Detection of Aflatoxigenic Fungi in Poultry Feed

Mycotoxins are poisonous biomolecules produced as secondary metabolites by some fungal species, as they grow on various substrates under suitable growth conditions. Approximately, 83% of these deaths occur in East Asia and sub-Saharan Africa. It has been estimated that more than five billion people in developing countries are at risk of chronic exposure to aflatoxins, through contaminated foods/feeds Several studies revealed that A. flavus and A. parasiticus are of significant concern in poultry contamination, being the most common producers of aflatoxins. In the current study, a total of 120 samples of poultry feeds were collected from different localities of Beni-Suef Governorate. The samples were examined for the existence of Aspergillus species. Moreover, the capacity to produce aflatoxins by the Aspergillus flavus was determined. The results revealed that the most predominant Aspergillus isolates was A. flavus (n= 75; 62.5%) followed by A. niger (n= 31; 25.9%) and A. fumigatus (n= 19; 15.8%). Out of 75 A. flavus isolates, 43 strains (57.3%) produced aflatoxins. These results concluded the potential exists for the production of mycotoxins by the Aspergillus flavus. The present study was designed to investigate the existence of Aspergillus species in poultry feed as well as determining the capacity of Aspergillus flavus isolates to produce aflatoxins. ـــــــــــــــــــــــــــــــــــــــــ


INTRODUCTION
Mycotoxins are poisonous biomolecules that are produced as secondary metabolites by some fungal species, as they grow on various substrates under suitable growth conditions (Tola and Kebede, 2016).
Several mold fungi are capable of contaminating various foods and feed with these toxic secondary metabolites, which have adverse effects on human and animal consumers following consumption of these contaminated food or animal feed (WHO, 2006; Maciorowski et al., 2007;Mostafa et al., 2012).
Moreover, Liu and Wu, (2010); Salim et al., (2011) added that the mycotoxins are responsible for many acute and chronic diseases in humans and animals such as; liver damage, esophageal cancer, reduced digestive enzyme activity, immune suppression, and various effects on children including stunted growth with many annual mortality cases. In addition to causing diseases, mycotoxigenic fungi and mycotoxins affect feed quality by reducing their nutritive value and producing an unpleasant smell.
They also affect poultry performance and health, leading to severe economic losses (Monson et al., 2014).
Several studies revealed that A. flavus and A. parasiticus are of significant concern in poultry contamination, being the most common producers of aflatoxins ( Generally, aflatoxins are the most common and most toxic primary mycotoxins of concern in poultry feedstuffs. Aflatoxins are composed of several types such as; Aflatoxin B1 (AFB1), Aflatoxin B2 (AFB2), Aflatoxin G1 (AFG1), and Aflatoxin G2 (AFG2). Which are the most commonly encountered (Monbaliu et al., 2010;Lereau et al., 2012). Of these aflatoxins, AFB1 is the most toxic type of aflatoxins and is the most commonly encountered natural carcinogens produced by these Aspergilli (Habib et al., 2015;Haruna et al., 2017).
Mycotoxins metabolized in the body into its hydroxylated form called Aflatoxin M1 (AFM1), which is excreted in the breast milk of humans and animals following the ingestion of contaminated food or feed with AFB1. (Xu et al., 2000). The most chronic form of aflatoxin exposure manifestation is hepatocellular carcinoma (HCC, or liver cancer), which has been described by WHO as the third-leading cause of cancer death globally (WHO, 2008), and with about 550,000 to 600,000 reported new cases annually (Habib et al., 2015). Approximately, 83% of these deaths occur in East Asia and sub-Saharan Africa. It has been estimated that more than five billion people in developing countries are at risk of chronic exposure to aflatoxins, through contaminated foods/feeds (Habib et al., 2015).
The present study was designed to investigate the existence of Aspergillus species in poultry feed as well as determining the capacity of Aspergillus flavus isolates to produce aflatoxins.

Samples:
A total of 120 samples of poultry feeds (90 Poultry ration, 10 Ration concentrates, 10 Yellow corn and 10 Soya bean) were collected from different farms in Beni-Suef Governorate that had a health problem in their flock. The samples were obtained from the feed at the time of the problem. The samples were representative of a lot of feed and included feed from the trough. The samples were examined for fungal contamination and toxin production.

Fungal Isolation:
The dilution of samples was carried out according to Dalcero et al., (1998). Approximately 10 g of each sample were diluted in 90 ml sterile distilled water, serial dilutions were done which 1 ml was transferred to a tube containing 9 ml sterile distilled water (10-fold serial dilutions). The tube was shaken and one ml was removed to a second tube till the 4 th dilution (10 -4 ). Then 0.1 ml is removed from each dilution into a sterile petri dish containing Sabouraud dextrose agar (SDA) with chloramphenicol (0.05g/l) using spread method and incubated at 25-28 o C for 7-10 days, all process was done under complete aseptic condition. After incubation, the plates examined visually and microscopically by making films. Individual suspected colonies were selected depending up on their morphological characters. Stock cultures were made from each isolate and maintained in SDA slopes in refrigerator for further identification.

Identification of fungal isolates:
The recovered Fungal mycelia were identified morphologically according to Rippon (1988) by examination of mycelial morphology, the reverse colour as well as the examination of colonial smears using lactophenol cotton blue stain.

5'-CCTGAGCCAGTCCGAAGGCC-3'
The reaction was performed in a volume of 25 µl consisting of 12.5 µl of 10X PCR master mix, 1µl of each 20 pmol primers, 5µl of DNA extract, and the volume was completed to 25 µl using sterile deionized water. The thermal profile consisted of primary denaturation at 94˚C for 5 min., 35 cycles of amplification; denaturation at 94˚C for 30 sec., primer annealing at 50˚C for 40 sec., and extension at 72˚C for 40 sec. followed by a final extension step at 72˚C for 10 min. PCR products were visualized using 1% agarose stained with ethidium bromide using a UV transilluminator.

Methods of DNA Extraction: Extraction of DNA:
According to QIAamp DNeasy Plant Mini kit instructions 1. 100 mg of the tissue were frozen in -80°C for 24 hrs for later processing. . 2. Fungal material and a tungsten carbide bead were added to a 2 ml safe-lock tube. 400 μl Buffer AP1 and 4 μl RNase A stock solution (100 mg/ml) were added. Tubes were placed into the adaptor sets, which are fixed into the clamps of the TissueLyser. Disruption was performed in two 1-2 minute high-speed (20-30 Hz) shaking steps. 3. The mixture was incubated for 10 min at 65°C and mixed 2 or 3 times during incubation by inverting tube. 4. 130 μl Buffer P3 was added to the to the lysate, mixed, and incubated for 5 min on ice. 5. The lysate was centrifugated for 5 min at 14,000 rpm. 6. The lysate was pipetted into the QIAshredder Mini spin column (lilac) placed in a 2 ml collection tube, and centrifugated for 2 min at 14,000 rpm. 7. The flow-through fraction from step 16 was transferred into a new tube without disturbing the celldebris pellet. 8. 1.5 volumes of Buffer AW1 was added to the cleared lysate, and mixed by pipetting. 9. 650 μl of the mixture from step 8 (including any precipitate that was Formed) were pipetted into the DNeasy Mini spin column placed in a 2 ml collection tube and centrifugated for 1 min 8000 rpm and the flow-through was discarded. 10.
Step 9 was repeated with the remaining sample. The flow-through and collection tube were discarded. 11. The DNeasy Mini spin column was placed into a new 2 ml collection tube. 500 μl Buffer AW2 was added and centrifugated for 1 min at 8000 rpm and the flow-through was discarded. 12. 500 μl Buffer AW2 was added to the DNeasy Mini spin column, and centrifugated for 2 min at 14,000 rpm to dry the membrane. 13. The DNeasy Mini spin column was transferred to a 1.5 ml or 2 ml microcentrifuge tube, and 50 μl Buffer AE were directly pipette onto the DNeasy membrane. It was incubated for 5 min at room temperature (15-25°C), and then centrifugated for 1 min at 8000 rpm to elute.

Preparation of PCR Master Mix:
According to Emerald Amp GT PCR mastermix (Takara) Code No. RR310A kit as shown in table (1): Cycling conditions of the primers during cPCR: Temperature and time conditions of the two primers during PCR are shown in Table (2) according to specific authors and Emerald Amp GT PCR mastermix (Takara) kit.

Measuring of Aflatoxin by using Fluorometer (Afla Test):
Aflatoxin was extracted, column chromatographed, and measured by using VICAM series-4 and 4ex All required kits were obtained from VICAM (Milford, MA , USA) according to AOAC (1990).

Incidence of fungi isolated from the examined samples other than Aspergillus spp.:
Mycological examination of 120 samples of poultry feed showed the presence of 2 genera of fungi (Table 3) other than Aspergillus spp. The two species were Penicillium spp. (n=37; 30.8 %) and Mucor (n=23; 28.3%).

Incidence of Aspergillus species in poultry feed.
Data illustrated in Table (4) showed that out of 120 poultry feed samples, 125 Aspergillus species were recovered and were included in three species. Aspergillus flavus was the most frequent as 75 isolates (62.5%), followed by A. niger (n=31; 25.9%) and finally A. fumigatus (n=19; 15.8%).

PCR confirmation for A. flavus isolates:
PCR was applied on all Aspergillus flavus as a confirmatory diagnosis. The results revealed detection of 357 bp Aspergillus 18S rRNA gene of A. flavus in all tested isolates (Fig.1).

Aflatoxin detection in the examined samples using Afla Test Fluorometer Method:
According to Table (6), mycotoxins analysis of the samples revealed that out of 75 examined samples, aflatoxin was detected in a total of 43 samples (57.3%) while 32 samples (42.7%) were negative for aflatoxin contamination.

DISCUSSION
Mycotoxins are considered unavoidable contaminants in foods and feeding stuff because agronomical technology has not yet advanced to the stage at which preharvest infection of susceptible crops by fungi can be eliminated. These Feeds in storage conditions with more than12-15% moisture suitable to grow fungi. Because aerobically growth of most molds, increasing of moisture concentrations can eliminate air and prevent mold growth. (Ghaemmaghami et al.,  2016).
Feed stuffs are liable to be contaminated by molds and or fungi producing toxins which make the food nasty, unpleasant and toxic. Feed is considered the major cost of poultry production that lies between 65 and 75%. , therefore , any effect on the feed leads to change on the performance of broilers and layers .The storage conditions are necessary to safe feed , so weather extremes unsuitable storage practices and improper feeding conditions can cause feedfungal contamination that increase mycotoxins production. (Nkukwana et al., 2018). So it is desirable from time to time to examine these stuff periodically either from the superficial looking and/or laboratory examination for the presence of any toxic materials. Table (3) showed the different fungi other than Aspergillus spp. recovered from 120 poultry feed samples. The most prevalent genus was Penicillium which appeared in 37 samples (30.8%) out of 120 examined samples and Mucor spp. Appeared in 34 samples (28.3%) out of 120 examined samples. These results agree with those of Abd El Hamid et al., (1989) who isolated Penicillium spp. At a rate of 31% out of 73 poultry diets, and disagree with Dalcero et al., (1997) who reported that the Penicillium spp. Occurred in high level in poultry feed reaching 87%. The results illustrated in Table (5) presented the incidence of different Aspergillus species in the different examined samples. Regarding A.s flavus, the highest prevalence of contamination was recorded in poultry ration samples (72.2%) followed by yellow corn (50%), ration concentrates (30%), and finally soya bean (20%). The high level of Aspergillus flavus in Poultry ration maybe due to unsuitable storage conditions or maybe that the feeding stuffs were used as raw materials for the preparation of compound feed (poultry ration) and these feed stuffs were contaminated with fungi. These results agree with Abou El-Magd (1997) who reported that poultry ration was highly contaminated with Aspergillus flavus (10%) followed by ration concentrates (9%), yellow corn (8.7%), and soya bean (1.5%). Meanwhile, A. niger and A. fumigatus were isolated at a high level in poultry ration followed by ration concentrates, then yellow corn and soya bean. This result agreed with Abou El-Magd (1997) who reported that poultry ration was highly contaminated with Aspergillus spp.

Data illustrated in
PCR technique was applied for confirmatory diagnosis of all Aspergillus flavus using Aspergillus 18S rRNA gene of A. flavus. The results revealed detection of the gene in all tested isolates (Fig.1). Table (6) represented the mycotoxins analysis of the examined samples. A total of 43/75 samples (57.3%) were aflatoxin contaminated meanwhile 32/75 samples (42.7%) were negative for aflatoxin contamination. This result agreed with Dalcero et al., (1997) who reported that aflatoxin was the predominant toxin detected in poultry feed samples, and Natour et al., (1985) who reported that aflatoxins were found in 63.9% of poultry feed samples due to unsuitable storage condition but in our studies the high level of aflatoxins is due to the samples have been collected from farms that had disease problems so the samples are suspected to be mycotoxin contaminated. Viegas et al., (2017) found that 23 (25.55%) samples from 100 examined samples were toxigenic.

CONCLUSION
The most predominant Aspergillus isolates from poultry feed was A. flavus About 57.3% of A. flavus isolates produced aflatoxins. These results concluded that the potential exists for the production of aflatoxins by the Aspergillus flavus.