Lactic acid bacteria in the inhibition of Fusarium graminearum and deoxynivalenol detoxification.

AIMS: Considering the agronomic and industrial damage that is caused by the fungus Fusarium graminearum, as well as the serious health risks it poses to humans and animals exposed to F. graminearum-produced mycotoxin deoxynivalenol (DON), this study evaluated the ability of different lactic acid bacteria (LAB) strains to inhibit fungal development and remove DON in vitro. METHODS AND RESULTS: The antagonistic effects of strains and commercial cultures of LAB were evaluated against F. graminearum IAPAR 2218 by the agar diffusion method. Additionally, the influence of the culture media, pH and the presence of lactic and acetic acid on these effects was tested. The capacity to remove DON by viable cells and heat-inactivated cells was analysed in liquid media and quantified by high performance liquid chromatography (HPLC). All isolated strains and commercial cultures inhibited the fungus and removed DON. The pH and culture media concentration did not influence these abilities, but heat inactivation had a strong effect on the ability of bacteria to remove mycotoxin. CONCLUSIONS: The isolated bacteria are able to inhibit F. graminearum growth and remove DON in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests potential application of the isolated LAB strains in the inhibition of F. graminearum IAPAR 2218 and DON removal in vitro.

Immunoassay based on monoclonal antibodies versus LC-MS: deoxynivalenol in wheat and flour in Southern Brazil.

An indirect competitive enzyme-linked immunosorbent assay (ELISA) method using a monoclonal antibody for deoxynivalenol (DON) detection in wheat and flour was standardised and validated (detection limit = 177.1 µg kg(-1)) and its performance was compared with LC-MS, quantification limit =140 µg kg(-1)). DON recovery ranged from 88.7% to 122.6% for wheat grain and from 70.6% to 139.3% for flour. Among the 38 wheat samples evaluated, DON was detected in 29 samples (76.3%) by ic-ELISA (281.6-12 291.4 µg kg(-1)) and in 22 samples (57.9%) by LC-MS (155.3-9906.9 µg kg(-1)). The 0.93 correlation coefficient between ic-ELISA and LC-MS data in 19 positive DON wheat samples demonstrated the reliability and efficiency of ic-ELISA. Results indicated that standardised ic-ELISA was suitable for DON screening in wheat samples and the need for continuous monitoring of mycotoxin levels in foodstuffs.

Effect of the time interval from harvesting to the pre-drying step on natural fumonisin contamination in freshly harvested corn from the State of Parana, Brazil.

Natural mycoflora and fumonisins were analysed in 490 samples of freshly harvested corn (Zea mays L.) (2003 and 2004 crops) collected at three points in the producing chain from the Northern region of Parana State, Brazil, and correlated to the time interval between the harvesting and the pre-drying step. The two crops showed a similar profile concerning the fungal frequency, and Fusarium sp. was the prevalent genera (100%) for the sampling sites from both crops. Fumonisins were detected in all samples from the three points of the producing chain (2003 and 2004 crops). The levels ranged from 0.11 to 15.32 microg g(-1)in field samples, from 0.16 to 15.90 microg g(-1)in reception samples, and from 0.02 to 18.78 microg g(-1)in pre-drying samples (2003 crop). Samples from the 2004 crop showed lower contamination and fumonisin levels ranged from 0.07 to 4.78 microg g(-1)in field samples, from 0.03 to 4.09 microg g(-1)in reception samples, and from 0.11 to 11.21 microg g(-1)in pre-drying samples. The mean fumonisin level increased gradually from < or = 5.0 to 19.0 microg g(-1)as the time interval between the harvesting and the pre-drying step increased from 3.22 to 8.89 h (2003 crop). The same profile was observed for samples from the 2004 crop. Fumonisin levels and the time interval (rho = 0.96) showed positive correlation (p < or = 0.05), indicating that delay in the drying process can increase fumonisin levels.

Post-harvest storage of corn: effect of beginning moisture content on mycoflora and fumonisin contamination.

The effect of storage on mycoflora profile was monitored bimonthly in 36 corn (Zea mays L.) samples, dividing the same sample into groups dried to 11 and 14% moisture content (1008 analysis). These groups were further subdivided based on the initial total count (moulds and yeasts) up to 10(4) CFU g(-1) (12 samples, range 1.6 x 10(4) to 9.0 x 10(4), mean 3.8 x 10(4) CFU g(-1)) and up to 10(5) CFU g(-1) (24 samples, range 1.0 x 10(5) to 5.0 x 10(5), mean 2.7 x 10(5) CFU g(-1)). In the corn group dried to 11%, the fumonisin content was analysed at the initial stage (freshly harvested) and at the end of 12-month storage. Fusarium spp. and Penicillium spp. prevailed at the freshly harvested stage (100%), maintaining this profile throughout 12 months, in corn dried to both 11 and 14%. Cladosporium spp., Aspergillus spp. and Phoma spp. were also detected at lower frequencies during the storage. Fusarium spp. and the total fungal colony count during 12-month storage carried out with samples dried to 11 or 14% moisture content were statistically evaluated using ANOVA for randomized complete block design. The correlation between storage time and Fusarium spp. and total fungal colony count data was analysed by Pearson's correlation test. There was no difference in Fusarium spp. and total counts in the 10(4) CFU g(-1) initial total count group throughout the storage time (p < 0.05). There was a negative correlation between fungal population and storage time (p < 0.05) in the 10(5) CFU g(-1) initial total count group. Fumonisins were detected in all freshly harvested corn, at a mean concentration of 9.9 +/- 6.0 micro g g(-1) (range 0.74-22.6 micro g g(-)1). These values did not change in the 12-month stored corn (mean of 9.9 +/- 5.8 micro g g(-1), range 0.81-23.7 micro g g(-1)). These post harvest data indicated the importance of moisture content at the crop harvesting/predrying stage to control fungal growth and further fumonisin production.

Evaluation of fumonisin-aflatoxin co-occurrence in Brazilian corn hybrids by ELISA.

The natural co-occurrence of fumonisins and aflatoxins was investigated in freshly harvested corn kernels (150 samples, 62 hybrids), acquired from the Central-Southern (27 samples, 21 hybrids), Central-Western (86 samples, 51 hybrids) and Northern (37 samples, 18 hybrids) regions of the State of Paraná, Brazil using enzyme-linked immunosorbent assay (ELISA). Fumonisins were detected in 147 (98%) samples at a concentration range of 0.096 to 22.6 microg/g, while aflatoxins were detected in 17 (11.3%). All the aflatoxin-positive samples (range 38.0-460.0 ng/g) came from the Central-Western region and were co-contaminated with fumonisins. Fumonisin contamination was higher in corn from the Northern (9.85 microg/g) and Central-Western regions (5.08 microg/g), when compared with the Central-Southern region (1.14 microg/g). The overall evaluation detected 62% samples with fumonisin levels < or = 5.0 microg/g. Regional differences affected fumonisin levels in the same hybrid, regardless of Fusarium count and moisture content, suggesting interference from climatic conditions, in addition to the local predominance of toxigenic strains of the Fusarium biotype.

Immunohistochemistry of fumonisin in poultry using avidin-biotin-peroxidase system.

Using monoclonal anti-fumonisin B1 antibody (anti-FB1) and avidin-biotin-peroxidase system, liver and kidneys of broiler chicks were evaluated for the detection and distribution of fumonisins (FBs). One hundred and fifty micrograms of FB1 or culture extract of Fusarium moniliforme str. 113F containing 150 microg of FB1 and 4 microg of FB2 were administered into the vitelline sac of 1-day old, specific pathogen-free chicks. The animals were killed 24 h after injection, and renal and hepatic tissues submitted for immunohistochemical analysis. FBs were detected in the epithelial cells of convoluted distal and proximal tubules of the kidneys, as well as in the cytoplasm of hepatocytes. This novel immunohistochemical method developed is expected to be an efficient way for monitoring the target of the FB toxins in tissues.

Effect of climatic conditions on natural mycoflora and fumonisins in freshly harvested corn of the State of Paraná, Brazil.

Natural mycoflora associated with fumonisins were analyzed in 150 samples of freshly harvested corn from Central-Southern, Central-Western and Northern regions of the State of Paraná, Brazil and correlated to climatic conditions. The corn samples were frequently contaminated with Fusarium sp. (98.7 to 100%) and Penicillium sp. (93 to 100%), when compared to Aspergillus sp. (not detected to 27.7%). The highest contamination with potentially mycotoxigenic fungi occurred in corn harvested in the Central-Western region, where total mould and yeast counts ranged from 5.5 x 10(3) to 5.2 x 10(6) CFU/g, with 98.7% contaminated by Fusarium sp. and 93% by Penicillium sp. In this region F. moniliforme (F. verticillioides) was the predominant Fusarium sp., and was isolated in 85.9% of the samples. Aspergillus sp. was isolated from 27.7% samples. FB1 was detected in 100% of the samples (mean of 2.39 micrograms/g) and FB2 in 97.7% (mean of 1.09 micrograms/g). Fumonisins were also detected in all samples from Northern region, with mean of 4.56 micrograms/g (FB1) and 2.20 micrograms/g (FB2). Considering 1.0 microgram/g as the threshold, 72% of the corn samples from the Central-West and 92% from the North were contaminated with concentrations above this value, in contrast to a 18.5% contamination rate from Central-Southern samples. Between corn planting to harvesting season, the average maximum temperature and relative humidity were 26 degrees C and 77.1% (Central-Southern), 27 degrees C and 69% (Northern) and 29.9 degrees C and 89.1% (Central-Western). Therefore, the higher fumonisins contamination of corn from Northern region when compared to the Central-South were due to the differences in rainfall levels (92.8 mm in Central-Southern, 202 mm in Northern) during the month preceding harvest.

Survey of microcystins in water between 1995 and 1996 in Paraná, Brazil using ELISA.

An enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody was used to determine microcystin (MC) concentrations in water supplies and water plant samples collected between November 1995 and October 1996, from five regions of Paraná, Brazil. In addition, the presence of Microcystis sp. was monitored. Of the 50 samples obtained, 12 were from an urban lake, 8 from human water supplies, 10 from recreational lakes, 13 from farm waters used for animal pasture and 7 from aquaculture facilities. M. aeruginosa was positive in all locations. MCs were positive (>50 pg ml(-1)) in 9 samples (2 samples from human water supplies, 5 from recreational lakes and 2 from animal pasture). Heavy contamination with MCs was observed in water samples collected in May 1996 from 2 recreation (swimming-fishing sites at Itaipu dam, 6380 and 10,000 pg ml(-1)) and human supplies (6627 pg ml(-1)) samples. At these sites, a large bloom of Microcystis sp. was detected. Treatment with 1 ppm Cl- reduced MCs levels, although 267 pg ml(-1) remained in the water plant samples. Our data showed frequent occurrence of Microcystis sp., which may be a hazard to humans and animals in the state of Paraná. More detailed investigations are required to evaluate the risk of natural MC contamination in the water supplied in this region.

The natural occurrence of fumonisins in Brazilian corn kernels.

Since animal intoxication related to corn-based feed is frequently observed in the State of Paraná, Brazil, natural contamination by fumonisins in 48 corn samples (39 from the State of Paraná, and 9 from the Brazilian tropical states, Mato Grosso do Sul and Goias) harvested in 1990-1991 was investigated, along with fungal flora. The total mould count ranged from 6.3 x 10(2) to 5.5 x 10(7) cfu/g, and Fusarium moniliforme and Aspergillus species belonging to section Flavi were detected in 41 and 33 samples, respectively. Regarding the samples from the State of Paraná, F. moniliforme was present in 33 samples at a count of 1.0 x 10(2) to 1.6 x 10(7) cfu/g and Aspergillus spp. in section Flavi in 27 samples at 1.0 x 10(2) to 1.0 x 10(6) cfu/g. HPLC analysis of fumonisins in the corn showed that fumonisins B1 (FB1) and B2 (FB2) were positive for 97.4% and 94.8% of samples respectively. All the corn from North Paraná was positive for fumonisins, with average FB1 levels of 4.79 micrograms/g and average FB2 levels of 3.95 micrograms/g: the Central-West region had average levels of 3.30 and 2.52 micrograms/g, and the Central-East had average of 3.25 and 2.34 micrograms/g, respectively. Except for one negative sample all the corn samples from the Central Region were positive for fumonisins, averaging FB1 levels being 5.45 micrograms/g and FB2 levels being 5.09 micrograms/g. Out of eight samples from the tropical state of Mato Grosso do Sul, F. moniliforme was detected in seven and Aspergillus spp. in section Flavi in five samples with average FB1 levels of 10.59 micrograms/g and average for FB2 levels of 10.31 micrograms/g. The samples from Goias were also contaminated with these two fungi, with the FB1 contamination being 5.83 and the FB2 contamination 3.62 micrograms/g.

A limited survey of fumonisins in corn and corn-based products in Asian countries.

Natural occurrence of fumonisins B1 (FB1) and B2 (FB2), a promoter for hepato-carcinogenesis, was investigated in corn and corn - based products sampled in Japan, Nepal, and China by high - performance liquid chromatographic method. From the 9 imported corn kernel and 6 gluten feed samples, FB1 was detected in 8 corn (0.6 ∼ 4.1µg/g) and all gluten feed (0.3 ∼ 2.4µg/g) samples, while FB2 was found in the same corn (0.3 ∼ 10µg/g) and 3 gluten feed (0.8 ∼ 8.5µg/g) samples. ELISA analysis also revealed the contamination of aflatoxin B1 in 2 corn and all gluten feed samples along with fumonisins. Of 17 corn grit samples, 14 and 5 samples were contaminated with fumonisin B1 and B2, with maximum levels of 2.6 and 2.8µg/g, respectively. As for corn-based foodstuffs marketed in Japan, no significant contamination of fumonisins was observed. Among 24 corn kernel samples in Nepal, 12 and 7 samples were positive for FB1 and FB2, and averaged to 0.6 and 1.6µg/g, respectively. One sample showed the highest fumonisin contents as 4.6 and 5.5µg/g, respectively. In corn samples harvested at Shanghai and Beijing, China, FB1 and FB2 were detected in various concentrations. Mycological survey has also revealed the presence of a fumonisin - producing fungus in a crop field of Japan. These findings have for the first time demonstrated high levels of contamination of fumonisins in corn and corn - based products in Asian countries. Natural co - occurrence of fumonisins and aflatoxin B1 was also detected in raw materials for mixed feed.

Enterotoxigenicity of Staphylococcus intermedius of canine origin.

Seventy-three staphylococcal strains isolated from pyrodermatitis in dogs were classified as Staphylococcus intermedius (52 strains) or Staphylococcus aureus (21 strains) on the basis of acetoin formation, anaerobic mannitol fermentation, aerobic maltose fermentation, pigmentation, coagulation of human plasma, and reaction on crystal violet agar. Enterotoxin was produced by 13 of the 52 S. intermedius strains and 6 of the S. aureus strains. The highest percentage of enterotoxigenic strains produced enterotoxins C (6 strains), D (7 strains), and E (6 strains). Four strains produced the toxic shock syndrome toxin-1. There was little difference in the antibiotic susceptibility between the enterotoxigenic and non-enterotoxigenic strains.