Intraspecific variability of growth and ochratoxin A production by Aspergillus carbonarius from different foods and geographical areas.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin naturally found in a wide range of food commodities throughout the world. Aspergillus carbonarius is the most important source of OTA in food commodities such as wine, grapes and dried vine fruits and is also responsible for the formation of OTA in coffee. The aim of this study was to determine the simultaneous effect of three culture media (Czapek Yeast Extract Broth (CYB); Synthetic Grape Juice Medium (SGM) and White grape juice (WGJ)) at three water activity (aw) levels (0.90; 0.95 and 0.98-0.99), and three incubation temperatures (15 °C, 25 °C and 35 °C) on the growth and OTA production by 16 strains of A. carbonarius. The strains were mainly isolated from grapes from areas with a Mediterranean climate. All the strains were confirmed for identity by sequencing of the calmodulin gene. The assay was performed in microtiter plates, determining the absorbance at 530 nm and the concentration of OTA after 1, 2, 4 and 10 days of incubation. No significant differences were observed in absorbance values between the strains. The highest absorbance values were recorded in CYB at 0.99 aw and at 0.95 aw after 10 days of incubation at 25 °C and 35 °C. None of the strains were able to grow at 0.90 aw and 15 °C in any culture media after 10 days of incubation. OTA concentration was statistically higher at 15 °C than at 25 °C or 35 °C. The highest significant OTA values were obtained at 0.98-0.99 aw and the best culture media for OTA production was CYB, followed by WGJ and SGM. While strains isolated from Mediterranean climate foods had a similar behavior despite being isolated from different geographical areas, OTA concentration produced by one Robusta coffee strain from Thailand was statistically higher at 25 °C than at 15 °C. This would suggest that the type of food matrices and consequently the adaptation of A. carbonarius strains to different climatic conditions would have a greater influence on the ecophysiology of the strains than only their geographical origin.

Impact of some environmental factors on growth and ochratoxin A production by Aspergillus niger and Aspergillus welwitschiae.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin which may contaminate various foods and feed products worldwide. Aspergillus niger is one of the species responsible for OTA contamination in grapes and derived products. This species has recently been split into A. niger and Aspergillus welwitschiae. Both species can not be distinguished by phenotypic or extrolite profiles and to date there is no ecophysiological information of A. welwitschiae. The aim of this study was to determine the effects of water activity (aw) (0.90; 0.95 and 0.98-0.99), culture media (Yeast Extract Sucrose Broth (YESB); Synthetic Grape Juice Medium (SGM); White grape juice (WGJ)) and temperature (15 °C, 25 °C and 35 °C) on the growth and OTA production of four strains of A. niger and six strains of A. welwitschiae. The assay was performed in microtiter plates, determining the absorbance at 530 nm and the concentration of OTA at 1, 2, 4 and 10 days. No significant differences were observed in absorbance and OTA values between the two species under study. The highest absorbance values were recorded in YESB, followed by SGM and WGJ. Absorbance values increased with increasing aw and temperature. The highest OTA values were obtained at 0.98-0.99 aw and the best culture media for OTA production was YESB, followed by WGJ and SGM. The studied strains of A. niger produced the highest mean OTA level at 25 °C whereas A. welwitschiae strains produced the highest mean OTA concentration at 15 °C, although not differing significantly from concentration produced at 25 °C. To our knowledge, this is the first report on the impact of some environmental factors on growth and OTA production by A. welwitschiae.

Trichophyton erinacei in pet hedgehogs in Spain: Occurrence and revision of its taxonomic status.

Hedgehogs have increased in popularity as pets in Spain but there are no data of infection rates of this exotic animal with dermatophytes in our country. During the period of 2008-2011 a total of 20 pet hedgehogs (19 African pygmy hedgehogs and 1 Egyptian long-eared hedgehog) suspected of having dermatophytoses were studied. This is the first survey of the occurrence of T. erinacei in household hedgehogs in Spain. The T. erinacei infection rate was 50% (9 out of 19 African pygmy hedgehogs, and the one Egyptian long-eared hedgehog surveyed). Morphological identification of the isolates was confirmed by molecular analysis. All the strains had the same ITS sequence and showed 100% sequence similarity to T. erinacei type strain CBS 511.73 (AB 105793). The Spanish isolates were confirmed as T. erinacei urease positive. On the basis of ITS sequences, T. erinacei is a species close to but separate from the taxa included in the A. benhamiae complex. Review of the current literature on DNA-based methods for identification of species included in this complex has highlighted the urgent need to reach a consensus in species circumscription and classification system accepted by all mycologists.

Six-Year Follow-up of Slaughterhouse Surveillance (2008-2013): The Catalan Slaughterhouse Support Network (SESC).

Meat inspection has the ultimate objective of declaring the meat and offal obtained from carcasses of slaughtered animals fit or unfit for human consumption. This safeguards the health of consumers by ensuring that the food coming from these establishments poses no risk to public health. Concomitantly, it contributes to animal disease surveillance. The Catalan Public Health Protection Agency (Generalitat de Catalunya) identified the need to provide its meat inspectors with a support structure to improve diagnostic capacity: the Slaughterhouse Support Network (SESC). The main goal of the SESC was to offer continuing education to meat inspectors to improve the diagnostic capacity for lesions observed in slaughterhouses. With this aim, a web-based application was designed that allowed meat inspectors to submit their inquiries, images of the lesions, and samples for laboratory analysis. This commentary reviews the cases from the first 6 years of SESC operation (2008-2013). The program not only provides continuing education to inspectors but also contributes to the collection of useful information on animal health and welfare. Therefore, SESC complements animal disease surveillance programs, such as those for tuberculosis, bovine cysticercosis, and porcine trichinellosis, and is a powerful tool for early detection of emerging animal diseases and zoonoses.

A new in vitro method to detect growth and ochratoxin A-producing ability of multiple fungal species commonly found in food commodities.

The aim of the study was to develop a new screening method to detect growth and ochratoxin A (OTA) production by multiple fungi growing in a small quantity of culture media, using microtiter plates. Eight ochratoxigenic species were included in the study. The strains were inoculated in sterile 96-well flat-bottom microtiter plates containing Yeast Extract Sucrose broth and Czapek Yeast Extract broth and incubated at 25 °C. Growth was daily monitored by absorbance measurements for 4 days and extended to 7 and 10 days for Penicillium spp. The entire experiment was repeated twice on different days. On each sampling time, five of the seven replicate wells inoculated for each strain and culture media were randomly selected and the content of each well was removed, extracted and injected into the HPLC. No statistically significant differences were observed for absorbance and OTA values, neither between replicates nor between experiments. Quantifiable OTA levels were detected after 48 h of incubation in Aspergillus alliaceus, Aspergillus carbonarius and Aspergillus niger, after 72 h in Aspergillus flocculosus, Aspergillus steynii and Aspergillus westerdijkiae and after 7 days in Penicillium nordicum and Penicillium verrucosum. The method offers the necessary tools for a rapid detection of growth and OTA production avoiding the use of plate cultures and can be very useful when many fungal isolates need to be screened.

Mycobiota and mycotoxin contamination of maize flours and popcorn kernels for human consumption commercialized in Spain.

Mycobiota and co-occurrence of aflatoxins, citrinin, ochratoxin A and zearalenone in 30 samples of maize flours and 30 of popcorn kernels purchased in Spain for human consumption were determined. The mycotoxin-producing ability of Aspergillus, Fusarium and Penicillium spp. was also studied. Total fungal counts of maize flours ranged from <10 to 8.4 × 10(4) CFU/g and predominant mycobiota belonged to Aspergillus spp. and Penicillium spp. In popcorn kernels samples the most frequent species were Aspergillus spp., Mucorales, Fusarium spp. and Penicillium spp. Aflatoxins were produced by Aspergillus flavus and Aspergillus parasiticus, citrinin by Penicillium citrinum and Penicillium verrucosum, ochratoxin A by Aspergillus niger and patulin by Aspergillus clavatus and Penicillium griseofulvum. Identification of all the mycotoxin-producing strains as well as some Aspergillus spp. difficult to identify using phenotypic characters only was also performed by molecular methods. Aflatoxins were detected in 14 maize flours and 2 popcorn kernels samples, while ochratoxin A was detected in 4 maize flours and 10 popcorn samples. Co-occurrence of aflatoxins and ochratoxin A was found in the 4 ochratoxin-positive maize flour samples. Citrinin and zearalenone were not detected. This is the first report of aflatoxins and ochratoxin A contamination in maize flours and popcorn kernels commercialized in Spain.

Pyogranulomatous pleuropneumonia and mediastinitis in ferrets (Mustela putorius furo) associated with Pseudomonas luteola Infection.

Between 2008 and 2009, three pet ferrets from different sources presented with acute episode of dyspnoea. Cytological examination of pleural exudates revealed severe purulent inflammation with abundant clusters of rod-shaped microorganisms with a clear surrounding halo. Treatment was ineffective and the ferrets died 2-5 days later. Two ferrets were subjected to necropsy examination, which revealed pyothorax, mediastinal lymphadenopathy and multiple white nodules (1-2mm) in the lungs. Microscopical examination showed multifocal necrotizing-pyogranulomatous pleuropneumonia and lymphadenitis with aggregates of encapsulated microorganisms, some of which were positively stained by periodic acid-Schiff and alcian blue. In-situ hybridization for Pneumocystis spp., Ziehl-Neelsen staining and immunohistochemistry for distemper, coronavirus and influenza antigen were negative in all cases. Electron microscopically, the bacteria were 2-3 µm long with a thick electron-lucent capsule. Microbiology from one ferret yielded a pure culture of gram-negative bacteria identified phenotypically as Pseudomonas luteola. This speciation was later confirmed by 16S RNA gene amplification.

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.

Temperature and incubation time effects on growth and ochratoxin A production by Aspergillus sclerotioniger and Aspergillus lacticoffeatus on culture media.

AIMS: As there is no knowledge of the influence of abiotic factors on the two new ochratoxin A (OTA)-producing species Aspergillus sclerotioniger and Aspergillus lacticoffeatus, the aim of this study was to evaluate the effect of temperature and incubation time on growth and OTA production by these species on culture media. METHODS AND RESULTS: The study was carried out on yeast extract sucrose agar (YES) and Czapek yeast extract agar (CYA) incubated at ten different temperatures from 5 to 50°C (at 5°C intervals). Growth assessment and OTA production were determined after 5, 10, 15, 20 and 30 days of incubation at each temperature. Aspergillus sclerotioniger grew from 10 to 35°C; OTA was detected from 10 to 35°C and the highest concentration was achieved at 15°C in CYA. Aspergillus lacticoffeatus grew from 10 to 45°C; OTA was detected from 15 to 45°C, and the maximum concentration was produced after 5 days at 25°C in YES. CONCLUSIONS: The studied species can produce OTA over a wide range of temperatures and significant amounts can be produced in only 5 days. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the influence of ecophysiological factors on these two ochratoxigenic species. The pattern of effects of temperature on growth and OTA production by A. sclerotioniger and A. lacticoffeatus was similar to those reported for the closely related species Aspergillus carbonarius and Aspergillus niger, respectively. The two new OTA-producing species have both been isolated from coffee beans, and the closely related ochratoxigenic species of section Nigri, A. carbonarius and A. niger are important sources of OTA in this substrate.

Chrysosporium guarroi sp. nov. a new emerging pathogen of pet green iguanas (Iguana iguana).

Chrysosporium guarroi sp. nov. represented by five strains isolated from cases of dermatomycosis in pet green iguanas (Iguana iguana) in Spain, is described and illustrated. This taxon is characterized by its ability to grow at temperatures from 15 to 37 degrees C and by the presence of arthroconidia and aleurioconidia. The latter are unicellular, smooth, pyriform or clavate, sessile or borne at the ends of narrow stalks. The analysis of the sequences of the D1/D2 and ITS regions confirm the separation of this new species from others of the genus Chrysosporium.

Dermatomycosis in a pet inland bearded dragon (Pogona vitticeps) caused by a Chrysosporium species related to Nannizziopsis vriesii.

A Chrysosporium sp. related to Nannizziopsis vriesii was isolated in pure culture from squames and biopsies of facial lesions in a pet inland bearded dragon (Pogona vitticeps) in Spain. The presence in histological sections of morphologically consistent fungal elements strongly incriminates this fungus as the aetiological agent of infection. Lesions regressed following treatment with oral ketoconazole and topical chlorhexidine and terbinafine until the lizard was lost to follow up 1 month later. The ITS-5.8S rRNA gene of the isolate was sequenced and a search on the GenBank database revealed a high match with the sequences of two Chrysosporium sp. strains recently isolated from green iguanas (Iguana iguana) with dermatomycosis, also in Spain. Phylogenetic analysis of the sequences revealed that all these strains are related to N. vriesii. This is the first report of dermatomycoses caused by a Chrysosporium species related to N. vriesii in a bearded dragon outside North America.

Comparison of methods to detect resistance of Penicillium expansum to thiabendazole.

AIMS: Because of the lack of a standard method, the aim of this work is to evaluate the suitability of the broth microdilution method CLSI M38-A in determining the resistance level of some Penicillium expansum isolates to thiabendazole (TBZ). The ability of the isolates to produce patulin (PAT) and citrinin (CIT) has been also assessed. METHODS AND RESULTS: Penicillium expansum isolates (128) were assayed (apples, pears, grapes and five reference strains). It was observed that 69.4% of the strains isolated from apples and pears were resistant to TBZ. Sensitive isolates were inhibited at 0.25-0.5 microg ml(-1) whilst resistant isolates still grew at 512 microg ml(-1). PAT was produced by all P. expansum isolates. CIT was detected in 98.8% of TBZ-resistant isolates and in 89.1% of the TBZ-sensitive isolates. CONCLUSIONS: The preliminary screening method combined with the adaptation of the method CLSI M38-A, can be a good strategy to be used in assessing the in vitro activity of TBZ against a large number of isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed methodology can be a contribution to the standardization of susceptibility tests to fungicides against P. expansum.

In vitro activity of imazalil against Penicillium expansum: comparison of the CLSI M38-A broth microdilution method with traditional techniques.

Penicillium expansum is one of the most important pathogens that cause blue mold in stored apples and is regarded as the major producer of the mycotoxin patulin. Imazalil is one of the fungicides used in Spain to control postharvest blue mold, but development of fungal resistance has been reported in P. digitatum and P. italicum. The most common used methods to detect antifungal susceptibility of fungal crop pathogens in vitro, are direct-plating isolates in media amended with various concentrations of fungicide and determining inhibition of growth and/or spore germination. These techniques are time- and labor-intensive and are not suitable if a large number of isolates has to be evaluated. On the other hand, the broth microdilution method M38-A is the reference method developed by the Clinical and Laboratory Standards Institute (CLSI) for antifungal susceptibility testing in some clinical fungi, but Penicillium spp. are not included. Due to the lack of a standard method, the aim of this work is to evaluate the suitability of an adaptation of the CLSI M38-A method to monitor P. expansum susceptibility to imazalil in comparison with other techniques. A total of 128 P. expansum strains have been studied (118 isolates from apples and pears, 5 from grapes and 5 reference strains). Imazalil has shown to be highly active in vitro against all the P. expansum isolates tested, as all the evaluated parameters were in the range reported for imazalil sensitive Penicillium spp. The mean minimum inhibitory concentration determined by broth microdilution method and by agar dilution method (48-72 h readings) was 0.0625 microg/ml and 0.11-0.12 microg/ml respectively. The mean concentration that inhibited the size of colonies (48-72 h) and spore germination by 50% was 0.05-0.06 and 0.04 microg/ml respectively. Our results highlight that the broth microdilution method CLSI M38-A is a good alternative to be used in screening the in vitro activity of imazalil against a large number of isolates.

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.

Cutaneous hyalohyphomycosis caused by a Chrysosporium species related to Nannizziopsis vriesii in two green iguanas (Iguana iguana).

This report describes the first isolation of a Chrysosporium species as the etiological agent of dermatomycosis in two green iguanas (Iguana iguana). The ITS-5.8S rRNA gene of the two strains was sequenced and a search on the GenBank database revealed that the closest match was Nannizziopsis vriesii. Treatment with oral ketoconazole, in combination with topical 2% chlorhexidine solution and terbinafine resulted in clinical cure.

Low occurrence of patulin- and citrinin-producing species isolated from grapes.

AIMS: To assess the ability of fungi isolated from grapes to produce patulin and citrinin. METHODS AND RESULTS: A total of 446 Aspergillus isolates belonging to 20 species and 101 Penicillium isolates were inoculated in Czapek yeast extract agar and yeast extract sucrose agar and incubated for 7 days at 25 degrees C. Extracts were analysed for patulin and citrinin by thin-layer chromatography. None of the isolates of Aspergillus spp. produced either patulin or citrinin. Patulin was produced by three isolates of Penicillium expansum and two of Penicillium griseofulvum. Citrinin was produced by five isolates of P. expansum, two of Penicillium citrinum and one of Penicillium verrucosum. CONCLUSIONS: Our results show that the Aspergillus and Penicillium species commonly isolated from grapes are not a source of the mycotoxins, patulin and citrinin. SIGNIFICANCE AND IMPACT OF THE STUDY: The possibility of co-occurrence of patulin and citrinin with ochratoxin A in grapes and grape products remain low, owing to the low frequency of isolation of potentially producing species.

Ochratoxin A-producing fungi from grapes intended for liqueur wine production.

The ochratoxigenic mycobiota of grapes intended for liqueur wines from four Spanish vineyards were studied. The specific wine-making technology of these wines requires overripening of the grapes on the vine or extended post-harvest exposure of the grapes in the sun. In every vineyard, samples were taken at three different developmental stages: veraison, harvesting time and after over-ripening. With the maturation of the berries there was a clear increase of Aspergillus spp. In the last sampling time studied, they were isolated from the 90.3% of the plated berries. Black aspergilli (mainly A. niger aggregate and A. carbonarius) were predominant among the different Aspergillus spp. isolated and constituted 98.5% of the total Aspergillus strains isolated. At harvesting time and after over-ripening, the percentage of colonized berries with A. carbonarius exceeded that of Aspergillus niger aggregate. Due to their low frequency of isolation, Penicillium spp. and Aspergillus spp. outside black aspergilli are not an important source of ochratoxin A in grapes for liqueur wine production. On the contrary, 98.5% of the A. carbonarius isolates screened were able to produce ochratoxin A. Although the possible participation of different ochratoxin A-producing species may occur, our results confirm that A. carbonarius is the most important source of ochratoxin A in liqueur wines, increasing its occurrence along the ripening of grapes.

Study of the effect of water activity and temperature on ochratoxin A production by Aspergillus carbonarius.

The effect of water activity (aw) (0.78-0.99) and temperature (15 and 30 degrees C) on growth and production of ochratoxin A (OTA) of six Aspergillus carbonarius strains was studied in two culture media: Czapek yeast autolysate (CYA) agar and yeast extract sucrose (YES) agar, during a period of 30 days. The strains were selected to include different sources and different reported abilities to produce OTA and were characterized by RAPD and ITS-5.8S rDNA sequencing. CYA showed to be better culture medium than YES for OTA production in the isolates tested. OTA concentration was higher at 15 degrees C than at 30 degrees C. At 30 degrees C, ranges for OTA production were more restrictive than those for growth. OTA was produced from 0.86, 0.90 or 0.94 aw depending on the strain. At 15 degrees C, growth and OTA production were detected only in the 0.94-0.99 aw range. The molecular study performed showed that five of the strains were conspecific and no correlation was found between molecular data and the OTA production level or origin. The remaining strain had never been able to produce OTA and will probably represent a new species in the Aspergillus section Nigri. Our results show that A. carbonarius is able to grow and produce OTA in a wide range of water activities at both high and low temperatures.

Effect of pH on ochratoxin A production by Aspergillus niger aggregate species.

The effect of pH (2-10) on growth and ochratoxin A (OTA) production by 12 Aspergillus niger aggregate strains was studied in two culture media: Czapek yeast autolysate agar (CYA) and yeast extract sucrose agar (YES), over 30 days. The strains were selected to include different sources, different reported abilities to produce OTA and different ITS-5.8S rDNA RFLP patterns. YES was a better culture medium than CYA for OTA production. In this medium, OTA was produced from pH 2 or 3 to 10 depending on the strain. The results show the ability of A. niger aggregate strains not only to grow, but also to produce OTA over a wide pH range. The results will lead to a better understanding of the role of A. niger aggregate strains in the OTA contamination of several food commodities.