Gliotoxin production by Aspergillus fumigatus strains from animal environment. Micro-analytical sample treatment combined with a LC-MS/MS method for gliotoxin determination.

In this study, gliotoxin production by Aspergillus fumigatus strains from animal environment is studied. Moreover, a rapid, easy and environment-friendly micro-analytical sample treatment procedure coupled with LC-MS/MS was applied for the determination of gliotoxin from A. fumigatus cultures. The ability of gliotoxin production by 143 strains was assayed in yeast extract sucrose agar, and 1 ml of chloroform was used for toxin extraction without further clean-up. Mean recoveries at two spiking levels (2500 and 7000 ng/g; n = 6) were 100.3 ± 6.6 % relative SD (RSD) and 92.4 ± 3.8 % RSD. Repeatability and within-laboratory reproducibility for different concentration levels of gliotoxin (25 to 1000 ng/ml; n = 12) ranged from 0.3 to 5.4 % RSD and from 3.9 to 12.7 % RSD, respectively. The detection limit of the analytical method was 3.5 ng/g. The ability for gliotoxin production by A. fumigatus revealed that 61.5 % of the strains were able to produce the toxin at levels ranging from LOQ to 3430.5 ng/g. However, all the tested samples had similar percentages of producing strains (81.8 to 86.6 %). The micro-analytical sample treatment coupled with LC-MS/MS detection is a precise and useful methodology for determining gliotoxin from fungal extracts of A. fumigatus and allows working both fast and safely and also reducing the effect on the environment. This toxin plays a critical role in the pathobiology of A. fumigatus, and its presence in animal environments could affect animal health and productivity; in addition, there are risks of contamination for rural workers during handling and storage of animal feedstuffs.

Typing clinical and animal environment Aspergillus fumigatus gliotoxin producer strains isolated from Brazil by PCR-RFLP markers.

UNLABELLED: Aspergillus fumigatus, a well-known human and animal pathogen causing aspergillosis, has been historically identified by morphological and microscopic features. However, recent studies have shown that species identification on the basis of morphology alone is problematic. The aim of this work was to confirm the taxonomic state at specie level of a set of clinical (human and animal) and animal environment A. fumigatus strains identified by morphological criteria applying a PCR-RFLP assay by an in silico and in situ analysis with three restriction enzymes. The A. fumigatus gliotoxin-producing ability was also determined. Previous to the in situ PCR-RFLP analysis, an in silico assay with BccI, MspI and Sau3AI restriction enzymes was carried out. After that, these enzymes were used for in situ assay. All A. fumigatus strains isolated from corn silage, human aspergillosis and bovine mastitis and high per cent of the strains isolated from cereals, animal feedstuff and sorghum silage were able to produce high gliotoxin levels. Also, all these strains identified by morphological criteria as A. fumigatus, regardless of its isolation source, had band patterns according to A. fumigatus sensu stricto by PCR-RFLP markers. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspergillus fumigatus is a well-known human and animal pathogen causing aspergillosis. In this study, clinical (human and animal) and animal environment strains were able to produce high gliotoxin levels and had band profiles according to A. fumigatus sensu stricto by PCR-RFLP markers. The results obtained here suggest that strains involved in human and animal aspergillosis could come from the animal environment in which A. fumigatus is frequently found. Its presence in animal environments could affect animal health and productivity; in addition, there are risks of contamination for rural workers during handling and storage of animal feedstuffs.

Fungi and mycotoxins in silage: an overview.

The present revision shows the early and current knowledge in the field of silage fungi and mycotoxins explaining the relevance of fungi and mycotoxins in silage. The problem does not end in animal disease or production losses as mycotoxins in feed can lead to the presence of their metabolic products in dairy products, which will be eventually affecting human health, mainly infants. Silage is green forage preserved by lactic fermentation under anaerobic conditions. This ecosystem maintains its quality and nutritional value depending on interactions among physical, chemical and biological agents. Forages used for ensilage are naturally in contact with yeasts and filamentous fungi, and the contamination often occurs in the field and can also occur during harvesting, transport, storage. Moreover, postharvest poor management can lead to a rapid spoilage. Studies on fungal contamination of dairy cattle feed have shown how corn silage influences the contamination degree of feed supplied to livestock. Increasing knowledge in this area will help elucidate the influence that this microbiota exerts on production and/or degradation of mycotoxins present in silage. Some of these fungi, although opportunist pathogens, are relevant epidemiologically and represent a high risk of contamination to farm workers who handle them improperly.

In vitro study on the effect of Saccharomyces cerevisiae strains on growth and mycotoxin production by Aspergillus carbonarius and Fusarium graminearum.

The effect of Saccharomyces cerevisiae RC008 and RC016 strains, previously selected based on their aflatoxin B1 mycotoxin binding ability and beneficial properties, against Aspergillus carbonarius and Fusarium graminearum under different interacting environmental conditions was evaluated. In vitro studies on the lag phase, growth rate and ochratoxin A/zearalenone and DON production were carried out under different regimens of a(w) (0.95 and 0.99); pH (4 and 6); temperature (25 and 37 °C) and oxygen availability (normal and reduced). Both yeast strains showed antagonistic activity and decreasing growth rate compared to the control. In general, the RC016 strain showed the greatest inhibitory activity. Except at the interacting condition 0.95 a(W), normal oxygen availability and 37 °C, at both pH values, A. carbonarius and F. graminearum were able to produce large amounts of mycotoxins in vitro. In general, a significant decrease in levels of mycotoxins in comparison with the control was observed. S. cerevisiae RC008 and RC016 could be considered as effective agents to reduce growth and OTA, ZEA and DON production at different interacting environmental conditions, related to those found in stored feedstuff. The beneficial and biocontrol properties of these strains are important in their use as novel additives for the control of mycotoxigenic fungi in stored feedstuffs.

Saccharomyces cerevisiae strains and the reduction of Aspergillus parasiticus growth and aflatoxin B1 production at different interacting environmental conditions, in vitro.

The effect of Saccharomyces cerevisiae RC008 and RC016, previously selected based on their aflatoxin B(1) binding ability and beneficial properties, against Aspergillus parasiticus under different interacting environmental conditions was evaluated. Studies concerning the lag phase, growth rate and aflatoxin B(1) production were carried out in vitro under different regimes of a (w) (0.95 and 0.99), pH (4 and 6), temperature (25 and 37°C), and oxygen availability (normal and reduced). Both yeast strains showed great antagonistic activity at pH 4, decreasing growth rate compared with the control. The RC008 strain showed the greatest inhibitory activity at all assayed conditions. A. parasiticus produced large amounts of AFB(1) in vitro. A significant decrease of AFB(1) levels in comparison with the control were observed with yeast interaction. Differences between control and treatment values ranged from 130 to 5400 ng ml(-1). S. cerevisiae RC008 and RC016 could be considered as effective agents in reducing growth and AFB(1) production at different interacting environmental conditions, related to that found in stored feedstuff. The importance of the present work lies in the search for live strains with both probiotic and biocontrol properties able to prolong the safe storage of feedstuff and exert beneficial properties after animal consumption and which could be included in a novel product for animal feed.

Gliotoxin contamination in and pre- and postfermented corn, sorghum and wet brewer's grains silage in Sao Paulo and Rio de Janeiro State, Brazil.

AIMS: The aim of this study was to determine total fungal counts and the relative density of Aspergillus fumigatus and related species in silage samples intended for bovines before and after fermentation as well as to monitor the natural occurrence of gliotoxin in silage samples (pre- and postfermentation). METHODS AND METHODS: The survey was performed in farms located in São Paulo and Rio de Janeiro States in Brazil. In addition, the ability of A. fumigatus strains and related species strains to produce gliotoxin was also evaluated. A total of 300 samples were taken, immediately after opening of the silo (3-5 months) and during the ensiling period. Fungal counts were done by the surface-spread method. Gliotoxin production ability of isolates and natural contamination were determined by HPLC. RESULTS: All postfermented samples had a total number of moulds exceeding 1 × 10(4) CFU g(-1), with Aspergillus sp. as the most prevalent genus. Frequency of strains, among A. fumigatus and related species, was able to produce gliotoxin was similar in pre- and postfermented samples, except for sorghum, which showed differences between both kinds of samples. The highest toxin levels were produced by strains isolated from postfermented samples. More than 50% of the samples showed gliotoxin contamination levels that exceeded concentrations known to induce immunosuppressive and apoptotic effects in cells. CONCLUSIONS: The present data suggest that care should be taken because gliotoxin contamination in feedstuffs could affect productivity and also present a health risk for herds. SIGNIFICANCE AND IMPACT OF THE STUDY: Gliotoxin was found at quite important concentrations levels in pre- and postfermented substrates and its presence could therefore probably affect the productivity and health of herds. Current conservation and management practices do not avoid contamination with A. fumigatus on silage. Therefore, farm workers should be adequately protected during its handling.

Fungi and Mycotoxins from Pre- and Poststorage Brewer's Grain Intended for Bovine Intensive Rearing.

The aim of the study was to determine the mycobiota and natural levels of mycotoxins such as aflatoxin B1 (AFB1), ochratoxin A (OTA), fumonisin B1 (FB1), and deoxynivalenol (DON) present in brewers grains pre- and poststored intended for bovine intensive rearing. Poststored (80%) samples had counts higher than 1 × 10(4) colony-forming units (CFU/g). Cladosporium spp. and Aspergillus spp. were isolated at high frequencies. Aspergillus flavus was the prevalent isolated species. Prestored (70%) and poststored (100%) samples showed AFB1 levels over the recommended limits (20 µ g/Kg), and OTA levels were below the recommended limits (50 µ g/Kg) while pre- and poststored samples did not show FB1 and DON natural contamination levels. The presence of mycotoxins in this substrate indicates the existence of contamination. Regular monitoring of feeds is required in order to prevent chronic and acute toxic syndromes related to this kind of contamination.

Ochratoxin A in wines from 2002 to 2008 harvest marketed in Rio de Janeiro, Brazil.

The occurrence of ochratoxin A (OTA) in wine from 2002 to 2008 harvest, traded in Rio de Janeiro State, was evaluated by analysing 43 national and 37 imported wines from Argentina (32) and Chile (5), adding up to 80 samples in total. OTA determination was performed using immunoaffinity columns and high-performance liquid chromatography. In 80 wine samples analysed, 25 (31.3%) were positive, presenting levels greater than 0.020 ng OTA mL⁻¹. It was not detected in imported wines. Within national wines, 58.1% of the samples were contaminated, with levels ranging from 0.020 to 0.050 ng mL⁻¹. The toxin was detected in 18 (69.2%) of 26 samples analysed of red table wine. Wines from 2008 harvest presented 84.6% of samples contaminated in 13 samples analysed. Despite the levels found in this study, they are below Brazilian tolerance limits. Nevertheless, the presence of OTA as found contributes to the human exposure to this toxin.

Saccharomyces cerevisiae strains retain their viability and aflatoxin B1 binding ability under gastrointestinal conditions and improve ruminal fermentation.

The aim was to evaluate both the ability of yeast strains to survive and bind AFB(1) under ruminant gastrointestinal conditions and the effect of these yeast strains on ruminal fermentation. Yeast viability was studied under simulated gastrointestinal conditions. AFB(1) binding ability was evaluated at different pH values as present in the ruminant gastrointestinal tract. The effect of yeast strains on cellulose digestion and volatile fatty acids production by ruminal bacteria was also evaluated. All yeast strains were able to survive under gastrointestinal conditions and to adsorb AFB(1) at the different pH assayed. The strain RC016 showed the highest binding percentage at the three tested pH. The number of cellulolytic bacteria in ruminal fluid increased in the presence of RC008 and RC016 yeast strains. The concentration of acetate and propionate after ruminal fermentation increased with the addition of RC008 and RC016 strains; this effect was less significant with RC009 strain. Strains RC008 and RC016 are potential probiotic to be included in animal feed: they help to increase fibber digestibility and could reduce AFB(1 )bioavailability in the gastrointestinal tract. Viable S. cerevisiae RC008 and RC016 strains with both probiotic and mycotoxins adsorption properties could be used as feed additives in ruminant feedstuff.

Mycobiota and mycotoxins in malted barley and brewer's spent grain from Argentinean breweries.

AIMS: To evaluate mycobiota and aflatoxins B(1) (AFB(1)), B(2) (AFB(2)), G(1) (AFG(1)), G(2) (AFG(2)) and fumonisin B(1) (FB(1)) contamination in different malted barley types and brands and brewer's grain collected from a major Argentinean brewery. METHODS AND RESULTS: Total fungal counts were performed using the plate count method. Aflatoxin B(1), AFB(2), AFG(1), AFG(2) and Zearalenone (ZEA) analyses were performed by thin-layer chromatography (TLC). Fumonisin B(1) was determined by HPLC. Eighty-three percentage of the malted barley (100% M1, 50% M2 and 100% M3) and 61% of brewer's grain samples had a count >1 × 10(4) CFU g(-1). Yeasts were isolated from all malt and brewer's grain samples. Genera containing some of the most important mycotoxin producer species--Fusarium ssp., Aspergillus ssp., Penicillium ssp. and Alternaria ssp.--were isolated from the analysed samples, along with other environmental saprophytic fungi such as Geotrichum ssp., Mucorales and Cladosporium ssp. All samples were contaminated with 104-145 µg kg(-1) FB(1). Eighteen per cent of brewer's grain samples were contaminated with 19-44.52 µg kg(-1) AFB(1). Aflatoxin B(2), AFG(1), AFG(2) and ZEA were not detected in any of the analysed samples. CONCLUSIONS: Fungal and mycotoxin contamination in malt and brewer's grain is an actual risk for animal and human health. SIGNIFICANCE AND IMPACT OF THE STUDY: This study may be useful for assessing the risk of mycotoxins in Argentinean beers and especially in animal feeds.

Sodium bentonite and monensin under chronic aflatoxicosis in broiler chickens.

Clay feed additives have been increasingly incorporated into animal diets to prevent aflatoxicosis. Due to the nonselective nature of the binding interaction, many important components of the diets could also be made unavailable because of these feed additives. The anticoccidial monensin (MON) could also be sequestered by these clays. The use of sodium bentonite (Na-B) from a mine in the province of Mendoza, Argentina, was investigated as a sequestering agent to prevent the effects of 100 µg/kg of dietary aflatoxin B(1) (AFB(1)). In vitro studies demonstrated that the above Na-B was a good candidate to prevent aflatoxicosis. They also showed that MON competes with AFB(1) for the adsorption sites on the clay surface and effectively displaces the toxin when it is in low concentration. Even though the levels of MON in diets, approximately 55 mg/kg, are high enough to not be significantly changed as a consequence of the adsorption, they can further affect the ability of the clays to bind low levels of AFB(1). An in vivo experiment carried out with poultry showed that 100 µg/kg of AFB(1) does not significantly change productive or biochemical parameters. However, liver histopathology not only confirmed the ability of this particular Na-B to prevent aflatoxicosis but also the decrease of this capacity in the presence of 55 mg/kg of MON. This is the first report stressing this fact and further research should be performed to check if this behavior is a characteristic of the assayed Na-B or of this type of clay. On the other hand, the presence of MON should also be taken into account when assaying the potential AFB(1) binding ability of a given bentonite.

Production of citreoviridin by Penicillium citreonigrum strains associated with rice consumption and beriberi cases in the Maranhão State, Brazil.

The aim of this study was to determine the levels of Penicillium citreonigrum and citreoviridin present in rice samples from Maranhão State, Brazil, where an outbreak of beriberi was reported and 32 deaths occurred (7% of the notified cases died in 2006). The ability of P. citreonigrum to produce citreoviridin was assessed, and a total of 420 samples of 21 different kinds of rice were collected. Mycobiota isolation and identification, the ability of citreoviridin strains to produce toxin, and the natural occurrence of citreoviridin were established. Rice samples were found to have high fungal counts and showed increasing levels from 2004 to 2007 harvest years. The most frequent genus was Aspergillus followed by Penicillium and Cladosporium. Ten out of eleven strains of P. citreonigrum were able to produce citreoviridin. Three rice samples had levels of citreoviridin ranging from 12 to 96.7 ng g(-1), and two bran samples had levels of 128 and 254 ng g(-1). These samples contaminated with P. citreonigrum and citreoviridin were involved in the beriberi cases from Maranhão State. Monitoring rice for mycotoxins in areas where this substrate is the basic food is crucial to prevent outbreaks like the one reported in this study, to improve management practice, and to diminish exposure risk of humans to these harmful toxins.

Aspergillus fumigatus toxicity and gliotoxin levels in feedstuff for domestic animals and pets in Argentina.

AIMS: To evaluate gliotoxin production by Aspergillus fumigatus strains isolated from feedstuff intended for domestic animals and pets, and to determine the amount of gliotoxin in these substrates. METHODS AND RESULTS: A total of 150 feedstuff samples were collected. They were composed of 30 samples each of five different feed types (pigs, poultry, cattle, horse and pets). Aspergillus fumigatus gliotoxin production ability and gliotoxin presence in feedstuff was determined by HPLC. Aspergillus fumigatus strains were isolated from all of the tested samples. Strains from cattle, horses and pet food were able to produce gliotoxin. Corn silage samples intended for cattle did not show gliotoxin contamination. All the other tested samples had gliotoxin levels ranging from 29 to 209 microg g(-1). Horse and poultry feed samples had the greatest contamination frequency. CONCLUSIONS: Feed samples contaminated with gliotoxin are potentially toxic to animals. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of gliotoxin could affect animal productivity and health. Moreover, there are risks of contamination to farm workers handling improperly stored animal feed. Aspergillus fumigatus strains isolated from different sources should be investigated to determine prevention and control strategies.

Mycological survey and ochratoxin A natural contamination of swine feedstuffs in Rio de Janeiro State, Brazil.

Mycotoxin contamination of animal feeds represents a hazard to human and animal health due to potential transmission to meat and milk. Barley by-products are alternative feeding supplies for animal production. The aims of this assay were to study the mycobiota of feedstuffs and finished swine feed, to determine the ability of Aspergillus and Penicillium isolates to produce ochratoxin A (OTA) and to evaluate OTA occurrence in these substrates. Corn, brewers' grains and finished swine feed samples were collected from different factories. Fungal counts were higher than 2.8x10(4)CFU g(-1). Fusarium, Aspergillus and Penicillium genera were isolated at high levels. A 23.7% of the isolates produced 9-116 microg kg(-1) of OTA in vitro. Corn samples (44%) were contaminated with 42-224 microg kg(-1) of OTA. Finished feed (31%) and brewers' grains samples (13%) were contaminated with 36-120 microg kg(-1) and 28-139 microg kg(-1) of OTA, respectively. This is the first scientific report on contamination by OTA-producer molds and OTA in swine feedstuffs from Brazil. The presence of OTA in raw materials and finished feed requires periodic monitoring to prevent mycotoxicoses in animal production, reduce economic losses and minimize hazards to human health.

Mycobiota and aflatoxins in raw materials and pet food in Brazil.

Commercial feedstuffs are a basic element in modern pet husbandry in the world. In dogs, the effect of mycotoxins is severe and can lead to death. Few reports on the influence of dietary mycotoxins were found in the scientific literature. The aims of this work were to isolate and identify the mycoflora and to determine the aflatoxins (AFs) natural occurrence in raw materials and ready dry pet food. Therefore, the aflatoxigenic capacity of Aspergillus flavus species was investigated. Aspergillus was the prevalent genera (65-89%) followed by Penicillium and Fusarium spp. Aspergillus flavus was the most prevalent species, followed by Aspergillus sydowii, Aspergillus fumigatus and Aspergillus versicolor. Aspergillus flavus frequencies ranged from 58% to 86% except in sorghum meal. All samples assayed (except corn grains and ready pet food) showed Fusarium spp. contamination. Corn meal and corn meal and gluten samples had 100% Fusarium verticillioides. Fusarium graminearum was isolated from sorghum meal. Aspergillus flavus strains (75%) isolated from raw materials and 57% from pet food were able to produce AFs. All samples showed AFs contamination percentages over 70%; corn and sorghum meal obtained the highest AFs levels. Ready pet food did not show quantitative levels of the tested toxins. This is the first report of the aflatoxigenic capacity by A. flavus from Brazilian pet food.

Determination of mycobiota and mycotoxins in pig feed in central Argentina.

AIMS: To evaluate the mycobiota and natural levels of aflatoxins, fumonisins and zearalenone present in compound feed and home-corn grains intended for fattening pigs. METHODS AND RESULTS: Total fungi, Fusarium and Aspergillus species occurrence were examined. Aflatoxins and zearalenone were detected by thin-layer chromatography and fumonisins by high-pressure liquid chromatography. Fungal counts were generally higher than 1 x 10(5) colony forming units (CFU) ml(-1). Aspergillus flavus, Aspergillus parasiticus and Fusarium verticillioides were the most prevalent species. FB(1) and FB(2) were detected in all feed and corn samples. Aflatoxin B(1) was detected in 33.33% of initial and growing feed and in 44.44% of final feed samples. It was not detected in corn samples. All feed and corn samples were negative for AFB(2), AFG(1), AFG(2) and ZEA presence during all growing stages tested. CONCLUSIONS: Fungal counts at all growing periods exceeded the levels proposed as feed hygienic quality limits. Aflatoxin levels in all feeds and fumonisin levels in many samples were higher than the established regulations. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of mycotoxins indicates the existence of contamination. This fact requires periodic monitoring to prevent the occurrence of mycotoxicosis in animal production, to reduce the economic losses and to minimize hazards to human health.

Fungi and selected mycotoxins from pre- and postfermented corn silage.

AIM: To determine fungal genera, Aspergillus and Fusarium species and aflatoxin B(1) (AFB(1)), zearalenone (ZEA), deoxynivalenol (DON), fumonisin B(1) (FB(1)) contamination from pre- and postfermented corn silage produced in the most important region of Argentina where silage practice is developed. METHODS AND RESULTS: Sampling of corn silos was performed manually through silos in transects at three levels: upper, middle and low sections. AFB(1) and FB(1) were quantified by high-performance liquid chromatography, zearalenone by enzyme-linked immunosorbent assay and DON by gas chromatography. Over 90% of the samples showed counts higher than 1 x 10(4) CFU g(-1). Aspergillus flavus and Fusarium verticillioides were the prevalent species. Some tested samples were contaminated with AFB(1), ZEA, DON and FB(1). CONCLUSIONS: This study demonstrates the presence of fungi and AFB(1), ZEA, DON and FB(1) contamination in corn silage in Argentina. SIGNIFICANCE AND IMPACT OF THE STUDY: This manuscript makes a contribution to the knowledge of mycotoxins in Argentinean silage in particular because the environmental conditions in this country differ from those of most reports. The comparison of pre- and postfermentation silage is also outstanding. Therefore, information on fungi and mycotoxins present in silage--an increasingly popular commodity--is useful to estimate potential risk for animal and human health.

The mycobiota and toxicity of equine feeds.

Feed contamination can lead to nutrient losses and detrimental effects on animal health and production. The purposes of this study were to investigate the mycobiota in equine mixed feeds and to determine natural contamination with aflatoxin B1 (AFB1) and fumonisin B1 (FB1). Fungal enumeration of equine feed samples was done. A commercially available enzyme-linked immunosorbent assay kit was applied to quantify AFB1 and FB1. A comparison between ELISA and HPLC was carried out. Feed mould counts ranged from <1 x 10(2) to 1 x 10(5) cfu/g. The most frequent genus isolated was Aspergillus (40.54%), followed by Penicillium (18.38%) and Fusarium (16.22%). The most prevalent Aspergillus sp. was A. flavus (36%). AFB1 values ranged between 0.01 and 99.4 microg/kg. FB(1) levels ranged between 0.01 and 7.49 microg/kg. HPLC and ELISA methods showed positive correlation for AFB1 and FB1 determinations (r = 0.9851 and r = 0.9791, respectively). The ELISA analytical method was efficient for AFB1 and FB1 detection. The scarcity of studies on natural fungal contamination and on the presence of AFB1 and FB1 in materials used as equine feed ingredients highlights the value and contribution of this study.

Influence of water activity, temperature and time on mycotoxins production on barley rootlets.

AIMS: The objective of this study was to determine the ochratoxin (OT) and aflatoxin (AF) production by three strains of Aspergillus spp. under different water activities, temperature and incubation time on barley rootlets (BR). METHODS AND RESULTS: Aspergillus ochraceus and Aspergillus flavus were able to produce mycotoxins on BR. Aspergillus ochraceus produced ochratoxin A (OTA) at 0.80 water activity (a(w)), at 25 and 30 degrees C as optimal environmental conditions. The OTA production varies at different incubation days depending on a(w). Aflatoxin B(1) (AFB1) accumulation was obtained at 25 degrees C, at 0.80 and 0.95 a(w), after 14 and 21 incubation days respectively. Temperature was a critical factor influencing OTA and AFB(1) production. CONCLUSIONS: This study demonstrates that BR support OTA and AFB(1) production at relatively low water activity (0.80 a(w)) and high temperatures (25-30 degrees C). SIGNIFICANCE AND IMPACT OF THE STUDY: The study of ecophysiological parameters and their interactions would determine the prevailing environmental factors, which enhance the mycotoxin production on BR used as animal feed.

Mycoflora of poultry feeds and ochratoxin-producing ability of isolated Aspergillus and Penicillium species.

In Brazil, commercial feedstuffs are an important component in modern animal husbandry, but there is no information available about fungal contamination and ochratoxin A (OTA) production. The aims of this study were to determine the mycoflora incidence in poultry feeds and evaluate OTA production. In addition, the ability to produce OTA by several Aspergillus and Penicillium species was investigated. A total of 96 samples of poultry feeds were collected from four factories in Rio de Janeiro. Samples were examined for total moulds, for Aspergillus and Penicillium spp. occurrence and for their relative densities on dichloran rose bengal chloramphenicol and dichloran 18% glycerol media. The capacity to produce ochratoxin A by selected Aspergillus and Penicillium species was determined by HPLC. Total mould counts were generally higher than 1 x 10(5 )CFU ml(-1). Aspergillus and Penicillium species were isolated in the highest numbers. Aspergillus flovus and Penicillium citrinum were the most prevalent species. There was a high percentage of potential OTA producers (46%). The amount of OTA produced on this substrate was enough to cause adverse effects in animals. Several strains isolated from poultry feeds were able to produce high levels of OTA on chloramphenicol yeast medium. OTA in raw materials needs to be surveyed and storage practices must be investigated to determine occurrence and establish the livestock toxicological risk in poultry feed.