Prevalence and detection of toxigenic a. flavus, a .niger and a. ochraceus by traditional and molecular biology methods in feeds

A total of one hundred feed samples were collected from Cairo and Giza governorates and screened for aflatoxigenic and ochratoxigenic fungal isolates. A total of 106 fungal isolates comprising, Aspergillus flavus, A. ochraceus and A. niger wererecovered from feed samples and tested for aflatoxins and ochratoxin A (OTA) production. The most predominant isolate was A. flavus, which was recovered at the range of (40-55%), followed by A. niger (30-50 %) and A. ochraceus (15-20%). Thirty three of 47 A. flavus isolates produced aflatoxin B1 and B2 at average levels of (170-750 ppb), while, 22 of 44 tested isolates of A. niger produced OTA with average levels of (100-550 ppb), whereas, 12 of 15 A. ochraceus isolatesproduced OTA at average levels of (300-700 ppb). Molecular identification of 16 toxigenic fungal isolates (5 A. flavus, 6 A. ochraceus and 5 A. niger) was carried out by PCR. The results of PCR of the DNA extracted from these isolates using ITS primer confirmed the identification of A. flavus, A. ochraceous and A. niger. The application of real-time PCR (RT-PCR) system directed against the nor-1 gene of the aflatoxins biosynthetic pathway was applied on the DNA extracted from the 5 selected strains of A. flavus. The amplification plot of the DNA samples indicated the presence of nor-1 gene in all aflatoxins-producing A. flavus isolates and in only one isolate of the negative aflatoxins-producing A. flavus. On the other hand, the use of primer set for amplification of omtB gene responsible for AF production amplified 611 bp fragments bands in all aflatoxigenic A. flavus, while, no band was detected in all negative aflatoxinogenic isolates. All the sequenced A. flavus isolates were confirmed to belong to A. flavus species and were 100% similar to the reference isolates of A. flavus. The application of PCR assays for detection of ochratoxigenic fungi using OCRA1/OCRA2 primers, amplified a single fragment of about 400 bp, when genomic DNA from ochratoxigenic A. ochraceus and non- ochratoxigenic A. ochraceus isolates were tested. No product was observed with genomic DNA from A. niger isolates. When, PCR assayswas applied using a pair of primers (Aopks1/Aopks2) for specific detection of ochratoxigenic fungi by targeting the metabolic pathway genes Polyketide Synthase (pks) specific to ochratoxin, a single fragment of about 549 bp was produced with all positive ochratoxigenic A. ochraceus isolates and 2 ochratoxigenic A. niger isolates. No product was observed with genomic DNA from all negative ochratoxigenic isolates of A. ochraceus and A. niger. Moreover, the combination of biochemical and molecular methods is needed to correctly evaluate the potential toxicological risk in feed caused by these fungi. Conclusion: the application of molecular biology technique was found to be rapid, highly specific, easy to perform and cost effective method to assist creation the programs used for reducing the risk of harmful effects of toxigenic fungi and their toxins to human and other farm animal's health.