With soybean [Glycine max (L.) Merr.] seed cost increasing in Alberta, understanding economic optimum plant density (EOPD) could help growers save on input expenses. A study was conducted at two irrigated locations in southern Alberta (Bow Island and Lethbridge), in three growing seasons (2014–2016), using two maturity group (MG) 00 soybean genotypes, two row spacings (RS; narrow, 17.5 cm; wide, 35 cm), and three seeding densities (SD; 30, 50 and 80 seeds·m−2). Exponential plant density–yield relationships were used to estimate EOPD. The earlier MG 00.4 genotype compensated yield at lower plant density (39 vs. 43 plants·m−2) and emergence (74% vs. 80%) than the later MG 00.8 genotype. The EOPD gaps between environments, genotypes, and RS were minimal (from 1–3 plants·m−2), resulting in only 1.3%–2.0% differences in grain yield (37–56 kg·ha−1), and gross revenue at EOPD ($16–24·ha−1). The overall EOPD estimate was 46 plants·m−2, regardless of environment, genotype or RS. The study highlighted the difference between agronomic production and profit maximization in choosing an optimum plant density, and the need to establish a seeding density calculator for irrigated soybean in southern Alberta.

Sod-seeding of depleted pastures with non-bloating legumes can be a low-cost pasture rejuvenation strategy for ranchers in the North American prairies. A study was conducted to determine if sainfoin (Onobrychis vicifolia subsp. vicifolia) or cicer milkvetch (CMV) (Astragalus cicer L.) populations or cultivars (hereinafter populations) can be used to rejuvenate alfalfa (Medicago sativa L.) or grass pastures. The study included sainfoin populations with increased fitness to grow with alfalfa and CMV cultivars that were selected for early emergence and rapid seedling growth. These populations were seeded using three seeding methods: traditional re-seeding, drilling seed with Great Plains drill, or Pan drill, using split-plot randomization at three locations in Alberta in 2015 or 2016. Lethbridge had pre-existing alfalfa while Ponoka and Red Deer were predominantly grass pastures. The sod-seeded rejuvenation, as indicated by percentage dry matter (DM) contributed by newly planted species succeeded at Lethbridge (>= 15), failed at Ponoka (similar to 1), and partially succeeded at Red Deer (>= 9). Successful establishment of new plants did not increase the total forage mass of the rejuvenated pasture. Sainfoin populations established better and contributed greater forage mass compared to CMV cultivars when sod-seeded on alfalfa pasture, but the improvement was not consistent on grass pasture. Forage nutritive values were not different among populations within species, but pasture seeded with CMV cultivars had greater crude protein than unseeded plots. Sod-seeded grass-legume pasture had greater crude protein than unseeded plots at Red Deer. Sod-seeding alfalfa pasture with sainfoin populations can be an efficient pasture rejuvenation strategy.

Microbiological surveillance of the food chain plays a critical role in improving our understanding of the distribution and circulation of food-borne pathogens along the farm to fork continuum toward the development of interventions to reduce the burden of illness. The application of molecular subtyping to bacterial isolates collected through surveillance has led to the identification of strains posing the greatest risk to public health. Past evidence suggests that enrichment methods for Campylobacter jejuni, a leading bacterial foodborne pathogen worldwide, may lead to the differential recovery of subtypes, obscuring our ability to infer the composition of a mixed-strain sample and potentially biasing prevalence estimates in surveillance data. To assess the extent of potential selection bias resulting from enrichment-based isolation methods, we compared enrichment and non-enrichment isolation of mixed subtype cultures of C. jejuni, followed by subtype-specific enumeration using both colony plate-counts and digital droplet PCR. Results differed from the null hypothesis that similar proportions of C. jejuni subtypes are recovered from both methods. Our results also indicated a significant effect of subtype prevalence on isolation frequency post-recovery, with the recovery of more common subtypes being consistently favored. This bias was exacerbated when an enrichment step was included in the isolation procedure. Taken together, our results emphasize the importance of selecting multiple colonies per sample, and where possible, the use of both enrichment and non-enrichment isolation procedures to maximize the likelihood of recovering multiple subtypes present in a sample. Moreover, the effects of subtype-specific recovery bias should be considered in the interpretation of strain prevalence data toward improved risk assessment from microbiological surveillance data.

Cercospora leaf spot (CLS), caused by Cercospora traversoana, is an important phyto-pathological problem of self-pollinated fenugreek (Trigonella-foenum graecum). Developing resistant genotypes in crop plants has been considered the best option to control diseases for economic, environmental, and social reasons. However, before this can be accomplished, knowledge about the inheritance of disease-resistant genes is necessary for creating high-yielding resistant genotypes. One susceptible fenugreek cultivar, Tristar, and two resistant accessions L3717 and PI138687 were used in two-way crosses using hand emasculation and pollination technique in a greenhouse. F1 plants were grown in a greenhouse and allowed to grow till maturity to produce F2 seeds. Some flowers from F1 plants were crossed back to both resistant and susceptible parents separately to generate backcross (BC1) seeds. Parents, F1, F2, and BC1 populations were grown in the greenhouse using a RCBD with four replications. Plants were inoculated 30 d after sowing with a suspension of C. traversoana at 2 × 105 conidia mL−1. Symptoms were observed and rated on individual plants 25 d after inoculation, and plants were categorized according to susceptible or resistant reactions based on rating scores. Mean disease score was significantly different (p < 0.0001) among generations. In both the cross combinations, results showed CLS resistance in fenugreek (from L3717 and PI138687) was governed by a single dominant gene which is moderately heritable (46% narrow sense heritability). This indicates a relatively simple pathway for transfer of genes to adapted fenugreek cultivars.

Soybean [Glycine max (L.) Merr.] production has moved rapidly westward on the Canadian prairies, most recently arriving in southern Alberta. Adjusting row spacing (RS) and seeding density (SD) to maximize soybean productivity is well-documented for rainfed conditions but not where irrigation is obligatory. A 3 yr study was conducted at two irrigated locations in southern Alberta using two early-maturity [maturity group 00] soybean genotypes planted at two RSs and three SDs. Soybean reached 95% maturity in 114–132 d and only one of six growing environments experienced a killing frost prior to maturity. Wide rows led to 1 d earlier maturity for one genotype in all six environments and increased grain yield (5%–20%) in four out of six environments compared with narrow rows. Increasing SD from 30 to 80 seeds m−2 generally led to increased pod clearance (from 5.0 to 8.4 cm in one environment) and grain (mean increase of 33%, from 2100 to 2800 kg ha−1) and straw yield, but decreased seeds plant−1 (from 94 to 46). Notwithstanding 9% lower cumulative corn heat units during the study, and an average 5 d longer maturity requirement at Lethbridge, soybean performance was equal to Bow Island in many parameters including grain yield. Our findings will help develop recommendations for new soybean growers in the irrigated region of southern Alberta.

Cercospora leaf spot (CLS) caused by Cercospora traversiana is an important phyto-pathological problem of fenugreek (Trigonella foenum-graecum L.), a multiuse legume crop. Field screenings for resistant plants, although accurate and effective, demand significant time and a sizable workforce to accomplish. Also, weather conditions in the field may not always be favourable for uniform disease spread, which eventually may lead to failure of the overall experiment. Whole-plant assays (WPA) and detached leaf assays (DLA) with artificial inoculation not only help in scaling up the number of plants screened but also reduce the space, time, and amount of inoculum needed for the experiment. The results from our two experiments indicate that both the WPA and DLA methods can be used reliably to differentiate resistant and susceptible genotypes of fenugreek. In addition, the correlation coefficient between WPA and DLA (r = 0.875, P < 0.01), derived from the mean disease score of each genotype, shows that they can be used interchangeably while screening fenugreek for CLS. DLA was found to be temperature-sensitive for the development of CLS symptoms and wounded leaves developed symptoms faster than non-wounded leaves. These indoor methods can be used for the development of CLS-resistant fenugreek cultivars in areas where disease development is difficult under field conditions.

Often Escherichia coli are harmless and/or beneficial bacteria inhabiting the gastrointestinal tract of livestock and humans. However, Shiga toxin-producing E. coli (STEC) have been linked to human disease. Cattle are the primary reservoir for STEC and STEC "super-shedders" are considered to be a major contributor in animal to animal transmission. Among STEC, O157:H7 is the most recognized serotype, but in recent years, non-O157 STEC have been increasingly linked to human disease. In Argentina and Germany, O178 is considered an emerging pathogen. Our objective was to compare populations of E. coli O178, O157, shiga toxin 1 and 2 in western Canadian cattle feces from a sampling pool of ~80,000 beef cattle collected at two slaughterhouses. Conventional PCR was utilized to screen 1,773 samples for presence/absence of E. coli O178. A subset of samples (n = 168) was enumerated using droplet digital PCR (ddPCR) and proportions of O178, O157 and shiga toxins 1 & 2 specific-fragments were calculated as a proportion of generic E. coli (GEC) specific-fragments. Distribution of stx1 and stx2 was determined by comparing stx1, stx2 and O157 enumerations. Conventional PCR detected the presence of O178 in 873 of 1,773 samples and ddPCR found the average proportion of O178, O157, stx1 and stx2 in the samples 2.8%, 0.6%, 1.4% and 0.5%, respectively. Quantification of stx1 and stx2 revealed more virulence genes than could be exclusively attributed to O157. Our results confirmed the presence of E. coli O178 in western Canadian cattle and ddPCR revealed O178 as a greater proportion of GEC than was O157. Our results suggests: I) O178 may be an emerging subgroup in Canada and II) monitoring virulence genes may be a more relevant target for food-safety STEC surveillance compared to current serogroup screening.

Shiga toxin-producing Escherichia coli (STEC) are a subgroup of E. coli causing human diseases. Methods to control STEC in livestock and humans are limited. These and other emerging pathogens are a global concern and novel mitigation strategies are required. Habitats populated by bacteria are subjected to competition pressures due to limited space and resources but they use various strategies to compete in natural environments. Our objective was to evaluate non-pathogenic E. coli strains isolated from cattle feces for their ability to out-compete STEC. Competitive fitness of non-pathogenic E. coli against STEC were assessed in competitions using liquid, agar, and nutrient limiting assays. Winners were determined by enumeration using O-serogroup specific quantitative PCR or a semi-quantitative grading. Initial liquid competitions identified two strong non-pathogenic competitors (O103F and O26E) capable of eliminating various STEC including O157 and O111. The strain O103F was dominant across permeable physical barriers for all tested E. coli and STEC strains indicating the diffusion of antimicrobial molecules. In direct contact and even with temporal disadvantages, O103F out-competed STEC O157E. The results suggest that O103F or the diffusible molecule(s) it produces have a potential to be used as an alternative STEC mitigation strategy, either in medicine or the food industry.

A multi-year, multi-location grazing study with alfalfa (Medicago sativa L.) and sainfoin (Onobrychis viciifolia Scop.) mixed pastures was undertaken to determine pasture productivity and quality. At Lethbridge, mixed pastures of 3 experimental sainfoin populations (LRC-3401, LRC-3432, and LRC-3519) and Nova sainfoin with AC Blue J alfalfa were established in a replicated experiment in 2008. A similar experiment with Beaver alfalfa was established in 2009 at Swift Current, Saskatchewan, Canada (SC). The experimental populations were developed to persist in mixtures with alfalfa under a multiple-cut system. Pastures were grazed by steers using a rotational grazing system at a stocking rate of 4.0 animal unit month ha/yr (8.6 steers/ha per season). Four-year mean DM yields of LRC-3432 (10,713 kg/ha) and LRC-3519 (10,530 kg/ha) mixed pastures were higher (P < 0.05) than Nova (9,668 kg/ha) pastures. Percentages of DM in LRC-3432 (28–30%) and LRC-3519 (30–43%) in the mixed pastures were higher (P < 0.05) than Nova (5–8%) after the first rotation of grazing in all years at Lethbridge and in 2010 at SC. In continuous stocking experiments with different alfalfa mixtures, the same 2 sainfoin lines produced higher (P < 0.05) DM forage yields than Nova at Lethbridge but not at SC. Average daily gains of steers varied between 0.7 and 1.2 kg/d at both Lethbridge and SC, but the differences among pastures were not significant. New sainfoin populations, LRC-3432 and LRC-3519, can be used in alfalfa pastures for high ADG and bloat prevention, because they produced high DM yields and proportions in the mixed pastures.

A fundamental assumption in the use and interpretation of microbial subtyping results for public health investigations is that isolates that appear to be related based on molecular subtyping data are expected to share commonalities with respect to their origin, history, and distribution. Critically, there is currently no approach for systematically assessing the underlying epidemiology of subtyping results. Our aim was to develop a method for directly quantifying the similarity between bacterial isolates using basic sampling metadata and to develop a framework for computing the epidemiological concordance of microbial typing results. We have developed an analytical model that summarizes the similarity of bacterial isolates using basic parameters typically provided in sampling records, using a novel framework (EpiQuant) developed in the R environment for statistical computing. We have applied the EpiQuant framework to a data set comprising 654 isolates of the enteric pathogen Campylobacter jejuni from Canadian surveillance data in order to examine the epidemiological concordance of clusters obtained by using two leading C. jejuni subtyping methods. The EpiQuant framework can be used to directly quantify the similarity of bacterial isolates based on basic sample metadata. These results can then be used to assess the concordance between microbial epidemiological and molecular data, facilitating the objective assessment of subtyping method performance and paving the way for the improved application of molecular subtyping data in investigations of infectious disease.

Campylobacter jejuni is a leading human enteric pathogen worldwide and despite an improved understanding of its biology, ecology, and epidemiology, limited tools exist for identifying strains that are likely to cause disease. In the current study, we used subtyping data in a database representing over 24,000 isolates collected through various surveillance projects in Canada to identify 166 representative genomes from prevalent C. jejuni subtypes for whole genome sequencing. The sequence data was used in a genome-wide association study (GWAS) aimed at identifying accessory gene markers associated with clinically related C. jejuni subtypes. Prospective markers (n = 28) were then validated against a large number (n = 3,902) of clinically associated and non-clinically associated genomes from a variety of sources. A total of 25 genes, including six sets of genetically linked genes, were identified as robust putative diagnostic markers for clinically related C. jejuni subtypes. Although some of the genes identified in this study have been previously shown to play a role in important processes such as iron acquisition and vitamin B5 biosynthesis, others have unknown function or are unique to the current study and warrant further investigation. As few as four of these markers could be used in combination to detect up to 90% of clinically associated isolates in the validation dataset, and such markers could form the basis for a screening assay to rapidly identify strains that pose an increased risk to public health. The results of the current study are consistent with the notion that specific groups of C. jejuni strains of interest are defined by the presence of specific accessory genes.

ImplicationsShiga toxin-producing Escherichia coli (STEC) are bacterial pathogens responsible for deadly foodborne outbreaks and sporadic illnesses globally. Children under five are most susceptible to severe complications and death. Seven main serogroups (O157 and top six non-O157: O26, O45, O103, O111, O121, O145) have been identified as causing the majority of STEC infections in humans.Beef products are one frequent source of infection, necessitating robust surveillance programs. However, detection and isolation methods for clinically relevant serogroups have several inherent limitations, making routine screening for these pathogens difficult and time consuming.These pathogens are constantly evolving, further allowing them to evade current detection methods. Developments in technology and genomic sequencing may improve our knowledge of these pathogens, thereby enhancing surveillance systems. With intensive beef production systems and a growing global demand for food, such advances are essential to improve food safety.

Field studies with two types of alfalfa (Medicago sativa L.) cultivars were conducted at Lethbridge in 2012 and 2013 and at Picture Butte in 2012 to determine the effects of irrigation on the dry matter (DM) yield and on net returns. The irrigated cultivars (Longview and Blue J) and dryland cultivars (Rangelander and Rambler) were arranged on plots in a randomized complete block design with four irrigation treatments and replicated five times. For the optimal irrigation treatment (W1), soil water content was maintained between 60 and 90% of available water in the designated root zone. Other irrigation treatments received 75% (W2), 50% (W3), and 25% (W4) of the irrigation water applied to the optimal treatment. The mean DM yields of irrigated alfalfa cultivars were greater than one of the dryland cultivars in both locations. The mean total DM yields for W2and W3at Lethbridge for Blue J, Longview and Rambler were greater than those of W1, although the differences were not always significant. The net returns, calculated by using the same price for all alfalfa harvests were similar across the cultivars and irrigation treatments excepting Rangelander, where the returns were lower. The results obtained from this study indicated a trend towards comparable yields and net returns between the optimal and the 75% irrigation treatment with 40% depletion of available water at the root zone, for the irrigated alfalfa cultivars and a dryland type Rambler.

Deadly outbreaks and illnesses due to Shiga toxin-producing Escherichia coli (STEC) occur worldwide; however, the cultivation methods required for adequate monitoring and traceback investigations are inefficient at best. Detection of STEC relies heavily on enrichment; yet no standard media or protocols exist. Furthermore, whether enrichment may bias detection of multiple STEC serogroups from complex samples is unknown. Thus, 14 STEC strains of serogroups O157 and the top six non-O157s (O26, O45, O103, O111, O121, and O145) were enriched in pairs for 6–78 h in broth and evaluated by quantitative polymerase chain reaction (qPCR). Here we show that a conventional 6-h enrichment protocol did not result in intra-species culture bias for the isolates tested. However, subsequent enrichments often produced biased cultures, with differences in the qPCR gene copy number ≥2 log10 apparent in 12%, 38%, and 52% of competitions after 30, 54, and 78 h of consecutive enrichments, respectively. Some strains were able to prevail and (or) out-compete the opponent strain in 100% of competitions. Our results suggest that culture bias should be considered and (or) evaluated further due to the potential implications during routine pathogen screening and outbreak investigations.

Cercospora leaf spot is a destructive and widespread disease of fenugreek (Trigonella foenum-graecum L.). To identify resistant germplasms and to morphologically characterize the disease pathogen on fenugreek plants, 48 accessions adapted to western Canada were screened by artificially inoculating plants with 1x104 spores ml-1 conidial suspensions of C. traversiana in growth cabinets. Out of these, nine accessions were characterized as resistant (R) or moderately resistant (MR) and the rest were susceptible. A more detailed study with 20 selected (9 resistant and 11 other) accessions using the artificial inoculation method revealed that the accessions were significantly (p = 0.01) different in their reaction to the disease. In the later test only two (L3717 and PI138687) had R and two (L3698 and F86) had MR ratings. An ANOVA for the agronomic traits measured indicated that the interaction effect of accession and treatment (with or without inoculation) was significant and the R or MR rated accessions were less affected by inoculation than the rest. The western Canada adapted accessions recovered from the fungal inoculation and produced a reasonable seed and biomass yield. Although further studies are warranted to verify the findings, the disease symptoms described, the disease effect on agronomic traits observed, and morphology of the pathogen may serve as a reference for future studies involving plant reaction to C. traversiana.

Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens responsible for outbreaks of human infections worldwide. Ruminant livestock harbor STEC in their intestinal tract, and through fecal contamination possess the potential to compromise the safety of food and water. As a human health safety risk, STEC detection methods on beef carcasses and trim are needed as mandated by the USDA-FSIS. In order to monitor STEC prior to harvest and human consumption, our goal was to evaluate and/or improve detection of seven STEC serogroups in cattle feces. In comparison to traditional approaches, sample processing methods in bovine feces were evaluated using a multi-factorial Latin square design which involved freezing or freeze drying feces. Autoclaved versus non-autoclaved feces were spiked with O26:H11 or O157:H7 serotypes in various dilutions and enriched for up to 6h. Each hour, enriched aliquots were compared using traditional culture methods and quantitative polymerase chain reaction (qPCR). Furthermore, a 7-serogroup multiplex PCR (mPCR) was developed to detect O26, O45, O103, O111, O121, O145 and O157 serogroups simultaneously. The diagnostic sensitivity of our mPCR assay following 6h enrichment was superior (10CFU/g across all serogroups) compared to a previously established PCR assay (10CFU/g for O26, and O103; ≥10(4)CFU/g for all other serogroups). Obtaining viable isolates appeared to be limited by the efficiency of current immunomagnetic separation (IMS) methods, which ranged from 20 to 100% effectiveness at retrieving colonies depending on serogroup. After IMS, 70 putative STEC isolates were screened for Shiga toxin and attachment genes by mPCR. Sixty-five isolates contained one or both Shiga toxin genes.

Background. Recent outbreaks caused by bacterial contaminants in food, including sprouts by E. coli O104:H4 in Germany and processed meats by Listeria in Canada highlight the need for rapid and accurate characterization of bacterial pathogens. Current sequencing platforms have revolutionized the amount and quality of data available to epidemiologists, public health officials and microbiologists, who now require powerful yet intuitive tools to make sense of the underlying biology in these large datasets. In this study, we developed bioinformatics tools to: automate whole-genome analyses, make the data broadly accessible via novel reporting functions, and provide a dynamic computational platform for genomic analyses online at http://76.70.11.198/bacpath.Methods. A PHP-based web front end and PostgreSQL database display the pre-computed data. Genomic comparisons are performed using updates to our previously created pan-genomic software suite, Panseq (http:://lfz.corefacility.ca/panseq/). New genomic sequences are analyzed and added to the database without the need for recomputing previous analyses. Phylogenetic trees are created with MrBayes. Statistical calculations are performed using R.Results. A pathogen-specific genomic database encompassing all publicly available E. coli strains was created as a proof of concept. Pre-computed comparisons for the hundreds of bacterial genomes including phylogeny, presence/absence of virulence markers, group-specific biomarkers and geospatial information were generated. Data reporting tools were created to summarize the complexity of the data and to provide biologically pertinent results including genotype, phenotype (eg. anti-microbial resistance), and geospatial information.Discussion. The database provides rapid and accurate identification and characterization of E. coli. Output is formatted specifically for end users describing virulence, phylogeny and group-specific markers. Uptake of a global surveillance system with near real time analysis will provide an effective early warning system and allow for a faster response to pathogen-related outbreaks.

Previous research showed that the consumption of heat-killed E. coli O157:H7 bacteria resulted in an increase in the level of DNA damage in intestine, liver and spleen cells. We hypothesized that certain bacterial components released from heat-killed bacteria trigger this response. We analysed the possibility that bacterial components [such as lipopolysaccharides (LPS)] could induce changes in the level of proteins involved in cell proliferation, DNA repair and DNA methylation in distal spleen tissues of mice. Four-week-old male mice were provided water supplemented with whole heat-killed E. coli O157:H7 bacteria or components of bacteria (DNA, RNA, proteins and LPS). Spleen cells responded to exposure to whole heat-killed bacteria and LPS with an alteration in the level of PCNA proteins, DNA methylation proteins (DNMT1, DNMT3A, DNMT3B, and MeCP2) and DNA repair proteins (APE1 and KU70). Other bacterial components analysed in this study mostly did not alter protein expression. The data suggest that LPS is a bacterial component capable of inducing molecular changes in naïve spleen cells of hosts exposed to it.

Whole-genome sequence (WGS) data can, in principle, resolve bacterial isolates that differ by a single base pair, thus providing the highest level of discriminatory power for epidemiologic subtyping. Nonetheless, because the capability to perform whole-genome sequencing in the context of epidemiological investigations involving priority pathogens has only recently become practical, fewer isolates have WGS data available relative to traditional subtyping methods. It will be important to link these WGS data to data in traditional typing databases such as PulseNet and PubMLST in order to place them into proper historical and epidemiological context, thus enhancing investigative capabilities in response to public health events. We present MIST (Microbial In Silico Typer), a bioinformatics tool for rapidly generating in silico typing data (e.g. MLST, MLVA) from draft bacterial genome assemblies. MIST is highly customizable, allowing the analysis of existing typing methods along with novel typing schemes. Rapid in silico typing provides a link between historical typing data and WGS data, while also providing a framework for the assessment of molecular typing methods based on WGS analysis.