An investigation of horizontal transfer of feed introduced DNA to the aerobic microbiota of the gastrointestinal tract of rats

Background: Horizontal gene transfer through natural transformation of members of the microbiota of the lower gastrointestinal tract (GIT) of mammals has not yet been described. Insufficient DNA sequence similarity for homologous recombination to occur has been identified as the major barrier to interspecies transfer of chromosomal DNA in bacteria. In this study we determined if regions of high DNA similarity between the genomes of the indigenous bacteria in the GIT of rats and feed introduced DNA could lead to homologous recombination and acquisition of antibiotic resistance genes. Results: Plasmid DNA with two resistance genes (nptII and aadA) and regions of high DNA similarity to 16S rRNA and 23S rRNA genes present in a broad range of bacterial species present in the GIT, where constructed and added to standard rat feed. Six rats, with a normal microbiota, were fed DNA containing pellets daily over four days before sampling of the microbiota from the different GI compartments (stomach, small intestine, cecum and colon). In addition, two rats were included as negative controls. Antibiotic resistant colonies growing on selective media were screened for recombination with feed introduced DNA by PCR targeting unique sites in the putatively recombined regions. Conclusions: The analyses showed that extensive ingestion of DNA (100 \ub5g plasmid) per day did not lead to increased proportions of kanamycin resistant bacteria, nor did it produce detectable transformants among the aerobic microbiota examined for 6 rats (detection limit <1 transformant per 1.1 x 108 cultured bacteria). The key methodological challenges to HGT detection in animal feedings trials are identified and discussed

Demographic responses of Daphnia magna fed transgenic Bt-maize

The food/feed quality of a variety of genetically modified (GM) maize expressing Cry1Ab Bt-toxin was tested over the life-cycle of Daphnia magna, an arthropod commonly used as model organism in ecotoxicological studies. Demographic responses were compared between animals fed GM or unmodified (UM) near isogenic maize, with and without the addition of predator smell. Age-specific data on survival and birth rates were integrated and analysed using life tables and Leslie matrices. Survival, fecundity and population growth rate (PGR) data generally disfavoured transgenic Bt-maize as feed for D. magna compared to animals fed the unmodified (UM) near isogenic line of maize. Decomposition of age-specific effects revealed that the most important contributions to a reduced PGR in the GM-fed group came from both fecundity and survival differences early in life. We conclude that juvenile and young adult stages are the most sensitive experimental units and should be prioritized in future research. These stages are often omitted in toxicological/ecotoxicological studies and in feeding trials

Submission on Application A549 Food Derived from High Lysine Corn LY038: to Permit the Use in Food of High Lysine Corn

In the IAR, FSANZ invited “individuals and organizations to assist FSANZ in preparing the Draft Assessment for” this application A549 (FSANZ 2004, p. 3). Significant new evidence and analysis that is specifically relevant to the evaluation of LY038 is provided to support FSANZ in their preparation of a Draft Assessment. Our submission relates to “the scientific aspects of this Application, in particular, information relevant to the safety assessment of food from corn line LY038” (FSANZ 2004, p. 7). It also addresses the consistency of the IAR with “the objectives of FSANZ as set out in section 10 of the FSANZ Act” and provides “details of potential costs and benefits of the proposed change to the Code” (FSANZ 2004, p. 3). In Part One, we describe important structural differences between mDHDPS and cDHDPS that produce novel challenges for studies on allergenicity, developments in food safety science regarding novel aggregates that could be produced by transgenic cDHDPS, the importance of characterizing post-processing and cooking effects on lysine, lysine catabolites and cDHDPS itself, novel regulatory RNAs that may have physiological effects on human consumers and species of DNA that could have biological effects if taken up by human cells through food. In Part Two, we provide the Authority with a detailed examination of the molecular biological data supplied by the Applicant. We find suggestions in the Applicant’s data that secondary insertions into the LY038 genome may have been overlooked, and find deficiencies in protein identification protocols that could lead to false confidence in the low number of novel proteins and post-translational modifications reported in A549. Most importantly, new evidence suggests that the nos terminator sequence used in LY038 is a recombination hotspot, prone to read-through and may contain a cryptic cis-acting splice sequence that could generate novel RNA molecules and proteins at any place it is inserted into the genome. The lack of holistic proteomic and microarray analysis is a serious deficiency of this application. In Part Three, we evaluate the cost/benefit analysis conducted by the Authority. One of our chief concerns with this analysis is that the costs and benefits included are largely concerned with feed while the Authority is considering whether to amend the food Code. Only when the place of feed-related impacts in a food assessment is clarified can one accurately assess the costs and benefits of Options 1 and 2. We also note that significant costs and benefits have been omitted from the analysis, and evidence is lacking to support those that have been included. In addition, the premises underlying some of the listed costs and benefits require further scrutiny. Our conclusion is that significant additional information should be provided by the Applicant before the Australia New Zealand Food Code is amended. There have been important advances in biosafety and risk assessment science that are not uniformly reflected in the standard of reporting in A549. The studies submitted in support of A549 no longer uniformly meet what we see as the standard of the science. We have raised bona fide issues of safety that have not been addressed by the Authority or the Applicant, which also increases the cost of amending the Code. We have not found in the economic analysis any evidence of how amending Standard 1.5.2 will directly reduce food costs. In fact, we see no evidence that the consumer stands to receive any food benefit form amending the Code, while some consumers will certainly bear additional costs from doing so. We therefore must reject most of the Authority’s speculation on benefits in opting for Option 2. Should the Authority come to recommend that the Code be amended to include event LY038, then it is our opinion that a special condition be imposed upon event LY038. In Column 2 of the Table to Clause 2 of Standard 1.5.2, LY038 but not any other members of the LY038 line currently in existence or that may arise through breeding, hybridization or transformation in the future may benefit from a favorable assessment of LY038. In other words, approval of LY038 does not extend to other lines of maize that share parentage or the I-DNA with LY038