Clinicopathological characteristics and papillomavirus types in cutaneous warts in bovine.

Thirty-one bovine cutaneous warts were submitted to macroscopic and histological analyses and to molecular analyses to partial amplification and sequencing of the L1 gene of bovine papillomavirus (BPV). Viral types detected were BPV1 (52%), BPV2 (29%), BPV6 (16%) and BPV10 (3%). BPV2 had lower frequency in papilloma in comparison to that in fibropapilloma (p = 0.002).

Nasal swab real-time PCR is not suitable for in vivo diagnosis of bovine tuberculosis / A PCR em tempo real de swab nasal não é adequada para o diagnóstico in vivo de tuberculose bovina

Bovine tuberculosis (bTB) is a zoonosis causing economic losses and public health risks in many countries. The disease diagnosis in live animals is performed by intradermal tuberculin test, which is based on delayed hypersensitivity reactions. As tuberculosis has complex immune response, this test has limitations in sensitivity and specificity. This study sought to test an alternative approach for in vivo diagnosis of bovine tuberculosis, based on real-time polymerase chain reaction (PCR). DNA samples, extracted from nasal swabs of live cows, were used for SYBR® Green real-time PCR, which is able to differentiate between Mycobacterium tuberculosis and Mycobacterium avium complexes. Statistical analysis was performed to compare the results of tuberculin test, the in vivo gold standard bTB diagnosis method, with real-time PCR, thereby determining the specificity and sensitivity of molecular method. Cervical comparative test (CCT) was performed in 238 animals, of which 193 had suitable DNA from nasal swabs for molecular analysis, as indicated by amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and were included in the study. In total, 25 (10.5%) of the animals were CCT reactive, of which none was positive in the molecular test. Of the 168 CCT negative animals, four were positive for M. tuberculosis complex at real time PCR from nasal swabs. The comparison of these results generated values of sensitivity and specificity of 0% and 97.6%, respectively; moreover, low coefficients of agreement and correlation (-0.029 and -0.049, respectively) between the results obtained with both tests were also observed. This study showed that real-time PCR from nasal swabs is not suitable for in vivo diagnosis of bovine tuberculosis; thus tuberculin skin test is still the best option for this purpose.(AU)

A tuberculose bovina (bTB) é uma zoonose que causa perdas econômicas e riscos à saúde pública em muitos países. O diagnóstico da doença em animais vivos é realizado pelo teste intradérmico da tuberculina, que é baseado em reações de hipersensibilidade tardia. Como a tuberculose tem resposta imunológica complexa, este teste tem limitações em termos de sensibilidade e especificidade. Este estudo procurou desenvolver uma abordagem alternativa para o diagnóstico in vivo da tuberculose bovina, com base na reação em cadeia da polimerase (PCR) em tempo real. As amostras de DNA, extraídas de suabes nasais de vacas vivas, foram usadas para PCR em tempo real com SYBR® Green, capaz de diferenciar os complexos Mycobacterium tuberculosis e Mycobacterium avium. A análise estatística foi realizada para comparar os resultados de teste de tuberculina, padrão ouro para o diagnóstico in vivo da bTB, com PCR em tempo real, determinando-se assim a especificidade e sensibilidade do método molecular. O teste cervical comparativo (TCC) foi realizado em 238 animais, dos quais 193 tiveram DNA dos suabes nasais adequados para análise molecular, como indicado pela amplificação do gene gliceraldeído-3-fosfato-desidrogenase (GAPDH), e foram incluídos no estudo. No total, 25 (10,5%) animais foram reativos no TCC, dos quais nenhum foi positivo no teste molecular. Dos 168 animais negativos no TCC, quatro foram positivos para o complexo M. tuberculosis na PCR em tempo real a partir dos suabes nasais. A comparação destes resultados gerou valores de sensibilidade e especificidade de 0% e 97,6%, respectivamente; além disso, baixos coeficientes de concordância e correlação (-0,029 e -0,049, respectivamente) entre os resultados obtidos com ambos os testes também foram observados. Este estudo mostrou que a PCR em tempo real a partir de suabes nasais não é adequada para o diagnóstico in vivo da tuberculose bovina; portanto, o teste da tuberculina ainda é a melhor opção para este fim.(AU)

Versican expression in myoepithelial cells from carcinomas in canine mixed mammary tumors.

The matrix of canine mixed mammary tumors (CMMTs) consists of proliferating spindle cells of possible myoepithelial origin, as well as myxomatous tissue, cartilage matrix and/or bone. Among the multiple components of this tumor extracellular matrix, versican probably plays a prominent role due to its importance in tumor progression, cell proliferation and differentiation. However, there are few data related to a possible association between versican expression and the state of myoepithelial cell differentiation in CMMTs. Using immunohistochemistry and histochemistry, the objective of this study was to evaluate the expression of versican, sulfated proteoglycans and mucopolysaccharides in myoepithelial cells at different stages of differentiation and to explore a potential relationship with p63 and α-smooth muscle actin (SMA) expression. A significant difference in versican expression was observed among the different stages of myoepithelial cell differentiation with an inverse correlation between versican and p63/SMA expression. These results suggest that at an early stage of proliferation, myoepithelial cells acquire a phenotype consistent with a role in chondrogenesis. Moreover, myoepithelial cells showed an affinity for safranin and periodic acid-Schiff staining at different stages of proliferation supporting the myoepithelial origin of spindle cells from CMMTs.

Versican expression in canine carcinomas in benign mixed tumours: is there an association with clinical pathological factors, invasion and overall survival?

BACKGROUND: Components of the extracellular matrix have been studied in an attempt to elucidate the mechanisms involved in the biological behaviour of tumours. The presence of the proteoglycan versican has been strongly associated with cancer development and progression. However, relationship between versican expression and clinical pathological factors and overall survival has not been previously studied in veterinary medicine. Carcinomas in benign mixed tumours (CBMTs) are one of the most common malignant tumours in female canines and can serve as models for studies of tumour progression. The aim of this study was to evaluate the expression of versican in in situ and invasive carcinomatous areas of canine CBMTs and to evaluate possible associations of versican expression with other classic prognostic factors and overall survival. RESULTS: Clinical staging; histological grade determination; immunohistochemical staining for versican, E-cadherin and Ki-67; and confirmation of invasion areas by staining for p63 and smooth muscle α-actin (α-SMA) were performed on 49 canine cases of CBMT. Tumour invasion was considered when suspicious Haematoxylin-Eosin (HE)-stained areas showed a total loss of α-SMA and p63 immunoreactivity. Versican immunoreactivity was less intense in the areas adjacent to the in situ carcinomatous regions, compared to invasive regions, which showed extensive and strong staining. CONCLUSIONS: Our data reveal that in canine CBMTs, versican expression differs significantly between invasive and in situ areas, suggesting a role for this molecule in tumour progression. Although a direct relationship exists between versican and invasiveness, our results indicate that the isolated evaluation of this proteoglycan does not represent an independent prognostic factor in canine CBMTs.

Evaluation of adverse effects in tamoxifen exposed healthy female dogs.

BACKGROUND: Mammary tumors are among the most frequent neoplasms in female dogs, but the strategies employed in animal treatment are limited. In human medicine, hormone manipulation is used in cancer therapy. Tamoxifen citrate is a selective inhibitor of oestrogen receptors and exerts a potent anti-oestrogen effect on the mammary gland. The aim of this study was to evaluate the adverse effects when exposing healthy female dogs to tamoxifen. METHODS: Tamoxifen was administered for 120 days at a dose of 0.5 or 0.8 mg/kg/day to either intact or spayed female dogs. The effects were assessed through clinical examination, haematology, serum biochemistry, ophthalmology and bone marrow aspirate examination. Ovariohysterectomy was performed and the uterus examined by histopathology. RESULTS: Vulva oedema and purulent vaginal discharge developed with 10 days of tamoxifen exposure in all groups. Pyometra was diagnosed after around 90 days of exposure in intact females with frequencies increasing during the following 30 days of exposure. Up to 50% of dogs within the groups developed retinitis but none of the dogs had signs of reduced visual acuity. The prevalence of retinitis in each group was similar after 120 days of exposure. Haematological, biochemical and bone marrow changes were not observed. Due to the high risk of developing pyometra after prolonged exposure to tamoxifen, only spayed animals should be given this medication. CONCLUSIONS: A dose of 0.8 mg tamoxifen/kg body weight/day is recommended when treating tamoxifen-responsive canine mammary tumors. Due to the high risk of developing pyometra, ovariohysterectomy is recommended.

A simple ductal mammary papilloma in a male maned wolf (Chrysocyon brachyurus).

A 1-cm-diameter nodule was identified in the left inguinal mammary gland of a 9-year-old male maned wolf (Chrysocyon brachyurus). The mass was surgically excised and examined histologically. Microscopically, the neoplasm consisted of papillary proliferations of epithelial cells on well-defined fibrovascular stalks. A myoepithelial layer was located between the single layer of epithelial cells and the fibrovascular stalk. This histologic appearance was compatible with a diagnosis of simple ductal mammary papilloma. Immunohistochemical staining was positive for p63, cytokeratins AE1/AE3, and estrogen receptors. The clinical and histologic observations in the present case indicate that male maned wolves may develop mammary tumors that are similar to those observed in domestic dogs and humans.

An assessment of the clonality of the components of canine mixed mammary tumours by mitochondrial DNA analysis.

The aim of this study was to investigate if mutations in the mitochondrial DNA (mtDNA) D-loop fragment control region of canine mammary mixed tumours could be used as clonal markers that identified the cell population of origin. Ten benign mixed mammary tumours and nine carcinomas arising from benign mixed tumours were microdissected and DNA from epithelial and mesenchymal tumour cells and from normal mammary tissue was examined for sequence variations in a fragment of the hypervariable control region. Identical sequence variants in both the epithelial and mesenchymal components (as well as in the corresponding normal tissue) were found in 80% of the benign mixed tumours and in 89% of the carcinomas arising from benign mixed tumours suggesting a shared clonal origin. The distinctive sequence alterations identified in the epithelial and mesenchymal components of 15.8% of all 19 tumours examined, suggests the possibility that a minority of mammary tumours are polyclonal in origin or that early clonal divergence occurs. Increased mutation within the mtDNA D-loop fragment of mixed tumour components was not observed.

Optimization of methods to assess mitochondrial DNA in archival paraffin-embedded tissues from mammary canine tumors / Otimização dos métodos para avaliar o DNA mitocondrial obtido a partir de tumores mamários caninos incluídos em parafina

In this study we describe the alterations used to extract and amplify mitochondrial desoxyribonucleic acid (DNA) from formalin-fixed paraffin-embedded samples of canine mammary tumors. The epithelial and mesenchymal components (chondromyxoid and chondroid) of each tumor, as well as the normal mammary gland tissues, were manually microdissected from 19 mixed canine mammary tumors (10 benign mixed tumors and nine carcinomas arising in mixed tumors). DNA was extracted by Invisorb® Spin Tissue Mini Kit, with protocol changes proposed by the manufacturer. A 273-bp fragment was amplified by polymerase chain reaction (PCR) and submitted to automatic sequence analysis. The fragment was successfully analyzed in 100 percent of the samples. However, an additional lysis step, the reduction of volume in buffer solutions and PCR, a higher annealing temperature and an increase in the number of PCR cycles were required. The initial PCR products were diluted and re-amplified in six samples so that they could be successfully analyzed.

A presente comunicação descreve as modificações usadas para extrair e amplificar o DNA mitocondrial obtido de amostras de tumores mamários caninos fixados em formol tamponado a 10 por cento e incluídos em parafina. Os componentes epiteliais e mesenquimais (condromixóide e condróide), bem como a mama normal adjacente, foram microdissectados manualmente de 19 tumores mamários (10 tumores mistos benignos e nove carcinomas em tumores mistos). O DNA foi extraído utilizando-se o Invisorb® Spin Tissue Mini Kit com modificações do protocolo proposto pelo fabricante. Um fragmento de 273-pb foi amplificado por reação em cadeia da polimerase (PCR) e seqüenciado em seqüenciador automático. O fragmento foi analisado em 100 por cento das amostras, entretanto modificações como lise adicional, redução do volume das soluções de extração e PCR, aumento da temperatura de anelamento e do número de ciclos de amplificação foram necessárias. Em seis amostras os produtos iniciais de PCR foram diluídos e reamplificados para obtenção de sucesso.