F. J. Cabañes

An easy screening method for fungi producing ochratoxin A in pure culture.

A simple screening method has been developed for detecting ochratoxin production by fungi, based on high-performance liquid chromatographic determinations on extracts obtained from agar plugs cut from pure Petri dish cultures. Two culture media. Yeast Extract Sucrose agar and Czapek Yeast Extract agar, and three extraction solvents (methanol, methylene chloride/formic acid, and methanol/formic acid) were compared. All of the isolates tested produced ochratoxin A in one or both culture media after 7 or 14 days of incubation. Based on the results obtained, the use of both culture media is recommended. As extraction solvent, either methanol or methanol-formic acid could be used. This method also provides quantitative information on the level of ochratoxin produced by the cultures. The simplicity of the method makes it very useful when many fungal isolates need to be screened.

What is the source of ochratoxin A in wine

During a microvinification trial using natural mouldy grapes from a research experimental vineyard, ochratoxin A (OTA) contaminated white wine was obtained. Potential OTA-producing mycobiota of grape samples used in this microvinification process was assessed. Only Aspergillus carbonarius isolates were detected as producers of OTA. Our report is a strong evidence of the contribution of A. carbonarius in the OTA contamination in wine.

Aspergillus carbonarius as the main source of ochratoxin A contamination in dried vine fruits from the Spanish market.

Ochratoxin A (OTA) can occur in a wide range of foods, but unexpectedly high concentrations have been detected in dried vine fruits of various origins. The European Union has recently established a maximum OTA limit of 10 microg/kg for these foodstuffs. In order to determine the likely origin of OTA, a mycological study of 50 dried fruit samples (currants, raisins, and sultanas) representative of the Spanish market was conducted. Fungal contamination was detected in 49 of 50 (98%) samples. Black aspergilli were isolated from all of the positive samples. Aspergillus niger var. niger was isolated from 98% of the samples, and Aspergillus carbonarius was found in 58% of the samples. One hundred sixty-eight A. niger var. niger isolates and 91 A. carbonarius isolates were screened for their ability to produce OTA. Eighty-eight (96.7%) A. carbonarius isolates and one (0.6%) A. niger var. niger isolate were found to be OTA producers. Black aspergilli were the dominant fungi. Among black aspergilli, A. carbonarius has shown a consistent ability to produce OTA and is the most probable source of this mycotoxin in these substrates.

Current Importance of Ochratoxin A-Producing Aspergillus spp

Ochratoxin A (OA) is receiving attention worldwide because of the hazard it poses to human and animal health. OA contamination of commodities, such as cereals or pork and poultry meat, is well recognized. Nevertheless, there is an increasing number of articles reporting OA contamination in other food commodities, such as coffee, beer, wine, grape juice, and milk, in the last few years. This continuous and increasing exposure to OA that humans experience is reflected in the high incidence of OA in both human blood and milk in several countries. OA was believed to be produced only by Aspergillus ochraceus and closely related species of section Circumdati and by Penicillium verrucosum; however, in the genus Aspergillus, the production of OA has been recently reported by species outside the section Circumdati. Thus, it has been clearly established as a metabolite of different species of the section Nigri, such as Aspergillus niger and Aspergillus carbonarius. OA production ability by Aspergillus spp. is more widespread than previously thought; therefore, there is the possibility that unexpected species can be new sources of this mycotoxin in their natural substrates.

Dermatophytes isolated from domestic animals in Barcelona, Spain

This retrospective study deals with the main samples studied at the Mycology Diagnostic Service of the Faculty of Veterinary Science of Barcelona: animals with suspected dermatophytosis. Over a ten-year period from 1986 to 1995, 136 dermatophytes were identified from dog and cat cultures submitted for identification and from specimens submitted for mycological examination from a variety of other domestic animals. The most frequent dermatophytes isolated were Microsporum canis (55.9%), Trichophyton mentagrophytes var. mentagrophytes (27.2%), Microsporum gypseum (7.4%) and Trichophyton verrucosum (7.4%). The identity of the dermatophytes from dog and cat cultures submitted for identification was M. canis (77.8%), T. mentagrophytes (13.3%) and M. gypseum (8.9%). Dermatophytes were cultured from 15 of 105 (14.3%) canine specimens and 19 of 56 (33.9%) feline specimens submitted for mycological examination during this period. Microsporum canis was the most common species isolated (73.3% and 94.7% respectively). The percentage of positive microscopic examinations of the specimens of hair in culture positive submissions from dogs and cats was 58.8%. There was a high proportion of positive cultures from both dogs and cats less than 1 year of age, and in some breeds of dogs, but there was no significant difference between the sexes. Although dermatophytes were more frequently isolated in autumn and winter months, no significant difference was detected in the seasonal distribution of the canine and feline dermatophytosis. Trichophyton mentagrophytes was the most prevalent dermatophyte in rabbits and T. verrucosum in ruminants. Other isolated species were T. equinum and M. equinum from horses.

Collaborative Evaluation of Optimal Antifungal Susceptibility Testing Conditions for Dermatophytes

ABSTRACT A multicenter study was conducted to define the most suitable testing conditions for antifungal susceptibility of dermatophytes. Broth microdilution MICs of clotrimazole, itraconazole, and terbinafine were determined in three centers against 60 strains of dermatophytes. The effects of inoculum density (ca. 103 and 104 CFU/ml), incubation time (3, 7, and 14 days), endpoint criteria for MIC determination (complete [MIC-0] and prominent [MIC-2] growth inhibition), and incubation temperature (28 and 37°C) on intra- and interlaboratory agreement were analyzed. The optimal testing conditions identified were an inoculum of 104 CFU/ml, a temperature of incubation of 28°C, an incubation period of 7 days, and MIC-0.

Secondary metabolite profiling, growth profiles and other tools for species recognition and important Aspergillus mycotoxins

Species in the genus Aspergillus have been classified primarily based on morphological features. Sequencing of house-hold genes has also been used in Aspergillus taxonomy and phylogeny, while extrolites and physiological features have been used less frequently. Three independent ways of classifying and identifying aspergilli appear to be applicable: Morphology combined with physiology and nutritional features, secondary metabolite profiling and DNA sequencing. These three ways of identifying Aspergillus species often point to the same species. This consensus approach can be used initially, but if consensus is achieved it is recommended to combine at least two of these independent ways of characterising aspergilli in a polyphasic taxonomy. The chemical combination of secondary metabolites and DNA sequence features has not been explored in taxonomy yet, however. Examples of these different taxonomic approaches will be given for Aspergillus section Nigri.

Mycoflora and Aflatoxin-Producing Strains in Animal Mixed Feeds

The mycoflora of 69 samples of animal mixed feeds were studied. Fungal counts ranged from 102 to 108 CFU/g, the lowest counts corresponding to the samples of rabbit feeds. Seventy-one fungal species belonging to 26 genera were identified. The pre- dominant species were Aspergillus flavus , Fusarium moniliforme , and Penicillium chrysogenum . Thirty-six strains of A. flavus and one strain of A. parasiticus were screened for aflatoxin production in yeast extract-sucrose medium. The final pH, weight of mycelium, and production of aflatoxins were determined after 14 days of incubation. Five strains (13.5%) were aflatoxigenic. No statistical differences were observed in mycelial dry weights and final pH between aflatoxin-producing strains and nonaflatoxigenic strains.

Malassezia cuniculi sp. nov., a novel yeast species isolated from rabbit skin

Members of the genus Malassezia have rarely been associated with lagomorphs. During the course of an investigation of the lipophilic mycobiota of rabbit skin, two lipid-dependent isolates which could not be identified were recovered on Leeming and Notman agar medium from different animals. No growth of Malassezia yeasts was obtained either on Sabourauds glucose agar or modified Dixon agar media. In this study, we describe a new taxon, Malassezia cuniculi sp. nov., including its morphological and physiological characteristics. The validation of this new species was supported by analysis of the D1/D2 regions of the 26S rRNA gene and the ITS-5.8S rRNA gene sequences. The results of these studies confirm the separation of this new species from the other species of the genus Malassezia, as well as the presence of Malassezia yeasts on lagomorphs.

Molecular Analysis of Malassezia sympodialis-Related Strains from Domestic Animals

ABSTRACT Recently, several new lipid-dependent species belonging to the genus Malassezia have been described. Some of them, such as Malassezia dermatis, Malassezia nana, and the tentatively named “Malassezia equi,” have similar phenotypes and are genetically close to Malassezia sympodialis Simmons et Guého 1990. DNA characterization by D1/D2 26S rRNA gene and internal transcribed spacer (ITS)-5.8S rRNA gene sequencing analysis of lipid-dependent strains from different animal species close to M. sympodialis is described and illustrated. Phylogenetic analysis of both the D1/D2 regions of 26S rRNA gene and ITS-5.8S rRNA gene sequences showed four distinct clusters. Cluster I included isolates from different animal species (horse, pig, and lamb) and the type culture of M. sympodialis. Cluster II included isolates from horses grouping close to the “M. equi” AJ305330 sequence. Cluster III comprised isolates mainly from goats. Cluster IV contained isolates mainly from cats grouping together with the M. nana AB075224 sequence. This last cluster included isolates from healthy and external otitic ears. All of these strains had identical 26S rRNA gene and ITS regions. It is not clear whether the value of these genetic differences is for the definition of species or whether they only demonstrate genetic variation among strains from different origins within M. sympodialis, which are in the course of differentiation and probably adaptation to specific animal hosts.