G. Castellá

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.

Molecular characterization of ochratoxin A producing strains of the genus Penicillium

Sixty-six strains classified as P. verrucosum based on morphological criteria were characterized by molecular methods like RAPD, AFLP and ITS sequencing. Two groups could be identified by RAPD and AFLP analyses. The two RAPD as well as the two AFLP groups were completely coincidental. Strains in the two groups differed in their ability to produce ochratoxin A, with group I containing mainly high producing strains, and group II containing moderate to non-producing strains. The strains from group I originate from foods, such as cheeses and meat products, while the strains from group II originate from plants. The ribosomal ITS1-5.8S-ITS2 sequences were similar, except for two single nucleotide exchanges in several strains of each group. A chemotaxonomical analysis of some of the strains identified differences between the groups in secondary metabolite production. Strains from group I possessed the chemotype of P. nordicum and strains from group II that of P. verrucosum. The differences at the RAPD and AFLP level, which parallel the chemotypic differences, are consistent with the recent reclassification of ochratoxin A producing penicillia to be either P. verrucosum or P. nordicum. The homolgy between the ITS sequences however indicates phylogenetic relationship between the two species.

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.

Seasonal study of the fungal biota of the fur of dogs

During a one year period, 944 dogs from the Municipal kennel of Barcelona were examined to detect animals with suspected dermatophytosis. Only a few animals (1.8%) presented skin lesions but none of them had dermatophytosis. A representative number of dogs without visible skin lesions (n=172), selected at random, were used to carry out a seasonal study of the mycobiota of their fur. Fifteen isolates belonging to the genera Microsporum and Trichophyton were isolated from 14 of the 172 (8.1%) dogs without lesions. The identity of these fungi was Microsporum gypseum (6/15), Trichophyton terrestre (4/15), M. canis (2/15), M. cookei (2/15) and Trichophyton ajelloi (1/15) (one strain each of M. gypseum and T. ajelloi were isolated from one dog). Species of Penicillium (% prevalence=89.5%), Alternaria (86.6%), Cladosporium (84.9%), Aspergillus (77.3%), Scopulariopsis (65.7%) and Chrysosporium (64.5%) were the most prevalent. No significant differences in the fungal biota were observed with respect to age, gender, hair length or between mixed and pure breed dogs. A large number of isolates, including species belonging to the genera Beauveria, Chrysosporium, Malbranchea and Scopulariopsis, that macroscopically and/or microscopically resemble dermatophytes and may be mistaken for them, produced a red color change in Dermatophyte Test Medium. No significant seasonal difference was detected among the isolates belonging to the the most frequently encountered genera, with the exception of Scopulariopsis (higher in summer and autumn) and Chrysosporium (higher in summer). Species from other genera, with lower occurrence also presented significant differences in their seasonal distribution. Arthrinium, Aureobasidium, Chaetomium and Phoma spp. presented maximum prevalence peaks in spring, Fusarium, Paecilomyces, Phoma and Rhizopus spp. in summer and Geotrichum and Mucor spp. in autumn. The Microsporum and Trichophyton species were more frequently isolated in summer.

Occurrence of Penicillium verrucosum in retail wheat flours from the Spanish market

In Spain, low ochratoxin A (OTA) levels have been detected in wheat and different wheat products but no information has been published about the fungi involved in this OTA contamination. Some species of the genera Penicillium and Aspergillus are known to form OTA but few of them are known to contaminate foods with this mycotoxin. Penicillium verrucosum, an important OTA producer typical of temperate and cold climates, is much more frequently found on cereals in countries where they occasionally have OTA problems as in North European countries compared with South Europe, where levels of OTA generally seem to be lower or is not detected. The aim of this study was to determine, identify and characterize the occurrence of potential OTA-producing Aspergillus spp. and Penicillium spp. from retail wheat flours purchased in the Spanish market and used for human consumption. A total of 105 Aspergillus isolates were analyzed in order to know whether they are able to produce OTA and/or citrinin (CIT). None of these isolates were able to produce these mycotoxins. However, 17 suspected P. verrucosum isolates were recovered and confirmed by RAPD analyses. Eleven isolates were OTA producers and 14 isolates produced CIT. Our results confirm the potential risk of OTA and CIT production in wheat flours if stored improperly and the occurrence of P. verrucosum in South European countries. This was the only species able to produce these mycotoxins.

Ochratoxin A Producing Species in the Genus Penicillium

Ochratoxin A (OTA) producing fungi are members of the genera Aspergillus and Penicillium. Nowadays, there are about 20 species accepted as OTA producers, which are distributed in three phylogenetically related but distinct groups of aspergilli of the subgenus Circumdati and only in two species of the subgenus Penicillium. At the moment, P. verrucosum and P. nordicum are the only OTA producing species accepted in the genus Penicillium. However, during the last century, OTA producers in this genus were classified as P. viridicatum for many years. At present, only some OTA producing species are known to be a potential source of OTA contamination of cereals and certain common foods and beverages such as bread, beer, coffee, dried fruits, grape juice and wine among others. Penicillium verrucosum is the major producer of OTA in cereals such as wheat and barley in temperate and cold climates. Penicillium verrucosum and P. nordicum can be recovered from some dry-cured meat products and some cheeses.

Ochratoxin A and citrinin producing species of the genus Penicillium from feedstuffs

In Spain, low ochratoxin A (OTA) levels have been detected in several pork products but there is no information published about the fungi involved in this OTA contamination. It is well known that P. verrucosum is much more frequently found on cereals in countries where they occasionally have OTA problems as in North European countries compared with South Europe where levels of OTA generally seem to be lower or not detected. Much less information is available about citrinin (CIT) and CIT producing species in cereals and their by products. The aim of this study was to determine, identify and characterize the occurrence of potential OTA and CIT producing Penicillium spp. from mixed feeds and raw materials purchased in the Spanish market and used as feedstuffs. A total of 155 Penicillium spp. isolates belonging to 34 species were analyzed in order to know if they are able to produce OTA and/or CIT. From these isolates, 11 P. verrucosum which were characterized by RAPD analyses, produced OTA. Fourteen isolates were CIT producers, 10 isolates of P. verrucosum and 4 of P. citrinum. Although the occurrence and abundance of OTA and CIT Penicillium producing species have been low in our study, our results confirm the potential risk of OTA and CIT production in feeds if stored improperly. Our results also confirm the occurrence of P. verrucosum in South European countries and that it is the only OTA producing Penicillium species in these substrates.