Eur Food Res Technol (2003) 216:185–192 DOI 10.1007/s00217-002-0642-7
O R I G I N A L PA P E R
Tim Reuter · Karen Aulrich
Investigations on genetically modified maize (Bt-maize) in pig nutrition: fate of feed-ingested foreign DNA in pig bodies
Received: 15 July 2002 / Revised: 10 October 2002 / Published online: 26 November 2002 © Springer-Verlag 2002
Abstract The passage and fate of ingested DNA in 48 pigs fed with diets containing (n=12) parental or (n=36) transgenic (Bt) maize were examined. Pigs were fattened from an initial live weight of 24 kg to approximately 108 kg. Animals fed transgenic maize were slaughtered in groups (n=6) 4, 8, 12, 24, 48 and 72 h after feeding the last maize-containing diet. Those slaughtered at up to 12 h received no further feed, while those held for longer prior to slaughter received a diet in which maize was replaced by barley and wheat. Control animals were slaughtered at 4 and 8 h. DNA extracted from tissues and gut contents was examined by PCR for the presence of plant DNA and for any transgenic material. Recombinant DNA was detectable in the intestinal contents up to 48 h after the last feeding of a diet containing the transgenic maize. PCR amplification of plant gene spacers produced fragments of different sizes, dependent on feed source. The feed source of rectum samples depended on individual passage rate in the groups and their restriction analysis showed grain species-specific patterns. Recombinant or maize-specific DNA was not detectable in tissue samples of pigs. In contrast, plant DNA fragments were detectable in the investigated pig tissues. Keywords GMO · Pig · Bt maize · Bt corn · PCR · DNA transfer · passage rate
Introduction Genetic engineering of agriculture crops, and especially the development of Bt transgenic plants, has engendered a wide range of opinions concerning the economic value, and ecological, food safety and social consequences of their introduction [1, 2]. Major controversy exists about T. Reuter (✉) · K. Aulrich Institute of Animal Nutrition, Federal Agricultural Research Centre Braunschweig (FAL), Bundesallee 50, 38116 Braunschweig, Germany Phone: +49 531 596 3122, Fax: +49 531 596 3199 e-mail:
[email protected]
the relative benefits and risks of introducing genes into species where this would not be possible using conventional breeding [3, 4]. To estimate unexpected effects, safety management procedures are demanded and proposed by several authors [5, 6, 7] and the Commission of the European Communities (CEC) [8]. The introduction of genes, such as genes from Bacillus thuringiensis (Bt) encoding the delta-endotoxin, into the genomic DNA of pest-protected plants carries the risk of unexpected effects. Therefore, studies on the fate of DNA in men and animal seem to be necessary. DNA is a stable molecule that can survive extreme environmental conditions and has been detected in organic remnants after thousands of years [9, 10]. Forbes et al. [11] tested the stability of DNA present in plants used as feed-stuff for farm animals. They examined the effects of grinding and milling, heat treatment and steam pressure on commercial feed sources (all non-genetically modified). Different grinding treatments were without any detectable disruption of plant DNA. They concluded that only heat treatment with temperatures between 100 °C and 150 °C caused a disruption of plant DNA, resulting in fragments smaller than 100 base pairs (bp). Chiter et al. [12] also investigated the physical and chemical conditions necessary to ensure sufficient fragmentation of DNA (especially from genetically modified material) in plant tissues to make it unlikely that it could be stably transferred to strains of the bacterial gut microflora. They also concluded that temperatures above 95 °C and pressurised steam are necessary to fully degrade genomic DNA. Without processing, the feedstuff of farm animals contains plant DNA fragments of greater size than 21 kbp. In living organisms, the gastrointestinal tract (GIT) is constantly exposed to foreign DNA by the incessant flow of partly or completely digested feed components [13]. Feed-ingested foreign DNA is not completely degraded in the gastrointestinal tract of mammals and poultry. Extensive investigations [13] showed that ingested purified phage M13 DNA survives transiently in the gastrointestinal tract and enters the bloodstream of mice. In a fur-
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ther investigation [14], pregnant mice were given M13 DNA by an oral route. Fragments up to 830 bp of the M13 DNA could be detected in various tissues of foetuses and the newborn animals. Subsequently, Hohlweg and Doerfler [15] chose a more representative feed material (soybean leaves) and followed the fate of a plant-specific gene fragments (rubisco) in mice. Rubisco or fragments of it could be recovered in the intestine 2 h after ingestion and for a further 47 h and in the caecum up to 121 h after ingestion. Furthermore, rubisco gene fragments could be amplified from liver and spleen extracts using polymerase chain reaction (PCR) [16, 17]. After feeding conventional or transgenic feedstuff to target farm animals, Einspanier et al. [18] verified the presence of DNA fragments of 199 bp derived from plant chloroplasts in different tissues of the animals investigated. However, the Bt gene could not be detected in any of these samples. Aeschbacher et al. [19] also followed the fate of feed ingested Bt-maize DNA in broilers and detected a maize-specific gene fragment (226 bp) in the crop and gizzard as well as in liver, spleen and meat. The Bt gene, or rather a fragment of this gene (479 bp), could only be detected in the crop of broilers from the Bt group and not in any other samples. Examining the fate of ingested DNA in pigs as target farm animals, short chloroplast DNA fragments (199 bp) could be successfully amplified from the content of the small intestine up to 12 h after last feeding. Chloroplast-specific DNA, however, was not found in any investigated pig organ or tissue [20]. The authors concluded that these findings make the assumption of a DNA transfer through the epithelia cell layer of the GI tract into the blood stream for porcine species very unlikely. This is in contrast to the equivalent studies made with poultry and cattle where feed-derived DNA fragments have been detected in a variety of tissues. Consequently, there is a need for additional data on the fate of plant DNA (and transgenic DNA) in pigs to resolve this apparent discrepancy. In the present study, pigs were fed with diets containing parental or transgenic Bt-maize in order to investigate the degradation of nucleic acids in the digestive tract, the passage of DNA in the gut as well as the DNA transfer through the intestinal mucosa. The present paper is part of a comprehensive study to investigate: ●
●
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Composition of parental and transgenic (Bt) maize grains [21] Digestibility and nutritional value of both maize lines in pigs [21] Grower-finisher performance of pigs fed with diets rich in parental or transgenic maize [22]
Material and methods Maize and diets Diets were based on the parental maize line (Prelude) or a genetically modified maize line (NX6262) expressing the Bt trait [23], both derived from the parental line (Zea Mays L Line CG
Table 1 Composition of diets1 for pigs [%] Ingredients
Grower
Finisher3
Maize (parental or transgenic)1 Peas Potato protein Fish meal Wheat bran Mineral/vitamin premix2 Rape seed oil Lysine monohydrochloride DL-Methionine L-Threonine L-Tryptophan Ca-Carbonate
70.00 10.00 5.50 5.50 5.25 2.50 0.75 0.30 0.10 0.05 0.05 –
70.00 17.05 2.50 2.50 5.00 2.00 0.50 0.15 0.05 – 0.05 0.20
1 Composition of maize and diets, crude protein and energy contents, see [21] 2 Per kg premix: 245 g Ca, 60 g P, 55 g Na, 10 g Mg, 400 000 I.U. vit. A, 40 000 I.U. vit. D3, 1200 mg vit. E, 37.5 mg vit. B1, 100 mg vit. B2, 100 mg vit. B6, 750 µm B12, 52.5 mg vit. K3, 500 mg nicotinic acid, 337.5 mg pantothenic acid, 5000 mg choline chloride, 4000 mg Fe, 1000 mg Cu, 2000 mg Mn, 4000 mg Zn, 50 mg J, 15 mg Se 3 For maize-free diets: maize replaced by 45% barley and 25% wheat
00256–176). For the feeding study, two grower diets and two finisher diets were produced [22]. The diets were fed dry as ground particles with an approximate size of 1 mm. The grower and finisher diets contained 70% of the parental or the transgenic maize. The complete composition of the diets is shown in Table 1. The two diets (grower and finisher) containing the parental maize line were produced separately to exclude contamination with the genetically modified Bt-maize. The components of diets were selected to exclude influences by other possible genetically modified feedstuff components like soybeans. For this reason, potato protein and fish meal were used as a protein source. Rape seed oil was mixed into the diets to minimise dust from feedstuff in the pens. To exclude intermixing during the daily preparation of diets containing parental or transgenic maize, the diets were weighed and filled in separate storerooms. Animal experiments Forty eight female pigs with an initial body weight (BW) average of 23.9±3.0 kg were allotted to single boxes. The pigs were randomly divided into two groups. Twelve pigs (control group) were fed the parental maize diets and 36 pigs (Bt group) were fed the transgenic maize. The grower diet was fed until a BW of 80 kg, thereafter, the finisher diet was fed until slaughter. The capacity of the pigpen was 96 single boxes with 24 boxes in four rows. To exclude the possibility of contact between pigs of the control and Bt group, one row of boxes was kept free between treatment groups. Slaughtering The pigs from the control group were slaughtered with a final BW of 103.4±8.3 vs. 111.5±7.9 kg the pigs from Bt group. The control group was slaughtered first to exclude contaminations of the samples for DNA investigations with the tissues of pigs from the Bt group. To investigate the fate of feed-ingested DNA at selected times after feed intake, the pigs were divided in different groups. The 12 animals in the control group were split into two groups of six animals and the Bt group into six groups, each of six animals. The two control groups and two groups of the six Bt groups were slaughtered 4 and 8 h after morning feeding. The pigs from the re-
187 Table 2 Primer oligonucleotides Name
Sequence (5’–3’)
Amplicon
Specify
Reference
Plant 1-F Plant 1-R SW 01 SW 02 Ivr 1-F Ivr 1-R Rub 01 Rub 02 Cry 03 Cry 04
cga aat cgg tag acg cta cg ggg gat aga ggg act tga ac tca gtt tac act cac ctg ata gca tct ggg tgg tgg aga ggg gtg aat t ccg ctg tat cac aag ggc tgg tac c gga gcc cgt gta gag cat gac gat c ctt ggc agc att ccg agt a cct ttg taa cga tcaa gac tgg ctc tcg ccg ttc atg tcc gt ggt cag gct cag gct gat gt
532bp 642bp 108bp
Maize gene1 Barley gene1 Porcine growth hormone gene
[24] [26]
226bp
Maize invertase gene
[27]
140bp
Chloroplast gene
Aulrich2
211bp
Synthetic Bt gene
[29]
1 Chloroplast gene with plant-specific 2 Personal communication
product size
maining Bt groups were slaughtered 12, 24, 48 and 72 h after the last feeding of a maize-containing diet. The pigs from the groups slaughtered at 24, 48 and 72 h after last feeding a transgenic maize-containing diet were fed a diet in which the maize was replaced with 45% barley and 25% wheat. Where possible, all equipment used in the slaughtering process was heat sterilised. Other materials sensitive to heat were stored in ethanol (70% v/v C2H5OH). Facilities and work benches in direct contact with pig tissues or faeces were cleaned with ethanol and covered with single-use synthetic foils. Every tool was used only for one single sample. During the whole slaughtering process, all assistants wore single-use gloves replaced after each sample had been taken. The samples were placed into tubes (50 mL, Sarstedt, Nümbrecht, Germany) and rapidly frozen at –21 °C. Samples were taken from organs and tissues (blood, liver, spleen, kidney, lymphatic glands, ovary, musculus longissimus dorsi, musculus trapezius and gluteus maximus) and contents of GIT (stomach, duodenum, jejunum, ileum, caecum, colon and rectum). Extraction of DNA Samples of both maize lines and diets containing parental or transgenic maize were extracted for total DNA using the commercial kit NucleoSpin Plant (Macherey-Nagel, Düren, Germany). Additionally samples of both maize lines were extracted in parallel for total DNA, after fine grinding using liquid nitrogen and pestle and mortar. Tissue samples (25 mg solid samples, 200 µL blood) and contents of the small and large intestine (200 mg) were extracted for total DNA using two commercial kits (NucleoSpin Tissue, Macherey-Nagel, Düren, Germany and QIAamp DNA Stool Mini Kit, Qiagen, Hilden, Germany). The extractions were performed according to the manufacturer’s manual. The DNA concentration of solutions was determined by measuring the UV absorption at 260 nm. The extracted DNA was characterised by its 260/280 nm UV absorption ratios (Spectrophotometer: UV4, ATI UNICAM, Kassel, Germany) and by agarose gel electrophoresis. Oligonucleotide primers The primers (Table 2) were synthesised by MWG Biotech, Ebersberg, Germany and diluted with an appropriate volume of water to a final concentration of 50 pmol/µL and stored at -21 °C until use. Five primer pairs framing specific target sequences (Table 2) were used. The primer Plant1 F/R [24] frames a fragment from the chloroplast gene of plants with slightly differing product size dependent on biological origin [18]. The sequences of the chloroplast genes have been published in the NCBI GenBank data base (for maize: accession no. V00178 [25] and for barley: accession no. AF280795). The primer pair SW01/02 [26] was used to confirm the feasibility of PCR amplification of the extracted DNA from
tissues and frames a fragment of the porcine growth hormone gene. The primer pair Ivr1F/R [27] frames a fragment of the maize-specific invertase gene (accession no. U16123 [28]). The Ivr1F/R primer was chosen to monitor, beside the fragment of the genetic modification (primer Cry 03/04), an additional single copy gene with similar product size. The primer pair Cry 03/04 [29] frames a cross border sequence (calcium-dependent protein kinase promotor and cry1A(b)) in transgenic maize. The primer pair Rub 01/02 is a universal plant primer framing a fragment from the chloroplast (rbcL) gene encoding ribulose bisphosphate carboxylase (for maize: accession no. Z11973). PCR conditions PCR was done with 2 µL of extracted DNA (approximately 50 ng), 5 µL of 10×PCR buffer, 1 µL of dNTP mix (40 pmol/µL, Hybaid, Ulm, Germany), 1 µL of each primer (50 pmol/µL), 40 µL water (Eppendorf, Hamburg, Germany) and 0.2 µL (1 unit) of DNA polymerase (HotStarTaq, Qiagen, Hilden, Germany). HotStarTaq is provided in an inactive state with no polymerase activity at room temperature until an activation step (15 min, 95 °C) is taken. The cycler (T-Gradient, Biometra, Göttingen, Germany) conditions were an initial activation step at 95 °C for 15 min, 40 cycles consisting of denaturation at 95 °C for 30 s, primer annealing for Cry 03/04 at 63 °C for 30 s, for SW 01/02 at 54 °C for 40 s, for Rub 01/02 at 54 °C for 40 s, for IVR 1F/R at 64 °C for 30 s and for Plant1 at 54 °C for 40 s and followed by an extension step at 72 °C for 40 s and a final extension step at 72 °C for 3 min. The annealing temperatures were optimised for each primer system by using the cycler gradient function. Agarose gel electrophoresis Extracted DNA solutions (8 µL) or PCR amplifications were electrophoresed on agarose gel at constant voltage (50 V) in the TBEbuffer (89 mmol/L Tris, 89 mmol/L boric acid, 2 mmol/L EDTA). The agarose concentrations were 1% (for extracted DNA solution) or 2% (for PCR mixture) in TBE-buffer containing 0.5 µg/mL ethidium bromide. To evaluate the size of DNA or DNA fragments three DNA size markers (III, VIII and IX, Roche, Mannheim, Germany) were used. The gel was photographed with the BioDoc Analyze System (Biometra, Göttingen, Germany). Restriction enzyme digestion The PCR products were digested with HaeIII, for the Bt gene, with RsaI for the rubisco gene and with AluI for the grain species-specific genes. Restriction enzymes were supplied by MBI Fermentas, St. Leon-Rot, Germany. Digests were made by mixing 10 µL of
188 PCR product with 2 µL of ×10 restriction enzyme buffer, 1 µL of restriction enzyme (10 unit/µL) and 10 µl of water. The mixture then was incubated at 37 °C for 3 h and the reaction was stopped by thermal inactivation for AluI and RsaI at 65 °C and for HaeIII at 80 °C for 20 min. Restriction enzyme digestion products were visualised on 2% agarose gels. Southern analysis Additionally, the specificity of amplified Bt gene fragments as well as the amplified rubisco gene fragments was verified by southern analyses using 32P-labelled specific probes. Size-fractionated DNA was transferred onto nitrocellulose membranes by capillary blotting followed by hybridisation. Specific signals were detected using a P-imager (BioRad, Germany).
Results DNA extraction The measured ratio (A260/280) of the extracted DNA samples was between 1.7 and 1.9. Results of agarose gel electrophoresis are shown in Fig. 1. Extracted DNA from maize and diets, as well as maize additionally pre-treated with liquid nitrogen, was around 21 kbp (Fig. 1 A). No significant differences could be observed between the DNA extracted from the various compartments of the GI tract of single pigs or between single segments of the GIT from all pigs. Most DNA fragments were in the range below 21 kbp down to 5 kbp, with a minor fraction consisting of fragments from 5 kbp up to 500 bp and less (Fig. 1B. DNA, regardless of where in the GI samples were taken, was always substantially smaller (
