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.

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.

Paracoccidioides brasiliensis-associated dermatitis and lymphadenitis in a dog.

Paracoccidioidomycosis (PCM) is an endemic disease of humans from Latin America that is caused by Paracoccidioides brasiliensis and P. lutzii, with most cases of PCM in domestic animals being associated with P. brasiliensis. This study presents the clinical, cytological, mycological, serological, and molecular findings associated with P. brasiliensis in a dog from Southern Brazil. Fine needle biopsies were collected from the skin and several lymph nodes of a 5-year-old female Labrador dog that had enlargement of most superficial lymph nodes. Cytology of the skin and lymph nodes revealed pyogranulomatous dermatitis and lymphadenitis associated with fine-necked, budding fungal structures consistent with the Paracoccidioides genus of organisms; mycological culture derived from the lymph node aspirate demonstrated similar budding structures. Serological assays using exoantigens obtained from the fungal culture demonstrated that the fungal organisms derived from the lymph node were antigenically similar to P. brasiliensis by immunodiffusion and Western blot. A PCR assay, using the fungal culture as input, amplified a partial segment of the internal transcribed spacer 1 and 2 regions of P. brasiliensis; direct sequencing and phylogenetic analyses confirmed the PCR product as P. brasiliensis. The combined cytological, mycological, serological, and molecular findings confirmed a diagnosis of fungal dermatitis and lymphadenitis due to P. brasiliensis in this dog. This case represents the third description of clinical PCM in dogs and the first confirmation of mycotic dermatitis associated with P. brasiliensis in this species. The participation of dogs in the possible dissemination of PCM is reviewed, and it is proposed that dogs are probable accidental hosts in the epidemiological cycle associated with P. brasiliensis.

Paracoccidioides brasiliensis Infection in Small Wild Mammals.

Paracoccidioidomycosis (PCM) is a systemic mycosis prevalent in Brazil and other Latin American countries. The etiological agents of PCM are the thermo-dimorphic fungi Paracoccidioides brasiliensis and P. lutzii. Taking into account that the natural habitat of Paracoccidioides spp. is still undefined, domestic and wild animals could be useful as indicators of Paracoccidioides spp. presence in endemic areas. The objective of this study was to evaluate the infection of small wild mammals by P. brasiliensis in an endemic area for human PCM. Samples from 38 wild mammals from different species such as Akodon sp., Thaptomys nigrita, Euryoryzomys russatus, Oligoryzomys nigripes, Monodelphis sp., Sooretamys angouya, Abrawayaomys angouya, Abrawayaomys ruschii and Akodontinae sp. were evaluated by ELISA, immunodiffusion, histopathology, nested PCR and culture. The overall positivity to gp43 observed in the ELISA was 23.7%. Samples from heart and liver of one O. nigripes were PCR positive, and the animal was also seropositive to gp43 in ELISA. This study showed that wild animals living in endemic areas for PCM are infected with P. brasiliensis and can be valuable epidemiological markers of the fungus presence in the environment. This is the first evidence of PCM infection in Akodon sp., E. russatus, T. nigrita and O. nigripes.

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.

Aspergillus ochraceus biocontrol by Hanseniaspora opuntiae in vitro and on coffee fruits.

Aspergillus ochraceus is an ochratoxin-producing fungus which contaminates coffee. In this study the antifungal effect of the yeast Hanseniaspora opuntiae on three Aspergillus ochraceus strains (IOC 4417, IOC 4462, Ao 14) was evaluated in vitro and on coffee fruits. H. opuntiae (106 and 107 cells mL-1) reduced in vitro fungal growth from 82% to 87%, when co-cultivated with A. ochraceus. The yeast cell free supernatant (CFS) inhibited conidial germination from 76.5% to 92.5%, and hyphal growth from 54% to 78%. The yeast (107 and 109 cells mL-1) applied on coffee fruits delayed fruit decay by A. ochraceus (IOC 4417 and Ao 14) until the 9th day, and was significantly different (p < 0.05) from the controls. Furthermore, the ultrastructure of the yeast-fungus interaction on the coffee fruit surface showed yeast attachment to A. ochraceus hyphae, and morphological alterations in fungal structures, with hyphal abnormalities, such as tortuous hyphae with irregular, non-uniform surface compared to the control without yeast. H. opuntiae showed efficacy as biocontrol agent and, to the best of our knowledge, this is the first study on the antifungal activity of H. opuntiae against A. ochraceus on coffee fruits Nevertheless, application of H. opuntiae to the crop in the field requires further studies.