Differential role of gpaB and sidA gene expressions in relation to virulence in Aspergillus species from patients with invasive aspergillosis

The virulence genes in invasive aspergillosis (IA) have not been analyzed adequately. The present study was designed to evaluate the expression of gpaB and sidA genes, which are important virulence genes in Aspergillus spp. from bronchoalveolar lavage (BAL) samples. Direct examination and culture on Czapek Agar and Sabouraud Dextrose Agar media were performed for 600 BAL specimens isolated from patients with possible aspergillosis. A Galactomannan ELISA assay was also carried out. The expression levels of the gpaB and sidA genes in isolates were analyzed using quantitative real-time PCR (qRT-PCR). We identified 2 species, including Aspergillus flavus (A. flavus) and Aspergillus fumigatus (A. fumigatus) in 25 positive samples for invasive aspergillosis as validated using GM-ELISA. A. flavus is the main pathogen threatening transplant recipients and cancer patients worldwide. In this study, A. flavus had low levels of the gpaB gene expression compared to A. fumigatus (p = 0.006). The highest sidA expression was detected in transplant recipients (p = 0.05). There was no significant correlation between sidA expression and underlying disease (p = 0.15). The sidA and gpaB gene expression patterns may provide evidence that these virulence genes play important roles in the pathogenicity of Aspergillus isolates; however, there are several regulatory genes responsible for the unexpressed sidA and gpaB genes in the isolates.(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)

Serological monitoring of antibodies for an early diagnosis ofaspergillosis in captive penguins / Monitoramento sorológico com detecção de anticorpos para diagnóstico precoce da aspergilose em pinguins em cativeiro

This study aimed to evaluate the efficacy of detection of anti-Aspergillus fumigatus antibodies in captive penguins by double radial agar gel immunodiffusion (AGID) for the aspergillosis diagnosis. We included 134 Magellanic penguins (Spheniscus magellanicus) in rehabilitation at the Center for Recovery of Marine Animals (CRAM / FURG). All of them were monitored by AGID weekly until its final destination (death or release), totalizing 660 serum samples studied. All animals were clinically accompanied and post-mortem examinations was performed in penguins that died during the studied period. A total of 28% (37/134) of the penguins died, 89.2% (33/37) due to aspergillosis, 11% (4/37) by other causes and 97 were released. From the 33 animals with proven aspergillosis, 21 presented anti- A. fumigatus antibodies by AGID, being the average interval between death and positive AGID 16.4 days. Twelve animals with negative serology died of aspergillosis. The sensitivity and specificity rates were 63.6% and 95% respectively, and the positive and negative predictive values were 80.7% and 88.9% respectively. These data demonstrate that the serological monitoring for detection of antibodies by AGID can be an important tool for the diagnosis of aspergillosis in penguins.(AU)

Este estudo teve como objetivo avaliar a eficácia da detecção de anticorpos anti- Aspergillus fumigatus em pinguins em cativeiro por imunodifusão radial dupla em gel de ágar (IDGA) para diagnóstico da aspergilose. Foram incluídos 134 pingüins de Magalhães (Spheniscus magellanicus) em reabilitação no Centro de Recuperação de Animais Marinhos (CRAM/FURG), que foram monitoradas por IDGA, semanalmente, até o seu destino final (morte ou de liberação), totalizando 660 amostras de soro estudadas. Todos os animais foram acompanhados clinicamente e exames post mortem foram realizados em pingüins que vieram a óbito durante o período de estudo. Um total de 28% (37/134) dos pinguins foram a óbito, 89,2% (33/37) de aspergilose, 11% (4/37) de outras causas, e 97 foram liberados. A partir dos 33 animais com aspergilose comprovada, 21 apresentaram anticorpos anti- A. fumigatus por IDGA, sendo o intervalo médio entre a morte e IDGA positivas 16,4 dias. Doze animais com sorologia negativa vieram a óbito por aspergilose. As taxas de sensibilidade e especificidade foram de 63,6% e 95%, respectivamente, e os valores preditivos positivos e negativos foram de 80,7% e 88,9 %, respectivamente. Estes dados demonstram que o monitoramento sorológico para detecção de anticorpos por IDGA pode ser uma ferramenta importante no diagnóstico de aspergilose em pinguins.(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.

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.