Pathological and mycological characterization of pulmonary Aspergillus fumigatus infection producing gliotoxin in a captive African grey parrot (Psittacus erithacus)

Aspergillosis, the main causative agent of which is Aspergillus fumigatus, causes mortality in all types of birds. Gliotoxin (GT), one of the multiple virulence factors of A. fumigatus, has a variety of immunosuppressive effects. The corpse of an African grey parrot (Psittacus erithacus) was sent for necropsy and diagnostic rule-out. The lungs were enlarged, firm, and had dark-red coloration, on the parietal faces of both lungs, some semi-circular caseous necrosis areas were observed. The caudal thoracic and abdominal air sacs were thickened and contained a fibrin-heterophilic exudate. Histopathologically, a necrotic and granulomatous bronchopneumonia was observed with intralesional hyphae with characteristics compatible with Aspergillus sp. that were positive with Grocott´s staining. Fibrinous and heterophilic airsacculitis was found in the air sacs. A. fumigatus was isolated from lungs, characterized using serial microcultures, and confirmed using polymerase chain reaction. In addition, GT production was detected in vitro from the culture filtrate in which the isolate was grown; the organic extract was analysed via thin-layer chromatography. This is the first detection of GT in a case of pulmonary aspergillosis in a parrot, which could help to understand the pathogenesis of the disease in psittacines.(AU)

Antifungal susceptibility profile of Aspergillus fumigatus isolates from avian lungs / Perfil de suscetibilidade antifúngica de isolados de Aspergillus fumigatus provenientes de pulmões de aves

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.(AU)

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.(AU)

Outbreak of avian aspergillosis in colonial-bred chicks (Isa Brown) in southern Rio Grande do Sul - case report / [Surto de aspergilose aviária em pintainhas (Isa Brown) de criação colonial na região sul do Rio Grande do Sul - relato de caso]

In this study we describe the epidemiology, clinical signs, and pathology of an outbreak of avian aspergillosis in alternative breeding in the southern region of Rio Grande do Sul, Brazil. Between the fifth and tenth day of life, 360 chicks from a flock of 4000 developed unspecific clinical signs and died. The birds were housed in a reused aviary litter, without previous treatment. In 11 six-day-old female ISA Brown chicks (Gallus gallus domesticus), necropsy revealed firm, yellowish-white, multinodular lesions extending from the pleura to the lung parenchyma. Histologically, a granulomatous, multifocal to coalescent pneumonia was observed. Granulomas were characterized by central necrosis, with heterophil and epithelioid macrophage infiltration and presence of countless Y-shaped intralesional septate hyphae morphologically compatible with Aspergillus spp. The diagnosis through isolation confirmed Aspergillus fumigatus. We highlight the importance of aspergillosis as a primary cause of diseases in the respiratory tract of young birds in alternative breeding. Measures to prevent aspergillosis mainly regarding the reuse of aviary litter are essential in poultry husbandry to prevent economic losses, reduce environmental contamination and mitigate the potential risk to public health.(AU)

Descrevem-se os aspectos epidemiológicos e patológicos de um surto de aspergilose aviária em criação alternativa na região sul do Rio Grande do Sul, Brasil. De um lote de 4000 pintainhas, entre o quinto e o 10º dia de vida, 360 aves apresentaram sinais clínicos inespecíficos e morreram. As aves foram alojadas em cama reutilizada do aviário, sem tratamento prévio. Na necropsia de 11 pintainhas (Gallus gallus domesticus), fêmeas, seis dias de idade da linhagem Isa Brown, foram observadas no pulmão lesões multinodulares, branco-amareladas e firmes, que se estendiam da pleura ao parênquima. Histologicamente foi observada pneumonia granulomatosa, multifocal a coalescente. Os granulomas eram caracterizados por necrose central, com infiltrado inflamatório de heterófilos, macrófagos, células epitelioides com presença de inúmeras hifas septadas intralesionais, semelhantes à letra "Y", morfologicamente compatíveis com Aspergillus spp. O diagnóstico foi confirmado pelo isolamento de Aspergillus fumigatus. Alerta-se para a importância da aspergilose como causa primária de afecções no trato respiratório de aves jovens em criações alternativas. Medidas preventivas relacionadas ao manejo dessas aves são indispensáveis principalmente quanto à reutilização da cama dos aviários, a fim de evitar perdas econômicas, reduzir a contaminação ambiental e o potencial risco à saúde pública.(AU)

Antifungal susceptibility profile of Aspergillus fumigatus isolates from avian lungs / Perfil de suscetibilidade antifúngica de isolados de Aspergillus fumigatus provenientes de pulmões de aves

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.(AU)

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.(AU)

Outbreak of avian aspergillosis in colonial-bred chicks (Isa Brown) in southern Rio Grande do Sul - case report / [Surto de aspergilose aviária em pintainhas (Isa Brown) de criação colonial na região sul do Rio Grande do Sul - relato de caso]

In this study we describe the epidemiology, clinical signs, and pathology of an outbreak of avian aspergillosis in alternative breeding in the southern region of Rio Grande do Sul, Brazil. Between the fifth and tenth day of life, 360 chicks from a flock of 4000 developed unspecific clinical signs and died. The birds were housed in a reused aviary litter, without previous treatment. In 11 six-day-old female ISA Brown chicks (Gallus gallus domesticus), necropsy revealed firm, yellowish-white, multinodular lesions extending from the pleura to the lung parenchyma. Histologically, a granulomatous, multifocal to coalescent pneumonia was observed. Granulomas were characterized by central necrosis, with heterophil and epithelioid macrophage infiltration and presence of countless Y-shaped intralesional septate hyphae morphologically compatible with Aspergillus spp. The diagnosis through isolation confirmed Aspergillus fumigatus. We highlight the importance of aspergillosis as a primary cause of diseases in the respiratory tract of young birds in alternative breeding. Measures to prevent aspergillosis mainly regarding the reuse of aviary litter are essential in poultry husbandry to prevent economic losses, reduce environmental contamination and mitigate the potential risk to public health.(AU)

Descrevem-se os aspectos epidemiológicos e patológicos de um surto de aspergilose aviária em criação alternativa na região sul do Rio Grande do Sul, Brasil. De um lote de 4000 pintainhas, entre o quinto e o 10º dia de vida, 360 aves apresentaram sinais clínicos inespecíficos e morreram. As aves foram alojadas em cama reutilizada do aviário, sem tratamento prévio. Na necropsia de 11 pintainhas (Gallus gallus domesticus), fêmeas, seis dias de idade da linhagem Isa Brown, foram observadas no pulmão lesões multinodulares, branco-amareladas e firmes, que se estendiam da pleura ao parênquima. Histologicamente foi observada pneumonia granulomatosa, multifocal a coalescente. Os granulomas eram caracterizados por necrose central, com infiltrado inflamatório de heterófilos, macrófagos, células epitelioides com presença de inúmeras hifas septadas intralesionais, semelhantes à letra "Y", morfologicamente compatíveis com Aspergillus spp. O diagnóstico foi confirmado pelo isolamento de Aspergillus fumigatus. Alerta-se para a importância da aspergilose como causa primária de afecções no trato respiratório de aves jovens em criações alternativas. Medidas preventivas relacionadas ao manejo dessas aves são indispensáveis principalmente quanto à reutilização da cama dos aviários, a fim de evitar perdas econômicas, reduzir a contaminação ambiental e o potencial risco à saúde pública.(AU)

Aspergillosis in domestic and wild birds from Argentina / Aspergilose em aves domésticas e silvestres da Argentina

Aspergillus species are widely distributed throughout the world and can develop parasitic and saprophytic ways of life, allowing Aspergillus to infect living hosts, including plants, insects, birds and mammals. The most common form of aspergillosis in poultry and other birds is respiratory infection. Clinical manifestations depend on the infective dose, pre-existing diseases, and the immune response of the host. The aim of the present research was to study aspergillosis in domestic and wild birds from Argentina. We carried out morphological and molecular identification, and determination of antifungal susceptibility against seven antifungal drugs. Six birds from different cities of Buenos Aires Province of Argentina were studied. Three of the samples belonged to broiler chicks, while the other three belonged to an eagle, a pheasant, and a kelp gull. Two isolates were identified as Aspergillus fumigatus by morphological characteristics and growth at 50 C. Morphology and BenA sequencing enabled us to identify three isolates as Aspergillus flavus, and one as Aspergillus sydowii. All antifungal drugs tested showed low MIC values, ranging from 0.008 to 1 mg/L. Aspergillosis in birds causes high economic losses and could be controlled by sanitation, avoidance of moldy food, nest and litter and reducing stress factors.

As espécies de Aspergillus são amplamente distribuídas em todo o mundo e são capazes de desenvolver formas de vida parasitárias e saprófitas, permitindo que Aspergillus infecte hospedeiros vivos, incluindo plantas, insetos, pássaros e mamíferos. A forma mais comum de aspergilose em aves domésticas e outras aves são infecções respiratórias. As manifestações clínicas dependem da dose infecciosa, doenças pré-existentes e da resposta imune do hospedeiro. O objetivo da presente investigação foi estudar aspergilose em aves domésticas e silvestres da Argentina. Foi realizada a identificação morfológica e molecular, assim como a determinação do teste de suscetibilidade contra sete drogas antifúngicas. Foram estudadas seis aves pertencentes a diferentes cidades da província argentina de Buenos Aires. Três das amostras pertenciam a frangos de corte, enquanto as outras três amostras pertenciam a uma águia, um faisão e uma gaivota. Dois isolados foram identificados como Aspergillus fumigatus por características morfológicas e crescimento a 50 C. A morfologia e o sequenciamento de BenA permitiram identificar três isolados como Aspergillus flavus e um como Aspergillus sydowii. Todos os antifúngicos testados apresentaram baixos valores de CIM, variando de 0,008 a 1 mg/L. Aspergilose em aves causam altas perdas econômicas e podem ser controladas por práticas de higiene, evitando alimentos, ni

Elastin increases biofilm and extracellular matrix production of Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic saprobe fungus that accounts for 90% of cases of pulmonary aspergillosis in immunosuppressed patients and is known for its angiotropism. When it reaches the respiratory tract, A. fumigatus interacts with structural components and blood vessels of the lungs, such as elastin. To understand the effect of this structural component, we examined the effect of elastin on the production and development of the biofilm of A. fumigatus. In RPMI containing 10 mg/mL of elastin, a significant increase (absorbance p < 0.0001; dry weight p < 0.0001) in the production of biofilm was observed in comparison to when RPMI was used alone, reaching a maximum growth of 18.8 mg (dry weight) of biofilm in 72 h. In addition, elastin stimulates the production (p = 0.0042) of extracellular matrix (ECM) and decreases (p = 0.005) the hydrophobicity during the development of the biofilm. These results suggest that elastin plays an important role in the growth of A. fumigatus and that it participates in the formation of thick biofilm.(AU)

Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

An intronless endoglucanase from thermotolerant Aspergillus fumigatus DBINU-1 was cloned, characterized and expressed in the yeast Kluyveromyces lactis. The full-length open reading frame of the endoglucanase gene from A. fumigatus DBiNU-1, designated Cel7, was 1383 nucleotides in length and encoded a protein of 460 amino acid residues. The predicted molecular weight and the isoelectric point of the A. fumigatus Cel7 gene product were 48.19 kDa and 5.03, respectively. A catalytic domain in the N-terminal region and a fungal type cellulose-binding domain/module in the C-terminal region were detected in the predicted polypeptide sequences. Furthermore, a signal peptide with 20 amino acid residues at the N-terminus was also detected in the deduced amino acid sequences of the endoglucanase from A. fumigatus DBiNU-1. The endoglucanase from A. fumigatus DBiNU-1 was successfully expressed in K. lactis, and the purified recombinant enzyme exhibited its maximum activity at pH 5.0 and 60 °C. The enzyme was very stable in a pH range from 4.0 to 8.0 and a temperature range from 30 to 60 °C. These features make it suitable for application in the paper, biofuel, and other chemical production industries that use cellulosic materials.(AU)

Identification and characterization of Aspergillus fumigatus isolates from broilers / Identificação e caracterização de Aspergillus fumigatus isolados de frangos de corte

Aspergillosis is one of the main causes of mortality in birds. The pulmonary system is most frequently affected, with lesions observed in the air sacs and lungs of a wide variety of bird species. The aim of this study was to confirm by molecular methods the identification and the genetic diversity of Aspergillus fumigatus isolates of lung's samples from healthy broilers (Galus galus domesticus). Forty-four (9.5%) isolates of lung's samples were confirmed as A. fumigatus by polymerase chain reaction (PCR) multiplex (amplification of ß-tub and rodA gene fragments). Microsatellite typing for A. fumigatus was used to analyse all avian isolates. Among them, 40 genotypes (90.9%) were observed only one time. The results showed a high variability and multiple genotypes of de A. fumigatus collected from lung's samples of broilers.(AU)

Aspergilose é uma das principais causas de mortalidade em aves. O sistema pulmonar de uma grande variedade de espécies de aves é o mais frequentemente afetado, com lesões nos sacos aéreos e pulmões. Objetivou-se confirmar por métodos moleculares a identificação e a diversidade genética de Aspergillus fumigatus isolados de amostras pulmonares de frangos de corte sadios (Galus galus domesticus). Quarenta e quatro (9,5%) isolados foram confirmados como A. fumigatus através de reação em cadeia da polimerase (PCR) multiplex (amplificação de fragmentos dos genes ß-tub e rodA). Todos isolados foram tipificados, sendo quarenta (90,9%) observados apenas uma vez. Os resultados mostram uma alta variabilidade e múltiplos genótipos de A. fumigatus obtidos de amostras pulmonares de frangos, de corte.(AU)

Infecção por pox vírus e Aspergillus fumigatus em Bubo virginianus (Coruja jacurutu) / Avipoxvirus infecction in Bubo virginianus (great horned owl)

Este trabalho descreve um caso de infecção mista por pox vírus e Aspergillus fumigatus em Bubo virginianus (coruja jacurutu). A ave, um macho adulto, foi encaminhada ao Núcleo de Reabilitação da Fauna Silvestre do Instituto de Biologia da Universidade Federal de Pelotas (NURFS/CETAS/UFPEL). Apresentava bom estado corporal, estava ativa, porém com incapacidade de voo. Após três dias apresentou lesões crostosas e de aspecto verrucoso na superfície dorsal das patas. Havia, também, nódulos de mesmo aspecto na pálpebra esquerda e na cera. A ave morreu após 15 dias de sua chegada ao NURFS e foi necropsiada no Laboratório Regional de Diagnóstico da Faculdade de Veterinária da Universidade Federal de Pelotas (LRD/UFPel). Histologicamente, as lesões verrucosas caracterizavam-se por hiperplasia do epitélio e nas células das camadas basal, espinhosa, granular e córnea havia corpúsculos de inclusão intracitoplasmáticos do tipo Bollinger. Na microscopia eletrônica foram visualizadas partículas virais características de pox vírus, incluindo Bubo virginianus como um hospedeiro do vírus. Havia, ainda, infiltrado inflamatório de células mononucleares e focos de colônias bacterianas na derme. Nos pulmões havia congestão e presença de granulomas com hifas fúngicas, que pela técnica de Grocott, apresentaram ramificação dicotômica compatível com Aspergillus spp., identificado na cultura como A. fumigatus. O diagnóstico de infecção por avipoxvirus pode contribuir para estudos relacionados com a ocorrência desta doença nas populações de vida livre e como informação auxiliar para o manejo e conservação desta espécie. Sugere-se, ainda, a inclusão do uso de raios-X nos protocolos de centros de reabilitação como o diagnostico de aspergilose em aves rapinantes com bom estado corporal, porém incapazes de voar.(AU)

This paper describes a case of mixed infection by pox virus and Aspergillus fumigatus in Bubo virginianus (Owl Jacurutu). An adult male Bubo virginianus was referred to the Núcleo de Reabilitação da Fauna Silvestre, Instituto de Biologia, Universidade Federal de Pelotas (NURFS/CETAS/UFPEL). The owl was active and had a good body condition but with flight disability. After three days of their admission at NURFS the owl developed crusty and verrucous lesions at the dorsal surface of their feet. Also it had nodes on the left eyelid and cera with the same aspect. The owl died 15 days after its arrival. Necropsy and histopatological examination were carried out. The warty lesions had hyperplasia of the epithelium and intracytoplasmic Bollinger-like inclusion bodies in the basal, spinal, granulosa layer and cornea. Viral particles characteristic of pox viruses were shown by electron microscopy. This case includes Bubo virginianus as a host of the avipoxvirus. There were also a mononuclear inflammatory cell infiltrate and bacterial colonies in the dermis. In the lugs, there was congestion and presence of granulomas with intralesional fungal hyphae. With the Grocott stain those structures showed dichotomous branching which was later identified in mycological culture as characteristic for A. fumigates. The diagnosis of avipoxvirus infection can contribute to studies related to the occurrence of this disease in free-living populations and as auxiliary information for the management and conservation of this raptor species. It is also suggested to include the use of X-rays in rehabilitation center protocols as screening test to diagnose aspergillosis in birds of prey with good body condition but inability to fly.(AU)

Infecção por pox vírus e Aspergillus fumigatus em Bubo virginianus (Coruja jacurutu) / Avipoxvirus infecction in Bubo virginianus (great horned owl)

Este trabalho descreve um caso de infecção mista por pox vírus e Aspergillus fumigatus em Bubo virginianus (coruja jacurutu). A ave, um macho adulto, foi encaminhada ao Núcleo de Reabilitação da Fauna Silvestre do Instituto de Biologia da Universidade Federal de Pelotas (NURFS/CETAS/UFPEL). Apresentava bom estado corporal, estava ativa, porém com incapacidade de voo. Após três dias apresentou lesões crostosas e de aspecto verrucoso na superfície dorsal das patas. Havia, também, nódulos de mesmo aspecto na pálpebra esquerda e na cera. A ave morreu após 15 dias de sua chegada ao NURFS e foi necropsiada no Laboratório Regional de Diagnóstico da Faculdade de Veterinária da Universidade Federal de Pelotas (LRD/UFPel). Histologicamente, as lesões verrucosas caracterizavam-se por hiperplasia do epitélio e nas células das camadas basal, espinhosa, granular e córnea havia corpúsculos de inclusão intracitoplasmáticos do tipo Bollinger. Na microscopia eletrônica foram visualizadas partículas virais características de pox vírus, incluindo Bubo virginianus como um hospedeiro do vírus. Havia, ainda, infiltrado inflamatório de células mononucleares e focos de colônias bacterianas na derme. Nos pulmões havia congestão e presença de granulomas com hifas fúngicas, que pela técnica de Grocott, apresentaram ramificação dicotômica compatível com Aspergillus spp., identificado na cultura como A. fumigatus. O diagnóstico de infecção por avipoxvirus pode contribuir para estudos relacionados com a ocorrência desta doença nas populações de vida livre e como informação auxiliar para o manejo e conservação desta espécie. Sugere-se, ainda, a inclusão do uso de raios-X nos protocolos de centros de reabilitação como o diagnostico de aspergilose em aves rapinantes com bom estado corporal, porém incapazes de voar.(AU)

This paper describes a case of mixed infection by pox virus and Aspergillus fumigatus in Bubo virginianus (Owl Jacurutu). An adult male Bubo virginianus was referred to the Núcleo de Reabilitação da Fauna Silvestre, Instituto de Biologia, Universidade Federal de Pelotas (NURFS/CETAS/UFPEL). The owl was active and had a good body condition but with flight disability. After three days of their admission at NURFS the owl developed crusty and verrucous lesions at the dorsal surface of their feet. Also it had nodes on the left eyelid and cera with the same aspect. The owl died 15 days after its arrival. Necropsy and histopatological examination were carried out. The warty lesions had hyperplasia of the epithelium and intracytoplasmic Bollinger-like inclusion bodies in the basal, spinal, granulosa layer and cornea. Viral particles characteristic of pox viruses were shown by electron microscopy. This case includes Bubo virginianus as a host of the avipoxvirus. There were also a mononuclear inflammatory cell infiltrate and bacterial colonies in the dermis. In the lugs, there was congestion and presence of granulomas with intralesional fungal hyphae. With the Grocott stain those structures showed dichotomous branching which was later identified in mycological culture as characteristic for A. fumigates. The diagnosis of avipoxvirus infection can contribute to studies related to the occurrence of this disease in free-living populations and as auxiliary information for the management and conservation of this raptor species. It is also suggested to include the use of X-rays in rehabilitation center protocols as screening test to diagnose aspergillosis in birds of prey with good body condition but inability to fly.(AU)

Identification and characterization of Aspergillus fumigatus isolates from broilers / Identificação e caracterização de Aspergillus fumigatus isolados de frangos de corte

Aspergillosis is one of the main causes of mortality in birds. The pulmonary system is most frequently affected, with lesions observed in the air sacs and lungs of a wide variety of bird species. The aim of this study was to confirm by molecular methods the identification and the genetic diversity of Aspergillus fumigatus isolates of lung's samples from healthy broilers (Galus galus domesticus). Forty-four (9.5%) isolates of lung's samples were confirmed as A. fumigatus by polymerase chain reaction (PCR) multiplex (amplification of β-tub and rodA gene fragments). Microsatellite typing for A. fumigatus was used to analyse all avian isolates. Among them, 40 genotypes (90.9%) were observed only one time. The results showed a high variability and multiple genotypes of de A. fumigatus collected from lung's samples of broilers.(AU)

Aspergilose é uma das principais causas de mortalidade em aves. O sistema pulmonar de uma grande variedade de espécies de aves é o mais frequentemente afetado, com lesões nos sacos aéreos e pulmões. Objetivou-se confirmar por métodos moleculares a identificação e a diversidade genética de Aspergillus fumigatus isolados de amostras pulmonares de frangos de corte sadios (Galus galus domesticus). Quarenta e quatro (9,5%) isolados foram confirmados como A. fumigatus através de reação em cadeia da polimerase (PCR) multiplex (amplificação de fragmentos dos genes β-tub e rodA). Todos isolados foram tipificados, sendo quarenta (90,9%) observados apenas uma vez. Os resultados mostram uma alta variabilidade e múltiplos genótipos de A. fumigatus obtidos de amostras pulmonares de frangos, de corte.(AU)

Production of bioethanol using agricultural waste: banana pseudo stem

India is amongst the largest banana (Musa acuminata) producing countries and thus banana pseudo stem is commonly available agricultural waste to be used as lignocellulosic substrate. Present study focuses on exploitation of banana pseudo stem as a source for bioethanol production from the sugars released due to different chemical and biological pretreatments. Two fungal strains Aspergillus ellipticus and Aspergillus fumigatus reported to be producing cellulolytic enzymes on sugarcane bagasse were used under co-culture fermentation on banana pseudo stem to degrade holocellulose and facilitate maximum release of reducing sugars. The hydrolysate obtained after alkali and microbial treatments was fermented by Saccharomyces cerevisiae NCIM 3570 to produce ethanol. Fermentation of cellulosic hydrolysate (4.1 g%) gave maximum ethanol (17.1 g/L) with yield (84%) and productivity (0.024 g%/h) after 72 h. Some critical aspects of fungal pretreatment for saccharification of cellulosic substrate using A. ellipticus and A. fumigatus for ethanol production by S. cerevisiae NCIM 3570 have been explored in this study. It was observed that pretreated banana pseudo stem can be economically utilized as a cheaper substrate for ethanol production.

Production and partial characterization of serine and metallo peptidases secreted by Aspergillus fumigatus Fresenius in submerged and solid state fermentatio

Enzyme production varies in different fermentation systems. Enzyme expression in different fermentation systems yields important information for improving our understanding of enzymatic production induction. Comparative studies between solid-state fermentation (SSF) using agro-industrial waste wheat bran and submerged fermentation (SmF) using synthetic media were carried out to determinate the best parameters for peptidase production by the fungus Aspergillus fumigatus Fresen. Variables tested include: the concentration of carbon and protein nitrogen sources, the size of the inoculum, the pH of the media, temperature, and the length of the fermentation process. The best peptidase production during SSF was obtained after 96 hours using wheat bran at 30 ºC with an inoculum of 1 x 10(6) spores and yielded 1500 active units (UµmL). The best peptidase production using SmF was obtained after periods of 72 and 96 hours of fermentation in media containing 0.5% and 0.25% of casein, respectively, at a pH of 6.0 and at 30 ºC and yielded 40 UµmL. We also found examples of catabolite repression of peptidase production under SmF conditions. Biochemical characterization of the peptidases produced by both fermentative processes showed optimum activity at pH 8.0 and 50 ºC, and also showed that their proteolytic activity is modulated by surfactants. The enzymatic inhibition profile using phenylmethylsulfonyl fluoride (PMSF) in SmF and SSF indicated that both fermentative processes produced a serine peptidase. Additionally, the inhibitory effect of the ethylene-diaminetetraacetic acid (EDTA) chelating agent on the peptidase produced by SmF indicated that this fermentative process also produced a metallopeptidase.(AU)

Aspergillus fumigatus from normal and condemned carcasses with airsacculitis in commercial poultry / Pesquisa de Aspergillus fumigatus em carcaças de frango de corte normais e condenadas por aerossaculite

Carcass inspection is important for the detection of certain diseases and for monitoring their prevalence in slaughterhouses. The objective of this study was to assess the occurrence of aspergillosis caused by Aspergillus fumigatus in commercial poultry, through mycological and histopathological diagnosis, and to verify the causal association between the aspergillosis diagnosis criteria and condemnation due to airsacculitis in broilers through a case-control study. The study was carried out with 380 samples. Lungs were collected from broilers that were condemned (95) or not condemned (285) due to airsacculitis directly from the slaughter line. Forty-six (12%) lung samples were positive for A. fumigatus in mycological culture. Among all samples, 177 (46.6%) presented histopathological alterations, with necrotic, fibrinous, heterophilic pneumonia; heterophilic pneumonia and lymphoid hyperplasia being the most frequent. Out of the 380 lungs analyzed, 65.2% (30) showed histopathological alterations and isolation of fungi. The statistical analysis (McNemar's chi-square test) indicated a significant association between the presence of histopathological lesions and the isolation of A. fumigatus. Mycological cultivation and histopathological diagnosis increase the probability of detecting pulmonary alterations in birds condemned by the Final Inspection System, which suggests that such diagnostic criteria can improve the assessment and condemnation of birds affected by airsacculitis.(AU)

Nos abatedouros, a inspeção das carcaças é fundamental para a detecção e monitoramento da prevalência de certas doenças. Os objetivos do trabalho foram avaliar a ocorrência de aspergilose causada por Aspergillus fumigatus em aves comerciais através do diagnóstico micológico e histopatológico e verificar a possibilidade de associação causal entre os critérios de diagnóstico de aspergilose e condenação por aerossaculite em frangos de corte através de um estudo de caso-controle. O estudo foi realizado com 380 amostras. Foram coletados pulmões de frangos condenados (95) e não condenados (285) por aerossaculite, diretamente na linha de abate de um frigorífico. Quarenta e seis (12%) amostras de pulmão foram positivas na cultura micológica. Do total de amostras, 177 (46,6%) apresentaram alterações histopatológicas, sendo os mais frequentes pneumonia fibrinoheterofílica necrótica, pneumonia heterofílica e hiperplasia linfóide. Do total de 380 pulmões analisados, 65,2% (30) apresentaram alterações histopatológicas e isolamento fúngico. A relação entre a presença de lesões histopatológicas e isolamento de A. fumigatus testada por McNemar indicou que houve associação significativa entre a presença de alterações histopatológicas e o isolamento de A. fumigatus. O cultivo micológico e o exame histopatológico aumentam as chances de se detectar alterações pulmonares em aves condenadas pelo Sistema de Inspeção Final do que nas aves normais, sugerindo que tais critérios de diagnóstico são eficazes para aprimorar a avaliação e condenação de aves por aerossaculite.(AU)

Diagnóstico macro e microscópico de Aspergilose em frangos de corte / Macroscopic and microscopic diagnosis of Aspergillosis in poultry

Background: Aspergillus fumigatus is considered the major agent of mycotic diseases in birds, affecting mainly the respiratory tract. It is a disease of economic importance in the poultry industry, however it is not a zoonotic or contagious disease. Aspergillus spp. are an environment residents. Infection usually occurs by inhalation of conidia released by molds, which come off the diet or specific ingredients, the nest and contamination of eggs during incubation. The objective of this study is to relate the macro and microscopic diagnosis of aspergillosis in poultry. Case: In an intensive farming of poultry (Gallus gallus), it was observed mortality rate exceeding 20%, hoarseness and difficulty breathing in males of approximately two weeks of age. The batch was treated with Terramycin ® (oxytetracyclinehydrochloride) in the first week and Trissulfin® (sulfamethoxazole, trimethoprim and bromhexine hydrochloride) in the second week. Birds were sent for analysis at the Laboratório Central de Diagnóstico de Patologias Aviárias (LCDPA) of the Universidade Federal de Santa Maria (UFSM). Necropsy was performed in three affected birds and pulmonary aspergillosis was suspected due to local pulmonary and disseminated injuries in the coelomic cavity, associated to the clinical signs. In birds assessed by necropsy examination, it was common the visualization of nodules in the internal cavity and lungs, caseous masses in the air sacs, little pigmentation on the feet and beaks and fragile bones. Portions of lung and granulomas were sent for isolation and identification of fungi in the Laboratório de Pesquisas Micológicas (LAPEMI)-UFSM and histopathological analysis in the Laboratório de Patologia Veterinária (LPV)-UFSM, where the standard protocols for each exam were used. The result of mycological examination showed Aspergillus fumigatus as the agent. The histopathological lesions observed in the lung were consistent with aspergillosis, characterized by multifocal granulomas associated with intra-lesional dichotomously branched fungal hyphae, morphologically compatible with Aspergillus sp. Discussion: The occurrence of aspergillosis depends on the dose of inhaled conidia of the fungus and the susceptibility of the host, which occurs in birds in the first weeks of age, making it more resistant to infection in adults. The history of signs consisting of respiratory distress associated with stressful situations or the recent lack of response to antibiotics may provide support to the clinical diagnosis of aspergillosis. The isolation and identification of the fungus comprises the best method to confirm the disease agent. Histopathology provides an important contribution to the morphological diagnosis of the lesion and the fungus. The treatment of aspergillosis in poultry production is difficult and uneconomical, so that all attention is focused on prevention and control in poultry houses and hatcheries. Eggs for incubation must be cleaned and disinfected, dirty and cracked eggs should not be incubated. Care should be strict with hygiene in the hatchery. Once detected the source, it should be eliminated, and the implementation of antifungal agents according to the location of contamination and the substrate. The major difficulty for the prevention and control of aspergillosis is because these fungi can be present at all stages of poultry production.

Macroscopic and microscopic diagnosis of Aspergillosis in poultry / Diagnóstico macro e microscópico de Aspergilose em frangos de corte

Background: Aspergillus fumigatus is considered the major agent of mycotic diseases in birds, affecting mainly the respiratory tract. It is a disease of economic importance in the poultry industry, however it is not a zoonotic or contagious disease. Aspergillus spp. are an environment residents. Infection usually occurs by inhalation of conidia released by molds, which come off the diet or specifi c ingredients, the nest and contamination of eggs during incubation. The objective of this study is to relate the macro and microscopic diagnosis of aspergillosis in poultry.Discussion: The occurrence of aspergillosis depends on the dose of inhaled conidia of the fungus and the susceptibility of the host, which occurs in birds in the fi rst weeks of age, making it more resistant to infection in adults. The history of signs consisting of respiratory distress associated with stressful situations or the recent lack of response to antibiotics may provide support to the clinical diagnosis of aspergillosis. The isolation and identifi cation of the fungus comprises the best method to confi rm the disease agent. Histopathology provides an important contribution to the morphological diagnosis of the lesion and the fungus. The treatment of aspergillosis in poultry production is diffi cult and uneconomical, so that all attention is focused on prevention and control in poultry houses and hatcheri

Background: Aspergillus fumigatus is considered the major agent of mycotic diseases in birds, affecting mainly the respiratory tract. It is a disease of economic importance in the poultry industry, however it is not a zoonotic or contagious disease. Aspergillus spp. are an environment residents. Infection usually occurs by inhalation of conidia released by molds, which come off the diet or specifi c ingredients, the nest and contamination of eggs during incubation. The objective of this study is to relate the macro and microscopic diagnosis of aspergillosis in poultry.Case: In an intensive farming of poultry (Gallus gallus), it was observed mortality rate exceeding 20%, hoarseness and diffi culty breathing in males of approximately two weeks of age. The batch was treated with Terramycin ® (oxytetracyclinehydrochloride) in the fi rst week and Trissulfi n® (sulfamethoxazole, trimethoprim and bromhexine hydrochloride) in the second week. Birds were sent for analysis at the Laboratório Central de Diagnóstico de Patologias Aviárias (LCDPA) of the Universidade Federal de Santa Maria (UFSM). Necropsy was performed in three affected birds and pulmonary aspergillosis was suspected due to local pulmonary and disseminated injuries in the coelomic cavity, associated to the clinical signs. In birds assessed by necropsy examination, it was common the visualization of nodules in the internal

Aspergillosis in green-winged saltators (Saltator similis) participants in bird singing competitions / Aspergilose em trinca-ferros (Saltator similis) competidores de canto

Background: Aspergillosis is the most common mycosis in birds, which are considered potential host also susceptible to the infection by Aspergillus spp. The clinical signs of the disease are often related to the respiratory tract, although other organs or systems may be affected. Signs are generally either not specifi c or may be directly related to the extent of the lesions in the respiratory tract (rhinitis, changes in the vocalization and dyspnea). The aim of this study was to describe and characterize three cases of aspergillosis in Greenwinged saltators (Saltator similis), based on reference standard diagnostics of this mycosis.Cases: Three Green-winged adult male saltators that had been participating weekly for three months in bird singing contests were referred to a private veterinary clinic. The clinical evaluation revealed pronounced hoarseness (3/3) and intense dyspnea (1/3). The animals were allocated to a warmed (280 C) oxygen chamber at 65% relative humidity. All birds were nebulized with saline solution and hyaluronidase. In addition, the birds were treated orally with complex vitamins B, A, D and E and intravenously either with dexamethasone and doxycycline (Case 1), azithromycin (Case 2) or metronidazole-enrofl oxacin combination (Case 3), in association with the use of Arnica Montana CH6 in the drinking water, and fl uid therapy (5% glucose, Ringer-lactate and

Background: Aspergillosis is the most common mycosis in birds, which are considered potential host also susceptible to the infection by Aspergillus spp. The clinical signs of the disease are often related to the respiratory tract, although other organs or systems may be affected. Signs are generally either not specifi c or may be directly related to the extent of the lesions in the respiratory tract (rhinitis, changes in the vocalization and dyspnea). The aim of this study was to describe and characterize three cases of aspergillosis in Greenwinged saltators (Saltator similis), based on reference standard diagnostics of this mycosis.Cases: Three Green-winged adult male saltators that had been participating weekly for three months in bird singing contests were referred to a private veterinary clinic. The clinical evaluation revealed pronounced hoarseness (3/3) and intense dyspnea (1/3). The animals were allocated to a warmed (280 C) oxygen chamber at 65% relative humidity. All birds were nebulized with saline solution and hyaluronidase. In addition, the birds were treated orally with complex vitamins B, A, D and E and intravenously either with dexamethasone and doxycycline (Case 1), azithromycin (Case 2) or metronidazole-enrofl oxacin combination (Case 3), in association with the use of Arnica Montana CH6 in the drinking water, and fl uid therapy (5% glucose, Ringer-lactate and

Avaliação do teste platelia EIA® Aspergillus para diagnóstico da aspergilose em pinguins e interferência da contaminação ambiental

A aspergilose é uma doença infecciosa não contagiosa causada por fungos do gênero Aspergillus, especialmente aqueles pertencentes à secção Fumigati e já foi descrita em diversas espécies, dentre elas os pinguins. As altas taxas de mortalidade atribuídas a esta doença, são decorrentes especialmente da dificuldade de realização de um diagnóstico precoce. Técnicas diagnósticas diretas para detecção do antígeno fúngico galactomanana (GM) em amostras clínicas têm sido utilizadas de modo crescente para o diagnóstico de aspergilose invasiva (AI) em humanos. A GM constitui-se de um polissacarídeo presente na parede celular de fungos do gênero Aspergillus. Embora sua aplicabilidade já esteja estabelecida para o diagnóstico precoce da AI em humanos, uma única testagem sérica por ELISA sanduíche a partir do kit comercial Platelia® Aspergillus EIA, deve ser interpretada com cautela, pois a técnica apresenta algumas limitações. Em animais sua aplicabilidade ainda não é bem elucidada. Com isso, o estudo objetivou avaliar a eficácia do teste Platelia EIA® Aspergillus para diagnóstico da aspergilose em pinguins e a influência da contaminação ambiental nos resultados do mesmo. Foram incluídas no estudo diagnóstico, amostras séricas de 29 pinguins de Magalhães que vieram a óbito por aspergilose (grupo caso) e 23 hígidos (grupo controle), as quais foram testadas a partir do kit para ensaio imunoenzimático tipo sanduíche em microplaca (Platelia Aspergillus EIA®, Bio-Rad) conforme as instruções do fabricante. Para o experimento de interferência ambiental foram utilizados 12 isolados de A. fumigatus da micoteca do Laboratório de Micologia da Faculdade de Medicina da FURG/FAMED incluindo cepas padrão, isolados clínicos de pinguins, humanos e isolados ambientais. Foram utilizadas para padronização do inoculo etapa de filtração, sedimentação, Pour-plate e espectrofotometria de acordo com CLSI, 2008. A detecção de GM foi realizada em três diluições sucessivas dos inoculos seguindo instruções do fabricante. Ao final foi calculado o índice de GM dividindo o valor da média da DO da duplicata da amostra (clínica ou da cepa testada) pelo valor da média da DO da duplicata da amostra de cut-off fornecida pelo kit. O índice de GM sérica não diferiu entre os animais do grupo caso e controle (pKW=0,097). A partir dos valores determinados pelas coordenadas da curva ROC, quatro diferentes pontos de corte (0,5, 1,0, 1,5 e 2,0) foram analisados, resultando em taxas de sensibilidade variando de 86,2% a 34,5%, e de especificidade entre 87% e 26,1%. Já na avaliação da interferência ambiental, a menor concentração de propágulos fúngicos de Aspergillus fumigatus capaz de gerar um resultado positivo foi de 480 conidios, sendo a concentração mediana de 4,8x103. Conclui-se que a detecção sérica de GM pelo teste Platelia Aspergillus EIA® não parece ser útil para o diagnóstico da aspergilose em pinguins naturalmente infectados, resultando em muitos falso-positivos. E, em relação a quantidade de conídios de A. fumigatus necessária para determinar resultado falso-positivo no teste de detecção de GM, evidenciou-se que há necessidade de uma maciça contaminação ambiental, por no mínimo 500 conídios, para que haja interferência no Platelia Aspergillus EIA®.

Aspergillosis is a non-contagious infectious disease caused by fungi of the genus Aspergillus, especially those belonging to the Fumigati section and has been describe in several species, among them penguins. The high mortality rates attributed to this disease are due in particular to the difficulty of performing an early diagnosis. Direct diagnostic techniques for the detection of galactomannan (GM) fungal antigen in clinical samples have been increase used for the diagnosis of invasive aspergillosis (AI) in humans. GM is a polysaccharide present in the cell wall of fungi of the genus Aspergillus. Although its applicability from the demonstration of circulating GM kinetics is already establish for the early diagnosis of AI in humans, a single serum test by Platelia® Aspergillus EIA should be interpreted with caution, since the technique has some limitations. In animals, its applicability is still not well understood. The objective of this study was to evaluate the effectiveness of the Platelia EIA® Aspergillus test for the diagnosis of aspergillosis in penguins and the influence of environmental contamination on the results. Serological samples of 29 Magellanic penguins that died of aspergillosis (case group) and 23 healthy (control group) were included in the diagnostic study, which were tested from the micro plate sandwich immunoenzymatic assay kit (Platelia Aspergillus EIA ®, Bio-Rad) according to the manufacturer's instructions. For the environmental interference experiment, 12 isolates of A. fumigatus from the mycology laboratory of the FURG / FAMED School of Medicine including standard strains, clinical isolates of penguins, humans and environmental isolates were use. The filtration, sedimentation, Pour-plate and spectrophotometry steps was use to standardize the inoculum according to CLSI, 2008. GM detection was performing on three successive dilutions of the inoculums following the manufacturer's instructions. At the end, the GM index was calculated by dividing the mean OD value of the sample duplicate (clinical or strain tested) by the mean OD value of the duplicate cut-off sample provided by the kit. The serum GM index did not differ between the animals in the control and the case group (pKW = 0.097). From the values determined by the coordinates of the ROC curve, four different cutoff points (0.5, 1.0, 1.5 and 2.0) were analyzed, resulting in sensitivity rates ranging from 86.2% to 34, 5%, and specificity between 87% and 26.1%. In the evaluation of environmental interference, the lowest concentration of fungal propagates of Aspergillus fumigatus capable of generating a positive result was 480 conidia, with a median concentration of 4.8x103. It is conclude that the serum detection of GM by the Platelia Aspergillus EIA® test does not seem to be useful for the diagnosis of aspergillosis in naturally infected penguins, resulting in many false positives. And, in relation to the amount of A. fumigatus conidia required to determine false-positive results in the GM detection test for AI diagnosis, it was shown that the degree of environmental contamination capable of causing interference in the Platelia Aspergillus EIA® test Be robust, of about 500 conidia.

Aspergilose em trinca-ferros (Saltator similis) competidores de canto / Aspergillosis in Green-winged saltators (Saltator similis) participants in bird singing competitions

Background: Aspergillosis is the most common mycosis in birds, which are considered potential host also susceptible to the infection by Aspergillus spp. The clinical signs of the disease are often related to the respiratory tract, although other organs or systems may be affected. Signs are generally either not specific or may be directly related to the extent of the lesions in the respiratory tract (rhinitis, changes in the vocalization and dyspnea). The aim of this study was to describe and characterize three cases of aspergillosis in Greenwinged saltators (Saltator similis), based on reference standard diagnostics of this mycosis. Cases: Three Green-winged adult male saltators that had been participating weekly for three months in bird singing contests were referred to a private veterinary clinic. The clinical evaluation revealed pronounced hoarseness (3/3) and intense dyspnea (1/3). The animals were allocated to a warmed (28º C) oxygen chamber at 65% relative humidity. All birds were nebulized with saline solution and hyaluronidase. In addition, the birds were treated orally with complex vitamins B, A, D and E and intravenously either with dexamethasone and doxycycline (Case 1), azithromycin (Case 2) or metronidazole-enrofloxacin combination (Case 3), in association with the use of Arnica Montana CH6 in the drinking water, and fluid therapy (5% glucose, Ringer-lactate and saline solution). In spite of the therapeutic treatments, the birds died three days after the hospitalization. The necropsy examination revealed: syrinx with yellowish content ranging from 2 to 5 mm diameter occluding the lumen (3/3) and the lungs were diffusely reddened (3/3) with yellowish areas of 2 mm (1/3). The histologic findings included proliferation of connective tissue in the mucosa and submucosa of the syrinx and granulomas formation in the lumen with necrotic centers that had large amounts of branched and septate hyphae with radiated arrangement. A large amount of hyphae were observed in granulomas in the lungs. The mycological culture (Sabouraud Dextrose Agar and Malt Extract Agar) of lung fragments and syrinx from all three birds allowed the isolation of colonies of Aspergillus fumigatus. Discussion: In Brazil, there are sparse reports of aspergillosis, mainly for native species of birds in captivity. The disease has been often diagnosed in migratory marine birds (treated at specific rehabilitation centers) and, occasionally, in wild birds or in animals raised in commercial poultry farms. The presence of fungal propagules in the respiratory system may cause colonization (a potential latent infection), condition that allows the fungal isolation from the lung tissue. However, under stress conditions or in a fall in immunity, as observed in this case study involving birds that intensively participate in singing competitions, it is often observed an evolution from the colonizing stage to range of clinical symptoms indicative of aspergillosis. The adoption of preventive measures to reduce the spreading of fungal propagules in the environment is essential, as well as proper feeding and hydration, a minimization of stress-related issues during animal management and transport, the rational use of drugs, and conditions that may favor the development of opportunistic mycoses, such as aspergillosis, which is the most common fungal disease in the world. The reported cases of aspergillosis in the lungs and syrinx of Green-winged saltators emphasizes the importance of a conclusive diagnosis for the disease based on the detection of fungal structures at the histopathological examination associated with the isolation of the agent in a fungal culture. The occurrence of aspergillosis may cause a significant loss of native bird species maintained in captivity, which is a serious concern when related to the preservation of the diversity of the Brazilian avian fauna.