Fate of recombinant DNA and Cry1Ab protein after ingestion and dispersal of genetically modified maize in comparison to rapeseed by fallow deer ( Dama dama )

The fate of recombinant DNA in fallow deer (Dama dama) was investigated by feeding a diet of isogenic or genetically modified (GM) maize expressing Cry1Ab protein against the European corn borer (Ostrinia nubilalis). To study the degradability of ingested DNA, polymerase chain reaction (PCR) assays were introduced to detect fragments of the endogenous, highly abundant chloroplast-specific rubisco gene, the maize-specific zein gene and the recombinant cry1Ab gene. PCR analysis revealed that small chloroplast- and maize-specific DNA fragments were detectable in contents of rumen, abomasums, jejunum, caecum and colon and occasionally in visceral tissues. In contrast, no fragments of the recombinant cry1Ab gene were detectable in gastrointestinal (GI) contents. The Cry1Ab protein was analysed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting technique. Neither ELISA nor immunoblotting yielded positive signals of immunoactive Cry1Ab protein in GI contents and tissues of fallow deer fed with GM maize. In conclusion, after uptake of GM maize, neither cry1Ab-specific gene fragments nor Cry1Ab protein were detected in the GI tract of fallow deer, indicating complete digestion of the GM maize. Additional investigations on the germination capacity of conventional rapeseed and maize seed after ingestion by fallow deer and faecal excretion (endozoochory) were performed to draw conclusions regarding a potential spreading of germinable GM crop seed by deer. Germination tests revealed that germinable rapeseed kernels were detectable in faeces; in contrast, no intact maize seeds were found in faece

Puerperal influence of bovine uterine health status on the mRNA expression of pro-inflammatory factors

After parturition, uterine bacterial infections lead to inflammatory processes such as subclinical/clinical endometritis with high prevalence in dairy cows. Endometrial epithelial cells participate in this immune response with the production of pro-inflammatory factors. The objective of the present study was to evaluate the endometrial mRNA expression pattern of pro inflammatory factors during a selected postpartum (pp) period. Dairy cows with three different uterine health conditions on days 24-30 pp (healthy: n = 11, subclinical endometritis: n = 10, clinical endometritis: n = 10) were sampled using the cytobrush technique. Subsequently, each cow was sampled 3 more times in weekly intervals (days 31-37 pp; days 38-44 pp; days 45-51 pp). Samples were subjected to mRNA analysis performed by RT-qPCR. Additionally, an analysis of cultivable bacteria was performed at the early/late stage of the selected puerperal period. mRNA expression of 16 candidate genes was analyzed by using two different approaches. The first approach referred to the initial grouping on days 24-30 pp to reveal long-term effects of the uterine health on the subsequent puerperal period. The second approach considered the current uterine health status at each sampling to elucidate the impact of different points in time. Long-term effects seem to appear for chemokines, prostacyclin synthase and prostaglandin D2 synthase. If related to the current uterine health, the majority of candidate genes were significantly higher expressed in endometritic cows on days 45-51 pp in contrast to earlier stages of the puerperium. Microbiological analysis revealed the significantly higher prevalence of Trueperella pyogenes findings in cows with clinical endometritis on days 24-30 pp, but no correlations were found on days 45-51 pp. In conclusion, a strong immune response to subclinical/clinical endometritis in the late puerperium may be related to the negative impact of these conditions on reproductive performance in dairy cows

Deciphering the porcine intestinal microRNA transcriptome

<p>Abstract</p> <p>Background</p> <p>While more than 700 microRNAs (miRNAs) are known in human, a comparably low number has been identified in swine. Because of the close phylogenetic distance to humans, pigs serve as a suitable model for studying e.g. intestinal development or disease. Recent studies indicate that miRNAs are key regulators of intestinal development and their aberrant expression leads to intestinal malignancy.</p> <p>Results</p> <p>Here, we present the identification of hundreds of apparently novel miRNAs in the porcine intestine. MiRNAs were first identified by means of deep sequencing followed by miRNA precursor prediction using the miRDeep algorithm as well as searching for conserved miRNAs. Second, the porcine miRNAome along the entire intestine (duodenum, proximal and distal jejunum, ileum, ascending and transverse colon) was unraveled using customized miRNA microarrays based on the identified sequences as well as known porcine and human ones. In total, the expression of 332 intestinal miRNAs was discovered, of which 201 represented assumed novel porcine miRNAs. The identified hairpin forming precursors were in part organized in genomic clusters, and most of the precursors were located on chromosomes 3 and 1, respectively. Hierarchical clustering of the expression data revealed subsets of miRNAs that are specific to distinct parts of the intestine pointing to their impact on cellular signaling networks.</p> <p>Conclusions</p> <p>In this study, we have applied a straight forward approach to decipher the porcine intestinal miRNAome for the first time in mammals using a piglet model. The high number of identified novel miRNAs in the porcine intestine points out their crucial role in intestinal function as shown by pathway analysis. On the other hand, the reported miRNAs may share orthologs in other mammals such as human still to be discovered.</p

Fate of recombinant DNA and Cry1Ab protein after ingestion and dispersal of genetically modified maize in comparison to rapeseed by fallow deer (Dama dama

Abstract The fate of recombinant DNA in fallow deer (Dama dama) was investigated by feeding a diet of isogenic or genetically modified (GM) maize expressing Cry1Ab protein against the European corn borer (Ostrinia nubilalis). To study the degradability of ingested DNA, polymerase chain reaction (PCR) assays were introduced to detect fragments of the endogenous, highly abundant chloroplast-specific rubisco gene, the maize-specific zein gene and the recombinant cry1Ab gene. PCR analysis revealed that small chloroplast-and maize-specific DNA fragments were detectable in contents of rumen, abomasums, jejunum, caecum and colon and occasionally in visceral tissues. In contrast, no fragments of the recombinant cry1Ab gene were detectable in gastrointestinal (GI) contents. The Cry1Ab protein was analysed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting technique. Neither ELISA nor immunoblotting yielded positive signals of immunoactive Cry1Ab protein in GI contents and tissues of fallow deer fed with GM maize. In conclusion, after uptake of GM maize, neither cry1Ab-specific gene fragments nor Cry1Ab protein were detected in the GI tract of fallow deer, indicating complete digestion of the GM maize. Additional investigations on the germination capacity of conventional rapeseed and maize seed after ingestion by fallow deer and faecal excretion (endozoochory) were performed to draw conclusions regarding a potential spreading of germinable GM crop seed by deer. Germination tests revealed that germinable rapeseed kernels were detectable in faeces; in contrast, no intact maize seeds were found in faeces

Energy efficient plasma processing of industrial wastes

The paper presents the results of thermodynamic modeling of the process of joint plasma treatment of non-combustible and combustible industrial wastes. The compositions of water-salt-organic compositions based on these wastes and regimes providing their energy-efficient joint treatment in air plasma have been determined

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

Degradation of Cry1Ab protein from genetically modified maize (MON810) in relation to total dietary feed proteins in dairy cow digestion

To investigate the relative degradation and fragmentation pattern of the recombinant Cry1Ab protein from genetically modified (GM) maize MON810 throughout the gastrointestinal tract (GIT) of dairy cows, a 25 months GM maize feeding study was conducted on 36 lactating Bavarian Fleckvieh cows allocated into two groups (18 cows per group) fed diets containing either GM maize MON810 or nearly isogenic non-GM maize as the respective diet components. All cows were fed a partial total mixed ration (pTMR). During the feeding trial, 8 feed (4 transgenic (T) and 4 non-transgenic (NT) pTMR) and 42 feces (26 T and 18 NT) samples from the subset of cows fed T and NT diets, and at the end of the feeding trial, digesta contents of rumen, abomasum, small intestine, large intestine and cecum were collected after the slaughter of six cows of each feeding group. Samples were analyzed for Cry1Ab protein and total protein using Cry1Ab specific ELISA and bicinchoninic acid assay, respectively. Immunoblot analyses were performed to evaluate the integrity of Cry1Ab protein in feed, digesta and feces samples. A decrease to 44% in Cry1Ab protein concentration from T pTMR to the voided feces (9.40 versus 4.18 μg/g of total proteins) was recorded. Concentrations of Cry1Ab protein in GIT digesta of cows fed T diets varied between the lowest 0.38 μg/g of total proteins in abomasum to the highest 3.84 μg/g of total proteins in rumen. Immunoblot analysis revealed the extensive degradation of recombinant Cry1Ab protein into a smaller fragment of around 34 kDa in GIT. The results of the present study indicate that the recombinant Cry1Ab protein from MON810 is increasingly degraded into a small fragment during dairy cow digestion

Effects of Feeding Bt MON810 Maize to Pigs for 110 Days on Peripheral Immune Response and Digestive Fate of the cry1Ab Gene and Truncated Bt Toxin

peer-reviewedBackground: The objective of this study was to evaluate potential long-term (110 days) and age-specific effects of feeding genetically modified Bt maize on peripheral immune response in pigs and to determine the digestive fate of the cry1Ab gene and truncated Bt toxin. Methodology/Principal Findings: Forty day old pigs (n = 40) were fed one of the following treatments: 1) isogenic maize-based diet for 110 days (isogenic); 2) Bt maize-based diet (MON810) for 110 days (Bt); 3) Isogenic maize-based diet for 30 days followed by Bt maize-based diet for 80 days (isogenic/Bt); and 4) Bt maize-based diet (MON810) for 30 days followed by isogenic maize-based diet for 80 days (Bt/isogenic). Blood samples were collected during the study for haematological analysis, measurement of cytokine and Cry1Ab-specific antibody production, immune cell phenotyping and cry1Ab gene and truncated Bt toxin detection. Pigs were sacrificed on day 110 and digesta and organ samples were taken for detection of the cry1Ab gene and the truncated Bt toxin. On day 100, lymphocyte counts were higher (P<0.05) in pigs fed Bt/isogenic than pigs fed Bt or isogenic. Erythrocyte counts on day 100 were lower in pigs fed Bt or isogenic/Bt than pigs fed Bt/isogenic (P<0.05). Neither the truncated Bt toxin nor the cry1Ab gene were detected in the organs or blood of pigs fed Bt maize. The cry1Ab gene was detected in stomach digesta and at low frequency in the ileum but not in the distal gastrointestinal tract (GIT), while the Bt toxin fragments were detected at all sites in the GIT. Conclusions/Significance: Perturbations in peripheral immune response were thought not to be age-specific and were not indicative of Th 2 type allergenic or Th 1 type inflammatory responses. There was no evidence of cry1Ab gene or Bt toxin translocation to organs or blood following long-term feeding.The research leading to these results has received funding from the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 211820 and the Teagasc Walsh Fellowship programme

The Effect of Feeding Bt MON810 Maize to Pigs for 110 Days on Intestinal Microbiota

Objective: To assess the effects of feeding Bt MON810 maize to pigs for 110 days on the intestinal microbiota. Methodology/Principal Findings: Forty male pigs (,40 days old) were blocked by weight and litter ancestry and assigned to one of four treatments; 1) Isogenic maize-based diet for 110 days (Isogenic); 2) Bt maize-based diet (MON810) for 110 days (Bt); 3) Isogenic maize-based diet for 30 days followed by a Bt maize-based diet for 80 days (Isogenic/Bt); 4) Bt maizebased diet for 30 days followed by an isogenic maize-based diet for 80 days (Bt/Isogenic). Enterobacteriaceae, Lactobacillus and total anaerobes were enumerated in the feces using culture-based methods on days 0, 30, 60 and 100 of the study and in ileal and cecal digesta on day 110. No differences were found between treatments for any of these counts at any time point. The relative abundance of cecal bacteria was also determined using high-throughput 16 S rRNA gene sequencing. No differences were observed in any bacterial taxa between treatments, with the exception of the genus Holdemania which was more abundant in the cecum of pigs fed the isogenic/Bt treatment compared to pigs fed the Bt treatment (0.012 vs 0.003%; P#0.05). Conclusions/Significance: Feeding pigs a Bt maize-based diet for 110 days did not affect counts of any of the culturable bacteria enumerated in the feces, ileum or cecum. Neither did it influence the composition of the cecal microbiota, with the exception of a minor increase in the genus Holdemania. As the role of Holdemania in the intestine is still under investigatio