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Assessing the possibility of genetically modified DNA transfer from GM feed to broiler, laying hen, pig and calf tissues

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The aim of this study was to assess the possibility of genetically modified DNA transfer from feed containing RR soybean or/and MON810 maize to animal tissues, gut bacterial flora, food of animal origin, and the fate of GM DNA in the animal digestive
  DOI 10.2478/pjvs-2013-0061 Original article  Assessingthe possibility of genetically modifiedDNA transfer from GM feed to broiler, layinghen, pig and calf tissues Z. Sieradzki 1 , M. Mazur 1 , K. Kwiatek  1 , S. Świątkiewicz 2 , M. Świątkiewicz 2 , J. Koreleski 2 , E. Hanczakowska 2 , A. Arczewska-Włosek  2 , M. Goldsztejn 1 1 Department of Hygiene of Animal Feedingstuffs, National Veterinary Research Institute, Al. Partyzantów 57, 24-100 Puławy, Poland 2 Department of Animal Nutrition and Feed Sciences, National Research Institute of Animal Production,32-083 Balice n. Kraków, Poland  Abstract The aim of this study was to assess the possibility of genetically modified DNA transfer from feedcontaining RR soybean or/and MON810 maize to animal tissues, gut bacterial flora, food of animalsrcin, and the fate of GM DNA in the animal digestive tract. The experiment was carried out onbroilers, laying hens, pigs and calves. All animals were divided into four groups: I – control group(non-modified feed), II – GM soybean group (non-modified maize, RR soybean), III – GM maizegroup (MON810 maize, non-modified soybean), and IV – GM maize and soybean group (MON810maize, RR soybean). Samples of blood, organs, tissues, digesta from the gastrointestinal tract, andeggs were analysed for the presence of plant species specific genes, and transgenic sequences of CaMV 35S promoter and NOS terminator. PCR amplifications of these GM sequences were conduc-ted to investigate the GM DNA transfer from feed to animal tissues and bacterial gut flora. In noneof the analysed samples of blood, organs, tissues, eggs, excreta and bacterial DNA were plant refer-ence genes or GM DNA found. A GM crop diet did not affect bacterial gut flora as regards diversityof bacteria species, quantity of particular bacteria species in the animal gut, or incorporation of transgenic DNA to the bacteria genome. It can be concluded that MON810 maize and RR soybeanused for animal feeding are substantially equivalent to their conventional counterparts. Geneticallymodified DNA from MON810 maize and RR soybean is digested in the same way as plant DNA, withno probability of its transfer to animal tissues or gut bacterial flora. Key words :  GMO, maize, soybean, feed, transfer Introduction The first generation of genetically modified (GM)plants is widely used across the world as food andfeed. Following commercial release in 1996, the pro- Correspondence to: Z. Sieradzki, e-mail: portion of GM plants has grown rapidly, and in 2012170.3 mln hectares of genetically modified crops weregrown globally (James 2012). GM soybean has con-tinued to be the principal GM crop since 1996, oc-cupying, in 2012, more than 80 million hectares,  Polish Journal of Veterinary Sciences Vol. 16, No. 3 (2013), 435–441 Unauthenticated | 89 66 137 210Download Date | 2/7/14 8:59 PM  followed by GM maize (around 55 million hectares),cotton and canola. GM maize MON810 is one of thetransgenic maize varieties that can be grown in theEU. This maize has been modified by integration of a gene isolated from the  Bacillus thuringensis  (Bt) soilbacterium, in order to express the insecticidal Cry1 A(b) protein, which confers insect resistance to theEuropean corn borer ( Ostrinia numbilatis ), the pri-mary corn pest in Europe. Genetically modifiedsoybean is imported to EU countries as the main pro-tein source for animal feeding. Roundup Ready TM (RR) soybean (in EU registered as event GTS 40-3-2)is the dominant GM soybean variety grown globally.RR soybean is herbicide tolerant and has been devel-oped by introducing a gene isolated from the  Agrobac-terium  sp. CP4 strain of soil bacterium, expressing5-enolpyruvylshikimate-3-phosphate synthase inplants. This enzyme confers glyphosate toleranceupon plants, glyphosate being the active substance inherbicides. These two transgenic crops are mostlyused from among all GM crop varieties in animalfeeding in EU countries.To date, many articles about animal feeding stu-dies with GM crops, including RR soybean andMON810 maize, have been published (Aulrich et al.2001, Aulrich et al. 2002, Aumaitre et al. 2002, Reuteret al. 2003, Sanden et al. 2004, Deaville et al. 2005,Flachowsky et al. 2005, McCann et al. 2005, Rossi etal. 2005, Flachowsky et al. 2007, Sissener et al. 2009). All of these trials indicated that GM crops are safe foranimals, including poultry, pigs, cattle and fish. More-over, in all of these experiments there were no signifi-cant statistical differences in production indices, qual-ity of carcasses and food products of animal srcin, when the animals were fed diets containing conven-tional and GM crop varieties. Despite the number of studies with GM crops, the problem of GM feedraises many doubts and concerns, especially when in-terpretations of the test results contain suggestionsabout the negative impact of GM feed on the healthand safety of animals (Seralini et al. 2012). One of theproblems still raised by GM food and feed opponentsis the potential possibility of GM DNA transfer, ori-ginating from animals fed diets containing geneticallymodified crops, to animal gut bacterial flora and ani-mal tissues, then to animal products such as eggs,meat and milk.Therefore, the aim of this study was to assess thetransfer of genetically modified DNA from diets con-taining RR soybean or/and MON810 maize to animaltissues and food of animal srcin in a trial involvinga large number of animals of different species. More-over, much attention was paid to the aspect of GMDNA fate in the digestive tracts of poultry, pigs andcalves, with assessment of GM diet impact on bacteriaspecies diversity and incorporation of GM DNA se-quences to the bacteria genome. Materials and Methods Experiment design The experiment was carried out on broilers, layinghens, pigs and calves. Animals from each species weredivided into four groups: I – control group (non-modi-fied maize and soybean), II – GM soybean group(non-modified maize and genetically modified RRsoybean), III – GM maize group (genetically modifiedMON810 maize and non-modified soybean), and IV– GM maize and soybean group (genetically modifiedmaize MON810 and RR soybean). Maize grains con-sisted of GM maize plants expressing Cry 1A(b) pro-tein (MON810, YieldGard) and its non-modifiedisogenic parental line (DKC 3420). The GM soybeanRoundup Ready TM (GM event GTS 40-3-2) variety was used with its non-modified counterpart used ascontrol. In the case of control, the environmental con-ditions for growth of maize and soybean plants werethe same for both varieties. Broilers Six hundred and forty sexed Ross 308, one-day-oldanimals were obtained from a commercial hatchery.Food and water were available  ad libitum . Chickens were fed a mash, maize-soybean starter diet for thefirst 21 days and grower-finisher diet for the next 21days. All diets were formulated to meet nutrient re-quirements of growing broilers. Each treatment (ex-perimental group) was divided into four replicates(pens) of 40 birds (20 male and 20 female). At the endof the experiment, at day 43, six birds from each group were decapitated and bled at 43 days of age. Samplesof tissues (blood, whole liver and spleen, and breastmuscle) and digesta samples from different parts of the gastrointestinal tract (gizzard, duodenum, jejunum, ileum, caecum, and cloaca) were taken, col-lected and frozen at -20 o C in sterile plastic bags. Laying hens The study was carried out on 96 Bovans Brownhens aged 18 weeks, obtained from a commercialsource. Before the study (up to 25 weeks of age) theanimals were fed a commercial laying hen diet offered  ad libitum . At 25 weeks of age the hens were random-ly assigned to one of four treatments, each comprising 436 Z. Sieradzki et al. Unauthenticated | 89 66 137 210Download Date | 2/7/14 8:59 PM  24 individually caged layers. During the experiment,from 25 to 54 weeks of age, the layers were feda mash, maize-soybean meal based diet, formulatedto meet the nutrient requirements of laying hens. At48 weeks of age, one egg was collected from each hen(24 eggs from each treatment). At the end of theexperiment (54 weeks of age) six birds from eachgroup were decapitated and bled. Samples of tissues(blood, liver, spleen, and lungs) and digesta samplesfrom different parts of the gastrointestinal tract (giz-zard, duodenum, jejunum, ileum, caecum, andcloaca) were taken, collected and frozen at -20 o C insterile plastic bags. Fatteners Forty eight fatteners originated from (PolishLandrace x Large White Polish) sows mated with(Duroc x Pietrain) boar were chosen. The animals were fed isonitrogenous and isoenergetic feed mix-tures according to the requirements of growing (30-60kg BW) and finishing (60-110 kg BW) pigs. All fat-teners were fed individually restricted feed amountsaccording to body weight. At the start of the experi-ment the animal’s weight was about 30 kg, and finallythe fatteners reached about 110 kg body weight. Atthe end of fattening, all pigs were slaughtered andsamples of digesta from the stomach, duodenum, jejunum, caecum, and colon, as well as samples of tissues: liver, spleen, lung,  longissimus  muscle, andblood were taken from six pigs (three barrows andthree gilts) from each group. Samples were frozen andkept at -20 o C in sterile plastic bags. Calves The experiment was carried out on 40 young (7-10days of age) bulls of Black-White race. At the end of the experiment the animals were 90 days old. Untilday 56 of life the calves were fed milk replacers, andthen had free access to water and compound feed, toensure the whole nutrition requirements for calves.Ten calves were randomly selected for each experi-mental group. At the end of the experiment all calves were slaughtered and samples of digesta from thestomach, duodenum, jejunum, and colon, as well assample of tissues: liver, spleen, lung, kidney, pancreas,muscle, and blood were taken from five cattle fromeach group. Samples were frozen and kept at -20 o C insterile plastic bags. DNA isolation and PCR reactions DNA was extracted from homogenised samplesof feed and gastrointestinal tract digesta with CTABmethods (PN-EN ISO/IEC 21571:2007). DNA isola-tion from heparinised blood, tissues, and stool wasconducted with commercial extraction kits (BloodGenomic AX Kit, Genomic Tissue AX Kit, GenomicStool AX Kit all by DNA, Poland), according to thekit’s manuals. Methods used for GMO detection andidentification were based on EN ISO norm (PN-ENISO/IEC 21569:2005), which included PCR forCaMV 35S promoter and NOS terminator (regula-tory sequences used for transformation of both GMcrops; for MON810: CaMV 35; for RR soybean:CaMV 35S and NOS), and species specific PCR – in- vertase gene from maize and lectin gene fromsoybean. Analyses of feed for all trial groups andanimal species were done to check if the GMO com-position was consistent with the study assumptions.Tissue and blood analyses were done to assess thepossibility of GM DNA transfer from GM feed toanimal tissue. Digesta from the gastrointestinal tractand stool were also analysed to assess the degree of digestion of feed DNA. The Limit of Detection of allPCR methods was equal to 5 copies of proper DNA fragment per reaction. The PCR products were ana-lysed by electrophoresis on 2.0% agarose gels con-taining ethidium bromide intercaling dye. Gastrointestinal microflora  Additionally to PCR reaction of digesta from dif-ferent parts of the gastrointestinal tract, samples of ileum and/or colon content were also taken for micro-biological analysis. Some species of common gut bac-teria such as  Escherichia coli ,  Enterococcus faecalis and  Enterococcus faecium  were isolated and theirrelative quantity in the gut was measured, their DNA  was also investigated for the presence of transgenicDNA. Cultivation of bacteria was done on BrainHearth Infusion (BHI) medium. For detection andenumeration of   Escherichia coli  selective, chromo-genic medium Tryptone Bile X-Glucuronide medium(TBX) was used.  Enterococcus  was isolated on Slanetzand Bartley medium.The obtained data were subjected to analysis of  variance and the differences between mean values were estimated using the Duncan test (Statistica 5.1). A p-value of 0.05 was considered as significant.  Assessing the possibility of genetically modified DNA transfer... 437  Unauthenticated | 89 66 137 210Download Date | 2/7/14 8:59 PM  438 Z. Sieradzki et al. Table 1. Results of PCR reactions for the presence of CaMV 35S promoter, NOS terminator, species specific genes – invertase(maize) and lectin (soybean) DNA sequences in animal tissues, digesta from gastrointestinal tract and food of animal srcin.Group I Group II Group III Group IV     C   a    M    V    3    5    S    N    O    S    I   n   v   e    t   r   a   s   e    L   e   c    t    i   n .    C   a    M    V    3    5    S    N    O    S    I   n   v   e    t   r   a   s   e    L   e   c    t    i   n .    C   a    M    V    3    5    S    N    O    S    I   n   v   e    t   r   a   s   e    L   e   c    t    i   n .    C   a    M    V    3    5    S    N    O    S    I   n   v   e    t   r   a   s   e    L   e   c    t    i   n . SampleBlood – – – – – – – – – – – – – – – –Liver – – – – – – – – – – – – – – – –Spleen – – – – – – – – – – – – – – – –Lung 1 – – – – – – – – – – – – – – – –Kidney 2 – – – – – – – – – – – – – – – –Pancreas 2 – – – – – – – – – – – – – – – –Muscle** – – – – – – – – – – – – – – – –Gizzard 3 – – + + + + – + + – + – + + + +Stomach 4 – – + + + + – + + – + – + + + +Duodenum* –/– –/– –/+ –/+ –/+ –/+ –/– –/+ –/+ –/– –/+ –/– –/+ –/+ –/+ –/+Jejunum – – – – – – – – – – – – – – – –Ileum 3 – – – – – – – – – – – – – – – –Caecum 5 – – – – – – – – – – – – – – – –Cloaca 3 – – – – – – – – – – – – – – – –Colon 6 – – – – – – – – – – – – – – – –Excreta – – – – – – – – – – – – – – – –Eggs 7 – – – – – – – – – – – – – – – – 1 except broilers,  2 only calves,  3 only broilers and laying hens,  4 except broilers and laying hens,  5 except calves,  6 only pigs andcalves,  7 only laying hens, * positive results only for pigs, ** broilers – breast muscle, fatteners –  longissimus  muscle, calves– muscle tissue. Results In this study all animal species and feeding groupsachieved satisfactory performance indices, with nosignificant statistical differences in any of the par-ameters across dietary treatment. All feed used duringthe experiment for different groups and species of animals were analysed for chemical composition, in-cluding proximate analysis, amino acid content, NDF(neutral detergent fiber), ADF (acid detergent fiber),phosphorus and calcium level. Taking into account thequality of feed and nutrient requirements of theanimals, proper diets were formulated. The chemicalcomposition of the feed indicates that there is nosignificant difference between non-modified and gen-etically modified maize and soybean. Such parametersas average daily weight gain, feed utilisation, carcassmeasurement, laying performance indices, egg qualityindices  etc . were similar in all analysed dietary groupsfor all animal species. In this study we focused onassessing the possibility of GM DNA transfer fromGM feed to animal tissues and food of animal srcin,to bacterial gut flora and changes in diversity of bacteria species when using GM feed. Feed samples were analysed for the presence of GM crops to ch eckif all group feed were properly composed according tothe trial scheme. The results indicated that feed mix-tures for all species and feeding groups were preparedaccording to the scheme of the experiment. GM maizeMON810 was present only in feed for groups III andIV, GM soybean RR variety in feed for groups II andIV, and feed used for feeding animals from groupI was totally GMO free. Analysis of digesta samples from different parts of the gastrointestinal tract indicated that relatively smallfragments of reference genes (226 bp – invertase, 118bp – lectin) and/or transgenic sequences (123 bp– CaMV 35S, 118 bp – NOS) were detected in allsamples of bird gizzard and stomach content of allspecies, but also in the pig duodenum (Table 1). Thepresence of particular DNA sequences depended onthe feed used for feeding of animals from one of thefour different groups. There were no PCR products of plant reference genes and transgene sequences indigesta of the jejunum, ileum, caecum, cloaca, colon,and excreta taken from all animal species.Similar negative results of plant and transgenicDNA transfer were observed in animal tissues andeggs. In the samples of blood, liver, spleen, pancreas,kidney, lungs, eggs and muscles no copy of plantreference genes, CaMV 35S or NOS sequences werefound.We did not find any differences between all ex-periment groups in quantity and diversity of bacterialgut flora for investigated bacterial species. Moreover,DNA samples of particular  Enterobacteriaceae  species Unauthenticated | 89 66 137 210Download Date | 2/7/14 8:59 PM  isolated from all four feeding groups of broilers, lay-ing hens, pigs and calves did not contain any detect-able copy of reference plant genes, CaMV 35S orNOS sequences. Discussion The substantial equivalence of GM crops and itsconventional counterparts has been indicated in ear-lier publications of other authors (Padgette et al.1996, Gaines et al. 2001, Aulrich et al. 2001, 2002, Aumaitre et al. 2002, Flachowsky et al. 2005, 2005a,Rossi et al. 2005, Flachowsky et al. 2007). Althoughthe chemical composition of GM crops is the same asnon-modified plants, besides new expressed proteinscoded by inserted transgenes, substantial equival-ence was strongly criticized as not satisfactory for theassessment of the safety of GM crops, food and feed.Much more precise and careful studies are necessaryto estimate the safety of genetically modified organ-isms. One such parameter is the possibility of GMDNA transfer from GM plants to microorganismsliving in animal gut and to animal blood, tissues, andthen to food of animal srcin. In feed the animalstake DNA from plants, that DNA is quickly digestedby enzymes in the digestive tract. Our results showedthat DNA fragments of 118 bp or longer were notpresent in hundreds of samples of blood, tissues andeggs analysed during the experiment. All PCR reac-tions conducted on DNA isolated from such matrixesdid not give positive results. There were no PCRproducts in the case of maize and soybean referencegenes and in the case of transgenic sequences of CaMV 35S and NOS. Comparable results have beenpresented in many articles describing GM feed stu-dies carried out on various species of animals(Beever and Kemp 2000, Aulrich et al. 2001, Aulrichet al. 2002, Aumaitre et al. 2002, Reuter and Aulrich2003, Deaville and Maddison 2005). Jennings et al.(2003) did not detect the transgene of CP4 EPSPSenzyme in samples of pig muscle in an RR soybeanfeeding trial. The lack of detectable GM crop trans-genes in the blood and milk of cows was reported byKlotz et al. (2002), as well as in the meat, liver,spleen, stomach, kidneys, heart, and eggs of quails byFlachowsky et al. (2005). Comparison of many GMcrop feeding studies conducted on different speciesanimals was done by Flachowsky et al. (2007). Theseexperiments were done using Bt-corn, Bt-potato andGM soybean as feed components for broilers, layers,cattle, dairy cows, pigs, and quails. Authors of all thecompared experiments reported that transgenesfrom GM crops were not detectable by PCR in ani-mal tissues, blood or food of animal srcin. But re-sults have also shown that plant DNA fragments,other than transgenes from GM crops, were detect-able in samples of blood, organs and animal tissues. Aulrich et al. (2002) found short DNA fragments(shorter than 200 bp) of plant chloroplasts in cow white blood cells and very little positive signal inmilk. Other cows tissues did not give positive resultsfor the presence of plant DNA, nor for the presenceof transgenic sequences. Mazza et al. (2005) re-ported that in some cases plant DNA fragments werepresent in animal tissues and cells, mostly in organsrich in blood vessels and involved in filtration, suchas the liver and kidney. The authors concluded thatblood is the main tissue involved in the uptake of short DNA fragments since it collects macro-molecules directly absorbed by the intestinal epi-thelium and the cells of the immune system. Sandenet al. (2004) stated in their article that an explanationof DNA transfer to animal tissues and cells could bethe fact that DNA makes complexes with protein, in which protein is like a protective coat for the DNA.On the other hand, DNA sensitivity to inactivationand degradation is very high. As regards feed,deoxyribonuclease I produced by animal salivaryglands, pancreas and small intestine is a potentdegradative enzyme, and the low pH of the stomachor ruminant abomasal acts to remove adenine andguanine residues, thereby eliminating biological ac-tivity (Beever and Kemp 2000, Beever and Phipps2001). In the present study, fragments of transgenicDNA and plant DNA were detectable in the contentof the bird gizzard and stomach, and pig duodenum.Results of an experiment with pigs conducted byKlotz et al. (2002) indicated that fragments of chloroplast DNA from maize could be detected inintestinal digest contents up to 12 h, or, as otherauthors suggested, after 72 h after last feeding (Jan-nsen 1989). Deaville and Maddison (2005) reportedthat transgenic DNA from RR soybean meal or Btmaize was detectable in the broiler gizzard, but notin intestinal digesta. An experiment with broilers hasshown that the  bla  gene (part of the Bt constructfrom Bt176 GM maize) was present in the gizzardcontent, but not in small intestine, caecum and rec-tum digesta (Chambers et al. 2002). Such a highdegradation and digestion of plant DNA in the ali-mentary tract of animals makes the possibility of GMDNA transfer to gut bacterial very improbable. Mostexperiments focusing on the possibility of gene trans-fer from GM crops to microorganisms conclude thata such probability is extremely low. Beever andPhipps (2001) estimated that dairy cows fed with Btmaize ingest 57 g of total plant DNA per day, of  which only 54 ,g is transgenic Bt DNA (less than0.00094% of total DNA intake). De Vries et al.  Assessing the possibility of genetically modified DNA transfer... 439 Unauthenticated | 89 66 137 210Download Date | 2/7/14 8:59 PM
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