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Rabies is an encephalitis caused by rabies virus, whose transmission occurs upon contact with infected animals' saliva. The diagnosis is usually performed post-mortem through a direct fluorescent antibody test (DFAT). If the DFAT results are negative, they must be confirmed with an isolation test, usually the mouse inoculation test (MIT), which implies the suffering and death of the animals, high costs and most importantly, up to 28 days to confirm a negative result. Another issue related to rabies diagnosis is the sample collection and storage, which is critical for the rabies virus' RNA genome. Thus, this study aimed to evaluate (i) reverse transcriptase polymerase chain reaction (RT-PCR) and Rabies Tissue Culture Infection Tests (RTCIT) in comparison to DFAT and MIT and (ii) FTA

The objective of this study was to evaluate the occurrence of antibodies against Leptospira spp in horses used by communities of informal waste collectors who use horse-drawn cartsin Guaíba, Rio Grande do Sul. Additionally, interviews were conducted with owners to assess the characteristics of the horses, their environment, and work. Serological testing for Leptospira spp. was performed on blood samples collected from 54 horses using the microscopic agglutination test. Results showed that 50.0% of the horses were seropositive for Leptospira spp and the most frequent serogroup was LeptospiraCanicola (88. 8%), followed by Leptospira Pyrogenes (11.1%), Leptospira Bataviae (7.4%), Leptospira Hebdomadis (7.4%), Leptospira Icterohaemorrhagiae (3.7%), Leptospira Australis (3.7%), Leptospira Pomona (3.7%) and Leptospira Copenhageni (3.7%). There were no statistically significant associations between seropositivity for Leptospira spp and age, gender, body score, water source, type of food storage, cohabitation with dogs, rodent infestation, residential flooding, and waste removal services. The prevalence of the Canicola serogroup, as well as a large number of dogs in close relationship with thehorses and the waste collectors, draws attention to the potential of these animals to become a source of Leptospira spp. infection in humans and other animals.

Bovine tuberculosis (bTB) is a zoonotic disease caused by Mycobacterium tuberculosis var. bovis, for which the definitive diagnosis is accomplished by bacterial isolation, which has biosafety issues and requires long time. Thus, diagnostic methods with potential to be faster and more efficient can represent an advance in bTB epidemiological knowledge and decrease exposure to M. tuberculosis var. bovis. This study aimed to validate a molecular test for bTB post-mortem diagnosis, as a strategy to reduce waste in bovine production. A total of 185 tissues from animals of infected herds or with suspected lesions at abattoir were evaluated through bacterial isolation, PCR and histopathology. PCR and histopathology showed sensitivities of 45.1% and 71.2%, respectively, and specificities of 83.3% and 83.0%, respectively, when compared to bacterial isolation. The combination of both tests resulted in enhanced specificity and positive predictive values.Therefore, PCR in conjunction with histopathology may be used as screening, in which concordant results can be considered conclusive, and discordant results may be submitted to bacterial isolation.

Mammary neoplasms with malignant mesenchymal components are not common in female dogs, and they are poorly understood. As such, this study aimed to describe the clinical presentation, histological findings, and the COX-2 immunohistochemical expression of mammary neoplasms in female dogs with malignant mesenchymal components, as well as verify the relationships between the different neoplasm types and these aspects. We selected 41 female mammary neoplasms (23 carcinosarcomas, 16 sarcomas, and 2 sarcomas in mixed tumors). Medical records were reviewed to obtain clinical data. Subsequently, histological slides were analysed to establish histological parameters, and immunohistochemistry was used to assess the expression of COX-2 receptors. Carcinosarcomas and sarcomas developed as large tumours, mainly in the abdominal and inguinal mammary glands, with frequent intratumoral necrosis and a low frequency of nodal metastasis. Fifty-eight percent of the cases of malignant mesenchymal proliferation were identified as osteosarcomatous, and 24.5% chondrosarcomatous and fibrosarcomastous each. The osteosarcomatous pattern was the most predominant type in sarcomas and carcinosarcomas, and was the only one that resulted in vascular invasion, regional lymph node metastases, and higher histologic grades. High COX-2 expression was detected in 10% of the carcinosarcomas and 25% of the sarcomas. In conclusion, sarcomas and carcinosarcomas showed similar results regarding the clinical and pathological aspects. Discovering carcinosarcomas and sarcomas with high COX-2 expression suggests that, in some cases, these neoplasms may respond to therapy with COX-2 inhibitors.

Wild boar (Sus scrofa) is an exotic invasive species in Brazil and may be a reservoir for several pathogens, including those related to the porcine respiratory disease complex (PRDC), a critical infectious disease in pig production. The objective of this study was to investigate viral and bacterial pathogens related to PRDC in free-living wild boars from Brazil. Eighty animals were examined in search of genomes of porcine circovirus 2 (PCV2), Torque teno Sus virus 1a (TTSuV1a) and 1b (TTSuV1b), Influenza A virus (IAV), Actinobacillus pleuropneumoniae, Glaesserella parasuis, Pasteurella multocida, and Mycoplasma hyopneumoniae. The results demonstrated that 57.5% (46/80) of the animals had at least one detected pathogen, and 11.3% of them (9/80) were co-infected. TTSuV1a was the most prevalent genome, for which risk factors were associated with increased contact between wild boars and other animals. The other pathogens were detected at much lower frequencies or not detected (M. hyopneumoniae and IAV). An additional IAV serology search identified H1N1pdm09 antibodies in 35.5% (16/45) of the wild boars, bringing concern related to public health. In conclusion, wild boars are infected with pathogens that cause swine diseases, so their eventual contact with domestic pigs might risk animal production in Brazil.

Animal tuberculosis (aTB) is a zoonotic disease characterized by granulomatous lesions on affected tissues, occurring as a consequence of immunological response to infection. Mycobacterium bovis, the main causative agent of aTB, was investigated in Brazilian wild boars with 37.7 % (29/77) positivity. Among these animals, most had no macroscopic tuberculosis-like lesions (89.6 %; 26/29). The existence of co-infections, which may alter an individual's immune response to an immunological challenge, could influence the formation of tuberculosis lesions. Therefore, we investigated Metastrongylus sp. and aTB co-infection to seek an explanation for the absence of macroscopic lesions in aTB. Of the tested animals, 77.9 % (60/77) had Metastrongylus sp., however, there was no association between its occurrence and the pattern of aTB lesions. The absence of tuberculous lesions in infected animals is worrisome, especially to hunters who handle their carcasses, potentially assuming that the animal is healthy. Studies evaluating other possibilities that can explain the absence of lesions in infected animals should be carried out to better understand these findings.

Wild boars (Sus scrofa) are susceptible to mycobacterial infections, including tuberculous and non-tuberculous mycobacteria. Recently, Mycobacterium spp. infections were described in Brazilian wild boars, which can act as bacterial reservoirs. Here, we aim to characterize 15 Mycobacterium spp. isolates from Brazilian wild boars' tissues through partial sequencing of the heat shock protein 65 (hsp65) gene and phylogenetic analysis. The isolates were classified as M. tuberculosis (33.3%), M. colombiense (33.3%), M. avium subsp. hominissuis (13.3%), M. parmense (13.3%) and M. mantenii (6.66%). The isolates classified as M. tuberculosis were confirmed as variant bovis by PCR. At phylogenetic analysis some isolates formed separated clades, indicating genetic variability. Different Mycobacterium species were recovered from wild boars circulating in Brazil, including mycobacteria associated to zoonotic infections, such as M. tuberculosis. In addition, this is the first report in Brazilian wild boars on M. mantenii and M. parmense detection, two recently described pathogenic mycobacteria. However, the isolates’ genetic diversity—i.e. identities lower than 100% when compared to reference sequences—suggests that other genotyping tools would allow a deeper characterization. Nonetheless, the reported data contributes to the knowledge on mycobacterial infections in wild boars from Brazil.

Bovines and equines are the domestic animals with the highest incidence of rabies in Brazil. This study evaluated the data input in testing request forms for rabies in herbivores accompanying samples sent to a certified laboratory of rabies diagnosis in the state of Rio Grande do Sul, Brazil. Information was obtained systematically from all forms sent to the laboratory in 2013 and 2014 using a collection instrument that listed variables about the kind of form used, municipality of origin, description of the suspected animal, clinical signs, sample collection, and conservation procedure. The 11 most relevant variables (municipality of origin, geographic coordinates, species, gender, breed, age, herd size, clinical signs, persistence of clinical signs, biological material, and sample conservation) were assessed for the quality of the data entered, and percentage data input was calculated per form. In total, 603 forms were sent to the laboratory. The most used form was the FORM SN, (82.0%, 497/603). “Excellent” and “good” data inputs were observed only for one variable describing the location of suspected rabies cases or outbreaks and animals (species, gender, biological material). Quality of data input of variables describing geographic coordinates, age, breed, herd size, clinical signs, persistence of signs, and sample conservation method varied between “average” and “poor”. The data input in testing request forms for herbivores are not dully supplied in suspected cases neurological syndromes, pointing to the need to increase awareness and improve training of field staff. RESUMO: Bovinos e equinos são os animais domésticos com maior incidência de raiva no Brasil. Este estudo avaliou formulários de solicitação de testes para raiva em herbívoros acompanhando amostras enviadas a um laboratório certificado de diagnóstico de raiva no estado do Rio Grande do Sul, Brasil. As informações foram obtidas sistematicamente de todos os formulários enviados ao laboratório em 2013 e 2014 utilizando um instrumento de coleta que selecionou variáveis sobre o tipo de formulário utilizado, município de origem, descrição do animal suspeito, sinais clínicos, coleta de amostra e procedimento de conservação. As 11 variáveis mais relevantes (município de origem, coordenadas geográficas, espécie, gênero, raça, idade, tamanho do rebanho, sinais clínicos, persistência de sinais clínicos, material biológico e conservação da amostra) foram avaliadas quanto à qualidade dos dados inseridos, e os dados percentuais foram calculados por formulário. No total, 603 formulários foram enviados para o laboratório. O formulário mais utilizado foi o FORM SN, (82,0%, 497/603). Dados classificados como “excelentes” e “bons” foram observados apenas para uma variável que descreve a localização de casos suspeitos de raiva ou surtos e animais (espécie, gênero, material biológico). A qualidade da entrada dos dados das variáveis que descrevem coordenadas geográficas, idade, raça, tamanho do rebanho, sinais clínicos, persistência de sinais e método de conservação da amostra variou entre “regular” e “ruim”. A qualidade dos dados dos formulários de solicitação de testes para herbívoros não foi devidamente fornecida em casos suspeitos de síndromes neurológicas, apontando para a necessidade de aumentar a conscientização e melhorar o treinamento do pessoal de campo.

Canine mammary neoplasms (CMNs) are the most frequent lesions and in female dogs. However, studies correlating pathological criteria with clinical evolution in female dogs with mammary neoplasms are scarce. The present study aims to present epidemiological, clinical-pathological and overall survival data to help establish the prognosis and understand the biological behavior of CMNs. A total of 1539 cases were included (85% malignant and 13% benign). Tumor size was an important prognostic factor and was associated with overall patient survival (P< 0.0001). Most dogs diagnosed with malignant neoplasms (83%) had initial clinical staging, although 17% had regional or distant metastases at the time of diagnosis and lower overall survival (P< 0.0001). Carcinoma in mixed tumor was the most frequent histological type and had a better prognosis. Solid carcinomas, micropapillary carcinomas and carcinosarcomas were considered histological types with aggressive biological behavior and were associated with a worse prognosis and lower overall survival (P< 0.0001). RESUMO Neoplasias mamárias caninas (NMCs) são as lesões mais frequentes em cadelas. Estudos que correlacionam os critérios patológicos com a evolução clínica em cadelas com neoplasias mamárias são escassos. Este estudo objetiva apresentar dados epidemiológicos, clínico-patológicos e de sobrevida global fornecendo informações que auxiliam a estabelecer o prognóstico e a compreender o comportamento biológico de NMCs. Foram incluídos 1539 casos, 85% malignos e 13% benignos. O tamanho tumoral foi um importante fator prognóstico, sendo associado com a sobrevida global das pacientes (P

Bovine tuberculosis (bTB) is a zoonosis caused mainly by Mycobacterium bovis that affects domestic and wild animals. In Brazil, there are no epidemiological studies on tuberculosis in wild animal populations and their possible role in the disease maintenance in cattle herds; thus, the aim of this study was to evaluate the occurrence of tuberculosis in wild boars in Rio Grande do Sul, southern Brazil. Tissue samples of animals hunted under government consent were submitted to histopathology and M. bovis polymerase chain reaction (PCR) as screening tests; the positive samples were subsequently submitted to bacterial isolation, the gold standard diagnosis. Eighty animals were evaluated, of which 27.9% and 31.3% showed histopathological changes and M. bovis genome presence, respectively. Moreover, 23.8% of the animals had at least one organ with isolates classified as Mycobacterium tuberculosis complex (MTC). Three hunting points were risk factors for positive results on screening tests. This study shows the occurrence of tuberculosis in a wild boars' population, and raise the possibility of these animals to play a role as disease reservoirs in southern Brazil. These results may help to improve the Brazilian tuberculosis control programme, as well as elucidate the circulation of mycobacteria in this country.

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). ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00121-2) contains supplementary material, which is available to authorized users.

Highlights • Mixed tumors are common lesions in female dogs and present variable biological behavior. • Carcinomas in mixed tumors are neoplasms that present good biological behavior. • Even at an advanced stage, female dogs with carcinomas in mixed tumors exhibit a longer survival compared with bitches with carcinosarcomas. • Female dogs with carcinosarcomas undergoing surgery only presented a lower overall survival rate., Mixed tumors are the most frequent mammary gland neoplasms in bitches; however, studies that thoroughly describe their clinicopathological data, treatment approaches, and the survival of bitches with mixed tumors are scarce. This study evaluated the epidemiological and clinicopathological data, prognostic factors, and therapeutic approaches for bitches with mixed tumors. In all, 162 benign mixed tumors, 682 carcinomas in mixed tumors, and 60 carcinosarcomas were included. Regarding tumor size, T3 lesions were predominantly associated with carcinosarcomas, while T1 and T2 lesions occurred more frequently in benign mixed tumors and in carcinomas in mixed tumors. Based on clinical staging, most bitches with benign mixed tumors presented with stage I tumors; 92% of bitches with carcinomas in mixed tumors presented with stage I–III tumors, while 8% presented with stage IV–V tumors; and 70% of bitches with carcinosarcomas presented with stage I–III tumors, while 30% presented with stage IV–V tumors. Surgery was curative for bitches with benign mixed tumors and for those with stage I–III carcinomas in mixed tumors. Combination therapy in bitches with carcinomas in mixed tumors (IV–V) and carcinosarcomas resulted in a higher overall survival compared with bitches who underwent surgery only. Carcinosarcomas presented higher relapse rates and distant metastases than carcinomas in mixed tumors did.

The aim of the present prospective-retrospective study was to evaluate the response of high-risk canine mast cell tumours (MCTs) to tyrosine kinase inhibitors (TKIs) and to correlate this with prognostic factors. A total of 24 dogs presented with macroscopic cutaneous MCTs at disease stage II or III, and therefore, at high-risk of associ ated mortality, were included in the study and treated with masitinib (n=20) or toceranib (n=4). A total of 12/24 dogs achieved an objective response and the overall survival (OS) for all subjects was 113 days. Dogs responding to treatment had a significant increase in OS compared to non‑responders (146.5 days vs. 47 days, P=0.02). Internal tandem duplications in exon 11 of the c‑kit gene were identified in 6/24 cases. Ki67, KIT immunolabelling and c-kit mutation did not provide information regarding prognosis or prediction of response to TKIs in this population. Initial response to TKIs appears to be the most reliable prognostic factor for survival duration. O objetivo do presente estudo prospectivo-retrospectivo estudo foi avaliar a resposta do mastro canino de alto risco tumores celulares (MCTs) a inibidores de tirosina quinase (TKIs) e correlacionar isso com fatores prognósticos. Um total de 24 cães apresentaram MCTs cutâneos macroscópicos em doença em estágio II ou III e, portanto, com alto risco de mortalidade associada, foram incluídos no estudo e tratados com masitinibe (n=20) ou toceranibe (n=4). Um total de 12/24 cães alcançou uma resposta objetiva e a sobrevida global (OS) para todos os sujeitos foi de 113 dias. Cães que respondem ao tratamento teve um aumento significativo no OS em comparação com os não respondedores (146,5 dias vs. 47 dias, P=0,02). Duplicações em tandem internas no exon 11 do gene c‑kit foram identificados em 6/24 casos. Ki67, A imunomarcação KIT e a mutação c-kit não forneceram informações sobre prognóstico ou previsão de resposta a TKIs nesta população. A resposta inicial aos TKIs parece ser o fator prognóstico mais confiável para a duração da sobrevida.

bovine TB (bTB) is an infectious disease – caused by Mycobacterium tuberculosis variant bovis – that affects several species of animals. The World Health Organization (WHO) classifies bTB as a neglected zoonotic disease, which makes control programmes essential for protecting not only animals but also people.1 Most bTB control and eradication programmes are based on the diagnosis and slaughter of infected animals. However, the efficacy of this approach is hampered by factors such as the existence of wildlife reservoirs, which can act as a source of infection for cattle herds.2 Among the potential bTB reservoir species identified, wild boar ( Sus scrofa ) are important players in Mediterranean ecosystems.3 Their importance as reservoirs is also being studied in other areas of the world. Measures to control bTB in wildlife populations have been implemented in several countries, with the strategies used depending on knowledge of the reservoir species involved and the epidemiological characterisation of the disease.4-6 In most strategies …

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. RESUMO: 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.

Biomolecular evidence has shown that ductal carcinoma in situ(DCIS) may develop into invasive carcinoma of the canine mammary gland, and mutations in proto-oncogenes HER2 and EGFR; two members of the family of epidermal growth factor receptors, may be involved in this process. The purpose of this study was the characterization of the immunohistochemical expression of the EGFR and HER2 proteins in the process of neoplastic transformation, supposedly present in ductal carcinomas in situin canine mammary glands. Fifteen cases of DCIS were evaluated, with a higher expression of HER2 and EGFR being observed in low-grade carcinomas when compared with high-grade neoplasms, and with a high positive statistical correlation in the latter. Results suggest that aggressive tumors tend to lose the expression of EGFR and HER2 simultaneously. The loss of the expression of these markers may be related to the process of neoplastic progression in canine mammary tumors. Evidências biomoleculares sugerem que o carcinoma ductal in situ (CDIS) pode progredir para carcinoma invasor na mama canina e que mutações nos proto-oncogenes HER-2 e EGFR, dois membros da família de receptores para fatores de crescimento epidérmicos, podem estar envolvidas neste processo. A partir disso, este trabalho teve por objetivo caracterizar a expressão imuno-histoquímica das proteínas EGFR e HER-2 no processo de transformação neoplásica supostamente presente em carcinomas ductais in situ da glândula mamária canina. Foram avaliados 15 casos de CDIS, sendo observada maior expressão de HER-2 e EGFR em carcinomas de baixo grau em comparação às neoplasias de alto grau, com correlação estatística positiva alta nestes últimos. Os resultados sugerem que tumores mais agressivos tendem a perder, simultaneamente, a expressão de EGFR e HER-2. A perda na expressão desses marcadores pode estar envolvida no processo de progressão neoplásica em tumores mamários caninos.

O presente estudo apresenta o comportamento do gene HER2, a partir do uso da técnica de hibridização cromogênica in situ, em hiperplasias ductais atípicas associadas a carcinomas mamários caninos positivos para HER2. Aparentemente, uma fraca expressão da proteína HER2 foi observada nas hiperplasias ductais atípicas, bem como uma ausência de amplificação do seu gene codificador nessas hiperplasias e nos carcinomas mamários associados. O comportamento da proteína HER2 e do seu gene em carcinomas mamários caninos é similar ao observado em alguns subtipos histológicos de tumores mamários humanos, e a ausência dessas alterações sugerem que esse gene poderia aparentemente não estar envolvido com os estágios iniciais de proliferação celular atípica.

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.

RESUMO O objetivo do presente estudo foi descrever o estadiamento do câncer de mama em mulheres admitidas para atendimento em um centro de referência em oncologia localizado em Governador Valadares, Minas Gerais. Os prontuários de 257 pacientes atendidas entre novembro de 2009 a novembro de 2010 foram revisados para obtenção de dados do estadiamento e de características gerais das pacientes. As pacientes apresentavam em média 54,5 anos de idade, eram principalmente pardas (62,0%) e casadas ou com união estável. Predominaram pacientes atendidas pelo Sistema Único de Saúde (94,4%), sem história familiar de câncer de mama (78,2%) e com índice de massa corporal acima do ideal (57,5%). Os tumores in situ corresponderam a 2,3% dos casos, tumores no estádio I a 26,4%, no estádio II a 38,5%, no estádio III a 22,9% e estádio IV a 9,7%. Não houve relação significativa entre os estádios inicial ou precoce e a idade, cor da pele, estado civil, IMC e história familiar de câncer de mama. O percentual de casos diagnosticados em estádio tardio ainda é alto quando comparado com o encontrado em países desenvolvidos, o que aponta para necessidade de políticas que facilitem a detecção precoce da doença na região.

Mixed tumors are among the most frequent mammary neoplasms in female dogs. Some of these tumors present bone marrow associated with the newly formed osseous tissue, characteristic of myeloid metaplasia.To evaluate the occurrence of these lesions in a series of mixed tumors, and determine its histomorphological characteristics.In total, 384 canine mammary mixed tumors from 289 animals have been reviewed. The lesions were classified according to the presence of osseous metaplasia associated with myeloid metaplasia or extramedullary hematopoiesis. Myeloid metaplasia characterization was determined from the morphological characteristics and organization of the cells and adjacent tissues. Cytoplasmic staining for CD31 and Factor VIII were used as a criterion to confirm the presence of blood vessels and megakaryocytes, respectively.The 384 cases included 206 benign and 178 carcinomas in mixed tumors. Osseous metaplasia was present in 16.1% and calcified areas exclusively in 3.1% lesions. Among all osseous metaplasia, 33.9% presented some type of extramedullary hematopoiesis, of which 71.4% were classified as myeloid metaplasia and 28.6% as extramedullary hematopoiesis. Myeloid metaplasia cases consisted of 67% benign mixed tumors and 33% carcinomas in mixed tumors. CD31 and Factor VIII expression occurred in all myeloid metaplasia, confirming the presence of blood capillaries and megakaryocytes. Myeloid metaplasia was observed in 24% of mixed tumors containing osseous metaplasia and in 4% of all mixed tumors analyzed.Despite the low frequency of this lesion, additional studies are needed to understand the implications of myeloid metaplasia in canine mammary mixed tumors.

Static cytometric analysis of DNA content was performed on formalin-fixed paraffin wax-embedded samples of 56 canine mammary neoplasms (23 benign and 33 malignant) and histology, patient age and immunohistochemical markers were compared between diploid and aneuploid tumours. 10 benign tumours (43.47%) and 16 malignant (48.48%) were aneuploid. No association was found between age and ploidy (P > 0.05). Aneuploidy was not related to age or tumour malignancy (P < 0.05). The expression of the following markers: progesterone receptor, MIB-1, CD-31, p53, c-erbB2, and cyclin D1 did not show significant differences between diploid and aneuploid tumours (P < 0.05). Epithelial and mesenchymal components of benign mixed tumours, complex adenomas and carcinomas in mixed tumours had an identical DNA content in 74.0% of cases suggesting a common cell origin of both components.

Background Benign mixed tumours (BMTs) are frequently found in the mammary glands of female dogs, but the factors determining malignant transformation in these tumours are unknown. Objective To evaluate the expression of the oncoproteins, human epidermal growth factor receptor 2 (HER-2) and epidermal growth factor receptor (EGFR), in 46 carcinomas in BMTs (CBMTs) and to verify their possible association with the malignancy of the tumours. Methods Immunohistochemical expression was analysed in benign and malignant components separately, and then compared with 74 cases of BMTs. Results Among the CBMTs, positivity for HER-2 was found in the benign histological component of 4.3% (2/46), in the malignant epithelial non-invasive component of 14.8% (4/27) and in the malignant invasive epithelial component of 13.6% (6/44) of cases. Two of the 24 (8.3%) BMTs were positive for HER-2. There was no relationship between HER-2 and the tumour components. There was no significant difference between BMTs and CBMTs. Positivity for EGFR was found in the benign component of 17.4% (8/46) of the CBMTs, in the malignant epithelial non-invasive component of 40.7% (11/27%) and in the invasive epithelial malignant component of 45.4% (20/44). EGFR positivity was significantly associated with the invasive component of CBMTs. Conclusion EGFR may contribute to malignant epithelial transformation of BMTs. In contrast, HER-2 overexpression may not be associated with the acquisition of a malignant epithelial phenotype.

The correlation between microvessel density and mast cells density in canine mammary tumors was studied. Sixty-five samples of canine mammary tumors, being 24 benign and 41 malignant, were analyzed. The routine Toluidine Blue staining method was used to assess the mast cells. To evaluate angiogenesis, the immunohistochemical expression of CD31 was assessed. There was no significant difference in either mast cells (P=0.44) or microvessel density (P=0.77) between malignant and benign tumors. A positive correlation was observed between microvessel density and mast cells (r=0.39; P=0.011) in malignant tumors. These results suggest that mast cells may play a role in canine mammary malignant tumors development, promoting angiogenesis, similar to some tumors described in the human species Estimou-se a correlação entre a densidade de microvasos e a densidade de mastócitos em tumores mamários caninos. Sessenta e cinco amostras de tumores mamários caninos - 24 benignos e 41 malignos - foram analisadas, pela técnica rotineira de coloração com Azul de Toluidina para avaliação da densidade de mastócitos. Para a avaliação da angiogênese, foi utilizada a técnica de imunoistoquímica para expressão de CD31. Não foram observadas diferenças significativas de mastócitos (P=0.44) ou densidade microvascular (P=0.77) entre tumores malignos e benignos. A correlação entre densidade microvascular e densidade de mastócitos foi positiva (r=0,39; P=0,011) em tumores malignos. Estes resultados sugerem que os mastócitos podem exercer um importante papel no desenvolvimento de tumores mamários malignos caninos mediante promoção da angiogênese, similarmente a alguns tumores descritos na espécie humana

Dantrolene has been shown to be neuroprotective by reducing neuronal apoptosis after brain injury in several animal models of neurological disorders. In this study, we investigated the effects of dantrolene on experimental spinal cord injury (SCI). Forty-six male Wistar rats were laminectomized at T13 and divided in six groups: GI (n = 7) underwent SCI with placebo and was euthanized after 32 h; GII (n = 7) underwent laminectomy alone with placebo and was euthanized after 32 h; GIII (n = 8) underwent SCI with dantrolene and was euthanized after 32 h; GIV (n = 8) underwent SCI with placebo and was euthanized after 8 days; GV (n = 8) underwent laminectomy alone with placebo and was euthanized after 8 days; and GVI (n = 8) underwent SCI with dantrolene and was euthanized after 8 days. A compressive trauma was performed to induce SCI. After euthanasia, the spinal cord was evaluated using light microscopy, TUNEL staining and immunochemistry with anti-Caspase-3 and anti-NeuN. Animals treated with dantrolene showed a smaller number of TUNEL-positive and caspase-3-positive cells and a larger number of NeuN-positive neurons, both at 32 h and 8 days (P ≤ 0.05). These results showed that dantrolene protects spinal cord tissue after traumatic SCI by decreasing apoptotic cell death.

Mammary tumors are among the most common neoplastic processes in female dogs. Prostaglandin E2, the catalytic product of Cox-2, may promote tumor development and angiogenesis. It has been investigated in several human cancers and also correlated with the evolution of the disease. However, the clinical implications of tumor pathology require more investigation in veterinary medicine. Angiogenesis is essential for the growth and metastasis of major solid tumors and has been correlated with prognosis in human and canine breast cancer. The aim of this study was to evaluate Cox-2 expression and microvessel density in canine mammary carcinomas and to correlate them with overall survival of the animal. Cox-2 and angiogenesis were assessed by immunohistochemistry in 46 mammary carcinomas (19 ductal and 27 metaplastic) and in healthy mammary glands. To assess tumor angiogenesis, microvessel density (MVD) was determined by CD31 staining. Immunostaining revealed that 46/46 (100%) of the tumors were positive for Cox-2 and CD31, and there was no statistical difference among tumor types. Cox-2 protein expression correlated positively with CD31 staining ( r = 0.3742, P = .0104) but did not correlate significantly with tumor type. Longer overall survival was observed in metaplastic carcinomas ( P = .028), in tumors with low microvessel density ( P = .0002) and with low Cox-2 score ( P = .01). Our results demonstrate that increased microvessel density and increased Cox-2 expression were linearly related in the canine mammary tumors studied and were also related to worse prognosis and shorter overall survival. This suggests that Cox-2 inhibitors could be an alternative for the treatment and control of advanced neoplastic mammary disease in female dogs.

The presence of prorenin, renin, angiotensinogen, angiotensin-converting enzyme, angiotensin II (Ang II) and Ang II receptors in the ovary is suggestive of a functional ovarian renin-angiotensin system (RAS). In cattle, the expression of Ang II is greatest in large follicles, suggesting that it is important during follicular growth and maturation. The present study was designed to investigate the role of Ang II in bovine oocyte nuclear maturation. Bovine cumulus-oocyte complexes (COCs) were cultured with or without follicular cells and Ang II or saralasin (Ang II antagonist). In the absence of follicular cells, Ang II at 0, 10(-11), 10(-9) and 10(-7) M did not affect the percentage of oocytes reaching the germinal vesicle breakdown (GVBD), metaphase I (MI) and metaphase II (MII) stage after 7-h (41.3 +/- 4.3, 35.3 +/- 4.0, 31.3 +/- 9.7, 38.7 +/- 8.6), 12-h (31.6 +/- 7.0, 34.7 +/- 6.1, 31.7 +/- 5.3, 28.9 +/- 9.1; mean +/- S.E.M.) and 18-h (44.9 +/- 7.3, 58.4 +/- 8.4, 53.1 +/- 7.4, 44.9 +/- 7.3) of culture, respectively. Similarly, saralasin at 0, 10(-11), 10(-9) and 10(-7) M did not affect the percentage of oocytes reaching MII stage after 18-h of culture (37.6 +/- 7.4, 34.4 +/- 7.7, 30.0 +/- 10.8 and 31.2 +/- 5.1, respectively). The theca cells (MII = 22.9%) or medium conditioned with follicular cells (GV = 65.5%, MI = 23.6%) inhibited oocyte maturation; however, theca cells (MII = 35.5 +/- 4.9; P0.05) or medium conditioned with follicular cells (GV = 34.6%, MI = 52.7%; P0.01) were not able to inhibit nuclear maturation when Ang II (10(-11) M) was present in the culture system. Theca cells remained viable during the culture period when Ang II was present. Therefore, results supported the idea of a role of Ang II in blocking the inhibitory effect of theca cells on nuclear maturation of bovine oocytes.

Background: Rabies remains one of the most important zoonosis worldwide and in Brazil. In the Brazilian state of Rio Grande do Sul (RS) the disease is one of the main causes of death in bovine herds. The confirmation of suspected rabies cases upon clinical examination depends on specific laboratory diagnosis. The Instituto de Pesquisas Veterinárias Desidério Finamor (IPVDF) is the reference laboratory in RS to diagnose rabies. The objective of the present study was to present a spatial-temporal description of diagnostic procedures of rabies in cattle in the years 2011 to 2015 in RS.Materials, Methods & Results: The results of diagnosis procedures of rabies in cattle carried out in the Laboratory of Virology, IPVDF, between January 2011 and December 2015 were listed. Direct immunofluorescence (DIF) and biological assays for confirming rabies cases were used. Cattle population density per area of a municipality, as well as date, species involved, location of suspected case, and diagnosis were obtained from exams and from official rabies record, and input to a spreadsheet file. Spatial distribution maps of cattle rabies confirmed in laboratory and cattle population density per municipality were constructed using the software TerraView 4.2.2 (INPE). In the study period, 998 samples of brains of cattle presenting neurological symptoms compatible with rabies, of which 582 (58%) were confirmed in the laboratory. The frequency of positive cases on a yearly basis varied between 41 and 65%, with statistically lower number of positives in 2011 (P > 0.05). The annual incidence of rabies for a population of 10,000,000 bovines was 33, 88, 112, 108, and 79, respectively, for the years 2011 to 2015, in that order. A positive correlation was observed between the number of samples analyzed and the incidence of positive cases in the study period, and statistically significant difference in incidence between years (P > 0.05). Samples were from 175 municipalities, of which 114 (65%) had at least one confirmed rabies case and 61 did not have any. The five municipalities that sent most samples for analysis were Viamão (n = 66), Montenegro (n = 40), Camaquã (n = 39), Gravataí (n = 38), and São Lourenço do Sul (n = 37). The spatial distribution of rabies in cattle in a year did not follow a defined pattern, though most cases confirmed in the laboratory occurred in the greater Porto Alegre region and in southeast RS.Discussion: High incidence of rabies was observed in 2012 and 2013, and in the two following years the number of cases confirmed in laboratory remained high, compared with 2011. The high incidence of rabies cases confirmed in laboratory correlated with the number of samples analyzed. The fall in vaccine coverage during the study period may also have prompted the increase in suspected cases and in the number of samples examined in the laboratory. A large number of the municipalities presenting rabies cases in cattle was in the greater Porto Alegre region and in southeast RS, which are areas of high and intermediate risk for the disease, according to a previous epidemiological modeling study. The spatial distribution of positive cases was not associated with the cattle population density. The large number of rabies in cattle in RS underscores the importance of the disease and the high risk of exposure, both to humans and animals. The results presented herein may help define control and prevention actions against rabies in RS.

Surgery remains the treatment of choice for female dogs with mammary gland tumors. Chemotherapy is not commonly used as an adjuvant therapy. Cyclooxygenase 2 (COX-2) has been related to angiogenesis development in tumors, disease progression and worse prognosis. The aim of this prospective study was to compare overall survival periods of female dogs diagnosed with advanced mammary tumors submitted to different treatment protocols, including surgery, chemotherapy and cyclooxygenase inhibitors. Twenty-nine female dogs were evaluated and treated with four different protocols. The overall survival of patients with low COX-2 scores was longer when compared to patients with high COX-2 scores. Different proposed adjuvant treatments associated with surgery led to a statistically significant longer overall survival when compared to surgical treatment alone. Canine patients presenting malignant mammary gland neoplasms with advanced clinical staging should be submitted to complementary therapeutic medication based on clinical staging and immunophenotypical characteristics of the disease.

Mammary mixed tumours are the most frequent neoplasias in female dogs. In humans, mixed tumours are frequently found in the salivary glands and are known as pleomorphic adenomas. In addition to their histomorphologic similarities, mixed tumours and pleomorphic adenomas have the potential to become malignant and give rise to carcinomas in mixed tumours and carcinomas ex-pleomorphic adenoma, respectively. The factors associated with malignant transformation are still poorly known in the case of canine mixed tumours. However, this form of neoplasia tends to be associated with a better prognosis than other malignant histological types. This paper discusses the main features associated with female canine mammary mixed tumours.

Tumour size is considered one of the most important determinants of clinical staging in cancer patients. The aim of this study was to assess the value of tumour size as an indicator of the differentiation of mammary neoplasias in female dogs. The tumour, nodes metastates (TNM) system, based on primary lesion size, the extent of its dissemination to regional lymph nodes and the presence or absence of distant metastases, was applied to 120 female dogs diagnosed with mammary neoplasias. Paraffin blocks from 38 cases were selected and studied by immunohistochemical staining for prognostic and predictive markers of breast cancer. The Kaplan-Meier survival curve was estimated for 110 female dogs. Larger tumours (T3) were mostly malignant and showed lower expression of progesterone receptor and higher expression of cellular proliferation markers. Global survival time was shorter in female dogs with large tumour masses. This study highlights the importance of tumour size as a prognostic indicator of mammary neoplasias in female dogs.

The immunohistochemical expression of p63, DeltaNp63, and p53 was studied in mixed tumors of canine mammary glands (13 benign mixed tumors and 19 carcinomas arising from benign mixed tumors) to determine the role of p63 and its isoform DeltaNp63 in the development of mixed tumors, as well as to assess its relation with p53. P63 was expressed in myoepithelial cells of all benign mixed tumors and in 18 of 19 carcinomas in mixed tumors. The p63-negative carcinoma in mixed tumors was invasive, and a loss of p63 was detected in the other malignant tumors showing a discontinuous p63-stained myoepithelial layer. DeltaNp63 was expressed in all benign mixed tumors but only in p63-positive carcinomas in mixed tumors. Despite its positive correlation with p63 expression in carcinomas in mixed tumors (r = 0.8323, P < .00001), DeltaNp63 expression showed a decrease in benign tumors. Positivity for p53 was detected in 2 of 13 and 1 of 19 benign mixed tumors and carcinomas in mixed tumors, respectively. There was no correlation between p63 or DeltaNp63 and p53 expression. Our data support the notion that the decrease of p63 expression, in particular of its isoform DeltaNp63, seems to be an important factor in the development of carcinomas in mixed tumors.

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.

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% 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% 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% 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.

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.

Background Components of the extracellular matrix have been studied in the attempt to elucidate the mechanisms involved with biological behavior of tumours. The presence of the proteoglycan versican has been strongly associated to cancer development and progression, however its relationship with invasion and tumoral progression has been little studied in veterinary medicine. Carcinomas in benign mixed tumours (CBMT) are one of the most common malignant tumour in female dogs and can serve as a model for studies on tumour progression. The aim of this paper was to evaluate the expression of versican in in situ and invasive carcinomatous areas of CBMT.

Submitted by Alessandra Estrela-Lima (aestrela@ufba.br) on 2013-05-20T23:19:29Z No. of bitstreams: 1 Versican expression in canine carcinomas.....pdf: 2455158 bytes, checksum: 726f3cfd8d1b67b41aee82b52cc76492 (MD5) Approved for entry into archive by Alda Lima da Silva(sivalda@ufba.br) on 2013-05-28T20:01:13Z (GMT) No. of bitstreams: 1 Versican expression in canine carcinomas.....pdf: 2455158 bytes, checksum: 726f3cfd8d1b67b41aee82b52cc76492 (MD5) Made available in DSpace on 2013-05-28T20:01:13Z (GMT). No. of bitstreams: 1 Versican expression in canine carcinomas.....pdf: 2455158 bytes, checksum: 726f3cfd8d1b67b41aee82b52cc76492 (MD5) Previous issue date: 2012 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.

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.