PaPer 32 Ital. J. Food Sci., vol. 28 - 2016 - Keywords: Aspergillus flavus, Aflatoxin genes, Flourometer, molecular markers, genetic diversity - Molecular characterization of aflatoxigenic aspergilli-contaMinated poultry and aniMal feedstuff saMples froM the western region of saudi arabia youssuf a. gherbawy*1, 2, yassMin M. shebany1, 2 and helal f.alharthy1 1Biological Sciences Department, Faculty of Science, Taif University, Taif, Saudi Arabia 2Botany Department, Faculty of Science, South Valley University, Qena, Egypt *Corresponding author: youssufgherbawy@yahoo.com AbstrAct the aflatoxigenic abilities of 64 and 17 isolates of Aspergillus flavus and A. parasiticus isolated from poultry and animal feedstuff samples collected from the western region of saudi Arabia were studied. thirty-three (51.6%) and 13 (76.5%) isolates of A. flavus and A. parasiticus, respective- ly, were aflatoxigenic. the ranges of aflatoxins in A. flavus and A. parasiticus isolates were 4.4- 110 and 143.6-271.3 ppm (µg/g), respectively. A. parasiticus isolates generally produced a greater amount of aflatoxins than A. flavus. A. flavus isolates from poultry, cattle, and camel and cattle feeds produced aflatoxin amounts in the range 5.7-110, 4.4-19.0, and 7.0-28.5 ppm, respectively. From poultry feedstuff samples, A. parasiticus produced aflatoxins in the range 212.5-232.4 ppm. some aflatoxin biosynthesis genes (aflR, omt-1, ver-1, and nor-1) were detected with variable fre- quencies in all A. flavus and A. parasiticus isolates. the genetic diversity among 64 isolates of A. flavus using internal transcribed spacer sequence results and the amplification of some aflatoxin biosynthesis genes revealed that the investigated isolates showed high heterogeneity. mailto:youssufgherbawy@yahoo.com Ital. J. Food Sci., vol. 28 - 2016 33 INtrODUctION Aflatoxin contamination of agricultural com- modities has gained global significance as a re- sult of their deleterious effects on human and animal health as well as their importance in in- ternational trade. the contamination of foods by aflatoxigenic fungi, particularly in tropical countries, may occur during pre-harvesting, processing, transportation, and storage (EllIs et al., 1991; MANONMANI et al., 2005). regular monitoring of toxigenic mycobiota in agricultur- al-based feeds and foods is an essential pre-req- uisite in the development of strategies to control or prevent mycotoxin exposure of feed animals and human population. studies on the prev- alence of toxigenic mycobiota of animal/poul- try feeds have been regularly and frequently re- ported, including studies from brazil (OlIvEIrA et al., 2006; rOsA et al., 2006), Argentina (DAl- cErO et al., 1997), Nigeria (OshO et al., 2007), spain (AccENsI et al., 2004), and Pakistan (sAl- EEMI et al., 2010). the polymerase chain reaction (Pcr) first described by sAkI et al. (1985) requires the presence of specific target sequences. When genes involved in the biosynthetic pathway are known, they represent a valuable target for the specific detection of toxigenic fungi. the first re- searchers to use this approach for the detec- tion of toxigenic fungi were GEIsEN et al. (1996) and shAPIrO et al. (1996), describing a diag- nostic Pcr directed against DNA sequences in the aflatoxin biosynthetic gene cluster. how- ever, when the genes responsible for mycotox- in production are unknown, other sequences can function as a target. Examples are rDNA sequences, genes, or anonymous DNA mark- er sequences. GEIsEN (1998) and EDWArDs et al. (2002) reviewed available diagnostic Pcrs for toxigenic fungi. the advantages of the Pcr- based approach for the detection of toxigenic fungi compared with those of the classical my- cological or chemical analysis is mainly the time aspect. For the chemical analysis of mycotoxins in food, elaborated protocols for sample prepa- ration and expensive laboratory equipment are necessary. classical mycological analysis re- quires the isolation and cultivation of the fun- gi on different media and at least one week of growth for their reliable identification. In ad- dition, much expertise is required to recognize the species, particularly for the main genera of toxigenic fungi Fusarium, Penicillium, and As- pergillus. In contrast, DNA extraction from food samples and raw materials of food can be per- formed in a few minutes (kNOll et al., 2002a). Further, the use of modern thermocyclers can reduce analysis time to less than 1 h (kNOll et al., 2002b). the aflatoxin biosynthetic pathway involves approximately 25 genes clustered in a 70 kb DNA region (YU et al., 2004). A. flavus, A. parasiticus, and other Aspergillus section Flavi species share nearly identical sequences and conserved gene order in the cluster. In recent years, Pcr detec- tion of aflatoxin biosynthetic gene presence or expression has been used as a diagnostic tool for aflatoxigenic fungi in selected food commod- ities (GEIsEN, 2007; GAllO et al., 2012). Aflatoxins are regarded as potent hepatocar- cinogens and immunosuppressants, and there are reports showing that this group of mycotox- ins poses the biggest threat to the poultry and livestock industry through low productivity and death (vAN EGMOND, 1989; chUkWUkA et al., 2010; PEDrOsA and bOrUtOvA, 2011). there- fore, the potential risks of aflatoxicosis in sau- di poultry and livestock must be clearly evalu- ated in order to ensure prompt legislative ac- tion and mitigation of aflatoxin contamination in feed. this study was designed to determine and evaluate the aflatoxin-producing potentials of Aspergillus section Flavi isolated from poul- try and animal feedstuff samples collected from the western region of saudi Arabia. Further- more, the isolates were tested for the presence of four of the characterized aflatoxin biosynthet- ic genes in their genome in relation to aflatox- in production. MAtErIAls AND MEthODs Samples sixty-four A. flavus and 17 A. parasiticus iso- lates were used throughout this investigation. these isolates were retrieved from poultry and animal feedstuff samples collected from the western region of saudi Arabia (taif, Makkah, and Jeddah). the isolates were identified accord- ing to their morphological features as well as sequence results of internal transcribed spacer (Its) regions. the sequence results were depos- ited in the Genbank. Determination of total aflatoxin abilities of Aspergillus species isolates the aflatoxin-producing abilities of the iso- lates were determined by cultivating the fungal strains in czapek Yeast extract agar (bEN FrEDJ et al., 2009) medium for 5 days at 25±2°c. total aflatoxins were extracted by grinding the moldy agar (20 g) in a Waring blender for 5 min with methanol (100 ml) containing 0.5% Nacl. the mixture was then filtered through a fluted filter paper (Whatman 2v, Whatmanplc, Middlesex, Uk), and the filtrate was diluted (1:4) with wa- ter and re-filtered through a glass-fiber filter pa- per. two milliliters of the glass-fiber filtrate were placed on Aflatest® Wb sr column (vIcAM, Watertown, MA, UsA) and allowed to elute at 1-2  drops/s. the columns were washed twice with 5 ml of water, and aflatoxin was eluted from 34 Ital. J. Food Sci., vol. 28 - 2016 the column with 1 ml high performance liquid chromatography (hPlc)-grade methanol. A bro- mine developer (1 ml) was added to the metha- nol extract, and the total aflatoxin concentration was read in a recalibrated vIcAMseries-4 fluo- rometer set at 360 nm excitation and 450 nm emissions (lEWIs et al., 2005). Molecular detection of aflatoxin biosynthet- ic genes in Aflatoxigenic species of aspergilli the isolation of DNA from mycelia was per- formed according to the method described by FArbEr et al. (1997). Four published primer sets were used for the specific detection of nor- 1, ver-1, omt-A, and aflR genes (crIsEO et al., 2008). the 400, 537, 797, and 1032-bp frag- ments were amplified, respectively. A typical Pcr was carried out under the following conditions: 5 μl of genomic DNA were used as a template (2 μg/ml), 0.5 U Eurotaq polymerase (Euroclone, Pero-Milan, Italy), 1 × reaction buffer, 2.5 mM Mgcl 2 , 200 μM of each dNtP, and 7.5 pmol of each primer, in a total reaction volume of 50 μl. A total of 35 Pcr cycles with the following tem- perature regimen were performed: 95°c, 1 min; 65°c, 30 s; 72°c, 30 s for the first cycle; and 94°c, 30 s; 65°c, 30 s; 72°c, 30 s for the 34 re- maining cycles (crIsEO et al., 2008). Pcr prod- ucts were separated on a 1.3% (wt/vol) agarose gel stained with ethidium bromide. Statistical analysis of frequency of aflatoxin biosynthetic genes cluster analysis of data was performed by hi- erarchical cluster analysis (sPss software, sPss Inc., UsA; Norusis, 1993). rEsUlts AND DIscUssION Total aflatoxin potentials of Aspergillus species isolates thirty-three out of 64 (51.6%) and 13 out of 17 (76.5%) of A. flavus and A. parasiticus iso- lates were aflatoxigenic producers, respective- ly. the aflatoxin range in A. flavus and A. par- asiticus isolates was 4.4-110 and 143.6-271.3 ppm (µg/g), respectively (tables 1 and 2). DU- ttA and DAs (2001) carried out a groundwork study, in which 256 feed samples collected from different parts of Northern India were analyzed for aflatoxigenic strains of A. flavus/parasiticus and for detection of AFb1. Out of 198 A. flavus and 15 A. parasiticus strains isolated, 76% and 86%, respectively, were found to be toxigenic. rAzzAGhI-AbYANEh et al. (2006) surveyed the distribution of Aspergillus section Flavi in corn- field soils in Iran and their results indicated that only 27.5% of A. flavus isolates were aflatoxigen- ic (b1 or b2 or both), and all the A. parasiticus isolates produced aflatoxins of both b (b1 and b2) and G (G1 and G2) types. PItt (1993) also, reported that A. flavus isolates produced b1 and b2 or both types, while A. parasiticus produced the four aflatoxin types. these results support the present findings indicating that the level of aflatoxin production by A. parasiticus was higher than that by A. flavus isolates (tables 1 and 2). Further, kOEhlEr et al. (1975) reported that A. parasiticus isolates generally produced a great- er amount of aflatoxins than A. flavus. the range of aflatoxin production by A. fla- vus isolated from poultry, cattle, and camel and cattle was 5.7-110, 4.4-9.0, and 7.0-28.5 ppm, respectively. From poultry feedstuff samples, A. parasiticus produced aflatoxins in the range 212.5-232.4 ppm. In Pakistan, sAlEEMI et al. (2010) studied the mycoflora of poultry feed and mycotoxin-producing potential of Aspergillus species. they reported that the toxigenic fungi content among Aspergillus isolates was 73.58%, and that of aflatoxigenic isolates of A. flavus and A. parasiticus was 83.33% and 85.71%, respec- tively. Further, they recorded that, among tox- igenic A. flavus isolates (10/12), six produced four aflatoxins (AFb1, AFb2, AFG1, and AFG2), two produced AFb1, AFb2, and AFG1, one pro- duced AFb1, AFb2, and AFG2, and one pro- duced AFb1 and AFb2. Among aflatoxigenic iso- lates of A. parasiticus (6/7), five produced four aflatoxins (AFb1, AFb2, AFG1, and AFG2) while one produced three (AFb1, AFb2, and AFG1). the production range of aflatoxins from four isolates (tUh212, 221, 222, and 225) of A. parasiticus retrieved from cattle feed sam- ples was 143.6-271.3 ppm. Further, two iso- lates (tUht216 and 220) of A. parasiticus iso- lated from cattle and camel feed samples con- tained 195.5 and 211.2 ppm of aflatoxins (ta- ble 1). Among isolates of A. flavus collected from taif samples, tUht53 showed the lowest afla- toxin potential (7.0 ppm) and tUht44 showed the highest (106.8 ppm). Isolates tUht185 and tUht180 from feed samples collected from Jed- dah showed the lowest (5.7 ppm) and highest (33.0 ppm) levels of aflatoxins, respectively (ta- ble 1). For A. flavus isolates retrieved from feed samples collected from Makkah, tUht117 and tUht121 showed the lowest (5.0 ppm) and the highest (110 ppm) aflatoxin levels, respective- ly. the results shown in table 2 indicated that, from A. parasiticus isolates, the lowest aflatox- in producer was tUht212 (143.6 ppm), while the highest production was recorded in isolate tUht222 (269.5 ppm). Data from different ge- ographic areas demonstrated a great variabil- ity in the mycotoxin-producing potential of A. flavus and closely related species (hOrN and DOrNEr, 1999). these results are in accord- ance with previous reports showing that these two species have the ability to produce both b and G aflatoxins (PItt and hOckING, 1997; kUM- EDA et al., 2003; GhIAsIAN et al., 2004). In Al- Ital. J. Food Sci., vol. 28 - 2016 35 table 1 - total aflatoxins (PPM) and aflatoxigenic genes detected in 64 strains of Aspergillus flavus isolates collected from feedstuff samples. Strains code Source of isolation Location Total aflR omt-A ver-1 nor-1 AFs (PPM) TUHT43 Poultry Taif N.D. + + + + TUHT44 Poultry Taif 106.8 + + + + TUHT46 Poultry Taif 7.8 + + + + TUHT47 Poultry Taif N.D. + + + + TUHT53 Poultry Taif 7.0 + + + + TUHT59 Poultry Taif N.D. + - + + TUHT63 Poultry Taif N.D. - + + + TUHT84 Poultry Taif N.D. - - - + TUHT85 Poultry Taif N.D. + - - - TUHT86 Poultry Taif 11.0 + + + + TUHT87 Poultry Taif N.D. + + - + TUHT89 Cattle Taif N.D. - - - - TUHT91 Cattle Taif 10.0 + + + + TUHT92 Camel & cattle Taif N.D. + + - - TUHT93 Poultry Taif 16.0 + + + + TUHT94 Poultry Taif 28.4 + + + + TUHT98 Camel & cattle Taif N.D. + - - - TUHT99 Cattle Taif 19.0 + + + + TUHT100 Poultry Makkah N.D. - + - - TUHT104 Poultry Makkah N.D. + + - + TUHT106 Poultry Makkah N.D. - + - + TUHT107 Poultry Makkah 8.9 + + + + TUHT108 Poultry Makkah N.D. - + - + TUHT109 Poultry Makkah 20.0 + + + + TUHT110 Poultry Makkah 12.0 + + + + TUHT111 Cattle Makkah 13.0 + + + + TUHT115 Camel & cattle Makkah N.D. + + - + TUHT116 Cattle Makkah 19.0 + + + + TUHT117 Cattle Makkah 4.4 + + + + TUHT118 Cattle Makkah 15.0 + + + + TUHT119 Camel & cattle Makkah N.D. - + + - TUHT120 Camel & cattle Makkah 28.5 + + + + TUHT121 Poultry Makkah 110.0 + + + + TUHT123 Poultry Makkah N.D. + + - - TUHT124 Poultry Makkah 6.6 + + + + TUHT126 Poultry Makkah 13.2 + + + + TUHT152 Poultry Makkah N.D. + - - - TUHT154 Poultry Makkah 8.5 + + + + TUHT155 Poultry Makkah N.D. + - - + TUHT156 Poultry Makkah 12.6 + + + + TUHT157 Cattle Makkah N.D. + - - - TUHT158 Cattle Makkah 7.3 + + + + TUHT160 Poultry Jeddah N.D. - + - + TUHT112 Horses Jeddah N.D. + + - + TUHT161 Poultry Jeddah N.D. + + + + TUHT163 Poultry Jeddah N.D. + + - - TUHT164 Poultry Jeddah N.D. + - + - TUHT165 Camel & cattle Jeddah 7.0 + + + + TUHT166 Camel & cattle Jeddah N.D. - + - + TUHT168 Horses Jeddah 6.1 + + + + TUHT172 Camel & cattle Jeddah N.D. + + - - TUHT173 Camel & cattle Jeddah N.D. + + + + TUHT174 Camel & cattle Jeddah 7.5 + + + + TUHT176 Cattle Jeddah N.D. - + + - TUHT177 Cattle Jeddah 14.0 + + + + TUHT180 Poultry Jeddah 33.0 + + + + TUHT181 Poultry Jeddah 21.0 + + + + TUHT185 Poultry Jeddah 5.7 + + + + TUHT186 Camel & cattle Jeddah 8.0 + + + + TUHT187 Camel & cattle Jeddah N.D. - - - + TUHT188 Camel & cattle Jeddah 18.2 + + + + TUHT189 Camel & cattle Jeddah 9.13 + + + + TUHT190 Camel & cattle Jeddah N.D. + - - + TUHT193 Camel & cattle Jeddah 14.3 + + + + Total 33 53 53 42 51 36 Ital. J. Food Sci., vol. 28 - 2016 table 2 - total aflatoxins (PPM) and aflatoxigenic genes detected in Aspergillus parasiticus isolates collected from feedstuff samples. Strains code Source of isolation Location Total aflR omt-A ver-1 nor-1 AFs (PPM) TUHT226 Cattle Taif N.D. + + - + TUHT227 Cattle Taif N.D. + + + + TUHT228 Cattle Jeddah N.D. - + - + TUHT229 Poultry Jeddah N.D. + + + - TUHT26 Poultry Taif 212.7 + + + + TUHT211 Poultry Taif 232.4 + + + + TUHT212 Cattle Makkah 143.6 + + + + TUHT213 Poultry Makkah 231.2 + + + + TUHT214 Poultry Makkah 224.4 + + + + TUHT215 Poultry Jeddah 212.5 + + + + TUHT216 Camel & cattle Jeddah 195.4 + + + + TUHT219 Cattle Taif 265.5 + + + + TUHT220 Camel & cattle Taif 211.2 + + + + TUHT221 Cattle Makkah 271.3 + + + + TUHT222 Cattle Makkah 269.5 + + + + TUHT223 Poultry Jeddah 227.2 + + + + TUHT225 Cattle Jeddah 269.7 + + + + Total (17 Isolates) 13 16 17 15 16 geria, rIbA et al. (2010) determined the aflatox- in-producing capacity of 150 A. flavus isolates collected from wheat and its derivatives in 2004 and 2006, and the results showed that 72% of the strains produced aflatoxins. these strains produced amounts of AFb1 in the range 12.1- 234.6 mg/g of cYA medium. the results of the present study indicate that the aflatoxigenic species of Aspergillus vary in their aflatoxin potential according to the sub- strate and environmental factors. these results are in agreement with those reported by AbbAs et al. (2005). Detection of some of aflatoxin biosynthesis genes in Aspergillus species the production of aflatoxin involves a complex biosynthetic pathway consisting of at least 25 genes (YAbE et al., 1999; crIsEO et al., 2001a, bhAtNAGAr et al., 2003; YU et al., 2004; sch- ErM et al., 2005). All of the identified biosyn- thesis-related genes are located within a 75 kb DNA region in both A. parasiticus and A. flavus, and their relative positions in the cluster of both fungal species are similar (YU et al., 2000; Ehr- lIch et al., 2005). Pcr was used for the detec- tion of aflatoxigenic aspergilli based on the in- termediated enzymes, including norsolorinic acid reductase encoding gene nor-1, the versi- colorina dehydrogenase encoding gene ver-1, the sterigmatocystin 0-methyl transferase encoding gene omt-1, and the regulatory gene aflR (ErA- MI et al., 2009). representative aflatoxigenic and non-aflatox- igenic A. flavus and A. parasiticus isolates were subjected for detection of aflatoxin biosynthe- sis genes. Detection of aflatoxin biosynthesis genes in A. flavus isolates Pcr was applied using four sets of primers for different genes involved in the aflatoxin bio- synthetic pathway. bands of fragments of aflR, omt-1, ver-1, and nor-1 genes were visualized at 1032 bp, 797 bp, 537 bp, and 400 bp, respec- tively (Fig. 1). All examined A. flavus isolates yielded different DNA banding patterns with a number of bands ranging from zero to four (ta- bles 1 and 2). table 1 outlines the total aflatoxin and afla- toxigenic genes (aflR, omt-A, ver-1, and nor-1) detected in 64 strains of aflatoxigenic and non- aflatoxigenic A. flavus isolates collected from feedstuff samples. A. flavus isolates were rep- resented by 35 isolates from poultry feed sam- ples, 16 from camel and cattle feed, 11 from cat- tle feed, and two from horse feed. thirty-eight out of 64 (59.4%) A. flavus isolates contained all four aflatoxin biosynthesis genes; among them 21 isolates were retrieved from poultry feedstuff samples, eight from camel and cattle feed, eight from cattle feed, and one from horse feed (table 1). this result is in agreement with crIsEO et al. (2001a), who used specific Pcr-based methods to prove that aflatoxigenic A. flavus isolates al- ways contain the complete gene set. Among the 38 isolates tht showed the pres- ence of all four targeted genes, two isolates (tUht43 and 47) were not aflatoxigenic. there- fore, this result indicated clearly that the pres- ence of the four tested genes is not a sufficient marker for the differentiation between aflatoxi- genic and non-aflatoxigenic isolates. Other stud- ies (FlAhErtY and PAYNE, 1997; chANG et al., 1999a,b; 2000, cArY et al., 2002; tAkAhshI et Ital. J. Food Sci., vol. 28 - 2016 37 al., 2002; EhrlIch et al., 2003) have suggested that regulation of aflatoxin biosynthesis in As- pergillus spp. involves a complex pattern of pos- itive and negative acting transcriptional regula- tory factors affected by environmental and nu- tritional parameters. Furthermore, the lack of aflatoxin production apparently does not need to be related only to an incomplete pattern ob- tained in Pcr-based detection. Different muta- tions may be responsible for the inactivation of aflatoxin biosynthetic pathway genes in other A. flavus strains (GEIsEN, 1996). six isolates (9.4% of the tested isolates) with three gene amplicons were not aflatoxigenic (ta- ble 3). From these, four, one, and one isolates were retrieved from poultry, camel and cattle, and horse feeds, respectively. twelve isolates (18.8% of the tested isolates; six from poultry, five from camel and cattle, and one from cattle), contained two gene amplicons and seven iso- lates (10.9%) contained one gene amplicon (ta- ble 3). On the other hand, one non-aflatoxigen- ic isolate (tUht89) showed no bands, indicating a deletion of the targeted genes in this isolate. crIsEO et al. (2001a) proved that non-aflatoxi- genic isolates of A. flavus were lacking one, two, three, or four Pcr products, indicating that the genes do not exist in these strains or that the primer binding sites changed. Further, crIsEO et al. (2001b) reported that aflatoxin biosynthe- sis in A. flavus is strongly dependent on the ac- tivities of regulatory proteins and enzymes en- coded by the four genes aflR, nor-1, ver-1, and omt-A. GhErbAWY et al. (2012) reported on the presence of a complete set of these genes in sev- en aflatoxigenic isolates of A. flavus retrieved from date palm. the frequencies of the four aflatoxin biosyn- thesis genes aflR, omt-A, ver-1, and nor-1, in the tested isolates were 53, 53, 42, and 51, respec- tively (table 1). crIsEO et al. (2008) used 134 of non-aflatoxin producing strains of A. flavus isolated from food, feed, and officinal plants to study the different genes involved in the aflatox- in biosynthetic pathway. their results indicated that the nor-1 gene was the most representative (88%) of the four aflatoxin structural assayed genes, followed by ver-1 and omt-A, which were found at the same frequency (70.1%). A lower incidence (61.9%) was observed for aflR. Fur- ther, crIsEO et al. (2008) demonstrated that a high number of aflatoxin non-producing strains (61.9%) contain the aflR gene. this could im- pair the use of aflR to identify aflatoxigenic as- pergilli. Five out of ten A. flavus isolates were not aflatoxin producers (schErM et al., 2005), table 3 - Origin and genetic patterns of 64 aflatoxigenic Aspergillus flavus isolates collected from feedstuff samples in this study. values in brackets are percentages of the total samples analyzed. Sample name No isolates Complete Three Two One Zero set bands bands band band Poultry 35 21 4 6 4 - Camel & cattle 16 8 1 5 2 - Cattle 11 8 - 1 1 1 Horses 2 1 1 - - - Total 64 (100) 38 (59.4) 6 (9.4) 12 (18.8) 7 (10.9) 1 (1.6) Fig. 1 - Aflatoxin biosynthesis genes amplifications. lanes 1 - 7, Aspegillus flavus (tUht 43, 44, 47, 91, 168, 188 and 219); lanes 8 - 13, A. parasiticus (tUht 26, 212, 216, 221, 223 and 227); lanes 14 -16, A. flavus (tUht 87, 104, 115), lane 17 A. parasiticus (tUht 226), lane 18 A. parasiticus (tUh 229); lane 19, A. flavus (tUht 160); lane 20, A. parasiticus (tUht 228); lane 21 & 22, A. flavus (tUht 119 & 176); lane 23, A. flavus (tUht 163); lanes 24; A. flavus (tUht 164); lane 25 A. flavus (tUht84); lane 26 A. flavus (tUht157); lane, 27 negative control and lane 28, positive control. Asterisked lanes were non aflatoxigenic isolates. 38 Ital. J. Food Sci., vol. 28 - 2016 indicating that the frequencies of occurrence of aflR and omt-A, ver-1, and nor-1 genes were 8, 5, 9, and 5, respectively. Detection of some of aflatoxin biosynthesis genes in A. parasiticus isolates seventeen A. parasiticus isolates collected from different feedstuff samples from various cit- ies in saudi Arabia were examined for the pres- ence of aflatoxin biosynthesis genes using a spe- cific primer set as mentioned above. the results indicated the presence of four bands for aflR, omt-1, ver-1, and nor-1 genes at 1032 bp, 797 bp, 537 bp, and 400 bp, respectively (Fig. 1). All aflatoxigenic and non-aflatoxigenic isolates ex- amined yielded different DNA banding patterns with the number of bands ranging from 2 to 4 (tables 2 and 4). table 2 shows the total aflatoxin and aflatox- igenic genes detected in A. parasiticus isolates collected from three different feedstuff samples (poultry, camel and cattle, and cattle). thirteen out of 17 A. parasiticus isolates were aflatox- igenic. the frequencies of occurrence of aflR, omt-1, ver-1, and nor-1 genes in A. parasiticus isolates were 16 (94.1%), 17 (100%), 15 (88.2%), and 16 (94.1%), respectively. Gherbawy et al. (2014) reported that omt-A was the most prev- alent gene in A. flavus and A. parasiticus iso- lated from chili samples collected from taif city (saudi Arabia). Further, their results indicated that this gene was recovered from 27 out of 30 A. flavus isolates and two isolates of A. para- siticus, while nor-1, aflR, and ver-1 genes were recovered from 25, 26, and 24 isolates of afla- toxigenic and non-aflatoxigenic isolates of A. flavus. Out of seven A. parasiticus isolates col- lected from poultry feedstuff samples, 6 (85.7%) contained four genes, while two (14.3%) showed the amplicons of three genes. the two A. par- asiticus isolates collected from camel and cat- tle feedstuff samples showed a complete set of the targeted genes (tables 2 and 4). Amplifica- tion of the four targeted genes in eight A. par- asiticus isolates collected from cattle feedstuff samples showed that six (75%) had the four genes and one (12.5%) contained three genes (tables 2 and 4). Further, one isolate contained two genes. GEIsEN (1996) reported the presence of the abovementioned genes from two isolates of A. parasiticus. Additionally, schErM et al. (2005) indicated the presence of a complete set of genes (aflR, omt-1, ver-1, and nor-1 genes) in three isolates of A. parasiticus. the findings herein showed the presence of four targeted genes in all aflatoxigenic isolates of A. parasiticus and in one (tUht229) non-afla- toxigenic isolate. Further, all non-aflatoxigenic isolates were missing one or more of the target- ed genes. rAshID et al. (2008) studied the pres- ence of aflR, omt-1, ver-1, and nor-1 genes in 35 A. parasiticus isolates from stored wheat grains in Pakistan. their results revealed that only one isolate showed the complete set of genes. Addi- tionally, omt-1, ver-1, and nor-1 genes appeared in 8, 10, and 13 isolates. Deletion of aflR in A. parasiticus abolishes the expression of oth- er aflatoxin pathway genes (cArY et al., 2000). Finally, the regulation of aflatoxin biosynthe- sis genes in Aspergillus spp. is affected by en- vironmental and nutritional parameters (FlA- hErtY and PAYNE, 1997; chANG et al., 2000; cArY et al., 2002; tAkAhshI et al., 2002; Ehr- lIch et al., 2003). Genetic diversity among A. flavus strains isolated from feedstuff samples sixty-four aflatoxigenic and non-aflatoxigenic isolates of A. flavus represented different sources of isolation and different locations were used in this part. Using the Its region of rrNA sequenc- ing results and amplification of some aflatoxin biosynthesis genes, the genetic diversity among those strains was studied. Using Its sequencing results of 64 isolates of A. flavus, a neighbor joining tree was construct- ed (Fig. 2). the population of A. flavus split into several clades and sub-clades; the bootstrap val- ues for these clades and sub-clades ranged from 1 to 100, indicating a high heterogeneity in this population. Further, the clustering system did not correlate with the type of sample or its lo- cation. For example, A. flavus isolate tUth157 (isolated from cattle feedstuff sample collected from Makkah) clustered together with isolate tUth63 (isolated from poultry feedstuff sample collected from taif) in one sub-clade with a 98 bootstrap value. Additionally, isolates tUth154 and tUth193 constituted one sub-clad with a 69 bootstrap value, although the first one was table 4 - Origin and genetic patterns aflatoxigenic Aspergillus parasiticus isolates collected from feedstuff samples. values in brackets are percentages of the total samples analyzed. Sample name No isolates Complete set Three bands Two bands Poultry 7 6 1 - Camel & cattle 2 2 - - Cattle 8 6 1 1 Total 17 (100) 14 (82.4) 2 (11.8) 1 (5.9) Ital. J. Food Sci., vol. 28 - 2016 39 Fig. 2 - Phylogenetic tree based on the internal transcribed spacer (Its) region of rrNA of aflatoxigenic and non aflatoxigen- ic 64 isolates of Aspergillus flavus. the tree was constructed by neighbor-joining algorithm using maximum composite like- lihood model. bootstrap factors less than 55 were not shown. the tree was rooted with Aspergillus niger [hE649376] as the out-group. red rods indicated non aflatoxigenic isolates. isolated from poultry feedstuff samples from Makkah and the second from cattle and camel feedstuff samples from Jeddah (Fig. 2 and ta- ble 1). therefore, clustering according to the Its sequencing results did not indicate any re- lationship among the isolate clustering system and their geographical distributions and even the sources of isolation. Aflatoxigenic isolates spread all over the constricted phylogentic tree without separation of the clades into toxigen- 40 Ital. J. Food Sci., vol. 28 - 2016 Fig. 3 - the hierarchical cluster analysis using average linkage between groups form Aspergillus flavus isolates based on am- plification of aflatoxins biosynthesis genes. red bars indicated non aflatoxigenic species. ic and non-toxigenic. For example, aflatoxigen- ic isolate tUht189 clustered with non-aflatoxi- genic isolate tUht187 with a 96 bootstrap value (Fig. 2). since the clustering system was based on Its sequencing results, with non-function- al spacers, there is no correlation between clus- tering system and toxin production. the present results show that isolates identified as A. flavus had a polyphyletic origin, supporting the genet- ic heterogeneity of A. flavus as previously dem- Ital. J. Food Sci., vol. 28 - 2016 41 onstrated by other studies (GEIsEr et al., 1998, 2000; vAN DEN brOEk et al., 2001; chANG et al., 2007; GONcAlvEs et al., 2012). the genetic diversity among A. flavus iso- lates was studied using the results of amplifi- cation of some aflatoxin biosynthesis genes. the results were subjected to hierarchical cluster analysis using average linkage between groups to construct a dendrogram showing the corre- lation between the isolates (Fig. 3). A. flavus isolates did not follow any rule in their cluster- ing system. For example, aflatoxigenic isolates tUht121 and tUht126 (isolated from a poul- try feedstuff sample collected from Makkah) and non-aflatoxigenic isolates tUht85 (poul- try feedstuff samples from taif) and tUht98 (camel and cattle feeds from taif) were clus- tered together as shown in Fig. 3. On the con- trary, tUht112 (horse feeds from Jeddah), tUht160 (poultry feeds from Jeddah), tUht87 (poultry feeds from taif), and tUht104 (poul- try feeds from Makkah) were non-aflatoxigenic isolates clustered together (Fig. 3). Generally, these results indicate that the presence or ab- sence of Pcr products for the targeted aflatoxin biosynthesis genes was not correlated with the type of feedstuff or the location of sample col- lection. Previous authors (GEIsEr et al., 1998, 2000; MOOrE et al., 2009, GONcAlvEs et al., 2012), found that the aflatoxin cluster genes were useful tools for phylogenetic studies in the section Flavi. 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Yu J., chang P.-k., bhatnagar D. and cleveland r.E. 2000. cloning of a sugar utilization gene cluster in Aspergil- lus parasiticus. biochem. biophys. Acta. 1493: 211-214. Paper Received November 29, 2014 Accepted March 3, 2015 http://www.ncbi.nlm.nih.gov/pubmed?term=Wheeler KA%5BAuthor%5D&cauthor=true&cauthor_uid=8110599 http://www.ncbi.nlm.nih.gov/pubmed?term=Tanboon-Ek P%5BAuthor%5D&cauthor=true&cauthor_uid=8110599 http://www.ncbi.nlm.nih.gov/pubmed/8110599