In vitro aflatoxin B1 binding capacity by two Enterococcus faecium strains isolated from healthy dog faeces.

AIM: This study evaluated the binding capacity of aflatoxin B1 (AFB1 ) by two Enterococcus faecium strains (MF4 and GJ40) isolated from faeces from healthy dogs. MATERIALS AND METHODS: The binding assay was performed using 50 and 100 ppb of AFB1 analysing the effects of the viability, incubation time and pH on AFB1 binding. Binding stability was determined by washing three times the bacteria-AFB1 complexes with phosphate buffer saline. RESULTS: Both GJ40 and MF4 strains have the ability to remove AFB1 from aqueous solution. Viable cells were slightly more effective in AFB1 binding than nonviable ones for both strains. Enterococcus faeciumGJ40 removes 24-27% and 17-24%, and Ent. faeciumMF4 removes 36-42% and 27-32% of AFB1 (50 and 100 ppb, respectively) throughout a 48 h incubation period. In general, the removal of AFB1 was highest at pH 7.00 for both strains. The stability of the bacteria-AFB1 complex formed was found to be high (up to 50% of AFB1 remained bounded in bacterial cell after three washes with phosphate buffered saline). CONCLUSION: The Ent. faecium strains assayed are capable of removing AFB1 under different conditions in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first AFB1 binding assay performed with Ent. faecium strains isolated from dog faeces, being an interesting strategy for AFB1 decontamination of pet food.

Negligible effects of tryptophan on the aflatoxin adsorption of sodium bentonite.

The main objective of this study was to determine if the competitive adsorption of tryptophan (Trp) and aflatoxin B1 (AFB1) could potentially affect the ability of a sodium bentonite (NaB) to prevent aflatoxicosis in monogastric animals. The adsorption of Trp and AFB1 on this adsorbent is fast and could be operating on the same time-scale making competition feasible. In vitro competitive adsorption experiments under simulated gastrointestinal conditions were performed. A high affinity of the clay for Trp and NaB was observed. The effect of an excess of KCl to mimic the ionic strength of the physiological conditions were also investigated. A six-times decrease in the Trp surface excess at saturation was observed. A similar behaviour was previously found for AFB1 adsorption. Taking into account the amount of Trp adsorbed by the clay and the usual adsorbent supplementation level in diets, a decrease in Trp bioavailability is not expected to occur. Tryptophan adsorption isotherms on NaB were 'S'-shaped and were adjusted by the Frumkin-Fowler-Guggenheim model. The reversibility of the adsorption processes was investigated in order to check a potential decrease in the ability of NaB to protect birds against chronic aflatoxicoses. Adsorption processes were completely reversible for Trp, while almost irreversible for AFB1. In spite of the high affinity of the NaB for Trp, probably due to the reversible character of Trp adsorption, no changes in the AFB1 adsorption isotherm were observed when an excess of the amino acid was added to the adsorption medium. As a consequence of the preferential and irreversible AFB1 adsorption and the reversible weak binding of Trp to the NaB, no changes in the aflatoxin sorption ability of the clay are expected to occur in the gastrointestinal tract of birds.

Survey of Aspergillus section Flavi presence in agricultural soils and effect of glyphosate on nontoxigenic A. flavus growth on soil-based medium.

AIMS: To evaluate the cultivable mycobiota from agricultural soils exposed to pesticides, the aflatoxigenic capacity of Aspergillus section Flavi strains and the effect of glyphosate on lag phase and growth rates of native nontoxigenic Aspergillus flavus under different water potential (MPa) conditions on soil-based medium. METHODS AND RESULTS: Culturable mycobiota analysis from different agricultural soils was performed by the surface spread method. The effect of glyphosate (0-20 mmol l(-1)) on the growth of A. flavus strains was evaluated on a soil extract solid medium. Mycobiota analysis of crop soils showed the presence of twenty-one genera of filamentous fungi. Aspergillus flavus and Aspergillus niger aggregate strains were isolated from the three soil types. Ninety-two per cent of A. flavus strains were toxigenic. In vitro assay results showed that at -0·70 MPa, a significant increase in growth rate in all strains was recorded at 5 and 20 mmol l(-1) of glyphosate. At -2·78 MPa, this parameter remained constant at all glyphosate concentrations, except in GM4 strain where an increase in growth rate was recorded with increasing pesticide concentrations. At -7·06 MPa, a significant increase in growth rate has also been observed in GM 3 strain with 5 mmol l(-1) and in GM 4 strain with 10 and 20 mmol l(-1). CONCLUSIONS: This study showed that the imperfecti fungi Aspergillus spp., Penicillium spp., Trichoderma spp., Cladosporium spp. and Paecilomyces spp. are isolated as prevalent groups in agricultural soil exposed to pesticides, and the capacity of nontoxigenic A. flavus strains to tolerate different glyphosate concentrations under different water potential (MPa) conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This manuscript makes a contribution to the knowledge of cultivable fungal populations from agricultural soils exposed to pesticides and the glyphosate tolerance of A. flavus strains.

Fungal contamination and determination of fumonisins and aflatoxins in commercial feeds intended for ornamental birds in Rio de Janeiro, Brazil.

The purposes of this study were to determine the distribution of total mycobiota, to determine the occurrence of Aspergillus spp., Penicillium spp. and Fusarium spp. and to detect and quantify fumonisin B1 and aflatoxin B1 in birds' feedstuffs. Sixty samples from different commercial feeds were collected. Analysis of the total mycobiota was performed and total fungal counts were expressed as CFU g(-1). The isolation frequency (%) and relative density (%) of fungal genera and species were determined. Mycotoxins determination was carried out using commercial ELISA kits. The 48% of standard, 31% of premium and only 9% of super premium feed samples were found above of recommended limit (1 × 10(4) CFU g(-1)). Aspergillus (82%), Cladosporium (50%) and Penicillium (42%) were the most frequently isolated genera. Aspergillus niger aggregate (35%), Aspergillus fumigatus (28%) and Aspergillus flavus (18%) had the highest relative densities. Contamination with fumonisins was detected in 95% of total samples with levels from 0·92 to 6·68 µg g(-1), and the aflatoxins contamination was found in 40% of total samples with levels between 1·2 and 9·02 µg kg(-1). Feed samples contaminated with fumonisins and aflatoxins are potentially toxic to birds.

Natural co-occurrence of fungi and mycotoxins in poultry feeds from Entre Ríos, Argentina.

A total of 120 pelleted poultry feed samples from Entre Ríos Province, Argentina, were evaluated. The aims were to investigate (1) the presence of relevant toxigenic fungi, as well as to determine the ability to produce aflatoxins (AFs) by Aspergillus section Flavi isolated strains; and (2) the natural co-occurrence of AFs, fumonisins (FBs), gliotoxin, diacetoxyscirpenol (DAS), HT-2 and T-2 toxin by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Total fungal counts were below the established value (1 × 104 CFU g⁻¹). Aspergillus flavus and A. parasiticus were the only aflatoxigenic species isolated. Co-occurrence of fumonisin B1 (FB1), HT-2 and T-2 toxin was detected in 100% of the feeds, with mean levels from 4502 to 5813; 6.7 to 21.6 and 19.6 to 30.3 µg kg⁻¹, respectively. A large number of starter samples were co-contaminated with aflatoxin B1 (AFB1), FB1, HT-2 and T-2 toxins. Gliotoxin and DAS were not found in this survey.

Combined effects of aflatoxin B1 and corticosterone treatment on selected performance indices and liver histopathology in Japanese quail.

Animal feed may be contaminated with different mycotoxins, with aflatoxin B(1) (AFB(1)) being a very common and toxic compound. Considering that birds normally have to cope with different stressful situations at the same time, the present study aims to evaluate the effects of feed contamination with AFB(1) in combination with corticosterone treatment in drinking water (a model to induce physiological stress in birds) on selected performance indices: BW, feed conversion, egg production, and macroscopic and microscopic liver alterations. At 5 wk of age, quails were randomly assigned to 1 of 6 dietary treatment groups that resulted from the combination of the presence or absence of corticosterone in drinking water (5 mg/L) with the presence or absence of AFB(1) contamination (0, 100, or 500 µg/kg). The animals remained in these treatments from 5 to 11 wk of age. There were 6 replicates per treatment, each containing 2 males and 2 females. Contamination with 100 µg of AFB(1) per kilogram of feed induced no changes in BW, feed conversion, and egg production parameters. Quail fed with 500 µg of AFB(1) per kilogram of feed showed significant decreases in BW and feed consumption compared with their control counterparts. Corticosterone in combination with 500 µg of AFB(1) per kilogram of feed intensified the negative effects observed on BW and feed consumption and also had negative effects on feed conversion rate and egg production parameters, suggesting that the adverse effects of contamination with AFB(1) are intensified in situations of chronic stress. Quail treated with 500 µg of AFB(1) per kilogram showed hepatocytes with degree 1 and 2 lesions, and all quail treated with 500 µg of AFB(1) per kilogram of feed in combination with corticosterone showed degree 2 liver lesions (i.e., hepatocytes with fatty macro and microvacuoles and necrosis). This result is also consistent with the hypothesis that chronic stress exacerbates the effect of AFB(1) contamination. In conclusion, this study suggests that the negative effects of AFB(1) contamination are increased when overlapped with chronic stressful stimulation.

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.

Effect of low levels of aflatoxin B1 on performance, biochemical parameters, and aflatoxin B1 in broiler liver tissues in the presence of monensin and sodium bentonite.

Aflatoxins (AF) are a major problem in broiler production and are significant economic and public health burdens worldwide. A commercial sodium bentonite (Na-B) adsorbent was used to prevent the effect of AF [50 µg of aflatoxin B1 (AFB1)/kg of feed] in broiler productivity, biochemical parameters, macroscopic and microscopic liver changes, and AFB1 liver residues. The influence of Na-B (0.3%) and monensin (MON, 100 mg/kg), alone or in combination, was investigated in depth. The dietary treatments were as follows: treatment (T) 1: basal diet (B); T2: B + MON; T3: B + Na-B; T4: B + Na-B + MON; T5: B + AFB1; T6: B + AFB1 + Na-B + MON; T7: B + AFB1 + MON; T8: B + AFB1 + Na-B. Birds were fed dietary treatments for 28 d (d 18 to 46). No significant differences (P < 0.05) were observed among treatments with respect to broiler performance, biochemical parameters, or relative liver weights. With the exception of T8, all livers showed histopathological alterations, with accumulation of fat vacuoles. The normal appearance of livers from T8 showed the protective effect of Na-B against aflatoxicosis. The residual AFB1 levels in livers from T5 to T8 ranged from 0.2 to 1.0 ng/g and were higher in livers from T6 (P < 0.05). Results of this study indicate a competition between AFB1 and MON for adsorption sites on Na-B when feed contains low levels of the toxin, indicating a nonselective adsorption capacity of this particular Na-B. In addition, significant levels of AFB1 in livers indicate that this determination is an important technique not only for diagnosis of aflatoxicosis in broilers, but also for quality control of avian products.

Food-grade antioxidants and antimicrobials to control growth and ochratoxin a production by Aspergillus section Nigri on peanut kernels.

Each year, a significant portion of the peanuts produced cannot be marketed because of fungal disease at the postharvest stage and mycotoxin contamination. Antioxidants could be used as an alternative to fungicides to control ochratoxigenic fungi in peanuts during storage. This study was carried out to determine the effect of the antioxidant butylated hydroxyanisole (BHA) and the antimicrobial propyl paraben (PP) on the lag phase before growth, growth rate, and ochratoxin A (OTA) production by Aspergillus section Nigri strains in peanut kernels under different conditions of water activity (aw) and temperature. At 20 mM/g BHA, 18 degrees C, and 0.93 aw, complete inhibition of growth occurred. For PP, there was no growth at 20 mM/g, 18 degrees C, and 0.93, 0.95, and 0.98 aw. BHA at 20 mM/g inhibited OTA production in peanuts by Aspergillus carbonarius and Aspergillus niger aggregate strains at 0.93 aw and 18 degrees C. PP at 20 mM/g completely inhibited OTA production at 18 degrees C. The results of this work suggest that PP is more appropriate than BHA for controlling growth and OTA production by Aspergillus section Nigri species in peanut kernels.

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.

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.

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.

Occurrence of ochratoxin A in wine and ochratoxigenic mycoflora in grapes and dried vine fruits in South America.

Grape and wine production in South America represents about 6.6% and 10% respectively of the world grape and wine production. The available information on the ochratoxigenic mycoflora and ochratoxin A (OTA) presence in wine grapes, wines, grape juices and dried vine fruits is limited. Surveys have been carried out in Argentina and Brazil which showed that Aspergillus niger aggregate are predominant in the Argentinean varieties while from the Brazilian varieties the species A. niger, Aspergillus ochraceus and Aspergillus carbonarius were isolated. A mycobiota survey from wine grapes in Argentina showed that while Alternaria alternata was predominant, Aspergillus section Nigri species were isolated from 60% of samples. About 41% of black Aspergilli isolates produced OTA with levels ranging from 2 to 24.5 ng mL(-1). In another study, about 83% of A. carbonarius isolates from dried vine fruits produced OTA, with levels ranging from 2 to 5200 ng mL(-1). A survey of grape juices and wines of Brazilian, Argentinean and Chilean origin were found to contain very low levels of OTA. Studies are in progress in Latin America on the ecophysiology of ochratoxigenic fungi and OTA occurrence to reduce the impact of this toxin in the food chain.

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.

Occurrence of ochratoxin A in wine and grape juice marketed in Rio de Janeiro, Brazil.

Ochratoxin A (OA) is receiving attention world-wide because of the hazard it poses to human health. The aim was to test the distribution of OA in grape juice, pulps of frozen grapes, and national and imported table wine obtained from markets in Rio de Janeiro, Brazil. Analytical methodology using immunoaffinity column for OA extraction and clean-up with a final separation on a reversed-phase (C(18)) column and fluorescence detection in high-performance liquid chromatography showed a detection limit of 21 ng l(-1). The mean recovery was 91% for red wines and 82% for white wines; while the mean recoveries for juices and pulps of frozen grapes were 91.6 and 88%, respectively. Of 64 samples of grape juice and frozen pulps, 25% were positive for OA, being the mean content of 37 ng l(-1) with a maximum concentration of 100 ng l(-1). In wines, the mean concentration detected in 80 samples analysed was 34.4 ng l(-1) with 28.75% of positive samples. Red wines showed the highest percentages and levels of contaminated samples: 38% and 37 ng l(-1), respectively. The white wine contained levels above 26 ng l(-1) in 17.75% of the analysed samples. The levels of contamination detected in red wine sold in Río de Janeiro were not enough to surpass the virtually safe dose established as 5 n g kg(-1) body weight of daily intake.

Potential ochratoxin A producers from wine grapes in Argentina and Brazil.

The aim was to identify the normal mycoflora in wine grapes from Argentina and Brazil. We collected 50 grapes samples from Malbec and Chardonnay varieties in each country during the 1997-98 harvest. Yeasts were a major component of the fungal population, and the most frequent genera of filamentous fungi isolated were: Aspergillus, Penicillium and Botrytis. Other genera identified (in decreasing order) were: Phythophthora, Moniliella, Alternaria and Cladosporium. From grapes, the mean frequency of filamentous fungi ranged from 1.3 x 10(4) to 5.4 x 10(6) CFU g(-1). We isolated 48 Aspergillus niger strains from Argentinian grape, of which eight could produce ochratoxin A. Sixteen of 53 A. niger strains from Brazilian grapes produced ochratoxin A. The results indicate that similar mycobiota were isolated from Argentinian and Brazilian wine grapes and there could be ochratoxin A production in this substrate.

Natural occurrence of Fusarium species and fumonisin-production by toxigenic strains isolated from poultry feeds in Argentina.

Fusarium species and fumonisin production by toxigenic strains were investigated. During 1996-1998, 158 samples of poultry feeds were collected from a factory located in the department of Rio Cuarto Córdoba province, Argentina. The most common species of Fusarium were F. moniliforme (60.7%) and F. nygamai (35.4%) followed by F. semitectum, F. subglutinans, F. proliferatum, F. dlamini, F. solani, F. oxysporum and F. napiforme. Fungal counts ranged from 1 x 10(3) to 8 x 10(5) CFU/g with mean values from 1.5 x 10(3) to 2.3 x 10(5) CFU/g. The highest counts were for F. dlamini, F. subglutinans, F. moniliforme and F. nygamai. Strains of F. moniliforme, F. nygamai, and E. proliferatum were screened for their potential to produce fumonisin B1 (FB1), fumonisin B2 (FB2) and fumonisin B3 (FB3) in corn grain. The samples were analysed using a modified high performance liquid chromatography method. The strains assayed, 43 strains, produced three fumonisins. There was a high degree of variability in the quantities of FB1, FB2, and FB3 produced. The toxin produced in highest levels by the majority of the strains was FB1. The range of concentration varied from 5.4 to 3,991, 1.01 to 189 and 0.4 to 765 ppm per gram of corn for FB1, FB2 and FB3 respectively. The toxigenic pattern of strains was normal, although two strains of F. moniliforme produced exceptionally high concentrations of FB3 and minor concentrations of FB2 and FB1. This is the first report from Argentina on Fusarium species in poultry feeds and fumonisin production by these strains.

Fumonisin production on irradiated corn kernels: effect of inoculum size.

Production of fumonisins B1, B2, and B3 by Fusarium moniliforme was evaluated on irradiated corn kernels inoculated with different spore concentrations (10, 10(2), 10(3), 10(5), and 10(6)), a water activity of 0.97, and a temperature of 25 degrees C. There was a direct relationship between the level of toxin produced and inoculum size. The highest levels of total fumonisin produced after 35 days of incubation were 5,028 and 9,063 ng/g at 10(5) and 10(6) spores per ml, respectively. The pattern of fumonisin production (FB1 > FB2 > FB3) in cultures growing from different inocula was not affected during the 35 days of incubation. The ratio between FB2 and FB1 varied from 0.15 to 0.42, whereas the ratio between FB3 and FB1 varied from 0.34 to 0.87.