M. L. Abarca

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.

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.

Distribution of ochratoxin A producing strains in the A. niger aggregate.

Ochratoxin A (OA) production of 92 isolates belonging to the A. niger aggregate was tested. All these isolates were grouped into the two proposed species A. niger and A. tubingensis, according to their ITS-5.8S rDNA RFLP patterns. The distribution of the isolates into the two species was very similar since 52.2% were classified as pattern T (corresponding to A. tubingensis), and 47.8% were classified as pattern N (corresponding to A. niger). Six out of the 92 isolates studied produced OA. All the OA producing strains were classified as pattern N while none of the isolates classified as pattern T produced OA.

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.

Effect of water activity, temperature and incubation time on growth and ochratoxin A production by Aspergillus niger and Aspergillus carbonarius on maize kernels

The aim of this study was to determine the effects of water activity (a(w)) (0.92-0.98), temperature (5-45 °C) and incubation time (5-60 days) on growth and ochratoxin A (OTA) production by Aspergillus niger and Aspergillus carbonarius on maize kernels using a simple method. Colony diameters of both strains at 0.92 a(w) were significantly lower than those at 0.96 and 0.98 a(w) levels. The optimum growth temperature range for A. niger was 25-40 °C and for A. carbonarius 20-35 °C. A. niger produced OTA from 15 to 40 °C, and the highest OTA level was recorded at 15 °C. The concentration of OTA produced at 0.92 a(w) was significantly lower than those at 0.96 and 0.98 a(w). A. carbonarius produced OTA from 15 to 35 °C and the maximum concentration was achieved at 15 °C, although not differing statistically from the concentration detected at 20 °C. At 0.98 a(w) the OTA concentration was significantly higher than at 0.96 and 0.92 a(w). Our results show that maize supports both growth and OTA production by A. niger and A. carbonarius. The studied strains were able to produce OTA in maize kernels from the fifth day of incubation over a wide range of temperatures and water availabilities. Although the limit of quantification of our method was higher than that required for the analysis of OTA in food commodities, it has proved to be a useful and rapid way to detect OTA production by fungi inoculated onto natural substrates, in a similar way as for pure culture. Both species could be a source of OTA in this cereal in temperate and tropical zones of the world.