Evaluating the rate of aflatoxin contamination in livestock feed and milk and molecular indentification of their fungal strains in some dairy farms of East Azerbaijan provice

Aflatoxins are a large group of mycotoxins that are produced through Polyketide pathway by specific species of Aspergillus flavus</em>, Aspergillus parasiticus,</em> and  Aspergillus nomius</em>. These pesticides are known to be the most dangerous mycotoxins affecting human and livestock health (Pleadin et al. 2014). Several hundred mycotoxins have been identified, and more than 25% of the world annual grain production is contaminated with mycotoxin (Smith et al. 2016). Among the 400 known mycotoxins, Aflatoxin B1, B2, G1, and G2 are the most important food and feed mycotoxins (Costanzo et al. 2015). Aflatoxin M1 and M2 are the hydroxyl metabolite of aflatoxin B1 and B2 that can be found in milk or other animal products (Hussein and Brasel. 2001). At the first level, the main manifestations of mycotoxins exposure in animals are reductions in feed intake and weight gain. At the second level, mycotoxins affect the quantity of animal products. The third level of influence is the safety and quality of the products from exposed animals (Wang et al. 2019). The present study was designed to detect contamination of aflatoxin M1 in milkand, aflatoxin B1 in feed and subsequent molecular isolation and identification of Aspergillus flavus</em> species.

In this study, 10 milk samples from milk reservoirs and 10 feed samples from a total mixed ration of livestock from dairy farms of East Azarbaijan province (Tabriz and Marand) were collected. After preparation of samples, the experiment was conducted using competitive ELISA method. The principles were as follows: after adding standard solutions or samples to the wells, aflatoxin M1 was bonded from specimens or standards to specific antibody binding sites. After 30 minutes of  incubation step, unbound reagents were removed during a single wash step. Horseradish peroxidase (HRP) aflatoxin M1 was added to the wells and after one 15 minutes of incubation, the unlinked conjugate was removed during the washing step. Then, some aflatoxin M1-HRP was coherent by adding a substrate/chromogen (H2</sub>O2</sub>/TMB) solution. In the presence of colorless chromogen, mixed conjugated aflatoxin and M1-HRP agent was converted to colored product. The addition of sulfuric acid caused the suspension of the substrate reaction and finally, the light intensity was measured by a photometric method at 450 nm. Optical density had an inverse relationship with the concentration of aflatoxin in the sample. To isolate the fungus, first 2 g of the standardized feed  were weighed and milled  in  a falcon containing 18 ml of physiological serum and then, mixed well with a vortex for five minutes. A portion of the diluted feed was removed and cultured on plots containing a Potato Dextrose Agar medium at several locations. Plates were incubated for 7 days at 25 ℃. DNA was extracted from Potato Dextrose Broth (PDB) medium. The resulting mycelium mass was frozen and converted to a uniform powder by liquid nitrogen. DNA extraction was carried out by placing the samples in a buffer and purification with organic solvents such as chloroform/isoamyl alcohol and finally, curing with cold isopropanol. The resulting DNA was stored at -20 ° C. In order to evaluate the actuary of identification for Aspergillus flavus, </em>the primer sequence of AFLA-F and AFLA-R gene was aligned using the BLAST software (GenBank) to find similarity rate within resisted reference sequences. Each PCR reaction consists of: 6 μl of PCR Master Mix, 2 μl extracted DNA, 0.2 μl of each recipe primer, 1.6 μl of distilled water. Then, 10 μl of the final volume of reaction was placed on thermosecler device. A PCR program for amplification of the targeted PCR fragment was fixed based on following temperature: Initial denaturant at 95℃ for 10 min, {denaturant at 95℃ for 1 min, annealing at 66℃ for 2 min, extension at 72℃ for 2 min (total 34 cycle)} and the final amplification at 72℃ for 5 minutes (Zachová et al. </em>2003). Isolated strains of Aspergillus</em> strains were verified using the PCR method; its reaction products were detected in 1% agarose gel by electrophoresis.

The results showed that all milk and feed samples were contaminated with aflatoxin, but the contamination rate of milk samples was lower than the standard values of Iran, America, and the European Union (0.1, 0.5 and 0.05 μg / Kg). Among the 10 collected samples, only two edible samples with aflatoxin B1 contamination were higher than the Iranian and European standards (5 μg / kg). The contamination level of milk and feed samples were observed in the range of 0.021-0.05 and 1.1-6.9 μg / kg, respectively. Statistically, there was a significant difference between the mean of contamination of aflatoxin M1  in milk and B1 in feed in the region with national and international standards and the mean of M1 and B1 contamination was lower than these standards. The level of aflatoxin M1 in milk was detected by HPLC method, indicating that the infection rate of 10 samples was 0.02-0.31 μg / l (Besufekad et al. 2018). In another study, 178 wheat samples were collected in China and reported 18.8% of the samples contaminated with aflatoxin B1 (Liu et al. 2016). The results of the fungal species also showed that the analyzed samples did not show any bands in the 413bp range. As a result, it can be said that the dominant species and the main cause of contamination were not Flavus</em> species. Wang et al. (2016) reported that aflatoxins are mainly produced by the genus Aspergillus</em>, and are commonly found in food and feed in humid and warm environments. Research results in India show that among the 15 collected samples, only 9 samples (60%) were infected with Aspergillus</em>. Seven samples were detected as  Aspergillus flavus</em> and two samples as  Aspergillus niger</em> (Khare et al. 2018).

 Milk composition, body mass gain, immunity, and reproductive performance are affected in dairy ruminants by feeds contaminated with aflatoxins. It is expected that by controlling animal feed agaisnt aflatoxin and reducing its levels in feed by improving production and storage conditions, a suitable method for preventing contamination of milk and its products will be adopted to help improve the health of the community.