Molecular characterization of gliotoxin-producing Aspergillus fumigatus in dairy cattle feed

Abstract

Background and aim: Several strains of Aspergillus fumigatus produce mycotoxins that affect the health and productivity of dairy cattle, and their presence in dairy cattle feed is a serious concern. This study aimed to determine the densities of A. fumigatus and gliotoxin in commercial dairy feed.

Materials and methods: More than 60 dairy feed samples were examined for fungal contamination, specifically for A. fumigatus, using phenotypic approaches and DNA sequencing of the internal transcribed spacer (ITS) and β-tubulin regions. Thin-layer chromatography and high-performance liquid chromatography (HPLC) were used to assess gliotoxin production in A. fumigatus. Real-time polymerase chain reaction (RT-PCR) was used to investigate the expression of gliZ, which was responsible for gliotoxin production. High-performance liquid chromatography was used to detect gliotoxin in feed samples.

Results: Aspergillus was the most commonly identified genus (68.3%). Aspergillus fumigatus was isolated from 18.3% of dairy feed samples. Only four of the 11 A. fumigatus isolates yielded detectable gliotoxins by HPLC. In total, 7/11 (43.7%) feed samples tested had gliotoxin contamination above the threshold known to induce immunosuppressive and apoptotic effects in vitro. The HPLC-based classification of isolates as high, moderate, or non-producers of gliotoxin was confirmed by RT-PCR, and the evaluation of gliZ expression levels corroborated this classification.

Conclusion: The identification of A. fumigatus from animal feed greatly depended on ITS and β-tubulin sequencing. Significant concentrations of gliotoxin were found in dairy cattle feed, and its presence may affect dairy cow productivity and health. Furthermore, workers face contamination risks when handling and storing animal feed.

Keywords: Aspergillus fumigatus; cattle feed; gliZ; gliotoxin; high-performance liquid chromatography; real-time polymerase chain reaction.

Figure-1

Macroscopical features of Aspergillus fumigatus cultivated on different media; Dichloran rose Bengal chloramphenicol (a), Potato dextrose agar (b), and Sabouraud dextrose agar (c).

Figure-2

(a-c) Microscopical image of Aspergillus fumigatus isolates.

Figure-3

(a) The phylogenetic tree of the entire nucleotide sequence of ITS obtained from Aspergillus fumigatus strains isolated in this study compared with that of the reference strains obtained from GenBank. (b) Phylogram of β-tubulin showing the phylogenetic relationships among the Aspergillus fumigatus strains isolated in this study and strains belonging to section Fumigati and distinguishing the studied isolates from other Aspergillus spp.

Figure-4

The amplification profile of gliZ gene in Aspergillus fumigatus isolates. Lane (M): DNA ladder 100 bp, lane (P): Aspergillus fumigatus positive control, lanes (17–22): positive isolates, and lane (N) negative control.

Figure-5

The expression level of gliZ in the three studied Aspergillus fumigatus isolates. The expression levels of the three isolates showed ΔCT values ranging from 12.7 to −7.7. A low ΔCt value indicates a low expression level, and vice versa. Based on normalized values against β-tubulin expression, samples 20, 27, and 24 showed the highest, moderate, and lowest or almost no expression levels, respectively.

Supplementary Figure-1

Amplification plots of gliZ gene expression for three isolates Based on normalized values against β-tubulin expression, samples 20, 27, and 24 showed the highest, moderate, and lowest or almost no expression levels, respectively.