Isolation of Sporothrix schenckii from the claws of domestic cats (indoor and outdoor) and in captivity in São Paulo (Brazil)

Sporotrichosis is a subcutaneous mycosis and is also a zoonosis (sapro- and anthropozoonosis). The objective of the present study was to determine the occurrence of sporotrichosis in domestic cats and in wild or exotic felines in captivity through the isolation of Sporothrix spp. from claw impressions in a culture medium. The samples included 132 felines, of which 120 (91.0 %) were domestic cats, 11 (8.3 %) were wild felines, and one (0.7 %) was an exotic felid. Twenty-one (17.5 %) were outdoor cats. Of the total, 89 (67.4 %) had contact with other animals of the same species. It was possible to isolate Sporothrix schenckii from the claws of one (0.7 %) of the felids probed; this animal exhibited generalised sporotrichosis and had infected a female veterinarian. The potential pathogenic agents Microsporum canis and Malassezia pachydermatis were isolated in 12.1 and 5.3 % of the animals, respectively. The following anemophilous fungi, which were considered to be contaminants, were also isolated: Penicillium sp. (28 or 21.2 %), Aspergillus sp. (13 or 9.8 %), Rhodotorula sp. (5 or 3.8 %), Candida sp. (5 or 3.8 %), Trichoderma sp. (1 or 0.7 %), and Acremonium sp. (1 or 0.7 %). Due to the low magnitude of occurrence (0.7 %) of Sporothrix in feline claws, the potential of the cats evaluated in this study to be sources of infection in the city of São Paulo is considerably low.

Mycobiota, aflatoxins and cyclopiazonic acid in stored peanut cultivars

This study evaluated the presence of fungi and mycotoxins [aflatoxins (AFs), cyclopiazonic acid (CPA), and aspergillic acid] in stored samples of peanut cultivar Runner IAC Caiapó and cultivar Runner IAC 886 during 6 months. A total of 70 pod and 70 kernel samples were directly seeded onto Aspergillus flavus and Aspergillus parasiticus agar for fungi isolation and aspergillic acid detection, and AFs and CPA were analyzed by high-performance liquid chromatography. The results showed the predominance of Aspergillus section Flavi strains, Aspergillus section Nigri strains, Fusarium spp., Penicillium spp. and Rhizopus spp. from both peanut cultivars. AFs were detected in 11.4% of kernel samples of the two cultivars and in 5.7% and 8.6% of pod samples of the Caiapó and 886 cultivars, respectively. CPA was detected in 60.0% and 74.3% of kernel samples of the Caiapó and 886 cultivars, respectively. Co-occurrence of both mycotoxins was observed in 11.4% of kernel samples of the two cultivars. These results indicate a potential risk of aflatoxin production if good storage practices are not applied. In addition, the large number of samples contaminated with CPA and the simultaneous detection of AFs and CPA highlight the need to investigate factors related to the control and co-occurrence of these toxins in peanuts.

The capability of endophytic fungi for production of hemicellulases and related enzymes

Abstract BACKGROUND: There is an imperative necessity for alternative sources of energy able to reduce the world dependence of fossil oil. One of the most successful options is ethanol obtained mainly from sugarcane and corn fermentation. The foremost residue from sugarcane industry is the bagasse, a rich lignocellulosic raw material uses for the production of ethanol second generation (2G). New cellulolytic and hemicellulytic enzymes are needed, in order to optimize the degradation of bagasse and production of ethanol 2G. RESULTS: The ability to produce hemicellulases and related enzymes, suitable for lignocellulosic biomass deconstruction, was explored using 110 endophytic fungi and 9 fungi isolated from spoiled books in Brazil. Two initial selections were performed, one employing the esculin gel diffusion assay, and the other by culturing on agar plate media with beechwood xylan and liquor from the hydrothermal pretreatment of sugar cane bagasse. A total of 56 isolates were then grown at 29°C on steam-exploded delignified sugar cane bagasse (DEB) plus soybean bran (SB) (3:1), with measurement of the xylanase, pectinase, β-glucosidase, CMCase, and FPase activities. Twelve strains were selected, and their enzyme extracts were assessed using different substrates. Finally, the best six strains were grown under xylan and pectin, and several glycohydrolases activities were also assessed. These strains were identified morphologically and by sequencing the internal transcribed spacer (ITS) regions and the partial β-tubulin gene (BT2). The best six strains were identified as Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49. These strains produced glycohydrolases with different profiles, and production was highly influenced by the carbon sources in the media. CONCLUSIONS: The selected endophytic fungi Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49 are excellent producers of hydrolytic enzymes to be used as part of blends to decompose sugarcane biomass at industrial level.

Practical implications of aflatoxin contamination in corn

Aflatoxin (AFL) contamination of corn is a serious economic and food security issue. Although a variety of technical solutions for reducing AFL contamination of corn have been proposed, only a few have produced satisfactory results. A successful approach is a biocontrol strategy consisting of using non-flatoxigenic strains of Aspergillus flavus to replace indigenous AFL-producing isolates. The main objective of the present thesis was to investigate the dynamic and contamination of AFL/A. flavus in corn in Northern Italy. The study also investigated the role of the key-pest of corn, the European Corn Borer (ECB), on AFL contamination and dispersal of A. flavus propagules in corn. Finally, the study evaluated the feasibility of bioplastic-based granules entrapping a non-aflatoxigenic A. flavus strain for the biocontrol of this fungus in corn. The 2-year field study demonstrated the efficacy of the bioplastic formulation to reduce AFL contamination in corn. More precisely, although AFL contamination varied among the two years, application of 15 and 30 kg ha-1 of granules reduced AFL contamination to up 60 and 85% in 2009 and 2010 respectively. Microbiological analysis showed that the relative abundance of non-aflatoxigenic soil isolates significantly increased after 1 month from granules application (mid-May) and throughout the corn-growing season. These findings were consistent with data obtained using a bioplastic-based bait specifically developed to selectively isolate Aspergilli from soil and other environmental samples. In addition, field and laboratory evaluations showed that the level of damages produced by ECB larvae were not significantly correlated to A. flavus infestation and AFL contamination. Taking together, these findings demonstrated that AFL contamination of corn in Northern Italy was variable, but above the EU limit for human consumption. First proposed in the USA, this study showed the practical possibility of this formulation to be use for reducing AFL contamination in corn in the EU.

Untersuchungen mit Bodenpilzen aus der Rebstock-Rhizosphäre unterschiedlich bewirtschafteter Rebanlagen des Rheingaus unter besonderer Berücksichtigung ihrer Pathogenität

Ziel der Untersuchungen war, Pilze aus geschädigtem und ungeschädigtem Wurzelmaterial konventionell und ökologisch bewirtschafteter Weinbergsböden zu isolieren und diese auf ihre Durchsetzungsfähigkeit gegenüber den anderen Arten bzw. deren Pilzmetabolitsuspensionen unter unterschiedlichen Nahrungsbedingungen zu prüfen und eine eventuelle substratabhängige Verhaltensänderung bei den Spezies in Interaktion festzustellen. Zudem wurde in weiteren In-vitro- Versuchen das pathogene Potenzial der gefundenen Arten gegenüber Vitis spp. getestet. Hintergrund dieser Untersuchungen war die Hypothese, dass Absterbeerscheinungen in Rebanlagen nicht durch die Reblaus per se verursacht werden, sondern dass ein Zusammenhang zwischen der Bewirtschaftungsmethode und dem Schadbild in reblausbefallenen Rebanlagen besteht und dessen Entstehung auf pathogenkonduktive und –suppressive Eigenschaften des Bodens zurückgeführt werden kann. Aus rund 2400 Wurzelproben konnten insgesamt 49 Pilzarten isoliert und bestimmt und mehr als die Hälfte davon in Wurzeln beider Versuchsflächen nachgewiesen werden. Ein Großteil der Pilze wurde sowohl in geschädigten als auch in ungeschädigten Wurzelgeweben identifiziert. Darunter waren Arten, die in der Literatur als Parasiten und Saprobier beschrieben werden, aber auch Arten, die eine andere Lebensweise pflegen oder deren Lebensweise nicht bekannt ist. Mit Hilfe von Interaktionsversuchen auf unterschiedlichen Nährmedien (einem Voll- und einem Mangelmedium) konnte bei den untersuchten Arten teilweise starke substratabhängige Verhaltensänderung in Interaktion mit bestimmten Pilzkolonien festgestellt und auf die Verfügbarkeit von organischem Kohlenstoff zurückgeführt werden. Starke Konkurrenz um organischen Kohlenstoff und dadurch entstehende fungistatische und antibiotische Effekte können in diesem Zusammenhang pathogenkonduktive bzw. pathogensuppressive Bodeneigenschaften fördern oder hemmen. Weiterhin konnte gezeigt werden, dass alle 15 in vitro an Vitis spp. inokulierten Pilze (Absidia glauca, Acremonium kiliense, Aspergillus ustus, Cylindrocarpon magnusianum, Cylindrocarpon sp., Fusarium culmorum, F. detonianum, F. oxysporum, F. sacchari, F. semitectum, Gliocladium roseum, Leptosphaeria coniothyrium, Penicillium expansum, Trichoderma harzianum, T. pseudokoningii), unter denen sich auch als Saprobier bekannte Arten befanden (P. expansum, T. harzianum), selbst bei Verfügbarkeit organischer Kohlenstoffverbindungen im Substrat, gegenüber Vitis spp. ein fakultativ pathogenes Potenzial besitzen. Diese aus In-vitro-Interaktionsversuchen gewonnenen Erkenntnisse geben Hinweise darauf, welchen Einfluss die Bewirtschaftung, insbesondere die Versorgung der Weinbergsböden mit organischem Kohlenstoff, auf fakultativ pathogene Sekundärparasiten in Form von Bodenpilzen und folglich auf die Entwicklung von Schadbildern an durch die Reblaus prädispositionierten Rebpflanzen in vivo haben kann.

Self-sufficient control of urate homeostasis in mice by a synthetic circuit

Synthetic biology has shown that the metabolic behavior of mammalian cells can be altered by genetic devices such as epigenetic and hysteretic switches, timers and oscillators, biocomputers, hormone systems and heterologous metabolic shunts. To explore the potential of such devices for therapeutic strategies, we designed a synthetic mammalian circuit to maintain uric acid homeostasis in the bloodstream, disturbance of which is associated with tumor lysis syndrome and gout. This synthetic device consists of a modified Deinococcus radiodurans-derived protein that senses uric acids levels and triggers dose-dependent derepression of a secretion-engineered Aspergillus flavus urate oxidase that eliminates uric acid. In urate oxidase-deficient mice, which develop acute hyperuricemia, the synthetic circuit decreased blood urate concentration to stable sub-pathologic levels in a dose-dependent manner and reduced uric acid crystal deposits in the kidney. Synthetic gene-network devices providing self-sufficient control of pathologic metabolites represent molecular prostheses, which may foster advances in future gene- and cell-based therapies.

[Tremorgenic syndrome in a cattle herd after feeding silage contaminated with A. clavatus];;Tremorgenes Syndrom in einem Rinderbestand nach Verfütterung einer mit A. clavatus befallenen Silage

The clinical signs, pathological and laboratory findings of cattle suffering from a tremorgenic syndrome are described. Animals on a farm with a total of 22 cows, 18 heifers and 9 calves were fed mouldy grass and spent malt-grain silage. Five heifers were affected with muscular tremor, hyperexcitability and hypersensitivity. They were ataxic or in sternal recumbency, while their appetite remained normal. Haematology and blood chemistry in two heifers as well as cerebrospinal fluid from one sick animal were unremarkable. The pathological examination of one animal brought no macroscopic changes to light. Histological examination, however, revealed the degeneration of motor neurones in the midbrain, brain stem and spinal cord. Analysis of a silage sample provided evidence of the presence of Aspergillus clavatus, a mould capable of producing neurotoxic tremorgenic mycotoxins. Epidemiology, clinical findings, pathology and microbiological examination suggest that the five cattle were suffering from neuromycotoxicosis.

IgE, IgGa, IgGb and IgG(T) serum antibody levels in offspring of two sires affected with equine recurrent airway obstruction

Equine recurrent airway obstruction (RAO) is a chronic lower airway disease of the horse caused by hypersensitivity reactions to inhaled stable dust, including mould spores such as Aspergillus fumigatus. The goals of this study were to investigate whether total serum IgE levels and allergen-specific IgE and IgG subclasses are influenced by genetic factors and/or RAO and whether quantitative trait loci (QTL) could be identified for these parameters. The offspring of two RAO-affected sires (S1: n=56 and S2: n=65) were grouped by stallion and disease status, and total serum IgE levels and specific IgE, IgGa, IgGb and IgG(T) levels against recombinant Aspergillus fumigatus 7 (rAspf7) were measured by ELISA. A panel of 315 microsatellite markers covering the 31 equine autosomes were used to genotype the stallions and their offspring. A whole-genome scan using half-sib regression interval mapping was performed for each of the IgG and IgE subclasses. There was no significant effect of disease status or sire on total IgE levels, but there was a significant effect of gender and age. rAspf7-specific IgGa levels were significantly higher in RAO-affected than in healthy horses. The offspring of S1 had significantly higher rAspf7-specific IgGa and IgE levels than those of S2. Five QTLs were significant chromosome-wide (P<0.01). QTLs for rAspf7-specific IgGa and IgE were identified on ECA 1, for rAspf7-specific IgGa and IgGb on ECA 24 and for rAspf7 IgGa on ECA 26. These results provide evidence for effects of disease status and genetics on allergen-specific IgGa and IgE.

Invasive sphenoidal aspergillosis: successful treatment with sphenoidotomy and voriconazole

Treatment of invasive sphenoidal aspergillosis is surgical, followed by antifungal therapy, mostly amphotericin B. To optimize the adjuvant antifungal treatment, which is often limited by severe side effects, the new triazole antifungal agent voriconazole with broad coverage of fungal pathogens including Aspergillus was investigated in a study of 4 patients with clinical, radiological and histological signs of invasive sphenoidal aspergillosis. They first underwent endoscopic sphenoidotomy with drainage and extraction of the fungal mass. Postoperatively, 2 patients were immediately treated with voriconazole. Two patients initially received amphotericin B; but this treatment had to be stopped because of acute renal toxicity. Finally, all patients were treated orally with 200 mg voriconazole twice a day for 12-14 weeks. After this combined treatment all patients were asymptomatic and there were no endoscopic or radiological signs of residual fungal disease. The only side effects were nausea in one and transient visual disturbances in 2 other patients. In the 4 patients presented and treated, voriconazole was shown to be effective and less toxic than amphotericin B in adjuvant treatment of invasive sphenoidal aspergillosis. Copyright (c) 2007 S. Karger AG, Basel.

Chemokines indicate allergic bronchopulmonary aspergillosis in patients with cystic fibrosis

RATIONALE: Allergic bronchopulmonary aspergillosis (ABPA) is characterized by a Th2 immune response. Mouse models suggest a critical role for the Th2 chemokines thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) in ABPA. OBJECTIVES: To determine whether serum levels of TARC and MDC characterize ABPA in patients with cystic fibrosis (CF) and to examine longitudinally if levels of TARC and MDC indicate ABPA exacerbations in patients with CF. METHODS: Levels of TARC and MDC and levels of Th1 (IL-12 and IFN-gamma) and Th2 (IL-4, IL-5, and IL-13) cytokines were analyzed in serum of 16 patients with CF with ABPA, six non-CF patients with asthma with ABPA, 13 patients with CF colonized with Aspergillus fumigatus, six patients with CF sensitized to A. fumigatus, 12 atopic patients with CF, and 13 non-CF atopic control subjects by ELISA. The longitudinal course of TARC, MDC, and IgE levels was assessed during ABPA episodes. RESULTS: Patients with ABPA had significantly higher serum levels of TARC compared with the other patient groups. Cytokine levels did not differ among the patient groups. Longitudinally, levels of TARC indicated ABPA exacerbations in patients with CF more clearly than IgE levels. In patients with CF and ABPA, levels of TARC correlated positively with specific IgE to A. fumigatus and rAsp f4. CONCLUSIONS: Serum levels of TARC differentiate patients with CF or patients with asthma with ABPA from patients with CF colonized with or sensitized to A. fumigatus, atopic patients with CF, and atopic control subjects. Longitudinally, levels of TARC indicate ABPA exacerbations, suggesting TARC as a marker for identification and monitoring of ABPA in patients with CF.

Comparison of serum markers for allergic bronchopulmonary aspergillosis in cystic fibrosis

The diagnosis of allergic bronchopulmonary aspergillosis (ABPA) in cystic fibrosis (CF) is a challenge. Thymus- and activation-regulated chemokine (TARC) has recently been reported to play a role in ABPA. The aim of this study was to compare the diagnostic value of TARC with that of known serological markers for diagnosis of ABPA in CF patients. The present study longitudinally followed 48 CF patients, of whom 12 had a diagnosis of ABPA according to Nelson's criteria, for 1-8 yrs with repeated measurements of serum total immunoglobulin (Ig)E, specific Aspergillus fumigatus IgE and IgG, specific IgE against recombinant A. fumigatus allergens (rAsp f) 1, 3, 4 and 6, and TARC. Median (interquartile range) TARC levels were 589 (465-673) pg x mL(-1) in ABPA patients and 232 (189-289) pg x mL(-1) in non-ABPA patients. Receiver operating characteristic curves revealed that TARC was superior to the other markers for diagnosis of ABPA. Diagnostic accuracy was greater for TARC (93%) than for total IgE (74%), or rAsp f 4 (75%) or f 6 (79%). The present study indicates that thymus- and activation-regulated chemokine may be useful in the diagnosis of allergic bronchopulmonary aspergillosis in cystic fibrosis patients. However, larger studies are needed before thymus- and activation-regulated chemokine can routinely be used in diagnostic algorithms.

IgE-bearing cells in bronchoalveolar lavage fluid and allergen-specific IgE levels in sera from RAO-affected horses

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterized by small airway inflammation, airway neutrophilia and obstruction following exposure of susceptible horses to mouldy hay and straw and is thus regarded as a hypersensitivity reaction to mould spores. However, the role of immunoglobulin E antibodies (IgE) in the pathogenesis of RAO is unclear. We hypothesized that the number of cells with receptor-bound IgE in bronchoalveolar lavage fluid (BALF) and IgE levels in serum would be higher in RAO-affected than in healthy horses living in the same environment. Therefore, IgE-positive (+) cells were identified by immunocytochemistry on cytospins from BALF and counted. IgE levels against the mould extracts Aspergillus fumigatus (Asp. f.) and Alternaria alternata (Alt. a.) and the recombinant mould allergen Aspergillus fumigatus 8 (rAsp f 8) were measured by enzyme-linked immunosorbent assay (ELISA) in the sera of seven RAO-affected and 22 clinically healthy mature horses housed in the same conventional stable environment. After correcting for the number of neutrophils, there were no significant differences in IgE+ cells on cytospins from BALF between both groups of horses (5% versus 7%, P > 0.1). Serum IgE levels against the mould extracts were significantly higher in RAO-affected than in clinically healthy horses [median = 119 versus 66 relative ELISA units (REU), P < 0.05]. Furthermore, significantly more RAO-affected than healthy horses had detectable serum IgE against the recombinant allergen rAsp f 8 (4/7 and 3/22, respectively, P < 0.05). Age had no significant effect on BALF cell ratios or on specific serum IgE levels. These results show that high IgE levels against mould antigens are associated with RAO under controlled environmental conditions but ranges of mould-specific serum IgE levels overlapped too much between diseased and clinically healthy animals to be of any diagnostic value. Further studies are needed to assess whether IgE-mediated reactions contribute to the pathogenesis of RAO.

A model of cerebral aspergillosis in non-immunosuppressed nursing rats

Central nervous system aspergillosis is an often fatal complication of invasive Aspergillus infection. Relevant disease models are needed to study the pathophysiology of cerebral aspergillosis and to develop novel therapeutic approaches. This study presents a model of central nervous system aspergillosis that mimics important aspects of human disease. Eleven-day-old non-immunosuppressed male Wistar rats were infected by an intracisternal injection of 10 mul of a conidial suspension of Aspergillus fumigatus. An inoculum of 7.18 log(10) colony-forming units (CFU) consistently produced cerebral infection and resulted in death of all animals (n = 25) within 3-10 days. Median survival time was 3 days. Histomorphologically, all animals developed intracerebral abscesses (2-26 per brain) containing abundant fungal hyphae and neutrophils. Fungal culture of cortical homogenates yielded maximal growth on day 3 after infection (5.4 log(10) CFU/g, n = 15) that declined over time. Galactomannan concentrations in cortical homogenates, assessed as an index for hyphal burden, peaked on days 3-5. Fungal infection spread to peripheral organs in 83% of animals. Fungal burden in lung, liver, spleen and kidney was two orders of magnitude lower than in the brain. The successful establishment of a model of cerebral aspergillosis in a non-immunosuppressed host provides the opportunity to investigate mechanisms of disease and to develop novel treatment regimens for this commonly fatal infection.

Allergen motifs and the prediction of allergenicity

We have recently shown that the majority of allergens can be represented by allergen motifs. This observation prompted us to experimentally investigate the synthesized peptides corresponding to the in silico motifs with regard to potential IgE binding and cross-reactions with allergens. Two motifs were selected as examples to conduct in vitro studies. From the first motif, derived from allergenic MnSOD sequences, the motif stretch of the allergen Asp f 6 was selected and synthesized as a peptide (MnSOD Mot). The corresponding full-length MnSOD was also expressed in Escherichia coli and both were compared for IgE reactivity with sera of patients reacting to the MnSOD of Aspergillus fumigatus or Malassezia sympodialis. For the second motif, the invertebrate tropomyosin sequences were aligned and a motif consensus sequence was expressed as a recombinant protein (Trop Mot). The IgE reactivity of Trop Mot was analyzed in ELISA and compared to that of recombinant tropomyosin from the shrimp Penaeus aztecus (rPen a 1) in ImmunoCAP. MnSOD Mot was weakly recognized by some of the tested sera, suggesting that the IgE binding epitopes of a multimeric globular protein such as MnSOD cannot be fully represented by a motif peptide. In contrast, the motif Trop Mot showed the same IgE reactivity as shrimp full-length tropomyosin, indicating that the major allergenic reactivity of a repetitive structure such as tropomyosin can be covered by a motif peptide. Our results suggest that the motif-generating algorithm may be used for identifying major IgE binding structures of coiled-coil proteins.

Autoantibodies directed against bactericidal/permeability-increasing protein in patients with cystic fibrosis: association with microbial respiratory tract colonization

BACKGROUND: Cystic fibrosis (CF) is associated with the appearance of serum autoantibodies directed against bactericidal/permeability-increasing protein (BPI). OBJECTIVES: To determine the age-specific seroprevalence rates of anti-BPI-IgG and IgA in a population of patients with CF and to correlate anti-BPI antibody concentrations with microbial respiratory tract colonization and pulmonary function variables at the time of serum sampling and 6 years thereafter. METHODS: Determination of BPI antibodies of the IgG and IgA isotypes using a commercial enzyme-linked immunosorbent assay in sera of a CF serum bank of 1992; correlation of anti-BPI antibody concentrations with age, clinical score, pulmonary function variables in 1992 and 1998, total serum immunoglobulin isotype concentrations and respiratory tract colonization with Pseudomonas aeruginosa and Aspergillus spp. RESULTS: Seventy-one patients (age in 1992, 14.1 +/- 7.5 years) were studied. Reactivities for anti-BPI-IgG and IgA were found in 28 (39%) and 26 (37%) patients, respectively. The seroprevalence of anti-BPI-IgA, but not IgG, increased significantly with age. P. aeruginosa colonization was associated with elevated concentrations of anti-BPI-IgG (P = 0.003) and IgA (P = 0.037). There were significant negative correlations between pulmonary function variables (vital capacity, forced expiratory volume in 1 s) in 1992 and 1998, respectively, and concentrations of anti-BPI-IgG or IgA in a multiple regression analysis. Anti-BPI-IgG, but not IgA, remained significantly associated with P. aeruginosa colonization (P = 0.006) and with reduced vital capacity (P = 0.01) in 1998 after correction for total serum isotype concentration. CONCLUSIONS: Anti-BPI-IgG are strongly associated with concurrent P. aeruginosa colonization and with long term restrictive pulmonary function abnormalities.