The use of transgenic crops as feeds for ruminant animals has prompted study of the possible uptake of transgene fragments by ruminal micro-organisms and/or intestinal absorption of fragments surviving passage through the rumen. The persistence in buffered ruminal contents of seven different recombinant DNA fragments from GM rapeseed expressing the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) transgene was tracked using PCR. Parental and transgenic (i.e. glyphosphate-tolerant; Roundup Ready w , Monsanto Company, St Louis, MO, USA) rapeseed were incubated for 0, 2, 4, 8, 12, 24 and 48 h as whole seeds, cracked seeds, rapeseed meal, and as pelleted, barley-based diets containing 65 g rapeseed meal/kg. The seven transgene fragments ranged from 179 to 527 bp and spanned the entire 1363 bp EPSPS transgene. A 180 bp ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit fragment and a 466 bp 16S rDNA fragment were used as controls for endogenous rapeseed DNA and bacterial DNA respectively. The limit of detection of the PCR assay, established using negative controls spiked with known quantities of DNA, was 12·5 pg. Production of gas and NH 3 was monitored throughout the incubation and confirmed active in vitro fermentation. Bacterial DNA was detected in all sample types at all time points. Persistence patterns of endogenous (Rubisco) and recombinant (EPSPS) rapeseed DNA were inversely related to substrate digestibility (amplifiable for 48, 8 and 4 h in whole or cracked seeds, meal and diets respectively), but did not differ between parental and GM rapeseed, nor among fragments. Detection of fragments was representative of persistence of the whole transgene. No EPSPS fragments were amplifiable in microbial DNA, suggesting that transformation had not occurred during the 48 h incubation. Uptake of transgenic DNA fragments by ruminal bacteria is probably precluded or time-limited by rapid degradation of plant DNA upon plant cell lysis.

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.

The aim of this study was to examine the prevalence and distribution of Escherichia coli O157:H7 lineage-specific polymorphism assay (LSPA) 6 genotypes from cattle (n = 313) and clinical human (n = 203) isolates from northern and southern Alberta, Canada, to understand possible associations of genotypes with host and geographic location. The majority of cattle isolates (feedlot and dairy) typed as LSPA-6 111111 (72.2%), with proportionately higher LSPA-6 222222 (19.4%) than other LSPA-6 genotypes (10.7%). Clinical human isolates also typed primarily as LSPA-6 111111 (90.1%), but a higher percentage of genotypes (6.8%) other than LSPA-6 222222 (3.1%) was observed. A significantly higher frequency of LSPA-6 111111 in southern Alberta cattle (P < 0.0001) and a significant difference in LSPA-6 genotypes between human versus feedlot cattle from northern Alberta (P < 0.0001) were detected. LSPA-6 211111 genotype was third and second most common in cattle and humans, respectively, and several new LSPA-6 genotypes (n = 19) were also discovered. Despite avoiding over-representation of isolates from specific farms or outbreaks, higher strain diversity among cattle by pulsed-field gel electrophoresis (PFGE; 50 genotypes) in contrast to human (9 PFGE genotypes) isolates was observed. The majority of cattle (74.4%) and human (90.6%) isolates were susceptible to the antimicrobials tested. Within resistant cattle isolates, sulfisoxazole-tetracycline resistance was common (62.5%) and was accounted for by the presence of sul1 and sul2, and tet(A) and tet(B) determinants. An association between LSPA-6 and PFGE genotypes but not between geographic location and PFGE genotype for both hosts was evident.

Subacute ruminal acidosis (SARA) is a common digestive disorder in dairy cows characterized by prolonged periods of undesirably low rumen pH (<5.8) and is caused by the accumulation of volatile fatty acids in rumen. This disorder damages the ruminal mucosa, causes diarrhea, reduces dry matter intake (DMI), and can result in anorexia and death. In this study, nonlactating dairy cows were fed diets predisposing them to a high risk (HR; n = 6) or a low risk (LR; n = 6) for experiencing SARA. The goal was to investigate differences in antimicrobial resistance selection, proliferation, and characterization of Escherichia coli strain types among the two treatment groups. Fecal samples were used to isolate total, tetracycline-resistant (Tet(r)), and ampicillin-resistant E. coli, and selected isolates were examined. We found reduced total (1.2-fold) and Tet(r) (1.4-fold) E. coli in HR cows. Low ampicillin-resistant E. coli shedding was detected from both HR (0.22 colony forming unit/g) and LR (0.46 colony forming unit/g) cows. Overall, 39 pulsed-field gel electrophoresis (PFGE) profiles and 13 antibiotic resistance profiles (phenotypes) were identified from the total isolates examined (n = 144). The LR cows exhibited diverse genotypes (22 PFGE profiles) clustering into seven restriction endonuclease digestion pattern clusters (REPCs) within total and Tet(r) E. coli. In comparison, isolates from HR animals showed increased genotypic relatedness (16 PFGE profiles and 13 REPC with comparable phenotypes). From both HR and LR cows, no significant differences in the detection of a particular phenotype were observed (p > 0.05), and tet(A) allele was frequently detected among isolates from HR (45.2%) and tet(B) from LR (36.6%) cows. Changes in fecal E. coli genotypes should be explored further for its usefulness as an indicator for SARA since dairy cows are a reservoir of diverse E. coli strain types. Our results elucidate phenotypic and genotypic differences in fecal E. coli shed between HR and LR cows.

Real time PCR was used in this study to determine the effect of triticale dried distillers grains with solubles (TDDGS) as a replacement for grain or barley silage in finishing diets on the presence of six classical ruminal bacterial species (Succinivibrio dextrinosolvens, Selenomonas ruminantium, Streptococcus bovis, Megasphaera elsdenii, Prevotella ruminicola and Fibrobacter succinogenes) within the rumen contents of feedlot cattle. This study was divided into a step-wise adaptation experiment (112 days) that examined the effects of adaptation to diets containing increasing levels of TDDGS up to 30% (n = 4), a short-term experiment comparing animals (n = 16) fed control, 20%, 25% or 30% TDDGS diets over 28 days, and a rapid transition experiment (56 days) where animals (n = 4) were rapidly switched from a diet containing 30% TDDGS to a barley-based diet with no TDDGS. It was found that feeding TDDGS as replacement for barley grain (control vs. 20% TDDGS) decreased 16S rRNA copy numbers of starch-fermenting S. ruminantium and S. bovis (p<0.001 and p = 0.04, respectively), but did not alter 16S rRNA copy numbers of the other rumen bacteria. Furthermore, feeding TDDGS as a replacement barley silage (20% vs. 25% and 30% TDDGS) increased 16S rRNA copy numbers of S. ruminantium, M. elsdenii and F. succinogenes (p<0.001; p = 0.03 and p<0.001, respectively), but decreased (p<0.001) the 16S rRNA copy number of P. ruminicola. Upon removal of 30% TDDGS and return to the control diet, 16S rRNA copy numbers of S. ruminantium, M. elsdenii and F. succinogenes decreased (p = 0.01; p = 0.03 and p = 0.01, respectively), but S. dextrinosolvens and S. bovis increased (p = 0.04 and p = 0.009, respectively). The results suggest that replacement of TDDGS for grain reduces 16S rRNA copy numbers of starch-fermenting bacteria, whereas substitution for barley silage increases 16S rRNA copy numbers of bacteria involved in fibre digestion and the metabolism of lactic acid. This outcome supports the contention that the fibre in TDDGS is highly fermentable.