INCIDENCE OF GENETICALLY MODIFIED SOYBEAN AND MAIZE AS ANIMAL FEED IN EGYPT
M. A. Tony*+, J. Zagon**, H. Broll**, F. F. Mohamed*, B. M. Edrise*,
S. A. Awadalla*, M. Schauzu*, K. W. Bögl*
*Department of Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Cairo University, 12211 Giza, Egypt, **Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV), Thielallee 88 - 92, D-14195 Berlin, Germany
+Present address: Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV), Thielallee 88 - 92, D-14195 Berlin, Germany
As a consequence of rapid progress in agricultural biotechnology, more and more crops and products derived from genetically modified organisms (GMO’s) have entered the food and feed chain in recent years. In Europe a regulatory framework controls all aspects of placing on the market of products from GMO. Although Egypt depends mainly on imported food and feed, there is no comparable legislation to control material of GMO origin. Soybean and maize play an important role in both, monogastric and ruminant nutrition, and are mainly imported for both livestock and human nutrition in Egypt. The aim of our study was to monitor the incidence of Roundup Readyä soybean (RRS) and the genetically modified maize lines Bt176, Bt11, T25, Mon810 and StarLinkä in Egypt, all of which are widely spread and approved for use as food and/or feed in the US and other countries. Fifty-one soybean samples and sixty-one maize samples have been randomly collected from different localities in Egypt. A key factor in controlling such novel foods and feeds is the availability of methods to distinguish between genetically modified material and their traditional counterparts. The detection techniques applied here is based on Polymerase Chain Reaction (PCR) using the validated, official detection methods according to Article 35 of the German Federal Foodstuffs Act. The results of our survey illustrate that all Argentinean soybean samples so as 50% of American soybean samples were contaminated with RRS. With respect to maize samples imported from USA, 80% contained Bt176, 85% Bt11, 60% MON810, 95% T25 and 45% StarLinkä maize. Furthermore, in maize samples imported from Argentine 57% revealed Bt 176 and MON 810, 71% T25, 85% Bt 11 and 28% StarLinkä. In contrast to this all local breeds from both, soybean and maize, were non-transgenic crops.
Key words: Genetically Modified Feed, Soybean, Maize, Egypt
Biotechnology offers enormous potential gains for the world's agriculture, including the production of higher yields with decreased amounts of herbicides and pesticides. It offers a tempting new tool to assist plant breeders not only to introduce resistance to insects and diseases, but also traits sustaining harsh environmental conditions. Additional goals are to improve the quality of seed proteins during storage , sparing fertiliser by nitrogen retention or to increase the nutritive value e.g. by manipulating the free carbohydrate content in forages  or production of low-phytate maize . Practical examples for modified feed crops from the nutritional viewpoint are soybeans expressing fungal phytase (an enzyme that catalyses the release of phosphate from plants) to ameliorate the bioavailability of phosphorus in chickens and pigs . Also a brazil nut protein was introduced into soybean to increase the content of the essential amino acid methionine . Although this soybean line was never commercialised, the latter example shows that new traits may also give rise to special risks: the introduction of a gene into a plant may create a new or non-expected allergen thus causing adverse reactions in susceptible individuals. Another point of scientific debate is the possibility of gene flow to non-target species by cross-breed or transformation. Against this background the migration of genes coding for antibiotic resistance from GM crops into pathogenic bacteria via the gut flora has been discussed.
To date in the European Union a wide regulatory framework controls multiple aspects from the deliberate release of GM crops and seeds to the final food product. The principal legislation related to food is the European Regulation on novel foods and novel food ingredients (EC) No. 258/97 that entered into force in May 1997. Apart from a thorough safety evaluation, Europe now requires mandatory labelling of GM foods. Furthermore the EC established a 1% threshold for unintended contamination of unmodified foods with approved GMO. However, up to now no labelling is required for animal feeds. A comparable law for animal feeds derived from GMO actually is in preparation.
In Japan a threshold of 5% for frequently used GM crops was implemented. Aside from labelling, the Ministry of Health and Welfare announced that health testing of GM is required. In the United States, GM crops or products must not be labelled. However GMO plant varieties must get through a consulting procedure. The responsible competent authorities in the USA are the Centre for Food Safety and Applied Nutrition (CFSAN), the Centre for Veterinary Medicine (CVM) and the Food and Drug Association (FDA). Based on information provided by the applicant the safety of the new product is evaluated prior to marketing.
Today the most common transgenic crops over the world are soy and corn, which represent the main sources of protein and energy respectively for livestock. Although Egypt mainly depends on imported soy and maize, the control and evaluation of these crops only depends on its nutrient content and the acceptable level of mycotoxins without paying any attention to genetically manipulation. As a result of lacking controls, there is no idea about the presence or absence of GM crops for both human and/or animal consumption in Egypt. Our work aimed to monitor the incidence of genetically modified soybean and maize in Egypt especially used for animal nutrition purposes. To achieve this goal, fifty-one soybean samples and sixty-one maize samples have been randomly collected from different localities in Egypt and subjected to detection techniques based on Polymerase Chain Reaction (PCR) using the official detection methods according to Article 35 of the German Federal Foodstuffs Act.
Fifty-one soy samples and sixty-one maize samples were randomly collected from different localities and governments in Egypt throughout the years 2000 / 2001 as tabulated in tables (3) and (4). The soybean samples divided into 28 samples of soybean seeds - from which 27 samples were local Egypt breeds and one sample of US origin - and 22 samples soybean meals which were imported from either USA or Argentine. Additionally 1 sample of full fat soybean was collected from the Egyptian market. Unless not other specified in Tab. 3 soybean meals contained 44% crude protein. The maize material under investigation (Tab. 4) predominantly consisted of whole grains. 33 samples were maize grains from Egypt, 24 samples were imported grains from USA or Argentine and 4 additional samples consisted of corn gluten. One of the corn gluten samples was produced in Egypt whereas the other 3 samples were imported from the USA.
Certified reference materials (CRM), produced by the Institute for Reference Material and Measurements (Geel, Belgium) were used as negative and positive controls (1 or 5 % GMO). Because there is no CRM available for maize lines MON810 and T25, samples containing 1% GMO were in-house prepared from these lines and used as positive control. For StarLink maize the positive control was provided with the commercial detection kit used.
Extraction and purification of genomic DNA
Soy and maize samples were ground in an electric grinder. 200 mg of the resulting flour as well as 200 mg from the CRM were used for the extraction of the genomic DNA by the cetyltrimethylammonium bromide method (CTAB) according to [6, 7]. From each sample two independent extractions were performed. In addition to 200 µl of deionized water was used as a blank sample and subjected to extraction and further treated in the same way as samples to control the reagents used and the procedure of work. The extracted DNA pellet was air dried under vacuum and the pellet resuspended in 50 µl deionized water (Fluka, Germany). The concentration of the isolated DNA were measured fluorometrically using Dynaquant 200 system fluorimeter (Hoefer) according to the manufacturers instructions. The DNA concentration were adjusted by dilution using deionized water to 20 - 25 ng / µl prior to PCR.
Oligonucleotides primers used and PCR conditions
Primers used in this study are listed in table (1). All primers were synthesised by TIB MOLBIOL, Berlin-Germany and obtained in a lyophilized state.
For detection of StarLink maize (Aventis) a commercial kit purchased from GeneScan Europe, Freiburg, Germany (GMO/Ident Kit StarLinkTM maize) was used.
DNA amplification and PCR condition
PCR was carried out on a Gene Amp. PCR system 2400 (perkin Elmer, Germany). For each series, a master mix was prepared. Each PCR reaction mix (25 µl total volume) contained 2.5 µl PCR buffer (10x concentrate, Perkin Elmer), 2 µl Mgcl2 solution (25 mM Mgcl2), 1µl dNTP solution 0.2 mM each of dATP, dCTP, dGTP and dTTP, 0.5 µM of each primer, 1 Unit AmpliTaq Gold polymerase (Perkin Elmer), 2 µl of template DNA and completed to 25 µl with purified water. Table (2) explains the time/temperature profiles used in PCR. All amplicons were stored at 4°C until gel electrophoresis.
Amplicons together with 50 bp DNA marker (Gibco BRL, USA) were separated on 2% W/V Agarose LE (Roche) gels. The amplicons were made visible by ethidium bromide staining and documented using UV transillumination (254 nm) with a Phoretix workstation (Biostep, Germany).
RESULTS AND DISCUSSION
Amplifyability of prepared sample DNA
The primer pair GM 03 / GM 04 is specific for the single copy lectin gene LE1 and yields a PCR product of 118 bp  size. It is detectable in transgenic as well as in conventional soy bean (soy specific primer pair). The primer pair Ivr1-F / Ivr1-R is specific for the invertase gene and flanks part of exon number 3 of this gene. It gives rise to a 226 bp amplicon . This product is detectable in transgenic, as well as in conventional maize (maize specific primer pair). Soy and maize specific primer pairs served as a control for the amplification of the isolated DNA and PCR procedure (PCR quality control). All tested samples gave positive results (not shown) with the amplification control primer pairs.
The primer pair p35s-f2 / petu-r1 is specific for the genetic modification in Roundup ReadyTM soybean and amplifies a 172 bp segment . The primer pair attaches to the CaMV35 S promoter sequence and the petunia hybrida chloroplast transit-signal sequence. The amplicon is only detected in transgenic samples and GMO containing CRM as presented in the example in Fig.1.
For the specific identification of transgenic maize Event 176 by PCR the primer pair cry 03 / cry 04 was used. The resulting sequence of 211 bp size is amplified from a genomic region between two adjacent genetic elements, namely the CDPK promoter and the N-terminus of the synthetic cry IA (b) gene . This 211 bp amplicon appears only in transgenic maize samples, as well as GMO containing CRM (Fig.2).
Primer pairs IVS2-2 / PAT-B were used for the detection of the transition site from the intron IVS2 into the Pat-gene in BT11 maize. The bacterial PAT gene codes for the enzyme phosphinotricine N-acetyl transferase giving rise to the resistance of Bt11 maize to the herbicide phosphinotricine . Primer pair T25-F7 / T25-R3 is used for the detection of the transition site between the CaMV-terminator into the PAT gene in T25 maize. Primer pair VW01 / VW03 flanks the transition site from the genomic maize DNA into the CaMV- Promotor in MON810 maize  thus representing an event specific detection system. Fig. 3, Fig.4 and Fig.5 are showing the results obtained for maize lines Bt 11, MON 810 and T25. Positive samples as well as positive controls revealed amplicons of the the expected size of 189, 170 and 209 bp respectively, while the negative control and negative samples gave no amplification product after PCR. For the detection of StarLink maize the commercial kit was used. An amplicon of 133 bp is specific for the presence of DNA from genetically modified StarLink maize. It does not occur in negative samples (Fig.6).
Tables (3) and (4) summarise the results of the examined soy and maize samples respectively and the location where the samples have been collected in Egypt. Obviously all 27 locally bred soybean samples are negative in PCR when using primer pair p35s-f2 / petu and hence do not contain any genetically modified (Roundup ReadyTM ) soybean material (Tab. 3). In contrast to this, out of 23 imported soy samples from USA (10 samples) or Argentine (13 samples) only 5 turned out to be free from Roundup ReadyTM soybean. All of these were of USA origin which means that half of the US soybean samples investigated contained GMO. All 13 samples imported from Argentine (100%) were tested positive for Roundup ReadyTM.
Table (4) demonstrates the results of PCR for the maize samples examined. All native breeds cultivated in Egypt (33 grain samples) were negative to all primers used in this study which means that there is none of the five GM maize lines investigated present. On the other hand, all imported maize samples collected from the Egyptian feed market were tested positive for GMO maize. With respect to maize samples imported from USA, 80% contained Bt176, 85% Bt11, 60% MON810, 95% T25 and 45% StarLink maize. Furthermore, in maize samples imported from Argentine 57% Bt 176 and MON 810, 71% T25, 85% Bt 11 and 28% StarLink were found. Nearly all GMO-positive samples contained more than one GM maize line simultaneously. Four samples even contained a mixture of all five GM maize lines investigated. From these, one sample was from Argentine and three imported from the USA. The pattern of the distribution of the GM maize lines among the imported samples was considerably inhomogeneous thus indicating different lots taken from the sampling localities.
The results clearly show that imported maize and soybean intended for animal feed on the Egyptian market contains GMO to a high degree including mixtures of several lines. On the other hand, all local Egyptian breeds were free from GMO with exception of one corn gluten sample which was produce of Egypt but probably made from imported seeds. Since no quantitative analysis was carried out, the absolute percentage of each GM line in the samples was not determined. However, the primary aim of this investigation was to present a first overview on the situation using highly sensitive, reliable methods that are capable to detect even trace amounts. The positive controls used in this study contained 1% -5% GMO (Fig. 1-6). However, with respect to the official German detection method used here even 0.1 % GMO mixed with non-GMO is detectable . All GMO examined here have been authorised in other countries of the world and thus passed a safety evaluation. Nevertheless it cannot be excluded, that also non-approved GM breeds are going to enter in particular uncontrolled markets. Therefore qualitative, sensitive methods as used here would be well suited for monitoring programmes. The urgent need to control
feed and food for the presence of GMO is underlined by the example of StarLink maize. This GM maize line, produced by Aventis Crop Inc., has been assessed for animal feed use exclusively in the USA. Recently it entered the food chain unintentionally although it was suspected to reveal allergenic potential for humans. It is still unknown, how much material was distributed or exported to other countries. It should be outlined, that 11 samples of maize taken from the Egyptian feed market also contained StarLink. Egypt and other importing countries need to control GMO foods and feed in their market to protect both, human and animal health as well as to protect the environment and local agricultural breeds.
This study is part of the Ph.D. study under Egyptian-German Channel Programme funded by German Academic Exchange Service (DAAD).
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