"Effect of feed processing conditions on DNA fragmentation"
Angela Ryan and Dr. Mae-Wan Ho, Institute of Science in Society and Department of Biological Sciences, Open University,UK
A pilot study, commissioned by the UK Ministry of Agriculture, Fisheries and Food (MAFF), was carried out in Leeds University to determine the extent to which DNA in animal feed is degraded under various processing conditions. It addressed concerns over the transfer of foreign genes introduced into GM crops, especially antibiotic resistance marker genes, to bacteria, which would render common infectious diseases untreatable. The study involved analysis of non-GM material provided by the food industry directly, or subsequent to small-scale processing in the laboratory.
The main conclusions are that DNA is not degraded under most commercial processing conditions, nor in the silage, and that further studies on GM material should be undertaken.
Current animal feed is likely to contain substantial amounts of undegraded DNA, and secondary horizontal transfer of intact antibiotic resistance genes to bacteria and other organisms cannot be ruled out.
Other components of transgenic DNA may also have significant health impacts on livestock and human beings up the food chain.
In view of the potential health impacts due to the secondary horizontal transfer of transgenic DNA on livestock and human beings, all current animal feed should be withdrawn immediately. Steps should be taken to ensure that no GM material will be fed to animals directly or incorporated into commercial animal feed.
Key Words: GM animal feed, transgenic DNA in processed food, DNA degradation
*UK Ministry of Agriculture, Fisheries and Food, Report CS0116, London
MAFF Report CS0116 describes work carried out in Leeds University to determine the extent to which DNA is broken down in animal feed. Animal feed is either fed raw and unprocessed, or after being processed under a variety of conditions in commercial mills. The effect of several methods of processing on the degradation of DNA in animal feed was investigated. DNA degradation in raw foodstuffs from commercial and non-commercial sources was also determined.
This work addressed concerns regarding the transfer of transgenes introduced into GM crops, especially antibiotic resistance marker genes, to bacteria. This would exacerbate the problem of antibiotic resistant strains of bacteria associated with the recent resurgence of infectious diseases such as tuberculosis. The rationale for the research is presumably to identify conditions of treatment or processing of animal feed which degrade DNA and prevent the transfer of intact, functional antibiotic resistance genes.
The study has all the hallmarks of a pilot project, not intended to provide definitive data on the extent to which DNA of GM crops, and more particularly, transgenic DNA, would be degraded in different kinds of raw or processed GM animal feed. No GM materials were used in the study, nor was the presence of antibiotic resistance genes examined. Nevertheless, the results give strong indications that DNA is not degraded under most commercial processing conditions.
Three major UK animal feed companies were involved with this study, providing advice on processing specifications and a range of samples. It was agreed early in the project that it would be better to process the feed materials under closely monitored conditions at a semi-commercial scale feed plant in the Roslin Institute, rather than in commercial mills. However, there was considerable delay in commissioning the equipment at the Roslin Institute, so the original plan had to be abandoned.
Samples of animal feed ingredients obtained from the collaborating companies include:
In addition, fresh maize leaves and maize silage were obtained from the University Farm, and fresh maize cobs and maize grains from the local supermarket.
DNA was extracted by standard procedures. Two methods were used to monitor the extent of DNA fragmentation. The first, agarose gel electrophoresis, determines the size of the fragments, but does not give any indication of their composition. It is also limited in sensitivity, and will not detect undegraded DNA less than 1% of the total contained in the sample. The DNA was judged to be degraded when the size of DNA fragments is less than 100 base pairs. The second method uses the polymerase chain reaction (PCR) to amplify DNA sequences and can detect even trace amounts of undegraded DNA of particular gene sequences. The particular gene sequence investigated was the small subunit of the Rubisco enzyme from maize.
The results on DNA degradation estimated with agarose gel electrophoresis are summarised in Table 1
Table 1 The state of DNA in different samples and after different treatments
|Sample/Treatment||State of DNA|
|Wheat/dry heat at 90 deg.C or below for 30 mins||intact|
|Wheat/dry heat at 93 deg. C for 4 mins.||intact|
|Wheat/low pressure steam, 60 deg. C for 30 mins||intact|
|Wheat/dry heat at 93 deg. C for 5 to 15 mins||partially degraded|
|Wheat/low pressure steam at 85 deg. C for 10 mins||partially degraded|
|Wheat/dry heat at 95 deg. C for 5 mins||degraded|
|Wheat/low pressure steam, 95-100 deg. C for 30 sec. or longer||degraded|
|Wheat/high pressure steam at 100-125 deg. C for 1 min. or longer||degraded|
|Oilseed rape meal after extraction||degraded|
|Oilseed rape cake||degraded|
|Dried sugar beet pulp||degraded|
The PCR technique gave the expected amplification of the Rubsico subunit gene sequence from maize leaves, grains and silage of up to about 600 base pairs. It is not clear from the Report whether attempts have been made to amplify the sequence from the other maize samples in which DNA was judged to be degraded by agarose gel electrophoresis.
The results show that DNA remains intact in fresh plant leaves and grain as well as in silage. Under even small-scale laboratory conditions, temperatures of not less than 95 deg. C for at least 5 mins were required to degrade DNA. Most commercially processed animal feeds are subjected to temperatures not exceeding 85 deg. C. Where steam is used to condition material for pelleting, temperatures reach at least the mid-80s for an uncertain length of time, and the laboratory results show that DNA may only be partially degraded under those conditions. The Report recommends against using ensilaged GM material for animal feed, and concludes that most commercially produced animal feed contains intact DNA fragments of a size greater than 1200 base pairs comparable to the beta-lactamase antibiotic resistance marker gene used in many GM crops.
This work was a pilot study. It used non-GM material, and processings were carried out on small laboratory scales. No attempt was made to follow degradation of a specific antibiotic resistance marker gene.
No consideration was given to the transfer of other transgenic DNA, such as the cauliflower mosaic viral (CaMV) promoter. The CaMV promoter is about 350 base pairs in length and contains a recombination hotspot, which makes it prone to break and join up with other pieces of DNA. It also has several short sequences, less than 100 basepairs in length, with specific functions for replication of the virus, which are interchangeable with other viruses. Thus, the CaMV promoter may have an enhanced propensity to transfer horizontally, with the potential to reactivate dormant viruses, to regenerate new viruses and to cause cancers in mammalian cells.
The criterion of degraded DNA is the absence, on agarose gel electrophoresis, of any fragment longer than 100 base pairs. This is not sufficient to exclude short fragments which may have significant biological activities from being transferred horizontally (see above).
The Report states there is no reason to believe that the conditions necessary to fragment the DNA of GM plant material are different from unmodified material. However, in the absence of experimental data, this is mere conjecture. Transgenic DNA may have an enhanced resistance to degradation, or not.
The small laboratory scale processings done in this study are unlikely to reflect the effects of large-scale commercial processings. However, the conditions used were more favourable to DNA degradation than those that the materials are likely to encounter in the commercial setting. Thus, the results give strong indications that most commercial processings will leave DNA intact. We understand that the Chief Sciences Group of MAFF have commissioned follow-up research on the effects of commercial scale processing on the integrity of DNA in animal feed, and that the report will be available by the end of March next year.
The scientists involved in the study are now working on "Assessment of the risks of transferring antibiotic resistance determinants from transgenic plants to micro-organisms" due to be completed in April 2001. In this new study, GM material will be fed to animals and transfer of antibiotic resistance marker genes to micro-organisms in the animal gut will be monitored by the PCR technique.
After the CS0116 Report was written, MAFF produced its own report, "GM Materials in Animal Feed", which makes a reference to the work as a first look study. But it fails to mention any of its important conclusions. Nevertheless, from the data presented regarding the level of GM material in British animal feed, it is clear that current British animal feed comprises at least 20% GM material obtained from suppliers abroad which do not segregate GM from non GM varieties (p.5). It is also clear that up to 45% of raw material used in compound feed in Great Britain in 1997/1998 contained cereals, peas, beans and vegetable residues, all of which may have intact GMO-derived DNA.
The report gives no reference to other MAFF research conducted to assess the safety of using GM material - MAFF project (FS 0204) showed that the human mouth contains bacteria capable of taking up and expressing free DNA and that similar transformable bacteria are also present in the respiratory tract. Furthermore, in a letter from UK MAFF to the US FDA dated Dec. 1998, it was pointed out that transgenic DNA may be transferred not just by ingestion, but by contact with plant dust and air borne pollen during farm work and food processing.
Article first published 02/04/00
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