<4D6963726F736F667420576F7264202D20C7E1C8C7CDCB20D2C7E5CF20C7D3E3C7DAEDE1> Abdual-shahid et al. Diyala Agricultural Sciences Journal, 5( 2 ) 38 – 44 ,2013   38   ISOLATION AND CHARACTERIZATION OF FUNGI AND MYCOTOXINS (DEOXYNIVALENOL AND ZARALENONE) IN FISH FEED FROM BAGHDAD CITY. Dalia K. Abdual-shahid* Oday S. Abbas** ZahidE. Mohammad*** *College of Veterinary MedicineــUniversity of Baghdad . ** Iraqi Center for Cancer and Medical Genetics ResearchــUniversity of Al-Mustansiriyah *** College of Veterinary Medicine ــUniversity of Diyala. ABSTRACT This research provides a brief review of approaches for the early detection of fungi and its metabolites in feedof fish from some Baghdad farms. During a mycological analysis of complete feed mixes(15 samples), a total of five genera of moulds were identified. Penicillium spp. was present in considerably more samples than any other genus 36.4%, followed by the genera Fusarium spp.24.5%. Other fungi from the genera Aspergillus spp. 20%, Mucor spp. 11.1% and Alternaria spp. 8% were represented in a smaller amount.The mycotoxinsdeoxynivalenol and zearalenone were detected. Deoxynivalenol was detected in 10 samples in the concentration range 0.25–2.5 mg/kg. Zaralenone were detected in 8 samples in the concentration range 0.2–5.0 mg/kg.Thesefindings indicate that there may be a risk for animal exposure to mycotoxins through the consumption of moldy infected feeds. Key words:Fusariummycotoxins, Deoxynivalenol, Zaralenone, Fungal. INTRODUCTION The contamination of agricultural commodities with fungi able to produce toxic metabolites is a worldwide concern. Discoloration, quality deterioration, reduction in commercial value and mycotoxin production has been linked to moldy contaminated foods and feeds (Pardo et al., 2005). Mould contamination not only generates great economic losses, but also represents a threat to human and animal health, particularly through the synthesis of mycotoxins (Mishra and Sopori, 2012). Mycotoxins are a structurally diverse group of mostly small molecular weight compounds, produced by the secondary metabolism of fungi that contaminate the whole food chain, from the harvested products to the plate of consumers. Mycotoxins occur sporadically both seasonally and geographically (Pestka, 2011). The main mycotoxin classes of concern produced by fungi in the genera Aspergillus, Penicillium and Fusarium include the aflatoxins, ochratoxin A, trichothecenes and fumonisins(Danicke et al., 2011). Environmental factors ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ Received for publication Sept. 10 , 2013 . Accepted for publication Nov. 10 , 2013 . Abdual-shahid et al. Diyala Agricultural Sciences Journal, 5( 2 ) 38 – 44 ,2013   39   such as nutrients, light, temperature, pH and water activity (aw), either as single factors or in combination, are known to control mycotoxin production in many filamentous fungi (Georgianna and Payne, 2009). These environmental factors typically exert their influence on mycotoxin biosynthesis atthe level of gene transcription(Mostrom and Raisbeck, 2012). Limited information exists regarding the effects of low levels of multiple mycotoxins in fishes. It has been suggested that combination of mycotoxins at low concentrations may have negative effects on animals, even though the concentrations of individual mycotoxins are well below concentrations reported to cause negative effects. Therefore, rapid and specific detection of mycotoxogenicmoulds is important for ensuring both microbiological quality and safety of both feed and food (Sultana and Hanif, 2011).Deoxynivalenol (DON) is one of the most common Fusarium based mycotoxins found in human foodstuffs. It is also called vomitoxin, because of its impact on farm animals consuming contaminated feed. DON is much more frequently found in barley, corn, sunflower, wheat, and compound animal feeds. The fungal pathogens producing DON cause ear rot in corn and head blight in wheat, two of the most common sources of DON in the feed supply. Rain during the flowering period in small grains clearly increases the risk of DON contamination (Mosse, 2012). Zearalenone (ZON) is a secondary metabolite of Fusariumspecies. Unlike other mycotoxins, zearalenone is virtually non-toxic to mammals following acute ingestion(Shekhany, 2008). Nonetheless, it is extremely potent in other ways, since it resembles a key hormone produced by human ovaries, 17β- estradiol, and as a result, can disrupt the human endocrine system (Dombroski, 2012). As little as 0.0001 ppm of zearalenone has been shown to create a detectable, hormone-related uterogenic response in females (Bennett and Klich, 2003). In other words, zearalenone exposure has been shown to impact reproductive processes at a dose that is 100 million times less than the lethal dose in a mice LD50 study (Mosse, 2012).Therefore, the aims of this research were to: Isolation of fungi and detection of mycotoxins (Deoxynivalenol and Zearalenone) in fish feeds. MATERIAL AND METHODS Sample collection The research materials consisted of 15 representative fishfeed samples which were collected directly at fish farms from different parts of Baghdad city during a 2 month period.The samples (each about 1 kg) were stored at -4˚C andanalyzed the day after collection. Material Standards of zearalenone(ZON) and deoxynivalenol (DON) were purchasedfrom Sigma. All othersolvents and reagents were analytical grade. Abdual-shahid et al. Diyala Agricultural Sciences Journal, 5( 2 ) 38 – 44 ,2013   40   Isolation and identification of fungal strains Isolation and identification of fungal strains were done on solid media using the potato dextrose agar (PDA). Plates were incubated at 25˚C for 7 days. Each isolated mould colony was observed microscopically for morphological characterization and identification to genera/species level. This was done by their macro- and micro morphology features using appropriate identification keys (Samson et al., 2002). Extraction and Cleanup of Mycotoxins The procedure described by Sreenivasaet al.,(2009) was used for extraction and clean up ofmycotoxins. Mycotoxin analysis Analysis of twomycotoxins in all samples was performed by High Performance Liquid Chromatography (HPLC). The optimized instrumental conditions are summarized in Table 1.A standard chromatogram of the Fusariummycotoxins of major interest is shown in Figure 1.The mycotoxin concentration in the samples was calculated by comparing the area of chromatographic peak of the samples with that of the standard calibration curve by densitometry analysis. Table 1.Instrumental conditions of HPLC. Instrumental conditions LC Column Shimadzu Shim 150 mm x 2 mm x5µm Flow-rate 0.1mL/min Detection wavelength 230 nm Injection volume 10µL Column temperature 40 °C Mobile phase 10mM ammonium acetate – methanol Time (minutes) 10mM ammonium acetate % methanol % 0 90 10 5 50 50 20 0 100 Abdual-shahid et al. Diyala Agricultural Sciences Journal, 5( 2 ) 38 – 44 ,2013   41     Figure 1. Standard chromatogram of the deoxynivalenol (DON) andzearalenone(ZON)mycotoxins. Statistical analysis Differences in the mean levels of mycotoxin contamination across the two groups of positive samples were calculated by a Student’s t-test. RESULTS AND DISSCUSION The results obtained from the mycoflora analysis and incidence of mycotoxins contamination in thesamples originating from the region where the sampleswere collected is presented in Table 2 and Figure2. Mycoflora analysis The most frequently isolated fungus was Penicillium spp., a 36.4%. Other frequently isolated moulds included Fusariumspp. 24.5%,Aspergillusspp. 20%, Mucor spp.11.1% and Alternariaspp. 8% were represented in smaller amount Figure 2. Figure 2. Percentage of moulds contamination fish feed. Time (min) 0 5 10 15 20 25 30 35 40 P er ce nt ag e % Abdual-shahid et al. Diyala Agricultural Sciences Journal, 5( 2 ) 38 – 44 ,2013   42   Occurrence of mycotoxins in samples The results obtained from the analysis of mycotoxins in the fish feed samples are presented in Table 2. The predominant mycotoxin for all analyzed samples was DON. The incidence of DON and ZON in all the samples was 51.7% and 48.3%, respectively figure 3. No significant differences were found between the median DON and ZON contents for all feed items (P�0.05). Table 2. HPLC data for occurrence of mycotoxins in fish feeds. * SD Standard deviation Means with same letters within the same column are no significantly differences. Figure 3.Samples chromatogram of the deoxynivalenol (DON) andzearalenone (ZON) mycotoxins. Isolated species in our case are mostly storage contaminators, implicating that the high number of contaminated feed is most probably the result of manipulative mistakes (storage duration, temperature and humidity levels, etc.), during storage of feedstuffs or feed. Fungal colonization, growth and synthesis of toxins, results from the complex interaction of several factors (water availability, temperature and incubation time) and therefore, an understanding of each factor involved is essential for understanding the overall process and predicting fungal spoilage in agricultural and food products (Pardoet al., 2005). Improper storage accompanied by too high a temperature and elevated moisture content in the grain favour further mycotoxin production and lead to reduction in grain quality (Ramirez et al., 2004). It is well known that cereal infection with moulds and toxin production depends strongly on environmental conditions (damp climate, cool temperatures). However, these data must be interpreted with caution, as they were calculated from a limited number of Mycotoxins Positive samples (Incidence %) Rang (mg/kg) mean ± SD* (mg/kg) DON 10 (51.7%) 0.25-2.5 0.78 ± 0.85a ZON 8 (48.3%) 0.2-5.0 0.85 ±1.416a In te n si ty  ( C P S) Abdual-shahid et al. Diyala Agricultural Sciences Journal, 5( 2 ) 38 – 44 ,2013   43   samples. The results of the mycoflora analysis carried out in this study are similar to previous results found by other authors (Stankovicet al., 2007;Milicevic, 2008). This shows that the incidence of these various species was important, as the produce was stored for prolonged periods of time.The main advantages of the HPLC technique include its general applicability to a broad range of compounds, high sensitivity and outstanding selectivity. Several methods already have been reported for the simultaneous determination of mycotoxins, which offer significant advantages over conventional techniques (Scarlett et al., 2012). REFERENCES Bennett, J. and M. Klich. 2003. Mycotoxins. Clin.Microbiol.Rev., 16:497- 516. Danicke, T., S. Goyarts, H. Valenta and K.H. Ueberschar. 2011. Carry-over of Fusariumtoxins (deoxynivalenol and zearalenone) from naturally contaminated wheat to pigs. Food Addit.Contam., 25: 389-420. Dombroski, T. 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J., 29: 211-213. في عالئق االسماك ) الديوآسينيفالينول و الزيرالينون(لفطريات والسموم الفطرية عزل وتصنيف ا .لمدينة بغداد ***محمد إسماعيلزاهد **عدي ستار عباس * داليا عبد الكريم عبد الشهيد . جامعة بغداد -آلية الطب البيطري -مدرس * .الجامعة المستنصرية -ي لبحوث السرطان والوراثة الطبيةآز العراقالمر -مدرس ** . جامعة ديالى- آلية الطب البيطري -مدرس مساعد *** المستخلص تعرض ث اس ذا البح ر ه ر نموذج ًامختص ة عش ي خمس مومها ف ات وس د الفطري ن تحدي الف ًاع ن اع م من جناس حيث تم عزل خمسة أ . رينشه كية لمدينة بغداد خالل مدة االسماك من بعض المزارع السم آان الغالب في النماذج وبنسبة spp. Penicillium جنسحيث وجد ان , الفطريات وبنسب مختلفة % 20فكان بنسبة spp. Aspergillusجنس أما %.24.5 وبنسبة spp.Fusariumجنسيتبعه % 36.4 آما %. 8المرتبة االخيرة وبنسبة ب spp. Alternariaجنسوحل % 11.1بنسبة spp. Mucorجنسثم آغم /ملغم 5.2 ـ 0.25في عشرة نماذج وبترآيز تراوح بين Deoxynivalenolتم تحديد السم الفطري ما ان . آغم عليقة /ملغم 5_ 0.2نماذج وبترآيز تراوح بين يةفي ثمان Zaralenoneفيما حدد ،عليقة .رًا على الحيوانات وخصوصًا الثروة السمكيةتم تحديده من فطريات وسمومها ربما يمثل خط .اتالفطري الديوآسينيفالينول، الزيرالينون،سموم الفيوزاريم، : الكلمات المفتاحية