The persistence of plant-derived recombinant DNA in sheep and pigs fed genetically modified (Roundup Ready) canola was assessed by PCR and Southern hybridization analysis of DNA extracted from digesta, gastrointestinal (GI) tract tissues, and visceral organs. Sheep (n = 11) and pigs (n = 36) were fed to slaughter on diets containing 6.5 or 15% Roundup Ready canola. Native plant DNA (high- and low-copy-number gene fragments) and the cp4 epsps transgene that encodes 5-enolpyruvyl shikimate-3-phosphate synthase were tracked in ruminal, abomasal, and large intestinal digesta and in tissue from the esophagus, rumen, abomasum, small and large intestine, liver, and kidney of sheep and in cecal content and tissue from the duodenum, cecum, liver, spleen, and kidney of pigs. High-copy chloroplast-specific DNA (a 520-bp fragment) was detected in all digesta samples, the majority (89-100%) of intestinal tissues, and at least one of each visceral organ sample (frequencies of 3-27%) from sheep and swine. Low-copy rubisco fragments (186- and 540-bp sequences from the small subunit) were present at slightly lower, variable frequencies in digesta (18-82%) and intestinal tissues (9-27% of ovine and 17-25% of porcine samples) and infrequently in visceral organs (1 of 88 ovine samples; 3 of 216 porcine samples). Each of the five cp4 epsps transgene fragments (179-527 bp) surveyed was present in at least 27% of ovine large intestinal content samples (maximum = 64%) and at least 33% of porcine cecal content samples (maximum = 75%). In sheep, transgene fragments were more common in intestinal digesta than in ruminal or abomasal content. Transgene fragments were detected in 0 (esophagus) to 3 (large intestine) GI tract tissues from the 11 sheep and in 0-10 of the duodenal and cecal tissues collected from 36 pigs. The feed-ingested recombinant DNA was not detected in visceral tissues (liver, kidney) of lambs or in the spleen from pigs. Of note, however, one liver and one kidney sample from the pigs (different animals) were positive for a 278-bp fragment of the transgenic cp4 epsps (denoted F3). Examination of genomic libraries from these tissues yielded no conclusive information regarding integration of the fragment into porcine DNA. This study confirms that feed-ingested DNA fragments (endogenous and transgenic) do survive to the terminal GI tract and that uptake into gut epithelial tissues does occur. A very low frequency of transmittance to visceral tissue was confirmed in pigs, but not in sheep. It is recognized that the low copy number of transgenes in GM feeds is a challenge to their detection in tissues, but there was no evidence to suggest that recombinant DNA would be processed in the gut in any manner different from endogenous feed-ingested genetic material.

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DNA from rumen digesta has several diagnostic applications such as studying microbial community dynamics, transgene/ DNA stability, and population typing of various rumen bacteria. Several DNA extraction procedures are described in the literature for rumen digesta, which describe the removal of tannins, polysaccharides, and other PCR inhibitors. Some of these protocols are time-consuming and impractical when handling a large number of samples routinely. Here we describe a rapid method for the extraction of PCR-quality plant and microbial DNA from total rumen contents that is based on modifications in the cetyltrimethylammonium bromide procedure followed by cleanup using a Qiagen column. This procedure is highly reproducible and relatively short, once the initial grinding of the samples is performed, and it consistently yields PCR-quality DNA.