Occurrence of Fusaproliferin and Beauvericin inFusarium-Contaminated Livestock Feed in Iowa

Sample collection and fungal strain characterization.The samples used in this study were maize or feed specimens submitted by field veterinarians to the Iowa State University Veterinary Diagnostic Laboratory (ISUVDL), Ames, for mycotoxin analysis. We included only samples that had no detectable deoxynivalenol. Fusariumstrains were isolated from ground maize and feed samples by culturing 0.5-g subsamples on Nash-Snyder medium (22). A single spore of each putative Fusarium colony was transferred to carnation leaf agar for identification according to the criteria and synoptic keys of Nelson et al. (22). One or more strains from seven of the samples were chosen arbitrarily for further characterization. Chosen strains represented most of theFusarium species isolated from each sample. The mycelium and conidia from each strain were frozen in sterile 18% glycerol-water and stored at −75°C in the Istituto Tossine e Micotossine da Parassiti Vegetali (ITEM) culture collection at Bari, Italy.

Fusarium strains identified as F. moniliforme, F. proliferatum, or F. subglutinans were characterized in terms of their G. fujikuroi mating populations. Tester strains for mating populations were obtained from J. F. Leslie, Kansas State University (9). Strains in the present study were crossed on carrot agar as male parents with tester strains of mating populations A through F as described by Klittich and Leslie (8). Mated cultures were considered interfertile if perithecia formed within 6 weeks. All strains were crossed twice with both testers from each mating population.

In vitro mycotoxin production.Single-conidium isolates ofF. moniliforme, F. proliferatum, andF. subglutinans were cultured on 100 g of autoclaved yellow maize kernels that were adjusted to about 45% moisture in 500-ml Erlenmeyer flasks and inoculated with 2 ml of an aqueous suspension containing approximately 10⁷ conidia/ml. Cultures were incubated at 25°C for 4 weeks. The harvested culture material was dried in a forced draft oven at 60°C for 48 h, finely ground, and stored at 4°C until use. Controls were treated the same way, except that they were not inoculated.

Mycotoxin analyses.Analyses for deoxynivalenol were performed at ISUVDL. Analyses for fumonisins, beauvericin, and fusaproliferin were performed at ITEM and at the Dipartimento di Scienze degli Alimenti, Portici, Italy.

For beauvericin and fusaproliferin extraction, 10 g of each sample was ground and homogenized in a Waring blender for 5 min with 15 ml of methanol (99.5%). Samples were filtered through Whatman no. 4 filter paper, and methanol was removed under reduced pressure. This extraction procedure yielded 150 mg of raw organic extract that was used to quantify beauvericin and fusaproliferin. An aliquot of 100 μl of methanol extracts, corresponding to 100 mg of raw material, was filtered through an Acrodisk filter (pore size, 0.22 μm) before the high-performance liquid chromatography (HPLC) injection (20 μl, corresponding to 20 mg of culture).

For fumonisin B₁ extraction, the procedure of Shephard et al. (31), partially modified as follows, was adopted. Extracts were prepared by shaking 10 g of each sample in 100 ml of methanol-water (3:1 [vol/vol]) for 1 h, and the extracts were then filtered through rapid-flow Whatman no. 4 filter paper. A 10-ml aliquot of the extract was applied to a Bond Elut strong anion exchange cartridge, and fumonisin B₁ was eluted from the column by a 99.5:0.5 (vol/vol) chloroform (99%)–acetic acid (100%) solution (2). The dried residue was dissolved in 100 μl of methanol and stored at 4°C.

Standards of fumonisins and beauvericin were purchased from Sigma Chemical Co., St. Louis, Mo. The fusaproliferin standard was isolated in the laboratory of the Dipartimento di Scienze degli Alimenti from maize kernels inoculated with an F. proliferatum strain (26). The contaminated natural samples were examined for fumonisins B₁ and B₂ by liquid chromatography (LC)-mass spectrometry (MS). A benchtop API 100 (Perkin-Elmer Sciex) single-quadrupole MS equipped with an atmospheric pressure ionization source and ionspray interface was used for the MS analyses. All quantitative results were performed in positive-ion mode with the orifice voltage set at 40 V. The acquired data were processed by using Multiview and MacQuan software (Perkin-Elmer Sciex). The resolution was set at 0.5 atomic mass unit (measured at half height), and the mass calibration and resolution adjustments on the resolving quadrupole were performed in ionspray by using a PPG 10-4 solution introduced via a model 22 Harvard infusion pump. Initial MS spectra were collected in continuous-flow mode by infusion directly to the ionspray probe. A standard mixture of fumonisin B₁ and fumonisin B₂ was infused at 10 μl/min. All other experiments were recorded by using a Perkin-Elmer LC-200 pump fully controlled from the API 100 data system.

The LC-MS analyses were performed by using a Brownlee C₁₈3- by 30-mm HPLC column (Perkin-Elmer). Column flow was 0.8 ml/min, and an aqueous eluent of 5 mM ammonium acetate acidified with 1% formic acid containing 80% methanol was used. Only 40 μl/min was delivered into the ionspray source. Standards of fumonisins were purchased from Sigma, and a stock solution was made in methanol (1 mg/ml). Successive dilutions were made by using a 50% aqueous solution of methanol containing 2 mM ammonium acetate and 0.1% formic acid. The quantification was performed by using the protonated signal atm/z 722 and 706 for fumonisins B₁ and B₂, respectively.

The detection limit determined for the fumonisin B₁standard was estimated to be equivalent to 0.5 ppb, and the limit of quantification was equivalent to 1.2 ppb. The detection limit determined for the fumonisin B₂ standard at m/z706 was estimated to be equivalent to 4.5 ppb, and the limit of quantification was equivalent to 9.0 ppb.

Analysis of fumonisin B₁ in culture extracts was performed by comparing the extracts (spotted from 1 to 20 μl) with pure fumonisin B₁ (0.1, 0.5, 1, and 2 μg of standard) by high-performance thin-layer chromatography (HPTLC) on precoated silica gel 60 F₂₅₄ plates (10 by 20 cm; thickness, 0.25 mm; E. Merck, Darmstadt, Germany) with two different solvent systems: chloroform-methanol-water-acetic acid (55:36:8:1 [vol/vol/vol/vol]) and chloroform-methanol (60:40 [vol/vol]). A detection limit of 2.5 μg/g was obtained for fumonisin B₁ by using the extraction procedure described above and the HPTLC analysis.

The amounts of beauvericin and fusaproliferin in feed samples and culture material were determined by HPLC. Samples (10 g) were extracted with 15 ml of methanol in a Waring blender. The raw extract obtained from each sample, after the removal of solvent, was redissolved to obtain a constant concentration of 1 g of raw material/ml of solvent. Then all the samples, feed and culture, were filtered through an Acrodisk filter and analyzed by HPLC. In this way each injection of 20 μl corresponded to 20 mg of contaminated starting sample.

For fusaproliferin, HPLC analysis was carried out with LC10AD pumps and an SPO-M10A diode array detector (both from Shimadzu) equipped with a Shiseido Capcell Pak C₁₈ column (250 by 4.6 mm; 5 μm). The HPLC system was set up with a flow rate of 1.0 ml/min and with a CH₃CN–H₂O (65:35 [vol/vol]) eluent system. The retention time of the authentic standard of fusaproliferin was 4.11 min. Quantification by HPLC procedures was carried out by comparison of the peak areas of the investigated samples with the calibration curve of the authentic standard. There was a linear correlation between the sample concentration and the areas of the peaks in the HPLC profile from 0.005 to 14 μg of fusaproliferin.

The HPLC configuration for beauvericin included CH₃CN–H₂O (85:15 [vol/vol]) as the eluent system with a flow rate of 1.0 ml/min. The wavelengths used to quantify beauvericin and fusaproliferin were 205 and 261 nm, respectively. There was a linear correlation between the sample concentration and the areas of the peaks in the HPLC profile from 0.02 to 5 μg of beauvericin. The retention time of the authentic standard of beauvericin was 5.7 min. As with fusaproliferin, quantification of beauvericin by HPLC procedures was done by comparison of the peak areas of the investigated samples with the calibration curve of the authentic standard. To confirm mycotoxin occurrence, the standards were spiked with extracts and analyzed by HPLC. The detection limit was 0.1 μg/g for beauvericin and 0.025 μg/g for fusaproliferin. To confirm beauvericin occurrence, the positive natural samples were analyzed by¹H nuclear magnetic resonance spectra and by low-resolution electronic impact mass spectrometry (m/z 784) (17). All analyses were run in triplicate, and the mean values are reported. The calculated standard deviation was always less than 5%.