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Investigation into a possible transfer of genes from cows fed on Bt maize to gastrointestinal micro-organisms

(2001 – 2004) Technical University of Munich (TUM), Chair for Physiology and Bavarian State Research Center for Agriculture, Institute for Animal Breeding; Grub

Topic

The aim of the project is to investigate possible impacts of genetically modified plants – compared with conventional plants – on higher vertebrates using the example of the cow. This involves tracing the foreign DNA from genetically modified fodder plants (in this case Bt maize) from different points of view:

  • What happens in terms of breakdown of the foreign DNA and the Bt protein during production of the feedstuff?
  • What happens to the foreign DNA and Bt protein in the digestive tract of the cattle and can it enter the cow’s circulation?
  • Is there an intake of foreign DNA among micro-organisms of the gastrointestinal tract?
  • If there is DNA integration, is the foreign DNA integrated in the genome and expressed there, i.e. activated?
  • Is the foreign DNA and/or the Bt protein excreted again and dispersed in the environment in e.g. manure?

The data is to be used later on as the basis for research into these processes in different food chains.

Summary

Over the investigation period of 61 days, a significant breakdown of plant and transgenic DNA was recorded during silage-making. The DNA fragments were smaller than a functional gene.

The quantity of the Bt protein fell rapidly after two days. After two months of silage-making only about one quarter of the original amount was measured.

The DNA of the Bt maize was broken down in the cow’s gastrointestinal tract. Only plant DNA fragments of chloroplasts were found which occur frequently in plant cells, but not the Bt gene or the genetically introduced ampicillin resistance gene, which are integrated in the DNA of the cell nucleus.

Breakdown products of the Bt protein were found in the digestive juices, but not in the epithelial cells of the gastrointestinal tract. To what extent these are still active as a toxin should be investigated in follow-up studies. There was no evidence of horizontal gene transfer of plant DNA to the bacteria in the cow.

No differences were found between the composition of the bacterial populations in the rumen of cows fed on isogenic feedstuff and those fed on transgenic feedstuff.

Experiment description

Breakdown of DNA and protein during processing of transgenic plants for feedstuffs

Maize silage in preserving jars

In order to trace plant and transgenic DNA and the Bt protein during the processing of maize plants to make silage, isogenic and transgenic maize is chopped up into lengths of one to two centimetres on average, pressed into standard preserving jars and closed with air-tight lids in order to simulate the silage-making process (lactic acid fermentation for preservation and to improve the nutritional value, as in “sauerkraut”) under laboratory conditions. Representative samples are taken for each variety at defined intervals. The pH value of the content is measured to monitor the silage-making process in the jars.

Feeding experiments to trace the path of foreign DNA

Various feedstuffs (grain maize, silage and feed concentrate) are produced from transgenic maize (Bt176) and conventional maize, mixed and fed to eleven animals each (cows and fattening bullocks) per test. The animals are measured daily to observe their utilisation of the feed.

The path taken by the foreign DNA and Bt protein is monitored in the animals’ gastrointestinal tract and in their excrement. Samples are taken, DNA is extracted and pieces of conventional and transgenic plant DNA are analysed qualitatively and quantitatively using highly sensitive PCR detection methods. Of particular interest are the fragment lengths and concentrations of Bt and ampicillin-resistance genes. In order to follow what happens generally with plant material from feeding to excretion, a chloroplast gene is also studied.

Tracing the path of the Bt proteins

The path taken by the Bt protein is traced during feedstuff production and during the digestive process in the animal using a highly sensitive protein test, ELISA.

In order to check whether the ELISA test finds intact Bt toxin or breakdown products, another protein detection test (Immunoblot), which can measure the protein fragment sizes, is used as well.

Research into horizontal gene transfer

Various micro-organisms are being studied.

If the foreign gene is found in the micro-organisms the project will investigate (using RT-PCR) whether the genes are also integrated and expressed, i.e. activated. The exit of potentially transgenic micro-organisms via excrement (presence in liquid and solid manure) and their ability to survive on the field after manure application are also to be investigated.

To investigate gastrointestinal micro-organisms, the animals’ rumen/stomach content is removed. In the laboratory the potential uptake of the ampicillin gene fragment is observed in an artificial rumen with rumen micro-organisms, with and without selection pressure (i.e. with and without the addition of ampicillin).

The effect of the Bt toxin on the rumen micro-organisms is also investigated in the laboratory.

Spectrum of micro-organisms

In the final phase, the project is to investigate a possible distortion of the spectrum of gastrointestinal micro-organisms as a result of the use of Bt maize.

Results

Breakdown of DNA and protein in the processing of transgenic plants to make feedstuffs

The pH tests on the samples taken from the preserving jars showed a rapid fall in pH during the first few days – an indication of the success of the silage-making process under laboratory conditions.

Sensitive PCR detection methods measured a clear decrease in plant and transgenic DNA during the silage-making over the course of the 61-day trial. After two months of silage-making, the DNA fragments had been broken down to a size smaller than one functional gene.

The Bt toxin was measured using a special quantitative analysis process for proteins (ELISA). It emerged that the volume of the Bt protein falls rapidly after two days. After two months of silage-making, there was only about one quarter of the original volume left. These results were confirmed using another protein detection method (Immunoblot). These tests showed that, unlike in the digestive process in the cow’s gastrointestinal tract mentioned below, in the silage-making process the Bt protein is broken down without forming shorter fragments.

Feeding tests to trace the path of foreign DNA

In each test, eleven cows were fed on silage from either Bt maize or maize from the isogenic control variety. The results show that the DNA of the Bt maize is broken down in the cows’ gastrointestinal tract. Plant DNA fragments were found, but only those that are often present in the genome, because they are localised in the chloroplasts. A plant gene that occurs rarely in the genome (zein), the genetically introduced ampicillin resistance gene and the Bt gene were not found.

Path of Bt protein

The path taken by the Bt protein (toxin) was followed using the above-mentioned screening test (ELISA), which detects the presence of immunologically active structures of the Bt protein or its components. In these tests a signal was observed in the digestive juices of the cows’ gastrointestinal tract, but not in the epithelial cells. This signal appeared to increase as it passed through the digestive tract, so another protein detection process (Immunoblot) was used. This method clearly showed that as it passes through the gastrointestinal tract, ELISA measures breakdown products of the Bt protein, rather than intact, full-length Bt protein. This means that the ELISA measurements need to be interpreted with care. To what extent the smaller fragments of Bt protein found still exhibit biological activity as a toxin should be investigated in follow-up studies.

Research into the spectrum of rumen micro-organisms and horizontal gene transfer

Gene libraries were set up for rumen bacteria and DNA fingerprints were carried out to investigate the composition of micro-organisms in the stomach and intestine of the ruminants. The transgenic maize and the isogenic control variety were processed to make silage and each was fed to two cows. The fingerprints showed no differences in the patterns of the bacteria populations between the animals fed on isogenic feedstuff and those fed on transgenic feedstuff. No evidence was found of horizontal gene transfer from plant DNA to bacteria in the cow.