PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

Susceptibility testing is essential to inform the correct management of Aspergillus infections. In this study we present antifungal susceptibility profile of A. fumigatus isolates recovered from lungs of birds with and without aspergillosis. Fifty three isolates were tested for their antifungal susceptibility to voriconazole (VRC), itraconazole (ITZ), amphotericin (AMB) and caspofungin (CSP) using the M38-A2 broth microdilution reference method. Five isolates were resistant to more than one antifungal drug (CSP + AMB, VRC + ITZ and AMB + ITZ). Fifteen (28%) isolates with susceptible increased exposure (I) to ITZ were sensible to VRC. Resistance to AMB (>2μg/mL) was observed in only four isolates. Eleven (21%) A. fumigatus present resistance to ITZ (13%) and VRC (8%). Fungal isolation from respiratory samples has been regarded as being of limited usefulness in the ante mortem diagnosis of aspergillosis in birds. However, the results suggest that the detection and antifungal susceptibility profile may be helpful for monitoring of therapy for avian species and where antifungal resistance might be emerging and what conditions are associated to the event.

• Aspergillus fumigatus antifungal susceptibility isolates birds lungs

Abstract in Portuguese:

Os testes de suscetibilidade são essenciais para informar o correto manejo das infecções por Aspergillus. Neste estudo apresentamos o perfil antifúngico de isolados de A. fumigatus provenientes de pulmões de aves com e sem aspergilose. Cinqüenta e três isolados foram testados quanto à susceptibilidade antifúngica ao voriconazol (VRC), itraconazol (ITZ), anfotericina B (AMB) e caspofungina (CSP) pelo método de referência de microdiluição do caldo M38-A2. Cinco isolados foram resistentes a mais de um antifúngico (CSP + AMB, VRC + ITZ e AMB + ITZ). Quinze (28%) isolados suscetíveis - com exposição aumentada (I) ao ITZ foram sensíveis ao VRC. A resistência ao AMB (>2μg/mL) foi observada em apenas quatro isolados. Onze (21%) A. fumigatus apresentaram resistência a ITZ (13%) e VRC (8%). O isolamento de fungos de amostras respiratórias tem sido considerado de utilidade limitada no diagnóstico ante mortem de aspergilose em aves. No entanto, os resultados sugerem que a detecção e o perfil de suscetibilidade a antifúngicos podem ser úteis para o monitoramento da terapia de espécies aviárias, assim como a emergência da resistência antifúngica e quais condições podem estar associadas ao evento.

• Aspergillus fumigatus antifúngicos suscetibilidade isolados aves pulmões

Abstract in English:

ABSTRACT.- Zambrano C.G., Fonseca A.O.S., Valente J.S.S., Braga C.Q., Sallis E.S.V., Azevedo M.I., Weiblen C., Santurio J.M., Botton S.A. & Pereira D.I.B. 2017. [Isolation and characterization of Pythium species from swampy areas in the Rio Grande do Sul, Brazil, and evaluation of pathogenicity in an experimental model.] Isolamento e caracterização de espécies de Pythium de ambientes aquáticos no Estado do Rio Grande do Sul e avaliação da patogenicidade em modelo experimental. Pesquisa Veterinária Brasileira 37(5):459-464. Laboratório de Micologia, Instituto de Biologia, Departamento de Microbiologia e Parasitologia, Universidade Federal de Pelotas, Campus Universitário s/n, Pelotas, RS 96160-000, Brazil. E-mail: danielabrayer@gmail.com One hundred and eighty-six water samples from swampy areas were collected in 13 municipalities of South, Central and West regions of Rio Grande do Sul, Brazil, in order to isolate and characterize Pythium species and assess their pathogenicity using rabbits as experimental model. Different Pythium species were isolated from 22 (11.8%) water samples, including P. insidiosum (n=1), P. catenulatum (n=3), P. pachycaule voucher (n=1), P. rhizo-oryzae (n=3), P. torulosum (n=4) e Pythium spp. (n=10). Zoospores of these microorganisms were produced in vitro and inoculated subcutaneously into rabbits, which were assessed over 45 days. Only P. insidiosum showed pathogenicity, causing pythiosis in the experimental model.

• Pythium insidiosum oomycete pythiosis oomicetos pitiose

Abstract in Portuguese:

RESUMO.- Zambrano C.G., Fonseca A.O.S., Valente J.S.S., Braga C.Q., Sallis E.S.V., Azevedo M.I., Weiblen C., Santurio J.M., Botton S.A. & Pereira D.I.B. 2017. [Isolation and characterization of Pythium species from swampy areas in the Rio Grande do Sul, Brazil, and evaluation of pathogenicity in an experimental model.] Isolamento e caracterização de espécies de Pythium de ambientes aquáticos no Estado do Rio Grande do Sul e avaliação da patogenicidade em modelo experimental. Pesquisa Veterinária Brasileira 37(5):459-464. Laboratório de Micologia, Instituto de Biologia, Departamento de Microbiologia e Parasitologia, Universidade Federal de Pelotas, Campus Universitário s/n, Pelotas, RS 96160-000, Brazil. E-mail: danielabrayer@gmail.com Foram coletadas 186 amostras de água de ambientes pantanosos em 13 municípios das regiões Sul, Central e Oeste do Rio Grande do Sul, Brasil, com o objetivo de isolar e caracterizar espécies de Pythium e avaliar a sua patogenicidade empregando coelhos como modelo experimental. Em 11,8% (n=22) das águas coletadas foram isoladas diferentes espécies de Pythium incluindo: P. insidiosum (n=1), P. catenulatum (n=3), P. pachycaule voucher (n=1), P. rhizo-oryzae (n=3), P. torulosum (n=4) e Pythium spp. (n=10). Zoósporos desses micro-organismos foram produzidos in vitro e inoculados por via subcutânea em coelhos, os quais foram avaliados durante 45 dias. Dentre os oomicetos testados, apenas P. insidiosum evidenciou patogenicidade, causando pitiose no modelo experimental, evidenciando que, em nossas condições, apenas esta espécie de Pythium é patógena para mamíferos.

Abstract in English:

ABSTRACT.- Bernardo F.D., Conhizak C., Ambrosini F., Jesus F.P.K., Santurio J.M., Kommers G.D., Elias F. & Franciscato C. 2015. Pythiosis in sheep from Paraná, southern Brazil. Pesquisa Veterinária Brasileira 35(6):513-517. Curso de Medicina Veterinária, Universidade Federal da Fronteira Sul, Av. Edmundo Gaievski 1000, Realeza, PR 85770-000, Brazil. E-mail: carinafranciscato@yahoo.com.br This paper reports pythiosis in a sheep from southwestern Paraná, Brazil, confirmed by indirect ELISA (Enzime-Linked Immunosorbent Assay) and immunohistochemistry, as well as it describes the macro and microscopic injuries, in order to understand the pathogenicity. A 4-year-old ewe from a flock of 30 Santa Inês sheep, raised semi-extensively with access to a weir, showed cachexia, bilateral enlargement in nasal region, a serous and bloody secretion with a fetid odor from its nose and swollen submandibular and retropharyngeal lymph nodes. Blood collection was performed trough jugular vein puncture in order to make complete blood cell count (CBC) and to obtain serum for the subsequent serological examination. As the hematological counts were within the normal range for sheep, the animal was euthanized and submitted to necropsy. Indirect ELISA resulted positive for pythiosis. Necropsy revealed necrosis of the hard palate with a diameter of 3.5cm and extending up to the nasal cavity, forming a fistula. Submandibular and retropharyngeal lymph nodes were enlarged and edematous on section. Microscopic findings for submandibular and retropharyngeal lymph nodes consisted in moderate infiltration of eosinophils mainly in the subcapsular sinus, characterizing reactive eosinophilic lymphadenitis. The nasal cavity revealed rhinitis and oral cavity stomatitis with necro-eosinophilic and pronounced multifocal granulomatous infiltration and presence of hyphae. Hyphae found in palate and nasal cavity were positive for Pythium insidiosum by Grocott’s method and immunohistochemistry, the last one considered to be confirmatory for the pathogen diagnostic. This report has an important epidemiological aspect, as it is the first case of pythiosis in sheep confirmed by serology in South Brazil and an alert of possible infection by the pathogen in floodplains.

• Pythiosis Pythium insidiosum pitiose

Abstract in Portuguese:

RESUMO.- Bernardo F.D., Conhizak C., Ambrosini F., Jesus F.P.K., Santurio J.M., Kommers G.D., Elias F. & Franciscato C. 2015. Pythiosis in sheep from Paraná, southern Brazil. [Pitiose em ovinos no Paraná, Sul do Brasil.] Pesquisa Veterinária Brasileira 35(6):513-517. Curso de Medicina Veterinária, Universidade Federal da Fronteira Sul, Av. Edmundo Gaievski 1000, Realeza, PR 85770-000, Brazil. E-mail: carinafranciscato@yahoo.com.br Este trabalho tem como objetivo descrever um caso de pitiose confirmado por ELISA (Enzime-Linked Immunosorbent Assay) indireto e imuno-histoquímica em uma ovelha do sudoeste do Paraná, Brasil, bem como suas lesões macro e microscópicas, a fim de compreender a sua patogenicidade. Trata-se de um ovino, fêmea, de 4 anos de idade, de um rebanho de 30 animais, da raça Santa Inês, criados em sistema semiextensivo, com acesso à um açude. O animal vinha apresentando aumento de volume na região do focinho, associado a emagrecimento progressivo. No exame físico apresentou-se caquético, com aumento de volume bilateral na região nasal, e com uma secreção serosanguinolenta de odor fétido fluindo das narinas, além de possuir os linfonodos submandibulares e retrofaríngeos bilateralmente infartados. Foi realizada coleta de sangue por punção da veia jugular para realização do hemograma e obtenção do soro para posterior realização de exame sorológico. Sendo que todos os parâmetros hematológicos analisados estavam dentro dos valores normais para a espécie. Após a realização do exame clínico e da coleta de sangue, o animal foi eutanasiado e procedeu-se a necropsia. Através do Teste de ELISA indireto, a amostra apresentou resultado positivo para pitiose. Na ocasião da necropsia pode-se verificar uma área necrosada de aproximadamente 3,5 cm de diâmetro no palato duro, a qual se estendeu até o assoalho da cavidade nasal formando uma fístula. Os linfonodos submandibulares e retrofaríngeos estavam aumentados de volume e ao corte edematosos. Os achados microscópicos dos linfonodos submandibulares e retrofaríngeos consistiram de quantidade moderada de eosinófilos, principalmente nos seios subcapsulares caracterizando uma linfadenite eosinofílica (reativa). Na cavidade nasal e oral, observou-se, respectivamente, rinite e estomatite necro-eosinofílica e granulomatosa multifocal acentuada associada a hifas intralesionais. As hifas encontradas no palato e na cavidade nasal foram Grocott-positivas e positivas pela técnica de imuno-histoquímica para Pythium insidiosum, esta última técnica também foi considerada confirmatória para o diagnóstico do agente. Este relato possui impacto epidemiológico por ser o primeiro caso de pitiose sorologicamente confirmado na espécie ovina na região Sul do Brasil e também, serve de alerta para a população, principalmente do meio rural, visto a possibilidade de infecção com o agente quando em contato com áreas alagadiças.

Abstract in English:

ABSTRACT.- Lautert C., Ferreiro L., Zimmermann C.E.P., Castilhos L.G., Jesus F.P.K., Zanette R.A., Leal D.B.R. & Santurio J.M. 2014. [In vitro effects of ochratoxin A, deoxynivalenol and zearalenone on cell viability and E-ADA activity in broiler chickens lymphocytes.] Efeitos in vitro de ocratoxina A, deoxinivalenol e zearalenona sobre a viabilidade celular e atividade de E-ADA em linfócitos de frangos de corte. Pesquisa Veterinária Brasileira 34(12):1173-1180. Setor de Micologia, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil. E-mail: claudialautert@yahoo.com.br Mycotoxins are a group of chemically diverse naturally occurring substances resulting from the secondary metabolism of pathogenic filamentous fungi. They are produced mainly by the genera Fusarium, Alternaria, Aspergillus and Penicillium which can contaminate grains and cereals such as wheat, corn and soy. According to the nature and the concentration levels, mycotoxins can induce toxic effects in food-production animals and humans. An in vitro study was conducted to evaluate the susceptibility of broiler chickens lymphocytes to different concentrations of ochratoxin A, deoxynivalenol and zearalenone. Each toxin was added to the cell medium at different concentrations (0.001, 0.01, 0.1 and 1&#956;g/mL). Cell viability and ecto-adenosine deaminase activity were assessed at 24, 48 and 72 hours by colorimetric assays. Thus, it were used 0.7x105 lymphocytes/mL in RPMI 1640 medium, supplemented with 10% fetal bovine serum and 2.5 IU of penicillin/streptomycin per mL, incubated at 37°C in a 5% CO2 atmosphere. All the experiments were carried out in triplicate and the results were expressed as mean ± standard error of the mean. The results showed that OTA and DON induced lymphocyte proliferation and reduced enzymatic activity in vitro (P<0,05), whereas ZEA also promoted proliferation (P<0,05), but neither alteration on enzymatic activity (P>0,05). It was possible to correlate the results about viability cell and ecto-adenosine deaminase activity, suggesting that, at minimal concentrations, the evaluated mycotoxins do not stimulated the enzymatic activity, which has proinflammatory action and contributes for the immunosuppression process, thus, avoiding a decrease on the viability cell. This is the first in vitro study conducted with OTA, DON and ZON in broiler chickens lymphocytes evaluating these parameters.

• Ochratoxin A deoxynivalenol zearalenone mycotoxins ocratoxina A deoxinivalenol zearalenona micotoxinas

Abstract in Portuguese:

RESUMO.- Lautert C., Ferreiro L., Zimmermann C.E.P., Castilhos L.G., Jesus F.P.K., Zanette R.A., Leal D.B.R. & Santurio J.M. 2014. [In vitro effects of ochratoxin A, deoxynivalenol and zearalenone on cell viability and E-ADA activity in broiler chickens lymphocytes.] Efeitos in vitro de ocratoxina A, deoxinivalenol e zearalenona sobre a viabilidade celular e atividade de E-ADA em linfócitos de frangos de corte. Pesquisa Veterinária Brasileira 34(12):1173-1180. Setor de Micologia, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil. E-mail: claudialautert@yahoo.com.br Micotoxinas representam um vasto grupo de contaminantes químicos naturais originados a partir do metabolismo secundário de fungos filamentosos patogênicos. Elas são produzidas, principalmente, pelos gêneros Fusarium, Alternaria, Aspergillus e Penicillium, os quais podem contaminar grãos e cereais, como trigo, milho e soja. Conforme sua natureza e níveis de concentração, micotoxinas podem induzir efeitos tóxicos em animais de produção e humanos. Um estudo in vitro foi realizado para avaliar a susceptibilidade das células linfocitárias de frangos de corte a diferentes concentrações de ocratoxina A, deoxinivalenol e zearalenona. Cada micotoxina foi adicionada ao meio celular em diferentes concentrações (0,001; 0,01; 0,1 e 1&#956;g/mL). A viabilidade celular e atividade de ecto-adenosina desaminase foram analisadas em 24, 48 e 72 horas através de ensaios colorimétricos. Para isso, foram utilizados 0,7x105 linfócitos/mL em meio RPMI 1640, suplementado com 10% de soro fetal bovino e 2,5 UI de penicilina/estreptomicina por mL, incubados em atmosfera de 5% de CO2 a 37 °C. Todos os experimentos foram realizados em triplicata e os resultados foram expressos como média e erro padrão da média. Os resultados obtidos demonstraram que tanto ocratoxina A como deoxinivalenol induziram proliferação linfocitária e baixa atividade enzimática in vitro (P<0,05), enquanto zearalenona também induziu proliferação (P<0,05), mas nenhuma alteração na atividade enzimática (P>0,05). Foi possível correlacionar os dados referentes à viabilidade celular e atividade de ecto-adenosina desaminase, sugerindo que, em concentrações mínimas, as micotoxinas testadas não estimularam a atividade da enzima, que possui ação pró-inflamatória e contribui para o processo de imunossupressão e, portanto, evitando um decréscimo na viabilidade celular. Este é o primeiro estudo feito com OCRA, DON e ZEA sobre linfócitos de frangos de corte em cultivos in vitro na avaliação desses parâmetros.

Abstract in English:

ABSTRACT.- Carrera M.V., Peixoto R.M., Gouveia G.V., Pessoa C.R.M., Jesus F.P.K., Santurio J.M., Botton S.A. & Costa M.M. 2013. [Pythiosis in sheep from Pernambuco and Bahia States, Brazil.] Pitiose em ovinos nos estados de Pernambuco e Bahia. Pesquisa Veterinária Brasileira 33(4):476-482. Campus Ciências Agrárias, Universidade Federal do Vale do São Francisco, Rodov. BR 407 Km 12, Lote 543, Projeto de Irrigação Senador Nilo Coelho s/n, Petrolina, PE 56300-990, Brazil. E-mail: rodolfo.peixoto@ifsertao-pe.edu.br Pythiosis is a devastating infectious disease caused by an aquatic oomycete, Pythium insidioum, and affects animals and humans that inhabit wetlands. The disease is characterized mainly by granulomatous lesions in the hosts. The purpose of this study was to report the occurrence of pythiosis in sheep in the states of Pernambuco (PE) and Bahia (BA), Northeastern Brazil, as well as to evaluate the efficacy of an immunotherapic against ovine pythiosis. Blood samples were collected from 53 sheep, 49 from flocks in counties located in PE and four from BA. Seven sheep showed clinical signs of ovine pythiosis; one of them was submitted to euthanasia and its head and submandibular lymph node was collected and sent for histopathologic and mycological analyses. Other six sheep were treated with an immunotherapic. During the treatment the animals were kept in the Sheep Industry Sector facilities at Univasf/Petrolina-PE. ELISA, fungal culture and polymerase chain reaction (PCR) methods were used to confirm the diagnosis of clinical ovine pythiosis in the sheep flock. At microscopic examination of the material collected from the nasal cavity of a sheep euthanized was observed a focally extensive area of necrosis with presence of diffuse infiltration of intact and degenerated neutrophils bordering the cartilage. Only one sheep showed clinical cure, indicating efficiency in the pythiosis treatment of 16.7% (1/6). Ovine pythiosis has been increasing in several municipalities of PE and BA. In this context, the immunotherapy may be an alternative to be searched. Therefore, further studies are needed to investigate the effect of immunotherapy on ovine pythiosis.

• Pythiosis pitiose

Abstract in Portuguese:

RESUMO.- Carrera M.V., Peixoto R.M., Gouveia G.V., Pessoa C.R.M., Jesus F.P.K., Santurio J.M., Botton S.A. & Costa M.M. 2013. [Pythiosis in sheep from Pernambuco and Bahia States, Brazil.] Pitiose em ovinos nos estados de Pernambuco e Bahia. Pesquisa Veterinária Brasileira 33(4):476-482. Campus Ciências Agrárias, Universidade Federal do Vale do São Francisco, Rodov. BR 407 Km 12, Lote 543, Projeto de Irrigação Senador Nilo Coelho s/n, Petrolina, PE 56300-990, Brazil. E-mail: rodolfo.peixoto@ifsertao-pe.edu.br A pitiose é uma doença infecciosa causada pelo oomiceto aquático P. insidiosum que acomete animais e o homem, especialmente habitantes de áreas úmidas. A enfermidade apresenta como característica principal a formação de lesões com aspecto granulomatoso nos hospedeiros. Neste trabalho, relatou-se a ocorrência da pitiose em ovinos nos estados de Pernambuco (PE) e Bahia (BA), Nordeste do Brasil, bem como foi avaliada a eficácia de um imunoterápico frente a esta enfermidade. Amostras de sangue de 53 ovinos foram coletadas, sendo 49 animais oriundos de propriedades localizadas em PE e quatro animais provenientes da BA. Sete ovinos demonstraram sinais clínicos de pitiose ovina. Um dos animais foi submetido à eutanásia e sua cabeça e linfonodo submandibular foram coletados e enviados para análises laboratoriais. Seis ovinos foram submetidos à imunoterapia, sendo mantidos nas instalações do setor de ovinocultura da Univasf/Petrolina-PE durante o tratamento. As técnicas de ELISA, cultura fúngica e reação em cadeia da polimerase (PCR) foram utilizadas como métodos diagnósticos da pitiose ovina, sendo eficientes para confirmação dos casos clínicos no rebanho. Ao exame microscópico do material coletado da cavidade nasal de um animal eutanasiado, observou-se uma área focalmente extensa de necrose com presença de infiltrado difuso de neutrófilos íntegros e degenerados margeando a cartilagem. Somente um animal apresentou cura clínica, indicando uma eficiência no tratamento da pitiose de 16,7% (1/6). O aumento de casos de pitiose tem sido denotado em diversos municípios de PE e da BA. Neste contexto, o emprego do imunoterápico pode ser uma alternativa a ser pesquisada. Portanto, estudos futuros devem ser realizados para investigar o efeito da imunoterapia aplicada à pitiose em ovinos.

Abstract in English:

ABSTRACT.- Azevedo M.I., Pereira D.I.B., Botton S.A., Costa M.M., Mahl C.D., Alves S.A. & Santurio J.M. 2012. Pythium insidiosum: Morphological and molecular identification of Brazilian isolates. Pesquisa Veterinária Brasileira 32(7):619-622. Laboratório de Pesquisas Micológicas, Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria, Camobi, Santa Maria, RS 97105-900, Brazil. E-mail: janio.santurio@gmail.com Pythium insidiosum is an oomycete belonging to the kingdom Stramenipila and it is the etiologic agent of pythiosis. Pythiosis is a life-threatening infectious disease characterized by the development of chronic lesions on cutaneous and subcutaneous, intestinal, and bone tissues in humans and many species of animals. The identification of P. insidiosum is important in order to implement a rapid and definitive diagnosis and an effective treatment. This study reports the identification of 54 isolates of P. insidiosum of horses, dogs and sheep that presented suspicious clinical lesions of pythiosis from different regions in Brazil, by using morphological and molecular assays. Throughout the PCR it was possible to confirm the identity of all Brazilian isolates as being P. insidiosum.

• Oomycete Pythium insidiosum pythiosis Oomiceto pitiose

Abstract in Portuguese:

RESUMO.- Azevedo M.I., Pereira D.I.B., Botton S.A., Costa M.M., Mahl C.D., Alves S.A. & Santurio J.M. 2012. Pythium insidiosum: Morphological and molecular identification of Brazilian isolates. Pesquisa Veterinária Brasileira 32(7):619-622. Laboratório de Pesquisas Micológicas, Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria, Camobi, Santa Maria, RS 97105-900, Brazil. E-mail: janio.santurio@gmail.com Pythium insidiosum é um oomiceto pertencente ao Reino Stramenopila e agente etiológico da pitiose, uma doença infecciosa com riscos de morte. A pitiose é caracterizada pelo desenvolvimento de lesões crônicas sobre os tecidos cutâneos, subcutâneas, intestinal e ósseo em humanos e muitas espécies de animais. A identificação de P. insidiosum é importante, a fim de se obter um diagnóstico rápido e definitivo, bem como um tratamento eficaz. Este estudo relata a identificação de 54 isolados de P. insidiosum de cavalos, cães e ovelhas que apresentavam lesões compatíveis e suspeita clínicas de pitiose, provenientes de diferentes regiões do Brasil, através de métodos morfológicos e moleculares. Através da PCR foi possível confirmar a identidade de todos os isolados brasileiros como sendo P. insidiosum.

Abstract in English:

ABSTRACT.- Cavallini Sanches E.M., Pacheco S.M., Cericatto A.S., Melo R.M., Colodel E.M., Hummel J., Bianchi S.P., Spanamberg A., Santurio J.M. & Ferreiro L. 2009. Detection of Pneumocystis in lungs of bats from Brazil by PCR amplification. Pesquisa Veterinária Brasileira 29(6):469-473. Setor de Micologia Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 90540-000, Brazil. E-mail: cavallini.sanches@ufrgs.br Pneumocystis has been isolated from a wide range of unrelated mammalian hosts, including humans, domestic and wild animals. It has been demonstrated that the genome of Pneumocystis of one host differs markedly from that of other hosts. Also, variation in the chromosome and DNA sequence of Pneumocystis within a single host species has been observed. Since information about the occurrence and nature of infections in wild animals is still limited, the objective of this work was to detect the presence of Pneumocystis sp. in lungs of bats from two states from Brazil by Nested-PCR amplification. The bats, captured in caves and in urban areas, were obtained from the Program of Rabies Control of two States in Brazil, Mato Grosso and Rio Grande do Sul, located in the Mid-Western and Southern regions of the country, respectively. DNAs were extracted from 102 lung tissues and screened for Pneumocystis by nested PCR at the mtLSU rRNA gene and small subunit of mitochondrial ribosomal RNA (mtSSU rRNA). Gene amplification was performed using the mtLSU rRNA, the primer set pAZ102H - pAZ102E and pAZ102X - pAZY, and the mtSSU rRNA primer set pAZ102 10FRI - pAZ102 10R-RI and pAZ102 13RI - pAZ102 14RI. The most frequent bats were Tadarida brasiliensis (25), Desmodus rotundus (20), and Nyctinomops laticaudatus (19). Pneumocystis was more prevalent in the species Nyctinomops laticaudatus (26.3% = 5/19), Tadarida brasiliensis (24% = 6/25), and Desmodus rotundus (20% = 4/20). Besides these species, Pneumocystis also was detected in lungs from Molossus molossus (1/11, 9.1%), Artibeus fimbriatus (1/1, 100%), Sturnira lilium (1/3, 33.3%), Myotis levis (2/3, 66.7%) and Diphylla ecaudata (1/2, 50%). PCR products which could indicate the presence of Pneumocystis (21.56%) were identified in DNA samples obtained from 8 out of 16 classified species from both states (5 bats were not identified). This is the first report of detection of Pneumocystis in bats from Brazil.

• Pneumocystis sp. bats Nested-PCR

Abstract in Portuguese:

ABSTRACT.- Cavallini Sanches E.M., Pacheco S.M., Cericatto A.S., Melo R.M., Colodel E.M., Hummel J., Bianchi S.P., Spanamberg A., Santurio J.M. & Ferreiro L. 2009. Detection of Pneumocystis in lungs of bats from Brazil by PCR amplification. Pesquisa Veterinária Brasileira 29(6):469-473. Setor de Micologia Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 90540-000, Brazil. E-mail: cavallini.sanches@ufrgs.br Pneumocystis has been isolated from a wide range of unrelated mammalian hosts, including humans, domestic and wild animals. It has been demonstrated that the genome of Pneumocystis of one host differs markedly from that of other hosts. Also, variation in the chromosome and DNA sequence of Pneumocystis within a single host species has been observed. Since information about the occurrence and nature of infections in wild animals is still limited, the objective of this work was to detect the presence of Pneumocystis sp. in lungs of bats from two states from Brazil by Nested-PCR amplification. The bats, captured in caves and in urban areas, were obtained from the Program of Rabies Control of two States in Brazil, Mato Grosso and Rio Grande do Sul, located in the Mid-Western and Southern regions of the country, respectively. DNAs were extracted from 102 lung tissues and screened for Pneumocystis by nested PCR at the mtLSU rRNA gene and small subunit of mitochondrial ribosomal RNA (mtSSU rRNA). Gene amplification was performed using the mtLSU rRNA, the primer set pAZ102H - pAZ102E and pAZ102X - pAZY, and the mtSSU rRNA primer set pAZ102 10FRI - pAZ102 10R-RI and pAZ102 13RI - pAZ102 14RI. The most frequent bats were Tadarida brasiliensis (25), Desmodus rotundus (20), and Nyctinomops laticaudatus (19). Pneumocystis was more prevalent in the species Nyctinomops laticaudatus (26.3% = 5/19), Tadarida brasiliensis (24% = 6/25), and Desmodus rotundus (20% = 4/20). Besides these species, Pneumocystis also was detected in lungs from Molossus molossus (1/11, 9.1%), Artibeus fimbriatus (1/1, 100%), Sturnira lilium (1/3, 33.3%), Myotis levis (2/3, 66.7%) and Diphylla ecaudata (1/2, 50%). PCR products which could indicate the presence of Pneumocystis (21.56%) were identified in DNA samples obtained from 8 out of 16 classified species from both states (5 bats were not identified). This is the first report of detection of Pneumocystis in bats from Brazil.

Abstract in English:

ABSTRACT.- Gabriel A.L., Kommers G.D., Trost M.E., Barros C.S.L., Pereira D.B., Schwendler S.E. & Santurio J.M. 2008. [Outbreak of cutaneous pythiosis in cattle.] Surto de pitiose cutânea em bovinos. Pesquisa Veterinária Brasileira 28(12):583-587. Departamento de Patologia, Universidade Federal de Santa Maria, Camobi, Santa Maria, RS 97105-900, Brazil. E-mail: glaukommers@yahoo.com Seventy-six young mixed breed cattle of both sexes, presented multifocal ulcerated nodular cutaneous lesions localized in the medial and lateral aspects of fore and hindlimbs, ventral neck, sternum, and tail. The disease occurred during summer and lesions were observed on areas of the body which were in contact with water of irrigation channels for long periods. Histologically, there were multiple granulomas and pyogranulomas with few negative profiles of hyphae, which were better visualized throughout Grocott methenamine silver stain. Definitive etiologic diagnosis was based on immuno-histochemistry with anti-Pythium insidiosum polyclonal antibody. Additionally, an indirect ELISA test was performed. Bovine cutaneous pythiosis outbreaks are uncommon and, particularly as occurred in the cattle of this report, all affected animals had spontaneous healing within two to three weeks.

• Pythiosis Pythium insidiosum diseases of cattle immunohistochemistry ELISA test pathology

Abstract in Portuguese:

ABSTRACT.- Gabriel A.L., Kommers G.D., Trost M.E., Barros C.S.L., Pereira D.B., Schwendler S.E. & Santurio J.M. 2008. [Outbreak of cutaneous pythiosis in cattle.] Surto de pitiose cutânea em bovinos. Pesquisa Veterinária Brasileira 28(12):583-587. Departamento de Patologia, Universidade Federal de Santa Maria, Camobi, Santa Maria, RS 97105-900, Brazil. E-mail: glaukommers@yahoo.com Seventy-six young mixed breed cattle of both sexes, presented multifocal ulcerated nodular cutaneous lesions localized in the medial and lateral aspects of fore and hindlimbs, ventral neck, sternum, and tail. The disease occurred during summer and lesions were observed on areas of the body which were in contact with water of irrigation channels for long periods. Histologically, there were multiple granulomas and pyogranulomas with few negative profiles of hyphae, which were better visualized throughout Grocott methenamine silver stain. Definitive etiologic diagnosis was based on immuno-histochemistry with anti-Pythium insidiosum polyclonal antibody. Additionally, an indirect ELISA test was performed. Bovine cutaneous pythiosis outbreaks are uncommon and, particularly as occurred in the cattle of this report, all affected animals had spontaneous healing within two to three weeks.

Abstract in English:

Santurio J.M., Leal A.T., Leal A.B.M., Alves S.H., Lübeck I., Griebeler J. & Copetti M.V. 2006. [Indirect ELISA for the serodiagnostic of pythiosis.] Teste de ELISA indireto para o diagnóstico sorológico de pitiose. Pesquisa Veterinária Brasileira 26(1):47-50. Departamento de Microbiologia, Universidade Federal de Santa Maria, prédio 20, sala 4139, Lapemi, Santa Maria, RS 97105-900, Brazil. E-mail: santurio@smail.ufsm.br Pythiosis is a granulomatous disease caused by the oomycete Pythium insidiosum that affects humans and animals, especially horses. Deaths are very often the consequence of incorrect or late diagnosis when animals no longer respond to treatment. This study aimed standardization of the ELISA assay for the serodiagnostic of pythiosis in horses and rabbits, in order to minimize errors and delays in the diagnosis of the disease. Sera of 72 healthy and 44 of by pythiosis affected horses were used for development and evaluation of the test. The ELISA for equine diagnostic showed 97.72% sensitivity, 90.27% specificity, 86% positive predictive value, 98.4% negative predictive value, and 93.1% efficiency. The rabbit test was standardized with 48 sera of healthy rabbits and 24 sera of rabbits immunized with P. insidiosum antigens. The results were 91.66 % sensitivity, 95.83% specificity, 91.66% positive predictive value, 95.83% negative predictive value, and 94.44% efficiency. It can be concluded that ELISA is a reliable test for diagnostic and serological monitoring of pythiosis.

• Pythium insidiosum pythiosis ELISA horses rabbits.

Abstract in Portuguese:

Santurio J.M., Leal A.T., Leal A.B.M., Alves S.H., Lübeck I., Griebeler J. & Copetti M.V. 2006. [Indirect ELISA for the serodiagnostic of pythiosis.] Teste de ELISA indireto para o diagnóstico sorológico de pitiose. Pesquisa Veterinária Brasileira 26(1):47-50. Departamento de Microbiologia, Universidade Federal de Santa Maria, prédio 20, sala 4139, Lapemi, Santa Maria, RS 97105-900, Brazil. E-mail: santurio@smail.ufsm.br Pythiosis is a granulomatous disease caused by the oomycete Pythium insidiosum that affects humans and animals, especially horses. Deaths are very often the consequence of incorrect or late diagnosis when animals no longer respond to treatment. This study aimed standardization of the ELISA assay for the serodiagnostic of pythiosis in horses and rabbits, in order to minimize errors and delays in the diagnosis of the disease. Sera of 72 healthy and 44 of by pythiosis affected horses were used for development and evaluation of the test. The ELISA for equine diagnostic showed 97.72% sensitivity, 90.27% specificity, 86% positive predictive value, 98.4% negative predictive value, and 93.1% efficiency. The rabbit test was standardized with 48 sera of healthy rabbits and 24 sera of rabbits immunized with P. insidiosum antigens. The results were 91.66 % sensitivity, 95.83% specificity, 91.66% positive predictive value, 95.83% negative predictive value, and 94.44% efficiency. It can be concluded that ELISA is a reliable test for diagnostic and serological monitoring of pythiosis.

Abstract in English:

ABSTRACT.- Leal A.B.M., Leal A.T., Santurio J.M., Kommers G.D. & Catto J.B. 2001. [Equine pythiosis in the Brazilian Pantanal region: Clinical and pathological findings of typical and atypical cases] Pitiose eqüina no Pantanal brasileiro: aspectos clínico-patológicos de casos típicos e atípicos. Pesquisa Veterinária Brasileira 21(4):151-156. Laboratório de Pesquisas Micológicas, Depto Microbiologia e Parasitologia, Universidade Federal de Santa Maria, 97105- 900 Santa Maria, RS, Brazil. E-mail: santurio@ccr.ufsm.br Equine pythiosis is an endemic disease of horses and causes significant economic losses to equine breeding in the Brazilian Pantanal. This article describes 16 cases of subcutaneous pythiosis in horses from that region. The clinical cases were divided in typical (11) and atypical (5), according to the clinical features and duration of the disease. The clinical diagnosis was confirmed by detection of specific antibodies by ELISA, isolation of the agent and histopathology. The duration of the disease varie d from 1 to 6 months in the typical cases and was over 12 months in the atypical ones. The lesions in the typical cases were characterized by subcutaneous ulcerated granulomas with abundant serosanguineous secretion and itching. The atypical cases presented subcutaneous lesions characterized by large, circumscribed tumorous masses covered by a dark non ulcerated skin, associated with Iittle orno secretion. These lesions showed a well organized aspect, were sometimes pedunculate and the animals showed a good body condition. Histologically, the typical cases were characterized by granulation tissue with abundant eosinophils; whereas the atypical cases presented pseudoepitheliomatous hyperplasia of the epidermis and eosinophilic infiltrate. The complete description of the clinical and histopathological features and possible factors responsible for differences between the two clinical forms are presented and discussed.

• Pythiosis Pythium insidiosum equine granuloma Pantanal Pitiose granuloma eqüino Pantanal.

Abstract in Portuguese:

RESUMO.- Leal A.B.M., Leal A.T., Santurio J.M., Kommers G.D. & Catto J.B. 2001. [Equine pythiosis in the Brazilian Pantanal region: Clinical and pathological findings of typical and atypical cases] Pitiose eqüina no Pantanal brasileiro: aspectos clínico-patológicos de casos típicos e atípicos. Pesquisa Veterinária Brasileira 21(4):151-156. Laboratório de Pesquisas Micológicas, Depto Microbiologia e Parasitologia, Universidade Federal de Santa Maria, 97105- 900 Santa Maria, RS, Brazil. E-mail: santurio@ccr.ufsm.br A pitiose eqüina é doença endêmica no Pantanal Brasileiro e causa prejuízos significativos a eqüinocultura. Neste trabalho são relatados 16 casos de pitiose subcutânea em eqüinos no Pantanal Sul-Matogrossense, que foram divididos em onze casos típicos e cinco casos atípicos, de acordo com o quadro clínico e o tempo de duração das lesões. O diagnóstico foi confirmado pela detecção de anticorpos específicos pelo teste ELISA, isolamento do agente e histopatológico. A duração da doença variou entre 1 e 6 meses nos casos típicos e superior a 12 meses nos casos atípicos. As lesões dos casos típicos caracterizavam-se por granulomas subcutâneos, ulcerados, com abundante secreção serossanguinolenta e prurido. Nos casos atípicos foram observadas lesões subcutâneas caracterizadas por grandes massas "tumorais" circunscritas, recobertas por pele escura, sem ulcerações e com pouca secreção. Os animais estavam em bom estado nutricional e as lesões apresentavam-se de aspecto organizado, às vezes pedunculadas. Histologicamente, foi observado tecido de granulação com muitos eosinófilos nos casos típicos, enquanto os atípicos, se caracterizaram por hiperplasia pseudo-epiteliomatosa da epiderme e infiltrado eosinofilico. As características clínicas e histopato lógicas completas das duas formas clínicas e os possíveis fatores responsáveis pelas diferenças entre as duas formas são apresentados e discutidos.