Molecular characterization of ochratoxigenic fungi associated with poultry feedstuffs in Saudi Arabia

Abstract

Fungal and mycotoxins contamination of food and poultry feeds can occur at each step along the chain from grain production, storage, and processing. A total of 200 samples comprising of mixed poultry feedstuffs (n = 100) and their ingredients (n = 100) were collected from Riyadh, Alhassa, Qassium, and Jeddah cities in Saudi Arabia. These samples were screened for contamination by fungi. Penicillium chrysogenum was the predominant species taking into its account and frequency, respectively, in both mixed poultry feedstuff and barley samples (4,561.9 and 687 fungal colony-forming units (CFU)/g) and (66% and 17%). Moisture content was an important indicator for the count of fungi and ochratoxin A. Ochratoxin analysis of plate cultures was performed by a HPLC technique. Sample of mixed poultry feedstuff which was collected from Jeddah displayed the highest level of ochratoxin (14.8 µg/kg) and moisture content (11.5%). Corn grains samples were highly contaminated by ochratoxin A (450 and 423 µg/kg) and recorded the highest moisture contents (14.1 and 14.5%). Ochratoxin A production in fungal species isolated from mixed poultry feedstuff samples were high with P. verrucosum (5.5 μg/kg) and A. niger (1.1 μg/kg). In sorghum and corn grains, the highest ochratoxins producing species were P. viridicatum (5.9 μg/kg) and A. niger (1.3 μg/kg), respectively. Sixty-three isolates of A. niger were ochratoxigenic, and all of them showed the presence of pks genes using PKS15C-MeT and PKS15KS primer pairs. The detection technique of A. niger in poultry feedstuff samples described in the present study was successfully used as a rapid and specific protocol for early detection of A. niger without cultivation on specific media.

Keywords: Aspergillus; Penicillium; Poultry; genes; ochratoxin A.

Figure 1

Heatmap of CFU, calculated per g dry feedstuff sample of fungal species were isolated from samples of mixed (a) and ingredients (b) of poultry feedstuff (n = 100 of each), which were collected from Riyadh, Alhassa, Qassium, and Jeddah cities on DRBC media at 27°C

Figure 2

Number of cases of isolation (NCI) of fungal species which were isolated from samples of mixed (a) and ingredients (b) poultry feedstuff (n = 100 of each) were gathered from Riyadh, Alhassa, Qassium, and Jeddah cities on DRBC media at 27°C

Figure 3

Association between OTA production capability of each strain and presence of pks genes, as demonstrated by PCR performed with two primer pairs, PKS15KS (a) and PKS15C‐MeT (b). PCR detected a 776 bp band and a 998 bp band, respectively, in the ochratoxin producing strains only. The 554 bp‐amplicon corresponding to the βt2 region of the β‐tubulin gene confirms the presence of PCR‐compatible DNA in all strains

Figure 4

Agarose gel electrophoresis of PCR products of DNA fragments specific for Aspergillus niger using primer pairs ITS1 and NIG. Lane 1: Positive control; Lanes 2–5: DNA from samples collected from Riyadh, Alhassa, Qassium, and Jeddah. Lane 6 sample (free Aspergillus niger) and lane 7 negative control. M: DNA marker