Iranian Journal of Medical Microbiology

Isolation of Aspergillus Species
Collecting Rice Samples
In November 2015, 50 samples of rice husk from Baghmalek (23 samples of Champa), Shadegan (12 samples of Anbarboo) and Shooshtar (15 samples of Anbarboo) were collected through non-random convenience sampling. About 300 gr of rice husk was collected and transferred to the laboratory in sterile bags.

Isolation of Aspergillus Colonies from Rice Samples
One hundred rice seeds were randomly selected from each sample after washing and culturing on Sabouraud dextrose agar (SDA), based on colonial appearance and reproductive organs.

Identification of Aspergillus Strains Based on Morphological Traits
After 5 to 7 days of daily growth of the fungi and the type and shape of the colonies (Figure 1), Aspergillus fungi were identified by mycological and physiological methods according to John I. Pitt Diagnostic Key.
 Classification of this group based on morphological identification is a traditional way that is very difficult and can lead to misdiagnosis, especially for Aspergillus niger species, which are a group of morphologically indistinguishable species (8-10).

Identification of Aspergillus Strains Based on ITS Sequencing
Fungal Mycelium Production and DNA Extraction
The grown mycelium mass was collected in SDB medium, washed with sterile distilled water and then dehydrated. The mycelium mass was powdered in liquid nitrogen. DNA isolation was performed with the modification of Raeder & Broda (1985) (with minor modification) (11,12).
Mycelium powder was purified and protein degraded in three successive stages using phenol, chloroform, and isoamyl alcohol. The DNA was finally collected by ethanol precipitation and washed with 70% ethanol.

ITS Gene Amplification
The ITS1-F and ITS4-R primer pairs were used to amplify around 600 bp of ITS regions. The reaction was performed in a thermocycler (Biorad) with the following temperature program: 3 min at 95°C and then 35 cycles, 95°C for 30 sec., 52°C for 40 sec., 72°C for 1 min, and a final extension step at 72°C for 5 min.

Measurement and Determination of Mycotoxins Level and Type by HPLC
Sample Collection
The analysis of mycotoxins in imported cereal was carried on 50 samples out of 2750 samples referred from Imam Khomeini Port Customs in winter, spring, summer and autumn 2016. It should be noted that the basis for the determination and detection of mycotoxins by the HPLC method was the same in all cases according to the standards of the National Iranian Standards Organization.

Sample Preparation
Fifty grams of samples were weighed for testing mycotoxins according to Iranian National Standard No. 6872 “Human-Animal Feed, Measurement of Group B and G Aflatoxins”, No. 9238 “Cereals and its Products - Measurement of Ochratoxin A”, No. 9239 “Cereals and Products” Its weights - the zearalenone measurement”, and No. 9240 “doxylamine content determination”

Measuring the Amount and Type of Mycotoxins
The toxin was extracted from the samples by solvent extraction (methanol 80%). The extract was diluted with Watman filter paper to a certain concentration after being filtered through a sinter filter. Extracts obtained from the immunoaffinity column containing specific antibodies (ZearalaTest™, AflaTest™, OchraTest ™, DONTest ™) were passed at a drop per second rate and the antigen present in the extract was bound to specific antibodies in the column. Injection, isolation, detection, and determination of Mycotoxins was calculated by reversed-phase HPLC columns and derivative and fluorescence detector, through comparison of the standard substrate surface with an unknown specimen, taking into account the dilution factor in ng/g (HPLC device, KNAUER, Germany). In order to calculate the recovery rate of mycotoxin toxins with a concentration of 1.6 ppb in healthy specimens, spiked specimens were read and extracted in a similar manner. Recovery rates of mycotoxins were in the range of 0-6%. This range is acceptable by national standards and indicates that the extraction operations are well performed.

Investigation and Identification of Aspergillus Isolates from Rice
Of the 19 samples sequenced, 8 isolates (group I) were identified as Aspergillus flavus, 2 isolates (group II) as Aspergillus terreus, 1 isolate as Aspergillus nidulans (Group III), 3 isolates as Aspergillus tubingensis, 3 isolates as Aspergillus niger and 1 isolate as Aspergillus SP (Figures 2 and 3).
The ITS region sequences in the studied samples showed 99-100% similarity to the type strains of any gene in the NCBI database (Table 1).

Results of Mycotoxins Assay
The presence of mycotoxins in imported cereals was evaluated qualitatively and quantitatively. From winter to autumn 2016, the type and amount of mycotoxin were measured in 2750 samples of cereals referred to customs in three laboratories in Ahvaz. These included corn, wheat, and barley. The mycotoxins tested on these cereals were aflatoxins, zearalenone, deoxynivalenol, and ochratoxin, according to applicants.
The results of the mean cereal contamination of different mycotoxins and their comparison with the national standard of Iran (5925) are presented in Table 1, 2 and 3 respectively (one sample t-test) (Graph pad prism 8). The results of statistical analysis showed that the mean of mycotoxins in different grains had a significant difference with the maximum national standard of each mycotoxin (P<0.05)
Out of 2111 corn samples, 8.4% (689) were over contaminated (5 ppb) in AFB1 and 0.4 (9), 3.3 (2), and 0.1 (3) of samples were over contaminated in AFB2, AFG1, and AFG2, respectively.
The levels of ZEN, DON and OTA mycotoxins in 1139 corn samples were lower than the national standard and lower than 200 ppb, 1000 ppb, and 50 ppb, respectively. Of 166 wheat samples, 4.2% (1.2%) and 1.2% (2%) of samples were contaminated at concentrations below 5 ppb with AFB2 and AFG1, respectively. Also, 13.8% (16) of the samples were contaminated with OTA at less than 5 ppb.
None of the wheat samples showed contamination with DON, ZEN, AFB1, and AFG2. Of the 149 barley samples imported during the sampling interval of this study, only aflatoxins were detected in which no contamination was observed in any of the samples. ANOVA and LSD post hoc tests were used to compare the mean levels of mycotoxins in different months of the year (January to December 2015). According to the results of this test, there was a significant difference between the mean of other mycotoxins in different months of the year (P<0.05).
Of the 50 rice samples tested for aflatoxins, 16 were contaminated with aflatoxin B1, of which only 1 showed more than 5 ppb. Samples 2 and 4 were contaminated with AFB2 and AFG1, respectively, (<5 ppb). The results of the study of mean rice contamination with aflatoxins and comparison with the national standard of Iran are presented in Table 4 (one sample t-test).

Table 2. Comparison of mean mycotoxin contaminants of imported corn with the national standard of Iran in winter and spring 2016

Table 3. Comparison of mean mycotoxins of imported corn contaminants with the national standard of summer and autumn 2016

Table 4. Comparison of mean mycotoxins contaminated by imported wheat with Iranian national standard in spring 2016

Table 5. Comparison of the average Aflatoxins of Khuzestan rice production contaminants with the national standard of Iran

In this study, Aspergillus susceptible to toxin production was identified and isolated from rice seeds of Khuzestan province (Champa and Anbarboo). Results showed high percentages of Aspergillus flavus (42.1%) followed by Aspergillus niger, Aspergillus tubingensis, Aspergillus terreus, and Aspergillus nidulans with 21.5%, 15.7%, 10.52%, and 5.26%, respectively. The findings of Nyongesa et al. (2015), Riba et al. (2010) and Gao et al. (2007) showed that Aspergillus flavus was the main pollutant in oilseeds (pistachio, almond, hazelnut), corn, wheat, cereals, and beans, respectively. The results of the studies of Makun et al. (2007), Amadi et al. (2009) were also consistent with this study (13, 14, 15, 16, 17). Because of the saprophyte and widespread toxinogenic fungi such as Aspergillus, rice and cereal products have some relative contamination from the beginning.
The presence of spores and their propagation during transport and storage of grains or contamination of the storage environment with these fungi can lead to the spread of contamination. As Magnussen & Parsi (2013) found out, drought stress is one of the factors increasing the susceptibility of plants to Aspergillus and as a result of aflatoxin infection (18), the results of this study also suggest that the rate of these fungi can be increased with high heat conditions and increased storage time.
By stage of infection, mycotoxins are “field mycotoxins” produced mainly by Fusarium species and “Mycotoxins in storage”, which are produced by Aspergillus and Penicillium species in the pre-harvest period or immediately after harvest during storage (19,20).
The growth of fungi and the accumulation of mycotoxins in food and feed are influenced by various factors which in general relative humidity and temperature are critical factors in the drying and storage period (21). Findings from the study of the amount and type of cereal mycotoxins imported from Imam Khomeini port, mainly from South American countries, showed that the contaminations were not nearly acceptable by Iranian national standard, and corn samples showed the highest levels of mycotoxins, especially aflatoxins. The findings of a study by Mazaheri et al. (2018) also confirmed that corn is most susceptible to mycotoxins, especially aflatoxins (22). The findings of this study indicate that the maximum concentration of mycotoxins is in September and November. These results illustrate the impact of the duration of storage of grain in shipments or in warehouses. This is confirmed by Najafian et al. (2014) (25).
In a study, Ranjbar et al. (2010) showed that Aspergillus and Aflatoxin levels decrease in spring and summer and due to the use of stocked livestock feed and the lack of proper storage conditions, the rate of milk contamination with aflatoxin M1 increases in the autumn and winter. They stated that inappropriate storage, in addition to contaminating livestock feed with Aspergillus and subsequent aflatoxin production, also transmitted it to packaged milk (26).
Cano-Sancho et al. (2013) also stated that the presence of mycotoxins in food depends on many conditions such as season, weather (temperature, humidity), target area, harvesting method, storage and processing (27). Lahouar et al. (2015) studied the effect of temperature and incubation time on the growth rate of Aspergillus flavus and AFB1 production on sorghum seeds, and reported that the flavus isolate from Tanzanian sorghum has the ability to grow over a wide range of temperatures (15-37°C), however, aflatoxin production occurs at a lower temperature range (25-37°C) (28). Ghali et al. (2010) showed that the highest aflatoxin production occurred at 24°C but the highest flavus strain growth occurred at -35°C (19).
In this study, the presence of Aspergillus did not indicate the presence of aflatoxins and, conversely, the presence of aflatoxins was not a reason for the presence of Aspergillus.
 
 

Given the widespread presence of mycotoxins in foods and their harms, especially AFB1, which is a contaminant in food products, control of AFB1 contamination in foodstuffs requires monitoring different factors such as raw materials and food supply, food processing, finished products, and storage.

The authors would like to express their gratitude to the Research Deputy of Khuzestan Agricultural Research Center and Parham Jonoub Company for their cooperation with this study.

Authors declared no conflict of interests.