Antimicrobial use (AMU) in the livestock industry has been associated with increased levels of antimicrobial resistance. Recently, there has been an increase in the number of “natural” feedlots in the beef cattle sector that raise cattle without antibiotics. Shotgun metagenomics was employed to characterize the impact of AMU in feedlot cattle on the microbiome, resistome, and mobilome. Sequenced fecal samples identified a decline (q < 0.01) in the genera Methanobrevibacter and Treponema in the microbiome of naturally vs. conventionally raised feedlot cattle, but this difference was not (q > 0.05) observed in catch basin samples. No differences (q > 0.05) were found in the class-level resistome between feedlot practices. In fecal samples, decreases from conventional to natural (q < 0.05) were noted in reads for the antimicrobial-resistant genes (ARGs) mefA, tet40, tetO, tetQ, and tetW. Plasmid-associated ARGs were more common in feces from conventional than natural feedlot cattle. Interestingly, more chromosomal- than plasmid-associated macrolide resistance genes were observed in both natural and conventional feedlots, suggesting that they were more stably conserved than the predominately plasmid-associated tetracycline resistance genes. This study suggests that generationally selected resistomes through decades of AMU persist even after AMU ceases in natural production systems.

This study aimed to better understand the potential public health risk associated with zoonotic pathogens in agricultural fairs and petting zoos in Canada. Prevalence of Salmonella, Shiga toxin-producing Escherichia coli (STEC) O157:H7, and top six non-O157 STEC serogroups in feces (n = 88), hide/feather (n = 36), and hand rail samples (n = 46) was assessed, as well as distributions of antimicrobial resistant (AMR) broad and extended-spectrum β-lactamase (ESBL)-producing E. coli. Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in pig nasal swabs (n = 4), and Campylobacter, Cryptosporidium, and Giardia in feces was also assessed. Neither Salmonella nor MRSA were detected. Campylobacter spp. were isolated from 32% of fecal samples. Cryptosporidium and Giardia were detected in 2% and 15% of fecal samples, respectively. Only one fecal sample was positive for STEC O157, whereas 22% were positive for non-O157 STEC. Multi-drug resistance (MDR) to antibiotics classified as critically and highly important in human medicine was proportionally greatest in E. coli from cattle feces. The β-lactamase-producing E. coli from pig, horse/donkey feces, and hand rail samples, as well as the STEC E. coli from handrail swabs were MDR. The diversity and prevalence of zoonotic pathogens and AMR bacteria detected within agricultural fairs and petting zoos emphasize the importance of hygienic practices and sanitization with respect to reducing associated zoonotic risks.

Ensiling of forages was recognized as a microbial-driven process as early as the late 1800s, when it was associated with the production of "sweet" or "sour" silage. Classical microbiological plating techniques defined the epiphytic microbial populations associated with fresh forage, the pivotal role of lactic acid-producing bacteria in the ensiling process, and the contribution of clostridia, bacilli, yeast, and molds to the spoilage of silage. Many of these classical studies focused on the enumeration and characterization of a limited number of microbial species that could be readily isolated on selective media. Evidence suggested that many of the members of these microbial populations were viable but unculturable, resulting in classical studies underestimating the true microbial diversity associated with ensiling. Polymerase chain reaction-based techniques, including length heterogeneity PCR, terminal RFLP, denaturing gradient gel electrophoresis, and automated ribosomal intergenic spacer analysis, were the first molecular methods used to study silage microbial communities. Further advancements in whole comparative genomic, metagenomic, and metatranscriptomic sequencing have or are in the process of superseding these methods, enabling microbial communities during ensiling to be defined with a degree of detail that is impossible using classical microbiology. These methods have identified new microbial species in silage, as well as characterized shifts in microbial communities with forage type and composition, ensiling method, and in response to aerobic exposure. Strain- and species-specific primers have been used to track the persistence and contribution of silage inoculants to the ensiling process and the role of specific species of yeast and fungi in silage spoilage. Sampling and the methods used to isolate genetic materials for further molecular analysis can have a profound effect on results. Primer selection for PCR amplification and the presence of inhibitors can also lead to biases in the interpretation of sequence data. Bioinformatic analyses are reliant on the integrity and presence of sequence data within established databases and can be subject to low taxonomic resolution. Despite these limitations, advancements in molecular biology are poised to revolutionize our current understanding of the microbial ecology of silage.

Escherichia coli O157:H7 is a foodborne pathogen that colonizes ruminants. Cattle are considered the primary reservoir of E. coli O157:H7 with super-shedders, defined as individuals excreting > 104 E. coli O157:H7 CFU g-1 feces. The mechanisms leading to the super-shedding condition are largely unknown. Here, we used 16S rRNA gene pyrosequencing to examine the composition of the fecal bacterial community in order to investigate changes in the bacterial microbiota at several locations along the digestive tract (from the duodenum to the rectal-anal junction) in 5 steers previously identified as super-shedders and 5 non-shedders. The overall bacterial community structure did not differ by E. coli O157:H7 shedding status; but several differences in the relative abundance of taxa and OTUs were noted between the two groups. The genus Prevotella was most enriched in the non-shedders while the genus Ruminococcus and the Bacteroidetes phylum were notably enriched in the super-shedders. There was greater bacterial diversity and richness in samples collected from the lower- as compared to the upper gastrointestinal tract (GI). The spiral colon was the only GI location that differed in terms of bacterial diversity between super-shedders and non-shedders. These findings reinforced linkages between E. coli O157:H7 colonization in cattle and the nature of the microbial community inhabiting the digestive tract of super-shedders.

Antimicrobial resistance (AMR) is a global health threat, and a standstill in the discovery and design of new antibiotics has been linked to the growing number of human deaths attributed to AMR infections. Intensive beef production utilizes antimicrobials to promote health and growth efficiency. To understand the magnitude and risk of AMR in beef production, it is important to assess the prevalence and diversity of antimicrobial resistant genes (ARGs) within microbial populations. Antimicrobial resistant bacteria are traditionally identified by isolation and growth in the presence of selective antibiotics. Whole-genome, metagenomic, and RNA sequencing provide new avenues to detect and identify novel ARGs in both culturable and unculturable bacterial communities. Some of these approaches place ARGs within the context of mobile genetic elements, gauging their likelihood of transfer across genomes. Genomics can also mitigate AMR, contributing to rational drug design or the development of alternatives to antimicrobials such as vaccines and probiotics. RNA-seq-based transcriptomics and Tn-seq may provide new ways to examine mechanisms that promote or prevent AMR. Finally, clustered regularly interspaced short palindromic repeats (CRISPR) - Cas gene editing could directly reduce AMR by killing AMR-resistant bacteria without harming beneficial bacteria. Together, these technologies may provide new opportunities to identify, quantify, and mitigate AMR while developing alternatives to antimicrobials for beef production.

J. Anim. Sci Vol. 94, E-Suppl. 5/J. Dairy Sci. Vol. 99, E-Suppl. 1 multiparous from d 2 to 5, d 2 to 14, d 2 to Peak, d 5 to 10, d 5 to 14, d 5 to Peak and d 10 to 14 (0.2, 1.0, 1.0, 1.2, 1.6, 1.4 and 2.2 vs. -0.1, 0.8, 0.9, 0.9, 1.4, 1.3 and 2.0, respectively). Cows that conceived, regardless of whether or not they suffered pregnancy loss, had greater P4 concentrations (except on d 2) and a greater rate of P4 increase than open cows. Also, primiparous cows consistently had greater P4 concentrations and rate of increase than multiparous cows.

Cattle are the primary reservoir of the foodborne pathogen Escherichia coli O157:H7, with the concentration and frequency of E. coli O157:H7 shedding varying substantially among individual hosts. The term ''super-shedder" has been applied to cattle that shed ≥10(4) cfu E. coli O157:H7/g of feces. Super-shedders have been reported to be responsible for the majority of E. coli O157:H7 shed into the environment. The objective of this study was to determine if there are phenotypic and/or genotypic differences between E. coli O157:H7 isolates obtained from super-shedder compared to low-shedder cattle. From a total of 784 isolates, four were selected from low-shedder steers and six isolates from super-shedder steers (4.01-8.45 log cfu/g feces) for whole genome sequencing. Isolates were phage and clade typed, screened for substrate utilization, pH sensitivity, virulence gene profiles and Stx bacteriophage insertion (SBI) sites. A range of 89-2473 total single nucleotide polymorphisms (SNPs) were identified when sequenced strains were compared to E. coli O157:H7 strain Sakai. More non-synonymous SNP mutations were observed in low-shedder isolates. Pan-genomic and SNPs comparisons did not identify genetic segregation between super-shedder or low-shedder isolates. All super-shedder isolates and 3 of 4 of low-shedder isolates were typed as phage type 14a, SBI cluster 3 and SNP clade 2. Super-shedder isolates displayed increased utilization of galactitol, thymidine and 3-O-β-D-galactopyranosyl-D-arabinose when compared to low-shedder isolates, but no differences in SNPs were observed in genes encoding for proteins involved in the metabolism of these substrates. While genetic traits specific to super-shedder isolates were not identified in this study, differences in the level of gene expression or genes of unknown function may still contribute to some strains of E. coli O157:H7 reaching high densities within bovine feces.

Escherichia coli O157:H7 is a foodborne pathogen that causes illness in humans worldwide. Cattle are the primary reservoir of this bacterium, with the concentration and frequency of E. coli O157:H7 shedding varying greatly among individuals. The term “super-shedder” has been applied to cattle that shed concentrations of E. coli O157:H7≥104 colony-forming units/g feces. Super-shedders have been reported to have a substantial impact on the prevalence and transmission of E. coli O157:H7 in the environment. The specific factors responsible for super-shedding are unknown, but are presumably mediated by characteristics of the bacterium, animal host, and environment. Super-shedding is sporadic and inconsistent, suggesting that biofilms of E. coli O157:H7 colonizing the intestinal epithelium in cattle are intermittently released into feces. Phenotypic and genotypic differences have been noted in E. coli O157:H7 recovered from super-shedders as compared to low-shedding cattle, including differences in phage type (PT21/28), carbon utilization, degree of clonal relatedness, tir polymorphisms, and differences in the presence of stx2a and stx2c, as well as antiterminator Q gene alleles. There is also some evidence to support that the native fecal microbiome is distinct between super-shedders and low-shedders and that low-shedders have higher levels of lytic phage within feces. Consequently, conditions within the host may determine whether E. coli O157:H7 can proliferate sufficiently for the host to obtain super-shedding status. Targeting super-shedders for mitigation of E. coli O157:H7 has been proposed as a means of reducing the incidence and spread of this pathogen to the environment. If super-shedders could be easily identified, strategies such as bacteriophage therapy, probiotics, vaccination, or dietary inclusion of plant secondary compounds could be specifically targeted at this subpopulation. Evidence that super-shedder isolates share a commonality with isolates linked to human illness makes it imperative that the etiology of this phenomenon be characterized.

Escherichia coli O157:H7 is a foodborne pathogen that causes illness in humans worldwide. Cattle are the primary reservoir of this bacterium, with the concentration and frequency of E. coli O157:H7 shedding varying greatly among individuals. The term "super-shedder" has been applied to cattle that shed concentrations of E. coli O157:H7 ≥ 10⁴ colony-forming units/g feces. Super-shedders have been reported to have a substantial impact on the prevalence and transmission of E. coli O157:H7 in the environment. The specific factors responsible for super-shedding are unknown, but are presumably mediated by characteristics of the bacterium, animal host, and environment. Super-shedding is sporadic and inconsistent, suggesting that biofilms of E. coli O157:H7 colonizing the intestinal epithelium in cattle are intermittently released into feces. Phenotypic and genotypic differences have been noted in E. coli O157:H7 recovered from super-shedders as compared to low-shedding cattle, including differences in phage type (PT21/28), carbon utilization, degree of clonal relatedness, tir polymorphisms, and differences in the presence of stx2a and stx2c, as well as antiterminator Q gene alleles. There is also some evidence to support that the native fecal microbiome is distinct between super-shedders and low-shedders and that low-shedders have higher levels of lytic phage within feces. Consequently, conditions within the host may determine whether E. coli O157:H7 can proliferate sufficiently for the host to obtain super-shedding status. Targeting super-shedders for mitigation of E. coli O157:H7 has been proposed as a means of reducing the incidence and spread of this pathogen to the environment. If super-shedders could be easily identified, strategies such as bacteriophage therapy, probiotics, vaccination, or dietary inclusion of plant secondary compounds could be specifically targeted at this subpopulation. Evidence that super-shedder isolates share a commonality with isolates linked to human illness makes it imperative that the etiology of this phenomenon be characterized.

The objective of this study was to determine the frequency and duration of super-shedding in cattle by enumerating Escherichia coli O157:H7 in feces and to compare lineage and pulsed-field gel electrophoresis (PFGE) subtypes from super- and low-shedders. E. coli O157:H7 was enumerated from fecal samples obtained from the rectums of 400 feedlot cattle. Super-shedding steers (N=11) were identified, transported, and penned individually. Freshly voided fecal pats were sampled 2 h before and 6 h after feeding for 7 d, then once daily for an additional 19 d. Isolates (N=126) were subtyped using PFGE, and lineage was typed using a lineage-specific polymorphism assay. Of the 11 super-shedders identified at the commercial feedlot, only five were confirmed as super-shedders at the research feedlot, with no super-shedders identified 6 d after sampling at the commercial feedlot. Super-shedding was not consistent in fecal pats collected from the same individual at different times of the day. Isolates exhibited three distinct PFGE subtypes, with most isolates (97.6%) displaying the same subtype, including those obtained from steers that transitioned from super- to low-shedding. The short duration of super-shedding and its lack of continuance suggest that these individuals may not play as great a role in the dissemination of E. coli O157:H7 within the feedlot as previously proposed.

ABSTRACT The objectives of this study were to identify endemic bacteriophages (phages) in the feedlot environment and determine relationships of these phages to Escherichia coli O157:H7 from cattle shedding high and low numbers of naturally occurring E. coli O157:H7. Angus crossbred steers were purchased from a southern Alberta (Canada) feedlot where cattle excreting ≥10 4 CFU · g −1 of E. coli O157:H7 in feces at a single time point were identified as supershedders (SS; n = 6), and cattle excreting <10 4 CFU · g −1 of feces were identified as low shedders (LS; n = 5). Fecal pats or fecal grabs were collected daily from individual cattle for 5 weeks. E. coli O157:H7 in feces was detected by immunomagnetic separation and enumerated by direct plating, and phages were isolated using short- and overnight-enrichment methods. The total prevalence of E. coli O157:H7 isolated from feces was 14.4% and did not differ between LS and SS ( P = 0.972). The total prevalence of phages was higher in the LS group (20.9%) than in the SS group (8.3%; P = 0.01). Based on genome size estimated by pulsed-field gel electrophoresis and morphology determined by transmission electron microscopy, T4- and O1-like phages of Myoviridae and T1-like phage of Siphoviridae were isolated. Compared to T1- and O1-like phages, T4-like phages exhibited a broad host range and strong lytic capability when targeting E. coli O157:H7. Moreover, the T4-like phages were more frequently isolated from feces of LS than SS, suggesting that endemic phages may impact the shedding dynamics of E. coli O157:H7 in cattle.

Escherichia coli O157:H7 is a major foodborne human pathogen causing disease worldwide. Cattle are a major reservoir for this pathogen and those that shed E. coli O157:H7 at >104 CFU/g feces have been termed "super-shedders". A rich microbial community inhabits the mammalian intestinal tract, but it is not known if the structure of this community differs between super-shedder cattle and their non-shedding pen mates. We hypothesized that the super-shedder state is a result of an intestinal dysbiosis of the microbial community and that a "normal" microbiota prevents E. coli O157:H7 from reaching super-shedding levels. To address this question, we applied 454 pyrosequencing of bacterial 16S rRNA genes to characterize fecal bacterial communities from 11 super-shedders and 11 contemporary pen mates negative for E. coli O157:H7. The dataset was analyzed by using five independent clustering methods to minimize potential biases and to increase confidence in the results. Our analyses collectively indicated significant variations in microbiome composition between super-shedding and non-shedding cattle. Super-shedders exhibited higher bacterial richness and diversity than non-shedders. Furthermore, seventy-two operational taxonomic units, mostly belonging to Firmicutes and Bacteroidetes phyla, were identified showing differential abundance between these two groups of cattle. The operational taxonomic unit affiliation provides new insight into bacterial populations that are present in feces arising from super-shedders of E. coli O157:H7.

Aims:  To characterize class 1 integrons and resistance genes in tetracycline‐resistant Escherichia coli originating from beef cattle subtherapeutically administered chlortetracycline (A44), chlortetracycline and sulfamethazine (AS700), or no antimicrobials (control).

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

Su, R., Munns, K., Beauchemin, K. A., Schwartzkopf-Genswein, K., Jin-Quan, L., Topp, E. and Sharma, R. 2011. Effect of backgrounding and transition diets on fecal concentration and strain types of commensal Escherichia coli in beef cattle. Can. J. Anim. Sci. 91: 449–458. The aim of the study was to assess the effect of four different backgrounding diets [55% corn silage in combination with 40% of either temper rolled barley-grain, wheat dried distillers' grain, corn dried distillers' grain or millrun (dry matter basis)] and a transition diet containing increasing amount of concentrate (60 to 90%) on fecal Escherichia coli in feedlot heifers. Heifers were fed backgrounding forage based diets for 91 d and then were shifted to a transition diet for an additional 18 d. Strain characterization of E. coli (n=224) was carried out at time time points over 109 d and indicated that heifers fed the barley–grain diet shed higher (P<0.001) total and tetracycline-resistant (Tetr) E. coli in the backgrounding phase. Of the total E. coli examined (n=224), 70.3% showed resistance to one or more of the 14 antimicrobials examined, which increased to 82.3% by day 109. Among the recovered E. coli, 23 phenotypes and 154 pulsed field gel electrophoresis patterns grouped into 38 clusters indicative of extensive E. coli diversity in heifers. Although supplementation of 40% barley-grain was correlated to higher total E. coli shedding in the backgrounding phase, the backgrounding diets did not influence strain clustering. Strains collected during the transition phase clustered more closely than those collected during the backgrounding phase. This increased strain clustering with high concentrate inclusion in the transition diet was observed within 18 d of the transition phase. Our study indicated variations in E. coli shedding rates and strain clustering in relation to time and diet.

Escherichia coli O157:H7 is a pathogenic, gram-negative bacterium that causes diarrhea, hemorrhagic colitis, and can lead to fatal hemolytic uremic syndrome in humans. We examined the persistence of E. coli O157:H7 lineages I and II in feces held at 4, 12, and 25 degrees C, from animals fed either grain or hay diets. Three strains of each lineage I and II were inoculated into grain-fed or hay-fed feces, and their persistence was monitored over 28 days. No significant differences in E. coli O157:H7 survival between the 2 lineages in both fecal types was found at the examined temperatures. Volatile fatty acids were higher in grain-fed than in hay-fed feces, resulting in consistently lower pH in the grain-fed feces at 4, 12 and 25 degrees C. Regardless of lineage type, E. coli O157:H7 CFUs were significantly higher in grain-fed than in hay-fed feces at 4 and 25 degrees C. Escherichia coli O157:H7 survival was highest in grain-fed feces at 25 degrees C up to 14 days. Our results indicate that the 2 lineages of E. coli O157:H7 do not differ in their persistence; however, it appears that temperature and feces type both affect the survival of the pathogen.

The area seeded to genetically modified crops has been steadily increasing since their introduction in the early 1990s. However, there have been concerns relating to the persistence and stability of transgenic DNA from genetically modified crops and subsequent outflow into the environment. The cp4 5-enolpyruvylshikimate-3-phosphate synthase (cp4epsps) gene confers glyphosate resistance in Roundup Ready(superscript ®) (RR) canola (Brassica napus event RT73). Sclerotinia sclerotiorum is an important pathogen causing stem blight and pod rot of canola. The aim of this study was to detect cp4epsps transgenic DNA on sclerotia of Sclerotinia sclerotiorum formed in diseased stems of RR canola grown in the field. In this 2-year study, sclerotia collected from naturally infected stems of RR canola and conventional canola at the time harvest were screened for the presence of 1363bp cp4epsps and smaller transgene fragments by PCR (limit of detection 50 pg) and Southern hybridization (limit of detection ＜5 pg). The complete cp4epsps transgene could not be detected in any of the analyzed sclerotia: however, a transgene fragment was detected in a single sclerotium tissue recovered from a RR canola plant. This fragment was not stably integrated as the transgene fragment could not be detected in mycelia produced from was not stably integrated as the transgene fragment could not be detected in mycelia produced from the germination of this positive sclerotium. Thus, trace levels of transgene fragments may be detected, albeit at an extremely low incidence rate, from surface of sclerotia of S. sclerotiorum produced on the transgenic plants of canola.

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.

Oilseed rape (Brassica napus L.) is an important agronomic crop used for oil, food and animal feed. Glyphosate resistant B. napus is tolerant to the herbicide glyphosate and has been widely adopted due to higher economic returns. However, a concern for growing transgenic B. napus is the potential escape of cp4 epsps transgene via cross fertilization with wild and/or weedy species including Brassica juncea, especially in China, where B. juncea is prevalent. We assessed transgene inheritance and quantification upon wild B. juncea × transgenic B. napus outcrossing from three backcross (BC) generations and their progenies using real-time PCR. Under in vitro conditions, a stable inheritance and similar cp4 epsps copy number between different generations (BC2, BC3 and BC4) and their progenies were observed. A marginally higher copy number was observed when B. juncea was used as the paternal as compared to when it was used as maternal parent. This study demonstrates stable inheritance of the glyphosate resistance trait and the production of fit out crossed offspring in cases of wild type B. juncea × transgenic B. napus.