Molecular Characterization of Aspergillus flavus Strains Isolated from Animal Feeds

Abstract

Aflatoxin (AF)-producing fungi such as Aspergillus flavus commonly contaminate animal feeds, causing high economic losses. A. flavus is the most prevalent and produces AFB1, a potent mutagen, and carcinogen threatening human and animal health. Aspergillaceae is a large group of closely related fungi sharing number of morphological and genetic similarities that complicate the diagnosis of highly pathogenic strains. We used here morphological and molecular assays to characterize fungal isolates from animal feeds in Southwestern Algeria. These tools helped to identify 20 out of 30 Aspergillus strains, and 15 of them belonged to the Aspergillus section Flavi. Further analyses detected four out of 15 as belonging to Aspergillus flavus-parasiticus group. PCR targeting the AF genes' aflR-aflS(J) intergenic region amplified a single 674 bp amplicon in all four isolates. The amplicons were digested with a BglII endonuclease, and three specific fragments were observed for A. flavus but A. parasitucus lacked two typical fragments. Sequencing data of four amplicons confirmed the presence of the two BglII restriction sites yielding the three fragments, confirming that all four strains were A. flavus. In addition, this analysis illustrated the genetic variability within the A. flavus strains.

Keywords: Aspergillus flavus; IGS; PCR-RFLP; aflR-aflS(J); diagnosis tools.

Fig. 1

Phenotypic characterization of A. flavus isolates grown on A. flavus/parasiticus agar (AFPA) medium. Colony color: white-green colony diameter 18 mm, colony reverse color: yellowish orange after seven days of incubation at 25°C.

Fig. 2

PCR amplification of intergenic sequences of the four isolates. IGS-F and IGS-R primers were used for PCR amplification of the intergenic sequences in total DNA isolated from the four fungal isolates as described in Material and Methods. A total of 5 μl of each PCR product was separated in a 1.5% agarose gel by electrophoresis. Lane 1 kb ladder (L) DNA marker, Aspergillus flavus isolates are indicated on the top of each lane. The sizes of selected DNA fragments are indicated on the sides of the panels.

Fig. 3

PCR and RFLP analyses of fungal amplicons. A total of 5 μl of PCR products of each of the isolates (FZM1, FAK45, FSZ47 and FDY50) was digested with Bgl II restriction endonuclease as described in Materials and Methods. Nondigested (ND) and digested (D) products were loaded and separated by electrophoresis in a 2% agarose gel. A 100 bp DNA ladder (L) was used as molecular weight marker, the positions of 100, 500 and 1,000 bp fragments are indicated. Positions of the 362, 210 and 102 bp fragments generated by BglII digestion are indicated.

Fig. 4

Nucleotide sequence alignment of the four isolates with four reference strains. The names of the (.) four isolates are in green. Nucleotide sequence alignment was performed using Clustal software (–) represent similarity (.), represent deletion. Nucleotide changes are shown in bold font.

Fig. 5

Maximum Likelihood phylogenic tree showing the relationships between the examined A. flavus isolates and reference strains, based on aflR/aflS(J) intergenic sequence.