Assessment of the Potential of a Native Non-Aflatoxigenic Aspergillus flavus Isolate to Reduce Aflatoxin Contamination in Dairy Feed

Abstract

Aspergillus species can produce aflatoxins (AFs), which can severely affect human and animal health. The objective was to evaluate the efficacy of reducing AF contamination of a non-aflatoxigenic isolate of A. flavus experimentally coinoculated with different aflatoxigenic strains in whole plant (WP), corn silage (CS), immature grains (IG) and in culture media (CM). An L-morphotype of A. flavus (CS1) was obtained from CS in a dairy farm located in the Mexican Highland Plateau; The CS1 failed to amplify the AFs biosynthetic pathway regulatory gene (aflR). Monosporic CS1 isolates were coinoculated in WP, CS, IG and CM, together with A. flavus strains with known aflatoxigenic capacity (originating from Cuautitlán and Tamaulipas, Mexico), and native isolates from concentrate feed (CF1, CF2 and CF3) and CS (CS2, CS3). AF production was evaluated by HPLC and fungal growth rate was measured on culture media. The positive control strains and those isolated from CF produced a large average amount of AFs (15,622 ± 3952 and 12,189 ± 3311 µg/kg), whereas A. flavus strains obtained from CS produced a lower AF concentration (126 ± 25.9 µg/kg). CS1 was efficient (p < 0.01) in decreasing AF concentrations when coinoculated together with CF, CS and aflatoxigenic positive control strains (71.6−88.7, 51.0−51.1 and 63.1−71.5%) on WP, CS, IG and CM substrates (73.9−78.2, 65.1−73.7, 63.8−68.4 and 57.4−67.6%). The results suggest that the non-aflatoxigenic isolate can be an effective tool to reduce AF contamination in feed and to minimize the presence of its metabolites in raw milk and dairy products intended for human nutrition.

Keywords: Mexico; aflatoxin biocontrol agents; biological control; dairy cows; mycotoxins.

Figure 1

Interaction between A. flavus strain isolated from corn silage 1 (CS1) and the aflatoxigenic strains Cuautitlán (C) and Tamaulipas (T). (A) Whole plant; (B) corn silage; (C) immature grain; (D) culture media. A negative control (sham without inoculum) and the non-aflatoxigenic strain AF-36 are included; ^a–e Different literals indicate significant statistical differences (p < 0.05) among means of aflatoxins (AFs) concentration per strain.

Figure 2

Interaction between A. flavus strain from corn silage 1 (CS1) and the aflatoxigenic strains isolated from concentrate feed (CF1, CF2, CF3). (A) Whole plant; (B) corn silage; (C) immature grain; (D) culture media. A negative control (sham without inoculum) and the non-aflatoxigenic strain AF-36 are included; ^a–e Different literals indicate significant statistical differences (p < 0.05) among means of aflatoxins (AFs) concentration per strain.

Figure 3

Interaction between A. flavus strain from corn silage 1 (CS1) and the aflatoxigenic strains isolated from corn silage (CS1, CS2). (A) Whole plant; (B) corn silage; (C) immature grain; (D) culture media. A negative control (sham without inoculum) and the non-aflatoxigenic strain AF-36 are included; ^a–e Different literals indicate significant statistical differences (p < 0.05) among means of aflatoxins (AFs) concentration per strain.

Figure 4

Interaction between nontoxigenic A. flavus strain isolated from corn silage 1 (CS1) and aflatoxigenic A. flavus strains in culture medium (5 days, coconut agar medium). Aflatoxigenic strains on the right of each image: (A) Cuautitlán; (B) Tamaulipas; (C) concentrate feed 1; (D) concentrate feed 3; (E) corn silage 2; (F) corn silage 3.

Figure 5

Gel electrophoretic analysis of PCR products using DNA obtained from Aspergillus flavus strains isolated from concentrate feed (CF), corn silage (CS) and control strains. (A) Internal transcribed spacer region; (B) calmodulin gene; (C) aflatoxins biosynthetic pathway regulator gene. Lanes: M: DNA molecular size markers (ladder in base pairs); 1: AF-36 (nontoxigenic control); 2: Cuautitlán (toxigenic control); 3: Tamaulipas (toxigenic control); 4: CF1; 5: CF2; 6: CF3; 7: CS1; 8: CS2; 9: CS3.

Figure 6

Design of interaction model between nontoxigenic A. flavus strain isolated from corn silage 1 (CS1) and toxigenic A. flavus strains on different substrates. (A) Whole plant (inoculation of the strains inside corn ear); (B) microsilage (application of vacuum on ground plant material inocu-lated with A. flavus strains); (C) immature grain (sham and Tamaulipas strains).