Antifungal effect and some properties of cell-free supernatants of two Bacillus subtilis isolates against Fusarium verticillioides.

Fusarium verticillioides causes significant decrease in corn yield and quality, and produces fumonisins, which represent a serious risk to human and animal health. Bacillus species can be an effective and environmentally friendly alternative for F. verticillioides biological control. In this study, some properties of cell-free supernatants (CFSs) of two Bacillus spp. identified as Bacillus subtilis (NT1, NT2) as well as the antifungal effect against F. verticillioides 97L were evaluated. B. subtilis NT1 and NT2 were isolated from commercially available fermented whole soybeans (Natto). Antifungal activity was observed in both CFSs of B. subtilis isolates (50-59 mm) obtained by co-culture suggesting that antifungal compound production depends on interaction between bacteria and fungi. Cell-free supernatants from the two B. subtilis isolates inhibited mycelial growth (77%-94%) and conidial germination (22%-74%) of F. verticillioides 97L. In addition, CFSs caused significant morphological changes such as distorted and collapsed hyphae with wrinkled surfaces and the presence of a large amount of extracellular material compared to the control without CFSs. Both B. subtilis isolates (NT1 and NT2) produced extracellular proteases, biosurfactants and polar low molecular weight compounds that probably act synergistically and may contribute to the antifungal activity. Antifungal compounds showed heat and pH stability and resistance to proteolytic enzymes. Furthermore, antifungal compounds showed high polarity, high affinity to water and a molecular weight less than 10 kDa. These results indicated that the two B. subtilis (NT1 and NT2) have potential as biocontrol agents for F. verticillioides.

An atypical case of chronic paracoccidioidomycosis in a dog caused by a fungus from the Paracoccidioides brasiliensis complex.

Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin American countries and one of the most important fungal diseases regarding incidence and mortality in humans. PCM has also been described in some animal species such as dogs. In this study we describe a new case of PCM disease in a dog that differed from previous records in the literature which includes a progressive evolution of fungal dermatitis causing a deforming lesion in the nose, like those found in human patients, and humoral response against gp70 instead of gp43, the major diagnostic antigen for human PCM. The clinical isolate through the ITS and partial gp43 gene phylogenetic analysis was grouped in the Paracoccidioides brasiliensis complex. This case describes several features which may contribute to improving diagnosis and understanding of canine paracoccidioidomycosis.

Re-Evaluation of the Cross-Reactions of the Antibody against the Causative Agent for Paracoccidioidomycosis Ceti; Paracoccidioides ceti and the Related Fungal Species.

Paracoccidioidomycosis ceti (PCM-C) is a chronic granulomatous keloidal dermatitis in cetaceans that has been reported worldwide and is caused by Paracoccidioides ceti. Serological cross-reactions among highly pathogenic fungal infections and related diseases have been reported. However, the true cross-reaction of antibodies against P. ceti has remained unknown due to the use of positive control sera from infected dolphins. This study aimed to re-evaluate antibodies from mechanically dislodged fungal cells in the infected tissue of a PCM-C case and demonstrate the actual cross-reaction. The results revealed a limited cross-reaction between PCM-C and paracoccidioidomycosis, while the antibodies did not react with other pathogens such as Coccidioides posadasii, Histoplasama capsulatum, and Arthrographis kalrae. Thus, the method for evaluation of the antibody against PCM-C is reliable, and there is potential for epidemiological study.

Yeasts as sustainable biocontrol agents against ochratoxigenic Aspergillus species and in vitro optimization of ochratoxin A detoxification.

AIMS: The aims of this study were to evaluate the potential of Hanseniaspora opuntiae, Meyerozyma caribbica, and Kluyveromyces marxianus for in vitro biocontrol of Aspergillus ochraceus, A. westerdijkiae, and A. carbonarius growth, the ochratoxin A (OTA) effect on yeast growth, and yeast in vitro OTA detoxification ability using an experimental design to predict the combined effects of inoculum size, incubation time, and OTA concentration. METHODS AND RESULTS: Predictive models were developed using an incomplete Box-Behnken experimental design to predict the combined effects of inoculum size, incubation time, and OTA concentration on OTA detoxification by the yeasts. The yeasts were able to inhibit fungal growth from 13% to 86%. Kluyveromyces marxianus was the most efficient in inhibiting the three Aspergillus species. Furthermore, high OTA levels (100 ng ml-1) did not affect yeast growth over 72 h incubation. The models showed that the maximum OTA detoxification under optimum conditions was 86.8% (H. opuntiae), 79.3% (M. caribbica), and 73.7% (K. marxianus), with no significant difference (P > 0.05) between the values predicted and the results obtained experimentally. CONCLUSION: The yeasts showed potential for biocontrol of ochratoxigenic fungi and OTA detoxification, and the models developed are important tools for predicting the best conditions for the application of these yeasts as detoxification agents.

Limosilactobacillus reuteri as sustainable biological control agent against toxigenic Fusarium verticillioides.

Corn contamination with Fusarium verticillioides (Sacc.) Nirenberg is a worldwide problem that affects yield and grain quality resulting in severe economic losses and implications for food safety. Control of F. verticillioides is a challenge, but lactic acid bacteria (LAB) has high potential as a biological control agent. In this study, the antifungal effect of Limosilactobacillus reuteri (formerly Lactobacillus reuteri) LR-92 against F. verticillioides 97L was investigated. Cell-free supernatant (CFS) from L. reuteri showed concentration-dependent fungicidal and fungistatic activity against F. verticillioides 97L. The antifungal compounds from CFS showed heat stability and pH dependence, and antifungal activity was not affected by treatment with proteolytic enzymes. High-performance liquid chromatography analysis indicated that L. reuteri LR-92 produces lactic and acetic acids. After liquid-liquid extraction, electrospray ionization mass spectrometry analysis of the active ethyl acetate fraction containing antifungal compounds revealed the production of 3-phenyllactic acid, cyclo-(L-Pro-L-Leu), cyclo-(L-Pro-L-Phe), and cyclo-(L-Phe-trans-4-OH-L-Pro). L. reuteri LR-92 has potential as a biocontrol agent for F. verticillioides and contributes to food safety.

First report of Paracoccidioides brasiliensis infection in fish.

The thermodimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM), a deep mycosis endemic in Latin American countries that affects mainly male rural workers. Infection by P. brasiliensis has also been reported in several species of terrestrial animals; however, the capacity of the fungus to infect aquatic organisms is poorly known. The aim of this study was to detect P. brasiliensis in a fish species, Nile tilapia (Oreochromis niloticus), the most farmed and widely distributed fish in endemic areas for human PCM in Brazil. As a first step, the humoral immune response against the fungus was evaluated in an experimental group of three fish immunized with inactivated P. brasiliensis yeast cells. For the seroepidemiological study, serum samples of Nile tilapia raised in cages (n = 109) and in ponds (n = 105), collected from a fish slaughterhouse, were analyzed for P. brasiliensis antibodies by ELISA using gp43 as antigen. All the inoculated fish produced antibodies against the fungus. The seropositivity observed in fish raised in cages and ponds was 17.4 and 5.7%, respectively. Due to the higher seropositivity observed in caged fish, 100 tissue samples (encephalon, liver, and kidney), from another group of tilapia raised in cages, were analyzed by polymerase chain reaction (PCR; Pb-ITSR and Pb-ITSE). Three tissue samples (liver n = 1, kidney n = 1, and enchepahlon n = 1) from three different fish resulted positive to PCR. This is the first report to show serological and molecular evidence of P. brasiliensis infection in a fish species.

Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay to Detect Fusarium verticillioides in Poultry Feed Samples.

Fumonisins are a group of toxic secondary metabolites that are produced by Fusarium verticillioides which are associated with poultry health hazard and great economic losses. The objective of the present study was to develop an immunological method to detect F. verticillioides in poultry feed samples. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a polyclonal antibody against 67 kDa protein of the F. verticillioides 97K exoantigen was developed to detect this fungus. Antibody anti-67 kDa protein showed cross-reactivity against F. graminearum (2⁻7%) and F. sporotrichioides (10%), but no or low cross-reactivity against Aspergillus sp. and Penicillium sp. exoantigens. The detection limit for the 67 kDa protein of F. verticillioides was 29 ng/mL. Eighty-one poultry feed samples were analyzed for Fusarium sp. count, 67 kDa protein of F. verticillioides and fumonisin concentrations. Eighty of the 81 feed samples (98.6%) showed Fusarium sp. contamination (mean 6.2 x 104 CFU/g). Mean 67 kDa protein and fumonisin concentration in the poultry feed samples was 21.0 µg/g and 1.02 µg/g, respectively. The concentration of 67 kDa protein, as determined by ic-ELISA correlated positively (p < 0.05) with fumonisin levels (r = 0.76). These results suggest that this ic-ELISA has potential to detect F. verticillioides and predict fumonisin contamination in poultry feed samples.