Isolation of coccidioides brasiliensis from armadillos (Dasypus noveminctus) captured in an endemic area of paracoccidioidomycosis

Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis (PCM), was first isolated from armadillos from the Amazonian region where the mycosis is uncommon. In the present study, we report on the high incidence of PCM infection in armadillos from a hyperendemic region of the disease. Four nine-banded armadillos (Dasypus novemcinctus) were captured in the endemic area of Botucatu, São Paulo, Brazil, killed by manual cervical dislocation and autopsied under sterile conditions. Fragments of lung, spleen, liver, and mesenteric lymph nodes were processed for histology, cultured on Mycosel agar at 37 degrees C, and homogenized for inoculation into the testis and peritoneum of hamsters. The animals were killed from week 6 to week 20 postinoculation and fragments of liver, lung, spleen, testis, and lymph nodes were cultured on brain heart infusion agar at 37 degrees C. Paracoccidioides brasiliensis was isolated from three armadillos both by direct organ culture and from the liver, spleen, lung, and mesenteric lymph nodes of hamsters. In addition, one positive armadillo presented histologically proven PCM disease in a mesenteric lymph node. The three armadillos isolates (Pb-AL, Pb-A2, and Pb-A4) presented thermodependent dimorphism, urease activity, and casein assimilation, showed amplification of the gp43 gene, and were highly virulent in intratesticularly inoculated hamsters. The isolates expressed the gp43 glycoprotein, the immunodominant antigen of the fungus, and reacted with a pool of sera from PCM patients. Taken together, the present data confirm that armadillos an a natural reservoir of P. brasiliensis and demonstrate that the animal is a sylvan host to the fungus.

Prostaglandin E-2 production by high and low virulent strains of Paracoccidioides brasiliensis

The production of prostaglandins (PGs) during fungal infections could be an important suppressor factor of host immune response. Host cells are one source of prostaglandin E-2 (PGE(2)); however another potential source of PGE(2) is the fungal pathogen itself. Thus, both host and fungal PGE2 production is theorized to play a role in pathogenesis, being critical for growth of the fungus and to modulate the host immune response. The purpose of this work was to investigate if high and low virulent strains of Paracoccidioides brasiliensis have the capacity to produce PGE(2) in vitro, and if this production was related to the fungal growth. The results demonstrated that both strains of P. brasiliensis produce high levels of PGE(2) and the treatment with indomethacin, a cyclooxygenase inhibitor, significantly reduced the production of this mediator, as well as the viability of the fungus. Thus, our data indicate that PGE(2) is produced by P. brasiliensis by a cyclooxygenase-dependent metabolic pathway, and its production is required for fungal survival. This discovery reveals an important factor that has potentially great implications for understanding the mechanisms of immune deviation during infection.

A critical analysis of isolation of Paracoccidioides brasiliensis from soil

The present review is a critical analysis of positive and negative reports of the isolation of Paracoccidioides brasiliensis from soil. The strains isolated from soil or soil-contaminated material (dogfood, penguin feces) by Batista et al. in Recife, Brazil, Negroni in the Argentinian Chaco, Albornoz in rural Venezuela, Silva-Vergara et al, in Ibia, Brazil, Ferreira et al, in Uberlandia, Brazil, and Gezuele et al. at the Uruguayan base in the Antarctic region, presented mycological characteristics consistent with P. brasiliensis. In most of these studies, morphological characterization was complemented with an evaluation of virulence and antigenicity, and biochemical or molecular analysis. These isolations, therefore, can be considered true, supporting the concept of soil as an important element in the ecology of the pathogen. The large number of negative reports in attempts involving soil samples and the low repeatability of isolation of the fungus from the same area indicate that the specific conditions supporting growth of the pathogen in soil have not been fully clarified.

High frequency of Paracoccidioides brasiliensis infection in armadillos (Dasypus novemcinctus): an ecological study

The fungus Paracoccidioides brasiliensis has been isolated from nine-banded armadillos (Dasypus novemcinctus) in different regions where paracoccidiodomycosis (PCM) is endemic. The link between PCM and these animals has provided the first valuable clue in the effort to elucidate the ecological niche of P. brasiliensis. The present study was aimed at correlating P. brasiliensis infection in armadillos with local ecological features and, if possible, the presence of the fungus in the soil in the Botucatu hyperendemic area of PCM. In this region the mean temperature ranges from 14.8 to 25.8degreesC and the annual average precipitation is 1520 mm. The sites where 10 infected animals (positive group) were collected were studied and compared with the sites where five uninfected animals were found. The occurrence of the fungus in soil samples collected from the positive armadillos' burrows and foraging sites was investigated by the indirect method of animal inoculation. Environmental data from the sites of animal capture, such as temperature, rainfall, altitude, vegetation, soil composition, presence of water and proximity of urban areas, were recorded. All 37 soil samples collected from the sites had negative fungal cultures. Positive animals were found much more frequently in sites with disturbed vegetation, such as riparian forests and artificial Eucalyptus Or Pinus forests, in altitudes below 800 m, near water sources. The soil type of the sites of positive animals was mainly sandy, with medium to low concentrations of organic matter. The pH was mainly acidic at all the sites, although the concentrations of aluminum cations (H+Al) were lower at the sites where positive animals were found. Positive armadillos were also captured in sites very close to urban areas. Our data and previous studies indicate that P. brasiliensis occurs preferentially in humid and shady disturbed forests in a strong association with armadillos.

A comparative study of the immunoantigenicity of eight Paracoccidioides brasiliensis isolates

Para se detectar diferenças imuno-antigênicas entre 8 amostras de P. brasiliensis isoladas de diferentes áreas endêmicas (Botucatu: Pb 1, 2 e 3; São Paulo: Pb: 18, 192 e 265; Venezuela: Pb 9 e 73), esutdaram-se: 1. A reatividade antigênica de cada amostra nas reações de imunofluorescência indireta (II) e de imunodifusão dupla em gel de agar (ID) contra painel de 20 soros controles positivos para paracoccidioidomicose; 2. A capacidade de induzir resposta imune humoral (medida por imunodifusão) e celular (medida pelo teste de coxim plantar) em camundongos imunizados com an-tígenos de cada amostra. Observamos: 1. As amostras Pb 265 e Pb 9 mostraram-se mais reativas na II; 2. Os antígenos das amostras Pb 192 e Pb 73 foram significativamente mais reativas na ID; 3. Estes dados demonstram diferenças de antigenicidade entre estas amostras; 4. A amostra Pb 18 mostrou baixo poder indutor de resposta imune celular e alta capacidade de indução de resposta imune humoral em camundongos imunizados, revelando dissociação de sua imunogenicidade. Estas diferenças podem indicar a existência de cepas distintas do fungo ou refletir modificações do parasita no hospedeiro ou du rante seu cultivo.

Serological follow-up of patients with paracoccidioidomycosis treated with itraconazole using Dot-blot, ELISA and Western-blot

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Slaframine toxicosis in Brazilian horses causing excessive salivation

Ingestion of food contaminated with slaframine, an alkaloid produced by Rhizoctonia leguminicola, causes a mycotoxicosis, characterised by excessive salivation. Twenty-eight horses demonstrated this clinical sign after the consumption of alfalfa hay which on inspection showed dark patches on many of the stems. The presence of slaframine (1.5 ppm) in this hay was confirmed by gas chromatography and mass spectroscopy. This is the first equine slaframine toxicosis case reported in Brazil.

Tratamento do feno de braquiária pelo fungo Pleurotus ostreatus

A inoculação de forragens com fungos lignocelulolíticos é uma opção para melhorar a qualidade destas sem adição de produtos químicos. O tratamento do substrato influencia a ação do fungo e a qualidade final do produto. Neste experimento, aplicaram-se quatro tratamentos (compostagem do feno inteiro, compostagem do feno picado, hidratação do feno em água fria e hidratação do feno em água quente) a um feno de Brachiaria decumbens. Aos tratamentos seguiu-se inoculação com o fungo Pleurotus ostreatus e incubação por 35 dias, sob temperatura controlada. Usou-se o delineamento inteiramente casualizado, com quatro repetições e medidas repetidas. Amostras foram colhidas semanalmente para acompanhar a degradação do substrato, mediante a análise química do feno. Observou-se aumento linear, com o decorrer do tempo, no teor de proteína bruta (PB) e na proporção de lignina na parede celular (LIG-FDN), e decréscimo linear nos valores de fibra em detergente neutro (FDN), celulose e hemicelulose. Não se observou efeito de tratamento no teor de FDA. Os tratamentos com compostagem apresentaram maiores valores de PB, lignina e LIG-FDN e menores de FDN e hemicelulose. Não se observou diferença entre os tratamentos com hidratação. O tratamento do feno de braquiária com o fungo propiciou degradação da fração fibrosa e aumento no teor de PB, com efeito mais intenso nos tratamentos que usaram compostagem. A ação do fungo foi mais efetiva sobre a hemicelulose que sobre os demais componentes da fibra.

Valor nutritivo do feno de braquiária amonizado com uréia ou inoculado com Pleurotus ostreatus

Verificou-se, mediante análises de composição química e ensaio de digestibilidade com ovinos, o efeito dos tratamentos químico (amonização com uréia) e biológico (inoculação com o fungo Pleurotus ostreatus) sobre o valor nutritivo do feno de Brachiaria decumbens. Ambos os tratamentos duraram 42 dias; após esse período, o feno foi seco e moído para fornecimento aos animais. As dietas experimentais foram: feno não tratado (FNT); feno não tratado + uréia (FNT + U); feno inoculado com fungo + uréia (FTB + U); e feno amonizado + feno não tratado (FTQ + FNT). As dietas FNT + U, FTB + U e FTQ + FNT foram isonitrogenadas. Tanto o tratamento químico como o biológico causaram mudanças na composição química do feno. A amonização elevou os teores de proteína bruta (PB) e fibra em detergente ácido (FDA) e reduziu os teores de hemicelulose (HEM) e a proporção de hemicelulose na parede celular (HEM-FDN). Já o tratamento biológico tendeu a aumentar o teor de PB; elevou os teores de FDA, lignina (LIG), a proporção de celulose na parede celular (CEL-FDN) e a proporção de lignina na parede celular (LIG-FDN); e reduziu os teores de fibra em detergente neutro (FDN), HEM e HEM-FDN. Entretanto, diminuiu a digestibilidade da matéria seca (MS), FDN, celulose (CEL) e FDA, mas aumentou o consumo, provavelmente em decorrência do menor teor de FDN e menor tamanho médio de partículas, o que causou maior velocidade de passagem. Os tratamentos biológico e químico são alternativas importantes no incremento do valor nutritivo de materiais lignocelulósicos, todavia, os resultados obtidos neste ensaio não foram satisfatórios.

Modelling the lethargic crab disease.

The lethargic crab disease (LCD) is an emergent infirmity that has decimated native populations of the mangrove land crab (Ucides cordatus, Decapoda: Ocypodidae) along the Brazilian coast. Several potential etiological agents have been linked with LCD, but only in 2005 was it proved that it is caused by an ascomycete fungus. This is the first attempt to develop a mathematical model to describe the epidemiological dynamics of LCD. The model presents four possible scenarios, namely, the trivial equilibrium, the disease-free equilibrium, endemic equilibrium, and limit cycles arising from a Hopf bifurcation. The threshold values depend on the basic reproductive number of crabs and fungi, and on the infection rate. These scenarios depend on both the biological assumptions and the temporal evolution of the disease. Numerical simulations corroborate the analytical results and illustrate the different temporal dynamics of the crab and fungus populations.

Genomic DNA microarray comparison of gene expression patterns in Paracoccidioides brasiliensis mycelia and yeasts in vitro

Paracoccidioides brasiliensis is a thermally dimorphic fungus, and causes the most prevalent systemic mycosis in Latin America. Infection is initiated by inhalation of conidia or mycelial fragments by the host, followed by further differentiation into the yeast form. Information regarding gene expression by either form has rarely been addressed with respect to multiple time points of growth in culture. Here, we report on the construction of a genomic DNA microarray, covering approximately 25% of the genome of the organism, and its utilization in identifying genes and gene expression patterns during growth in vitro. Cloned, amplified inserts from randomly sheared genomic DNA (gDNA) and known control genes were printed onto glass slides to generate a microarray of over 12 000 elements. To examine gene expression, mRNA was extracted and amplified from mycelial or yeast cultures grown in semi-defined medium for 5, 8 and 14 days. Principal components analysis and hierarchical clustering indicated that yeast gene expression profiles differed greatly from those of mycelia, especially at earlier time points, and that mycelial gene expression changed less than gene expression in yeasts over time. Genes upregulated in yeasts were found to encode proteins shown to be involved in methionine/cysteine metabolism, respiratory and metabolic processes (of sugars, amino acids, proteins and lipids), transporters (small peptides, sugars, ions and toxins), regulatory proteins and transcription factors. Mycelial genes involved in processes such as cell division, protein catabolism, nucleotide biosynthesis and toxin and sugar transport showed differential expression. Sequenced clones were compared with Histoplasma capsulatum and Coccidioides posadasii genome sequences to assess potentially common pathways across species, such as sulfur and lipid metabolism, amino acid transporters, transcription factors and genes possibly related to virulence. We also analysed gene expression with time in culture and found that while transposable elements and components of respiratory pathways tended to increase in expression with time, genes encoding ribosomal structural proteins and protein catabolism tended to sharply decrease in expression over time, particularly in yeast. These findings expand our knowledge of the different morphological forms of P. brasiliensis during growth in culture.

TNF-alpha activates human monocytes for Paracoccidioides brasiliensis killing by an H2O2-dependent mechanism

Human monocytes activated by recombinant tumor necrosis factor alpha (TNF-alpha) exhibited significant fungicidal activity on the yeast cells of a highly virulent strain of Paracoccidioides brasiliensis. This process was significantly inhibited in the presence of catalase (CAT - a scavenger of H2O2), but not in the presence of superoxide-dismutase (SOD - a scavenger of superoxide anion) or N-G-monomethyl-L- arginine (N-G-MMLA - a nitric oxide inhibitor). Furthermore, there was a direct association between the intracellular killing of the fungus and the production of H2O2 by activated cells. These results strongly suggest a role for H2O2 in the killing of highly virulent strains of P. brasiliensis by TNF-alpha-activated human monocytes.

Ecological study of Paracoccidioides brasiliensis in soil: growth ability, conidia production and molecular detection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of interleukin-10 on the Paracoccidioides brasiliensis killing by gamma-interferon activated human neutrophils

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytokine production in lungs and adrenal glands of high and low antibody producing mice infected with Paracoccidioides brasiliensis

Mice genetically selected for high (H) and low (L) antibody production (HIV-A and L-IV-A) were used in an experimental model of paracoccidioidomycosis. In a previous work, it was observed that male HIV-A animals were more susceptible to the infection due to adrenal gland damage. Male HIV-A and LIV-A animals were intravenously inoculated with Paracoccidioides brasiliensis (strain 18) and sacrificed 2, 4, 6, 8 and 10 weeks after inoculation. At each time interval, lungs and adrenals were removed to estimate recoverability of the fungus, as well as to determine Th1 (IFN-gamma, TNF-alpha) and Th2 (IL-4 and IL-10) cytokine profiles. While viable fungi recoverability from the lungs of HIV-A mice was higher after 4 and 8 weeks, there was less fungal recovery from the adrenals of LIV-A animals after the 2nd week, with total fungal elimination after the 8th week. With regard to Th2 cytokines, there was an inhibition in IL-4 production in the organs from infected animals, the extent of which varied according to the organ and the time period after initiation of infection. IL-10 production was found to be lower in both organs. Determination of Th1 cytokines revealed that IFN-gamma production increased in both organs, mainly in the adrenal of LIV-A after 8 and 10 weeks, when these animals showed a total fungal elimination. A significant difference was observed between HIV-A and LIV-A concerning TNF-alpha production in both organs and at all recovery times, in that LIV-A produced a higher level of this cytokine, mainly in the adrenal. These results may explain the high susceptibility of HIV-A to P. brasiliensis infection, is due, at least in part, to adrenal involvement. The higher production of Th1 cytokines by LIV-A in comparison to HIV-A mice may account for LIV-A resistance to P. brasiliensis infection. Our data reveal the importance of this experimental model in the study of the adrenal involvement in paracoccidioidomycosis, since this gland may be highly compromised in the patients, leading to the development of Addison's Disease.