesses a single-stranded RNA, and is classified into BVDV-1 and BVDV-2 genotypes accordingto the 5’UTR sequence. 21 genetic groups of BVDV-1 and four groups of BVDV-2 have been found. Diagnosis of viraldiarrhea is often relied on virus detection by isolation or detection of serum antibody. The main objective of the presentstudy was to establish a duplex real time PCR (qPCR) based on Taqman probe to detect synchronously BPV and BVDV.Materials, Methods & Results: TaqMan probe and primers were designed and synthesized from the sequences of conserved5′ - untranslated regions (5′ UTR) of Haden strain of BPV and NADL strain of BVDV. The cDNAs were transcribed invitro to make standard curves before optimizing the assay. DNA/PCR products were ligated into pMD18-T vector, andthen used to transfer BL-21 competent cells to acquire the recombinant plasmids of pMD18-T-BPV and pMD18-T-BVDV.Optimum reaction conditions were comparatively selected. The sensitivity, specificity and reproducibility of TaqMan probeqRT-PCR were evaluated respectively. The results showed the concentrations of pMD18-T-BPV or pMD18-T-BVDV were2.0 × 1010 DNA copies/μL, respectively. A duplex Taqman qPCR method was developed by optimizing the amplificationconditions to simultaneously detect BPV and BVDV. The assay targets at highly conserved VP2 gene of BPV and 5′ UTRgene of BVDV. This qPCR assay was assessed for specificity and sensitivity using DNA of BPV and cDNA of BVDV. Forclinical validation, 308 samples were tested from clinically diarrhea calves. The results showed that optimum annealingtemperature was achieved in 43.2 for duplex BPV and BVDV. Dynamic curves and standard...

Synchronous detection of BPV and BVDV with duplex Taqman qPCR method

Background: Bovine parvovirus (BPV) and bovine viral diarrhea virus (BVDV) are commonly etiologies causing diarrheain dairy herds. BPV is a member of Bocaparvovirus genus with a non-enveloped capsid. BVDV, belonging to Pestivirusgenus in Flaviviridae, possesses a single-stranded RNA, and is classified into BVDV-1 and BVDV-2 genotypes accordingto the 5UTR sequence. 21 genetic groups of BVDV-1 and four groups of BVDV-2 have been found. Diagnosis of viraldiarrhea is often relied on virus detection by isolation or detection of serum antibody. The main objective of the presentstudy was to establish a duplex real time PCR (qPCR) based on Taqman probe to detect synchronously BPV and BVDV.Materials, Methods & Results: TaqMan probe and primers were designed and synthesized from the sequences of conserved5′ - untranslated regions (5′ UTR) of Haden strain of BPV and NADL strain of BVDV. The cDNAs were transcribed invitro to make standard curves before optimizing the assay. DNA/PCR products were ligated into pMD18-T vector, andthen used to transfer BL-21 competent cells to acquire the recombinant plasmids of pMD18-T-BPV and pMD18-T-BVDV.Optimum reaction conditions were comparatively selected. The sensitivity, specificity and reproducibility of TaqMan probeqRT-PCR were evaluated respectively. The results showed the concentrations of pMD18-T-BPV or pMD18-T-BVDV were2.0 × 1010 DNA copies/μL, respectively. A duplex Taqman qPCR method was developed by optimizing the amplificationconditions to simultaneously detect BPV and BVDV. The assay targets at highly conserved VP2 gene of BPV and 5′ UTRgene of BVDV. This qPCR assay was assessed for specificity and sensitivity using DNA of BPV and cDNA of BVDV. Forclinical validation, 308 samples were tested from clinically diarrhea calves. The results showed that optimum annealingtemperature was achieved in 43.2 for duplex BPV and BVDV. Dynamic curves and standard...(AU)

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)

INCIDÊNCIA DE LESÕES EM VESÍCULA URINÁRIA DE BOVINOS ASSINTOMÁTICOS ABATIDOS NA MESORREGIÃO NORTE CENTRAL DO PARANÁ E A ASSOCIAÇÃO COM O PAPILOMAVÍRUS BOVINO

O papilomavírus bovino é amplamente disseminado entre as espécies de mamíferos e aves, caracterizado como espécie especifico, na qual possui capacidade de se replicar significativamente. O objetivo deste estudo foi avaliar a presença de lesões em vesícula urinária de bovinos assintomáticos abatidos na Mesorregião Norte Central do Paraná e associar as lesões com a presença do BPV. Foram analisadas 516 vesículas urinárias de animais de diversas raças, com predominância de bovinos Bos indicus taurus, provenientes de 87 propriedades situadas em 10 municípios da Mesorregião Norte Central e Centro-Sul do Paraná, que foram abatidos em abatedouro frigorífico localizado no município de Ivaiporã. A urina presente na vesícula urinária foi analisada, realizada a contagem de hemácias através do microscópio óptico, além da divisão em quadrantes da vesícula urinária em busca de lesões. Foram retirados fragmentos em duplicata, para análise molecular em busca do BPV e histopatológica em busca de lesões microscópicas. Encontrou-se 26 amplificações para o BPV, utilizando o par de primers genéricos (FAP 59/FAP 64). Com base na avaliação macroscópica vesical foi possível identificar 85 bovinos com lesões vesicais, correspondendo à 16,47% dos animais avaliados. Quanto à sede da lesões vesicais em quadrantes, foi observado que os quadrantes D e C foram os mais acometidos (p-valor<0,0001), totalizando 35,38% (69/195) e 34,36% (67/195). Dentre os achados histopatológicos, foram encontradas alterações indicativas de formação tumoral. Já na contagem de hemácias foi detectada micro hematúria (n=5/516) e hematúria (n=58/516) nas amostras analisadas. Contudo, não foi possível realizar a análise de 26,56% (137/516) amostras devido à ausência de urina, já na análise de urina pela fita reagente foi possível detectar que 87,60% (332/379) das amostras analisadas encontravam-se em seu estado fisiológico, a presença de hemoglobina foi detectada em 10,55% (40/379) e de hemácias em 1,85% (7/379). Conclui-se que a incidência de lesões vesicais em bovinos abatidos na mesorregião Norte Central do Paraná é moderada. No entanto, não é possível afirmar a correlação da lesão vesical com a presença do BPV.

Bovine papillomavirus is widely disseminated among species of mammals and birds, characterized as a specific species, in which it has the ability to replicate significantly. The aim of this study was to evaluate the presence of lesions in the urinary vesicle of asymptomatic cattle slaughtered in the North Central Mesoregion of Paraná and to associate the lesions with the presence of BPV. 516 urinary vesicles of animals of different breeds were analyzed, with predominance of Bos indicus taurus cattle, from 87 properties located in 10 municipalities in the North Central and Central-South Paraná regions, which were slaughtered in a slaughterhouse located in the municipality of Ivaiporã. The urine present in the urinary bladder was analyzed, the erythrocyte count was performed using an optical microscope, in addition to dividing the urinary bladder into quadrants in search of injuries. Duplicate fragments were removed for molecular analysis in search of BPV and histopathology in search of microscopic lesions. There were 26 amplifications for BPV, using the generic primer pair (FAP 59 / FAP 64). Based on the macroscopic bladder evaluation, it was possible to identify 85 cattle with bladder injuries, corresponding to 16.47% of the animals evaluated. As for the seat of bladder injuries in quadrants, it was observed that quadrants D and C were the most affected (p-value <0.0001), totaling 35.38% (69/195) and 34.36% (67/195 ). Among histopathological findings, alterations indicating tumor formation were found. In the red blood cell count, micro hematuria (n = 5/516) and hematuria (n = 58/516) were detected in the analyzed samples. However, it was not possible to perform the analysis of 26.56% (137/516) samples due to the absence of urine, in the analysis of urine by the reagent strip it was possible to detect that 87.60% (332/379) of the analyzed samples found if in their physiological state, the presence of hemoglobin was detected in 10.55% (40/379) and of red blood cells in 1.85% (7/379). It is concluded that the incidence of bladder injuries in cattle slaughtered in the North Central region of Paraná is moderate. However, it is not possible to affirm the correlation of bladder injury with the presence of BPV.

BLOQUEIOS LOCORREGIONAIS EM BUBALINOS: BLOQUEIO DO NERVO PUDENDO, PARAVERTEBRAL E TORÁCICO LATERAL

Com o objetivo de avaliar os efeitos dos bloqueios locorregionais: do nervo pudendo nos machos e paravertebral isolado e associado ao torácico lateral nas fêmeas, usando lidocaína 2% com vasoconstritor, foram utilizados 25 búfalos, sendo 13 machos, divididos em dois grupos (adultos e jovens) e 12 fêmeas. Observou-se a ocorrência ou não de bloqueio motor (BM), por meio da inspeção direta do relaxamento da musculatura, nos machos; para verificar o bloqueio sensitivo (BS) foi utilizada agulha hipodérmica nos machos e pinça dente de rato nas fêmeas no intuito de buscar respostas à dor. O período de bloqueio motor e sensitivo foi determinado a partir da apresentação de relaxamento e não reação aos estímulos dolorosos, avaliados a cada 20 minutos nos machos e a cada 10 minutos nas fêmeas até retorno do vigor e dor. Em machos no GJ e GA respectivamente, ocorreu a exposição do pênis em 83% e 85.7%, iniciaram 20.2±17 min. e 19.6±10.1min., durando 91.6±84.2 min. e 110±179.6 min. O BS do pênis não ocorreu em nenhum grupo. O BM do escroto ocorreu em 100% em ambos os grupos, iniciou 10.8±4.9 min e 9.8±5.6 min., durando 138.3±51.9 min. e 139.2±83.5 min., no GJ e GA, respectivamente. O BS do escroto ocorreu de forma U ou B em 83.2% e 100%, iniciou 11.5±7.7 min. e 17.5±7.2 min., durando 103.8±80.9 min. e 108.3±94.9 min., no GJ e GA, respectivamente. Nas fêmeas ambos os bloqueios foram eficientes em anestesiar as regiões do flanco e região ventral do abdômen. A anestesia ocorreu em 83% e 91.3% na fossa paralombar (FPL), de 91.3% e 91.3% na lateral (L) e 83% e 91.3% na região ventral (RV), nos bloqueios paravertebral (BPV) e paravertebral associado ao torácico lateral (BPV+BTL) respectivamente. Porém, ambos os bloqueios não foram eficientes em anestesiar os tetos anteriores (TA), a parte caudal do úbere (UCl) e os tetos posteriores (TP), anestesiando apenas 16.6% dos animais. Foi na porção cranial do úbere (UCr) que ocorreu a maior diferenças entres os bloqueios, sendo 24.9% no BPV e 58.1% no (BPV+BTL). Desta forma, verificou-se a eficiência das técnicas para exposição peniana e anestesia na região cranial do úbere.

In order to evaluate the effects of locoregional anesthesia blocks of the pudendal nerve in males, and isolated paravertebral and associated with the lateral thoracic in females, using 2% lidocaine with vasoconstrictor, 25 buffaloes were used, 13 males, divided into two groups (adults and young) and 12 females. Motor block (BM) was observed if present or not, through direct inspection of muscle relaxation in males. To check the sensory block (BS), a hypodermic needle was used in males and an Adson forceps in females in order to seek answers to pain. Motor block (BM) was observed if present not, through direct inspection of muscle relaxation in males; to check the sensory block (BS), a hypodermic needle was used in males and an Adson forceps in females in order to seek response to pain. The period of motor and sensory block was determined from the presentation of relaxation and non-reaction to painful stimuli, assessed every 20 minutes in males and every 10 minutes in females until the return of vigor and pain. In males in GJ and GA respectively, penis exposure occurred in 83% and 85.7%, starting at 20.2 ± 17 min. and 19.6 ± 10.1 min., lasting 91.6 ± 84.2 min. and 110 ± 179.6 min. The BS of the penis did not occur in any group. The scrotum BM occurred in 100% in both groups, starting at 10.8 ± 4.9 min and 9.8 ± 5.6 min., lasting 138.3 ± 51.9 min. and 139.2 ± 83.5 min., in GJ and GA, respectively. The scrotum BS occurred in a U or B form in 83.2% and 100%, starting at 11.5 ± 7.7 min. and at 17.5 ± 7.2 min., lasting 103.8 ± 80.9 min. and 108.3 ± 94.9 min., in GJ and GA, respectively. In females, both blocks were efficient in anesthetizing the flank and ventral regions of the abdomen. Anesthesia occurred in 83% and 91.3% in the paralumbar fossae (FPL), 91.3% and 91.3% in the lateral (L) and 83% and 91.3% in the ventral region (RV), in paravertebral (BPV) and paravertebral blocks associated with lateral thoracic (BPV + BTL) respectively. However, both blocks were not efficient in anesthetizing the anterior teat (TA), the caudal part of the udder (UCl) and the posterior teat (TP), anesthetizing only 16.6% of the animals. It was in the cranial portion of the udder (UCr) that the major differences occurred between the blocks, with 24.9% in BPV and 58.1% in (BPV + BTL). Thus, the efficiency of techniques for penile exposure and anesthesia in the cranial region of the udder was verified.

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.

Teat papillomatosis associated with bovine papillomavirus types 6, 7, 9, and 10 in dairy cattle from Brazil

This study describes the clinical, histopathological, and virological characterization of teat papillomatosis 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.(AU)

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)

Diversidade genética de papilomavírus bovino no Brasil

Resumo Os papilomavírus (PV) são vírus epiteliotrópicos que infectam um grande número de vertebrados, incluindo os mamíferos, aves, répteis e peixes. Eles são vírus não envelopados, compostos de um capsídeo estruturado pelas proteínas L1 e L2, que abriga uma molécula de DNA dupla fita e circular. Os PV são vírus oncogênicos que causam lesões benignas e malignas na epiderme e mucosas de seus hospedeiros. Em bovinos, o papilomavírus bovino (BPV) causa papilomas extensivos em animais suscetíveis. Apesar das infecções por BPV apresentarem alta morbidade e baixa mortalidade, elas causam prejuízos econômicos aos produtores, que frequentemente descartam animais precocemente devido à extensão e o local das lesões. Atualmente, 23 tipos de BPV foram classificados e caracterizados, contrastando com o alto número de papilomavírus humanos (HPV) sequenciados e caracterizados (204). Desta forma, acredita-se que a diversidade genética dos BPV seja provavelmente similar à diversidade genética dos HPV existentes. Este trabalho objetivou investigar a diversidade genética dos BPV existentes na região norte e sul do Brasil, assim como realizar o sequenciamento completo de novos tipos virais e tipos já descritos e, desta forma, contribuir para futuros estudos epidemiológicos, filogenéticos e de caracterização genética de BPV. Ainda, esta tese objetivou prover uma atualização da atual epidemiologia, classificação e características genéticas dos PV de ruminantes, com o foco principal nos BPV. Metodologias convencionais (baseadas em amplificação de ácidos nucléicos virais por PCR utilizando oligonucletotídeos específicos para PV) e de última geração (amplificação randômica de genomas circulares e sequenciamento de alta eficiência) foram empregadas, proporcionando a identificação dos vírus circulantes em rebanhos da região Norte e da região Sul do país. Nove novos tipos de BPV foram sequenciados e caracterizados neste estudo (BPV16, 17, 18, 19, 20, 21 e os prováveis BPV24, 25 e 26), evidenciando a grande diversidade genética de BPV presente na região amazônica quando comparada com amostras do sul do país. Além disso, o sequenciamento de genomas completos de outros tipos virais já descritos também foi realizado. Esses resultados proporcionaram uma visão geral dos tipos virais presentes em rebanhos de dois extremos do Brasil, assim como possibilitou a identificação de prováveis novos tipos e a caracterização de novos tipos de BPV. Esses resultados são importantes para entender a evolução e a distribuição dos papilomavírus, bem como para o desenvolvimento de estudos vacinais, que envolvem papilomavírus humano e bovino. Adicionalmente, podemos verificar que pesquisas envolvendo ruminantes selvagens são esporádicas, no entanto seriam importantes para o melhor entendimento da biologia e das relações intra e interespecíficas dos PV.

Papillomaviruses (PV) are epitheliotropic viruses that infect a large number of vertebrates, including mammals, birds, reptiles and fish. They are non-enveloped viruses composed of a capsid that is structured by the L1 and L2 proteins, which harbous a circular double-stranded DNA molecule. PVs are oncogenic viruses that cause benign and malignant lesions in the epidermis and mucosa of their hosts. In cattle, bovine papillomavirus (BPV) causes extensive papillomas in susceptible animals. Although BPV infections present high morbidity and low mortality, they cause economic losses to producers, who often discard animals prematurely because of the extent and location of the lesions. Currently, 23 types of BPVs have been classified and characterized, contrasting with the high number of human papillomaviruses (HPVs) sequenced and characterized (204). Therefore, it is believed that the genetic diversity of BPV is probably similar to the genetic diversity of existing HPV. This work aimed to investigate the genetic diversity of BPV in the Northern and Southern regions of Brazil, as well as complete sequencing of new viral types and types already described, and thus contribute to future epidemiological, phylogenetic and genetic characterization studies of BPV. Furthermore, this thesis aimed to provide an update of the current epidemiology, classification and genetic characteristics of ruminant PV, with the main focus on BPV. Conventional methodologies (based on amplification of viral nucleic acids by PCR using specific oligonucleotides for PV) and last generation methodologies (random amplification of circular genomes and high efficiency sequencing) were employed, providing the identification of circular viruses in herds of the North and South regions of the country. Nine new types of BPV were sequenced and characterized in this study (BPV16, 17, 18, 19, 20, 21 and the putatives BPV24, 25 and 26), evidencing the great genetic diversity of BPV present in the Amazon region when compared with samples from the South region. In addition, sequencing of complete genomes from other viral types already described has also been performed. These results provided an overview of the viral types present in Brazilian herds from two distant regions, as well as the identification of potential new types and the characterization of new types of BPV. These results are important to understand the papillomavirus evolution and distribution as well as to development of vaccinal studies, which involve human and bovine papillomaviruses. Additionally, we could verify that researches involving wild ruminants are sporadic, but it would be important for the better understanding of the biology as well as the intra and interspecific of PV relationship.

CARACTERIZAÇÃO MOLECULAR DO GENE L1 DE UM PROVÁVEL NOVO TIPO DE XIPAPILLOMAVIRUS BOVINO, BRASIL

Papilomavírus (PV) são vírus oncogênicos que acometem diversas espécies de mamíferos, incluindo o homem. São capazes de induzir tumores benignos (papilomas ou verrugas) e malignos (condilomas) em epitélio cutâneo e mucoso. O genoma viral é constituído por até 10 fases abertas de leitura (ORFs), sendo que a ORF L1, sequência de nucleotídeos mais conservada entre os PVs, é utilizada para a identificação de novos tipos virais. Este estudo teve como objetivo identificar os tipos de papilomavírus bovino (BPV) presentes em cinco amostras de papilomas provenientes de um rebanho bovino leiteiro da região do norte pioneiro do estado do Paraná e determinar a sua classificação taxonômica por meio de comparação dessas sequências com as sequências de nucleotídeos de outros PVs previamente identificados e depositados em base pública de dados (GenBank). O par de primers degenerados FAP54/59 foi utilizado em reação de PCR para a triagem das amostras de papilomas. Os fragmentos de 480 pb obtidos das cinco amostras analisadas, permitiram a identificação de cinco diferentes tipos de BPV no mesmo rebanho, sendo estes BPV10, BPV6 e dois subtipos mais similares a BPV11 e BPV3. A quinta amostra, denominada BPV/BRUEL8, foi identificada no terceiro animal analisado, a partir de uma amostra de papiloma cutâneo. A análise da sequência gerada com o emprego do primeiro par de primers indicou que a cepa BPV/BR-UEL8 apresentava apenas 77% de identidade com o isolado 7Z (KT315748). Adicionalmente, foram empregados primers específicos do gene L1 de BPV6 para a obtenção da sequência completa do gene L1 do provável novo tipo. O produto da PCR foi submetido à clonagem e sequenciamento. A análise filogenética envolvendo a sequência completa da ORF L1 com 1.521 pb possibilitou identificar que a cepa BPV/BR-UEL8 pertence ao gênero Xipapillomavirus com maior identidade (apenas 75,1% identidade de nt) com BPV15. Este resultado possibilitou caracterizar a cepa BPV/BR-UEL8 como um provável novo tipo viral. Nos últimos anos, estudos que avaliaram características moleculares de BPV revelaram grande diversidade de tipos virais envolvidos em infecções singulares e, principalmente, em infecções mistas. A identificação de novos tipos virais pode auxiliar na compreensão da patogenia da doença e na avaliação de novos tratamentos, assim como no desenvolvimento de vacinas eficazes contra a papilomatose bovina

Papillomaviruses (PV) are oncogenic viruses that affect various mammalian species, including man. They can induce benign tumors (papillomas or warts) and malignant tumors (condylomas) in cutaneous and mucosal epithelium. The viral genome consists of up to 10 open reading frames (ORFs), and the L1 ORF, the most conserved nucleotide sequence among the PVs, is used to identify new viral types. The aim of this study was to identify the bovine papillomavirus (BPV) types present in five samples of papillomas from a dairy cattle herd at pioneer northern region of the state of Paraná and to determine their taxonomic classification by comparing these sequences with the sequences of nucleotides from other PVs previously identified and deposited in a public database (GenBank). The degenerate primer pair FAP54/59 was used in PCR reaction for the screening of papillomavirus samples. The 480 bp fragments obtained from the five analyzed samples allowed the identification of five different types of BPV in the same herd, being these BPV10, BPV6 and two subtypes more similar to BPV11 and BPV3. The fifth sample, named BPV/BR-UEL8, was identified in the third animal analyzed from a cutaneous papilloma sample. Analysis of the sequence generated using the first pair of primers indicated that the BPV/BR-UEL8 strain showed only 77% identity with the 7Z isolate (KT315748). In addition, specific primers of BPV6 L1 gene were used to obtain the complete L1 gene sequence of the putative new type. The PCR product was subjected to cloning and sequencing. The phylogenetic analysis involving the complete sequence of the L1 ORF with 1.521 bp allowed to identify that the BPV / BR-UEL8 strain belongs to the genus Xipapillomavirus with identity (only 75.1% identity of nt) with BPV15. This result made it possible to characterize the BPV/BR-UEL8 strain as a putative new viral type. In recent years, studies that have evaluated molecular characteristics of BPV revealed a wide variety of viral types involved in unique infections and, especially, mixed infections. The identification of new viral types can help to understand the pathogenesis of the disease and the evaluation of new treatments, as well as in the development of effective vaccines against bovine papillomatosis.

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.

CARACTERIZAÇÃO GENÉTICA DE PAPILOMAVÍRUS ABRANGENDO TRÊS DIFERENTES GÊNEROS IDENTIFICADOS EM UMA LESÃO EPITELIAL DE BOVINO

Os papilomavírus constituem uma extensa família de vírus associados a lesões epiteliotrópicas, assim como acometem o tecido mucoso onde podem causar tumores benignos que podem evoluir para a malignização. A papilomatose bovina é uma doença economicamente relevante, já que há a desvalorização dos animais a serem comercializados devido à aparência e depreciação do couro. Em humanos, a identificação dos tipos de papilomavírus (HPV) envolvidos bem como o relato de co-infecções são bem estabelecidos. Já foram descritos na literatura mais de 200 tipos de HPVs, o que ainda é pouco explorado na área da Medicina Veterinária, em que somente 21 tipos de papilomavírus bovino (BPV) foram caracterizados até o momento. A presente dissertação visou investigar a diversidade de BPV presentes em um papiloma de um bovino oriundo do Estado de Rondônia. A partir do DNA extraído da lesão, foi realizada a rooling circle amplification (RCA) seguida do sequenciamento de última geração (Ilumina MiSeq). O emprego dessas metodologias culminou na identificação do BPV13, que atualmente é bem caracterizado, e de três prováveis novos tipos de BPVs que haviam sido descritos em um único estudo recente. Estes resultados confirmam a presença de co-infecção em lesões de papilomatose bovina e demonstram que estas técnicas possibilitaram a detecção de espécies que não são identificadas pelos métodos convencionais. Este conhecimento sobre a diversidade de BPV servirá de base para o melhor entendimento da biologia do vírus e para a geração de vacinas profiláticas ou terapêuticas eficazes.

Papillomaviruses are an extensive family of viruses associated with epitheliotropic lesions, as well as affecting mucosal tissue where they can cause benign tumors that may progress to malignancy. Bovine papillomatosis is an economically relevant disease, since there is a devaluation of the animals to be marketed due to the appearance and depreciation of the leather. In humans, the identification of the types of papillomavirus (HPV) involved as well as the reporting of co-infections is well established. More than 200 types of HPV have been described in the literature, which is still little explored in the area of Veterinary Medicine, in which only 21 types of bovine papillomavirus (BPV) have been characterized. This study aimed to investigate the diversity of BPV present in a papilloma of a bovine animal from the State of Rondônia. From the DNA extracted from the lesion, the rooling circle amplification (RCA) was performed followed by the last generation sequencing (Ilumina MiSeq). The use of these methodologies culminated in the identification of BPV13, which is currently well characterized, and of three putative new BPVs types that had been described in a single recent study. These results confirm the presence of co-infection in bovine papillomatosis lesions and demonstrate that these techniques enabled the detection of species that are not identified by conventional methods. This knowledge on BPV diversity will serve as a basis for a better understanding of the biology of the virus and for the generation of effective prophylactic or therapeutic vaccines.

OTIMIZAÇÃO DE PCR EM TEMPO REAL PARA DETECÇÃO E QUANTIFICAÇÃO DO ONCOGENE E6 DE PAPILOMAVÍRUS BOVINO EM SARCOIDES EQUINOS E NEOPLASIAS EM BEXIGAS DE BOVINOS

Os papilomavírus bovino (BPV) tipos 2 e 13 estão envolvidos na etiologia do sarcoide equinos e também da hematúria enzoótica bovina. O objetivo do presente estudo foi desenvolver a técnica PCR em tempo real (qPCR), utilizando SYBR Green, para quantificar o oncogene E6 do BPV2 e 13 em quatro amostras de sarcoides de um equino e 12 amostras de lesões proliferativas de bexiga de bovinos. Avaliações histopatológicas das amostras confirmaram a presença de tumores fibroblásticos tipo sarcoide e dos carcinomas de bexiga. Com o objetivo de otimizar a qPCR, dois pares de primers foram desenvolvidos para amplificar um fragmento de 120 pb do oncogene E6, sendo um par específico para o BVP2 e outro para o BPV13, os controles positivos foram obtidos do acervo de papilomas do laboratório de virologia animal da Universidade Estadual de Londrina. O produto da amplificação deste fragmento foi clonado utilizando kit comercial e quantificado para o desenvolvimento da curva padrão. A reprodutibilidade foi analisada por meio de diluições seriadas (6x) dos produtos da clonagem do BPV2 e do BPV13. O sequenciamento dos amplicons foi realizado para confirmar a especificidade das reações. Nenhuma reação inespecífica com relação a outros tipos de BPVs foi identificada. O limite de detecção (LOD) da PCR convencional e qPCR foi avaliado testando o produto da amplificação de seis diluições seriadas dos controles positivos padrões. Os LOD para a PCR convencional para o BPV2 e 13 foram de aproximadamente 114 e 154 cópias de DNA, respectivamente, enquanto para qPCR, foram de 11,4 e 15,4 cópias de DNA viral de BPV2 e 13, respectivamente. Quatro amostras de sarcoide de um equino macho e doze fragmentos de carcinoma de bexigas bovinas foram coletadas e testadas pela nova qPCR. Amostras de swab de pele equina e mucosa de bexiga bovina sem lesão também foram testadas. O fragmento do oncogene E6 foi amplificado em 8/12 amostras de tumores de bexiga, sendo seis positivas para o BPV2, duas para o BPV13 e uma para ambos os tipos. O valor de Cq variou de 23,84 a 26,32 (1,05 x 104 até 9,53 x 103 cópias/µL) para o BPV2, e 23,41 e 24,96 (1,30 x 104 e 1,23 x 104 cópias/µL) para o BPV13. Nas amostras de sarcoide, em duas foi amplificado o oncogene E6 do BPV2, com Cq 21,16 e 21,43 (1.18 x 104 e 1.17 x 104 cópias/µL), e nas outras duas o E6 de BPV13 com Cq 18,9 e 19,1 (1.62 x 104 e 1.60 x 104 cópias/µL). O presente estudo confirmou o envolvimento do BPV2 e 13 na etiologia do sarcoide equino e na formação de lesões proliferativas em bexiga de bovinos e reforça a importância da participação do oncogene E6 de BPV2 e 13 na etiopatogenia destes tipos tumores podendo abrir novas perspectivas para o tratamento e profilaxia dessas importantes neoplasias em saúde animal.

The bovine papillomavirus (BPV) types 2 and 13 are involved in the etiology of equine sarcoids as well as bovine enzootic hematuria. The aim of this study was to standardize a realtime PCR (qPCR), using SYBR Green, to quantify the E6 oncogene of BPV2 and 13 in four equine sarcoid samples and 12 samples of urinary bladder tumors in cattle. Histopathological evaluations confirmed the presence of fibroblastic proliferation in sarcoid tumors and bladder tumors. In order to standardize qPCR, two primer pairs were designed to amplify a 120 bp fragment of the E6 oncogene, a specific pair for BPV2 and another for BPV13, positives controls were obtained from the collection of laboratory of virology animal of State University of Londrina. The amplification product of this fragment was cloned using commercial kit, and quantified for development of the standard curve. Reproducibility was analyzed using six-fold serial dilutions of the cloning products of BPV2 and 13. Sequencing was performed to confirm the specificities of the reactions. No cross-reaction was detected with other BPVs. The limit of detection (LOD) of the standard PCR and qPCR was evaluated by testing the amplification product of six serial dilutions of the standard positive controls. The LODs for conventional PCR for BPV2 and 13 were approximately 114 and 154 copies of DNA, respectively, whereas for qPCR, LODs were 11.4 and 15.4 copies of BPV2 and 13 viral DNA, respectively. Four sarcoid samples from one male equine and twelve bovine bladder carcinoma fragments were collected and tested by the new qPCR. Equine skin swab samples and lesionless bovine bladder mucosa were also tested. The E6 oncogene fragment was amplified in 9/12 samples of urinary bladder tumors, six of which were positive for BPV2, two were positive for BPV13 and one for both BPVs types, the Cq ranged from 23.84 to 26.32 (1.05 x 104 to 9.53 x 103 copies/µL) for BPV2 and 23.41 to 24.96 (1.30 x 104 to 1.23 x 104 copies/µL) for BPV13. In the sarcoid samples, the oncogene E6 of BPV2 were amplified in two samples, the Cq were 21.16 and 21.43 (1.18 x 104 to 1.17 x 104 copies/µL), and the other two samples were obtained BPV13 amplified with Cq 18.9 and 19.1 (1.62 x 104 and 1.60 x 104 copies/µL). The present study confirmed the involvement of BPV2 and 13 in the etiology of equine sarcoid and in the formation of urinary bladder lesions in cattle and reinforces the importance of the participation of BPV2 and BPV13 E6 oncogene in the etiopathogenesis of these two types of tumors and may open new perspectives for the treatment and prophylaxis of these important neoplasia in animal health.

Relação entre a linfopenia e a persistência da papilomatose alimentar em bovinos intoxicados crônica e espontaneamente por samambaia (Pteridium aquilinum) / Relationship between lymphopenia and the persistence of alimentary papillomatosis in cattle chronically and spontaneously poisoned by bracken fern (Pteridium aquilinum)

Papilomavírus bovino tipo 4 (BPV-4) é responsável pelo desenvolvimento de papilomas no trato alimentar superior (TAS) de bovinos. Os passos necessários para o crescimento, o desenvolvimento e a regressão dos papilomas estão intimamente relacionados com o estado imunológico do animal. A ingestão de samambaia (Pteridium aquilinum) tem sido relacionada como o principal fator envolvido na persistência da infecção pelo BPV-4 no TAS. A teoria que estabelece a relação entre papilomatose alimentar e a formação de CCEs sugere a produção de um estado imunossupressivo crônico pela planta, permitindo a persistência dos papilomas no TAS. Os papilomas serviriam então como sítios de desenvolvimento dos CCEs através da interação entre as proteínas do BPV-4 e os carcinógenos da samambaia. O objetivo deste estudo foi avaliar a relação entre a quantidade de linfócitos circulantes e a papilomatose alimentar em casos de intoxicação espontânea crônica por P. aquilinum em bovinos com CCE no TAS. Quarenta bovinos com CCEs no TAS foram avaliados quanto à idade, à intensidade da papilomatose alimentar no TAS e ao leucograma. Três bovinos tinham leucopenia e um apresentava neutrofilia. A média de linfócitos foi de 5.395 (±1.696) na papilomatose leve, 4.560 (±1.561) na moderada e 5.007 (±1.786) na acentuada. Não houve diferença estatisticamente significativa entre o grau de papilomatose, a idade e a quantidade de linfócitos circulantes. Imunossupressão por linfopenia foi um achado esporádico (três casos) neste estudo. Os resultados indicam que a persistência da papilomatose alimentar em casos espontâneos de intoxicação crônica por samambaia em bovinos não tem relação com a quantidade de linfócitos circulantes e que talvez esteja relacionada a outros fatores imunológicos.(AU)

Bovine papillomavirus type-4 (BPV-4) is responsible for the development of papillomas in the upper digestive tract (UDT) of cattle. The necessary steps for the development, growth and regression of papillomas are intimately related with the immunological status of the animal. The consumption of bracken fern (Pteridium aquilinum) has been pointed out as the major factor involved in the persistence of BPV-4 infection of the UDT. The theory involving the relationship between alimentary papillomas and the formation of squamous cell carcinomas (SCCs) suggests the persistence of alimentary papillomatosis through a chronic immunosupressive lymphopenic state induced by bracken fern. The papillomas would serve then as a development site of SCCs through the interaction of BPV-4 oncoproteins and bracken carcinogens. The objective of this study was to evaluate the relationship between lymphopenia and alimentary papillomatosis in spontaneous cases of chronic bracken fern poisoning of cattle. The age, intensity of papillomatosis and the leukon were evaluated of 40 cattle with SCCs of the UDT. Three animals had leukopenia and one had neutrophilia. The mean lymphocyte count (and standart deviation) of mild, moderate and severe papillomatosis was 5,395(±1,696)/mm³, 4,560(±1,561)/mm³ and 5,007(±1,786)/mm³, respectively. There were no statistically significant differences between the intensity of alimentary papillomasis, age and the lymphocyte count. Immunosupression by lymphopenia was a sporadic finding (three cases) in the present study. The results demonstrate that the persistence of alimentary papilomatosis in spontaneous cases of bracken fern-related SCCs of the UDT is not related to lymphopenia and it may be related to other immunological factors.(AU)