Antimicrobial Activities of α-Helix and ß-Sheet Peptides against the Major Bovine Respiratory Disease Agent, Mannheimia haemolytica.

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in cattle raised in North America. At the feedlot, cattle are subject to metaphylactic treatment with macrolides to prevent BRD, a practice that may promote antimicrobial resistance and has resulted in an urgent need for novel strategies. Mannheimia haemolytica is one of the major bacterial agents of BRD. The inhibitory effects of two amphipathic, α-helical (PRW4, WRL3) and one ß-sheet (WK2) antimicrobial peptides were evaluated against multidrug-resistant (MDR) M. haemolytica isolated from Alberta feedlots. WK2 was not cytotoxic against bovine turbinate (BT) cells by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. All three peptides inhibited M. haemolytica, with WK2 being the most efficacious against multiple isolates. At 8-16 µg/mL, WK2 was bactericidal against Mh 330 in broth, and at 32 µg/mL in the presence of BT cells, it reduced the population by 3 logs CFU/mL without causing cytotoxic effects. The membrane integrity of Mh 330 was examined using NPN (1-N-phenylnaphthylamine) and ONPG (o-Nitrophenyl ß-D-galactopyranoside), with both the inner and outer membranes being compromised. Thus, WK2 may be a viable alternative to the use of macrolides as part of BRD prevention and treatment strategies.

Effect of continuous or intermittent feeding of ergot contaminated grain in a mash or pelleted form on the performance and health of feedlot beef steers.

This study evaluated the effect of feeding ergot contaminated grain continuously or intermittently through backgrounding (BG) and finishing (FN) in a mash or pelleted supplement on the growth performance, health and welfare parameters, and carcass characteristics of feedlot beef steers. Sixty black Angus steers (300 ±â€…29.4 kg BW) were used in a complete randomized 238-d study. Steers were stratified by weight and randomly assigned to four different diets (15 steers/treatment) and individually housed. Treatments included: (1) control [CON; no added ergot alkaloids (EA)], (2) continuous ergot mash (CEM; fed continuously at 2 mg total EA/kg of DM), (3) intermittent ergot mash (IEM; fed at 2 mg total EA/kg of DM, during the first week of each 21-d period and CON for the remaining 2 wk, this feeding pattern was repeated in each period), and (4) intermittent ergot pellet (IEP; fed at 2 mg of total EA/kg of DM as a pellet during the first week of each 21-d period and CON for the remaining 2 wk as described for IEM). Steers were fed barley based BG diets containing 40% concentrate:60% silage (DM basis) for 84 d (four 21-d periods), transitioned over 28 d (no ergot fed) to an FN diet (90% concentrate:10% silage DM basis) and fed for 126 d (six 21-d periods) before slaughter. In the BG phase, steer DMI (P < 0.01, 7.45 vs. 8.05 kg/d) and ADG (P < 0.01) were reduced for all EA diets compared to CON. The CEM fed steers had lower ADG (P < 0.01, 0.735 vs. 0.980 kg) and shrunk final BW (P < 0.01, 350 vs. 366 kg) than CON. CEM had lower gain:feed (P < 0.07, 0.130 vs. 0.142) than CON. In the FN phase, steer DMI (P < 0.01, 9.95 vs. 11.05 kg/d) and ADG (P = 0.04) were also decreased for all EA fed steers compared to CON. Total shrunk BW gain (P = 0.03, 202.5 vs. 225.2 kg), final BW (P = 0.03, 617.9 vs. 662.2 kg), and carcass weight (P = 0.06) decreased for all EA fed steers compared to CON. The percentage of AAA carcasses decreased for all EA fed steers (P < 0.01, 46.7 vs. 93.3%) compared to CON. EA fed steers had increased rectal temperatures (P < 0.01, 39.8 vs. 39.4 °C) compared to CON. Pelleting ergot contaminated grain did not reduce the impact of ergot alkaloids on any of the measured parameters during BG or FN. Continuously or intermittently feeding ergot contaminated diets (2 mg total EA/kg of DM) significantly reduced intake, growth performance, and carcass weight, with minimal impact on blood parameters in feedlot steers. Pelleting was not an effective method of reducing ergot toxicity.

Produced by the fungus Claviceps purpurea, ergot alkaloids (EA) are toxic to beef cattle when consumed and can lead to reduction in feed intake and growth performance, vasoconstriction of the blood vessels, hyperthermia, damage to extremities (ears, tails, and hooves) and in severe cases, death. Grain is often cleaned to meet quality standards, and the resulting screenings are often utilized for feeding livestock and can have high concentrations of EA. The application of heat during pelleting of EA contaminated grain has been suggested to reduce its toxicity. Backgrounding and finishing beef cattle feeding experiments were conducted to assess the effect of continuously or intermittently feeding EA contaminated grain (2 mg/kg of diet DM) either as a pellet or as mash on growth performance, health, and animal welfare. Feeding EA grain continuously or intermittently either as a mash or pellet drastically reduced growth performance of steers, with no difference between treatments.

In Silico Analysis of Shiga Toxin-Producing Escherichia coli O157:H7 Strains from Presumptive Super- and Low-Shedder Cattle.

Cattle are the primary reservoir for STEC O157, with some shedding >104 CFU/g in feces, a phenomenon known as super-shedding (SS). The mechanism(s) responsible for SS are not understood but have been attributed to the environment, host, and pathogen. This study aimed to compare genetic characteristics of STEC O157 strains from cattle in the same commercial feedlot pens with SS or low-shedding (LS) status. Strains from SS (n = 35) and LS (n = 28) collected from 11 pens in three feedlots were analyzed for virulence genes, Shiga toxin-carrying bacteriophage insertion sites, and phylogenetic relationships. In silico analysis showed limited variation regarding virulence gene profiles. Stx-encoding prophage insertion sites mrlA and wrbA for stx1a and stx2a, respectively, were all occupied, but two isolates had fragments of the stx-carrying phage in mrlA and wrbA loci without stx1a and stx2a. All strains screened for lineage-specific polymorphism assay (LSPA-6) were 111111, lineage I. Of the isolates, 61 and 2 were clades 1 and 8, respectively. Phylogenetic analysis revealed that pens with more than one SS had multiple distantly related clusters of SS and LS isolates. Although virulence genes and lineage were largely similar within and across feedlots, multiple genetic origins of strains within a single feedlot pen illustrate challenges for on-farm control of STEC.

Relationship between Desiccation Tolerance and Biofilm Formation in Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) is a major concern in the food industry and requires effective control measures to prevent foodborne illnesses. Previous studies have demonstrated increased difficulty in the control of biofilm-forming STEC. Desiccation, achieved through osmotic stress and water removal, has emerged as a potential antimicrobial hurdle. This study focused on 254 genetically diverse E. coli strains collected from cattle, carcass hides, hide-off carcasses, and processing equipment. Of these, 141 (55.51%) were STEC and 113 (44.48%) were generic E. coli. The biofilm-forming capabilities of these isolates were assessed, and their desiccation tolerance was investigated to understand the relationships between growth temperature, relative humidity (RH), and bacterial survival. Only 28% of the STEC isolates had the ability to form biofilms, compared to 60% of the generic E. coli. Stainless steel surfaces were exposed to different combinations of temperature (0 °C or 35 °C) and relative humidity (75% or 100%), and the bacterial attachment and survival rates were measured over 72 h and compared to controls. The results revealed that all the strains exposed to 75% relative humidity (RH) at any temperature had reduced growth (p < 0.001). In contrast, 35 °C and 100% RH supported bacterial proliferation, except for isolates forming the strongest biofilms. The ability of E. coli to form a biofilm did not impact growth reduction at 75% RH. Therefore, desiccation treatment at 75% RH at temperatures of 0 °C or 35 °C holds promise as a novel antimicrobial hurdle for the removal of biofilm-forming E. coli from challenging-to-clean surfaces and equipment within food processing facilities.

The Isolation and Characterization of Bacteriophages Infecting Avian Pathogenic Escherichia coli O1, O2 and O78 Strains.

Avian pathogenic Escherichia coli (APEC), such as O1, O2 and O78, are important serogroups relating to chicken health, being responsible for colibacillosis. In this study, we isolated and characterized bacteriophages (phages) from hen feces and human sewage in Alberta with the potential for controlling colibacillosis in laying hens. The lytic profile, host range, pH tolerance and morphology of seven APEC-infecting phages (ASO1A, ASO1B, ASO2A, ASO78A, ASO2B, AVIO78A and ASO78B) were assessed using a microplate phage virulence assay and transmission electron microscopy (TEM). The potential safety of phages at the genome level was predicted using AMRFinderPlus and the Virulence Factor Database. Finally, phage genera and genetic relatedness with other known phages from the NCBI GenBank database were inferred using the virus intergenomic distance calculator and single gene-based phylogenetic trees. The seven APEC-infecting phages preferentially lysed APEC strains in this study, with ECL21443 (O2) being the most susceptible to phages (n = 5). ASO78A had the broadest host range, lysing all tested strains (n = 5) except ECL20885 (O1). Phages were viable at a pH of 2.5 or 3.5-9.0 after 4 h of incubation. Based on TEM, phages were classed as myovirus, siphovirus and podovirus. No genes associated with virulence, antimicrobial resistance or lysogeny were detected in phage genomes. Comparative genomic analysis placed six of the seven phages in five genera: Felixounavirus (ASO1A and ASO1B), Phapecoctavirus (ASO2A), Tequatrovirus (ASO78A), Kayfunavirus (ASO2B) and Sashavirus (AVIO78A). Based on the nucleotide intergenomic similarity (<70%), phage ASO78B was not assigned a genus in the siphovirus and could represent a new genus in class Caudoviricetes. The tail fiber protein phylogeny revealed variations within APEC-infecting phages and closely related phages. Diverse APEC-infecting phages harbored in the environment demonstrate the potential to control colibacillosis in poultry.

Effect of increasing concentration of ergot alkaloids in the diet of feedlot cattle: performance, welfare, and health parameters.

This study was designed to evaluate the effects of feeding increasing dietary concentrations of ergot alkaloids from cereal grains (EA; 0, 0.75, 1.5, 3.0 mg/kg of dietary DM) to feedlot cattle over backgrounding (BG) and finishing (FS) phases on health, welfare, and growth performance. Two hundred and forty commercial steers (280 ±â€…32 kg BW) were stratified by weight and randomly allocated to 16 pens (15 steers/pen), 4 of which were equipped with the GrowSafe system (1 pen/treatment) to measure individual feed intake. Each pen was randomly assigned to a treatment (n = 4/treatment). Treatments included 1) control (CTRL), no added EA; 2) CTRL + 0.75 mg/kg EA (EA075); 3) CTRL + 1.5 mg/kg EA (EA150); and 4) CTRL + 3.0 mg/kg EA (EA300). Steers were fed barley-based BG diets containing 40% concentrate: 60% silage (DM basis) for 84 d. Steers were then transitioned over 28 d to an FS diet (90% concentrate: 10% silage DM basis) and fed for 119 d before slaughter. The diet fed to EA300 steers was replaced with the CTRL diet after 190 d on feed (DOF), due to EA-induced hyperthermia starting at 165 DOF. In the BG phase, average meal length (P = 0.01) and size (P = 0.02), daily feeding duration (P = 0.03), final body weight (BW; P = 0.03), and total BW gain (P = 0.02) linearly decreased with increasing EA levels, while gain to feed (G:F) responded quadratically (P = 0.04), with EA150 having the poorest value. Increasing concentrations of EA in the diet linearly increased rectal temperature (P < 0.01) throughout the trial. Over the full FS phase, a quadratic response was observed for ADG (P = 0.05), final BW (P = 0.05), total BW gain (P = 0.02), and carcass weight (P = 0.05) with steers fed EA150 having the lowest performance, as EA300 steers were transferred to CTRL diet after 190 DOF. Dressing percentage (P = 0.02) also responded quadratically, with the lowest values observed for EA300. Thus, EA reduced ADG during BG and FS phases, although more prominently in FS, likely due to increased ambient temperatures and high-energy diet in FS triggering hyperthermia. When EA300 steers were transferred to the CTRL diet, compensatory gain promoted higher hot carcass weight (HCW) when compared with steers fed EA150. In conclusion, feeding feedlot steers diets with > 0.75 mg/kg EA caused reductions in performance and welfare concerns, although this breakpoint may be affected by duration of feeding, environmental temperatures, and EA profiles in the feed.

Ergot alkaloids (EA) are produced by a parasitic fungus (Claviceps purpurea) during the cereal grain growth cycle. Feeding cereal grain containing EA to beef cattle can cause constriction of blood vessels, hyperthermia, gangrene of extremities (ears, hoof, and tail), reduced feed intake and growth, and even death. Feed cleaning and processing technologies have been developed to remove EA from the human food chain, thus diverting contaminated feed for livestock use. We performed a beef cattle feedlot experiment to evaluate the impact of increasing levels of EA (0, 0.75, 1.50, 3.00 mg/kg of diet DM) on performance, health, and welfare. Steers fed 3.0 mg/kg of EA were transferred to the control diet (without EA) in the last half of finishing due to toxicity (hyperthermia). As EA levels increased, growth rate throughout the backgrounding and finishing phases decreased, while rectal temperatures increased and altered feeding behaviors occurred. Steers removed from 3 mg/kg EA diet exhibited compensatory gain, but their respiratory rate remained elevated 50 d after EA were last consumed.

Influence of temperature and pH on induction of Shiga toxin Stx1a in Escherichia coli.

Shiga toxin-producing strains represent pathogenic group that is of concern in food production. The present study evaluated forty-eight E. coli isolates (11 with intact stx gene, while remaining isolates presented only stx-fragments) for Shiga toxin production. The four most expressive stx-producers (O26, O103, O145, and O157) were selected to evaluate effects of pH (3.5, 4.5, and 7) and temperature (35, 40, and 50°C). After determining acid stress effects in media on Stx-induction, we mimicked "in natura" conditions using milk, apple, and orange juices. Only isolates that showed the presence of intact stx gene (11/48) produced Shiga toxin. In addition, acid pH had a role in down-regulating the production of Shiga toxin, in both lactic acid and juices. In contrast, non-lethal heating (40°C), when in neutral pH and milk was a favorable environment to induce Shiga toxin. Lastly, two isolates (O26 and O103) showed a higher capacity to produce Shiga toxin and were included in a genomic cluster with other E. coli involved in worldwide foodborne outbreaks. The induction of this toxin when subjected to 40°C may represent a potential risk to the consumer, since the pathogenic effect of oral ingestion of Shiga toxin has already been proved in an animal model.

Bacteriophages for the Targeted Control of Foodborne Pathogens.

Foodborne illness is exacerbated by novel and emerging pathotypes, persistent contamination, antimicrobial resistance, an ever-changing environment, and the complexity of food production systems. Sporadic and outbreak events of common foodborne pathogens like Shiga toxigenic E. coli (STEC), Salmonella, Campylobacter, and Listeria monocytogenes are increasingly identified. Methods of controlling human infections linked with food products are essential to improve food safety and public health and to avoid economic losses associated with contaminated food product recalls and litigations. Bacteriophages (phages) are an attractive additional weapon in the ongoing search for preventative measures to improve food safety and public health. However, like all other antimicrobial interventions that are being employed in food production systems, phages are not a panacea to all food safety challenges. Therefore, while phage-based biocontrol can be promising in combating foodborne pathogens, their antibacterial spectrum is generally narrower than most antibiotics. The emergence of phage-insensitive single-cell variants and the formulation of effective cocktails are some of the challenges faced by phage-based biocontrol methods. This review examines phage-based applications at critical control points in food production systems with an emphasis on when and where they can be successfully applied at production and processing levels. Shortcomings associated with phage-based control measures are outlined together with strategies that can be applied to improve phage utility for current and future applications in food safety.

Efficacy of Quaternary Ammonium Compounds for Control of Individual and Mixed Cultures of Escherichia coli with High- and Low-Quaternary Ammonium Compounds Resistance.

Escherichia coli is a well-characterized micro-organism in scientific literature. Similarly, quaternary ammonium compounds (QACs) are historical sanitizers in food processing. However, the use of QACs has been questioned due to bacterial resistance in some studies. Therefore, this study aimed to compare effects of single and mixed cultures of E. coli strains of different serogroups with either high (six strains) or low (five strains) resistance to QACs. Twenty-five combinations of strains with either high (H)- or low (L)-QAC resistance were analyzed (H + H vs. L + L). After exposure to QAC, combinations with statistical differences (p < 0.05) compared with individuals were selected and an inactivation model determined using GInaFit®. Only one combination of two strains (C23 and C20) with low-QAC resistance (mixture T18) had greater resistance (p < 0.05) than the individual isolates. The combination T18 and individual strain C23 presented a Weibull model, whereas the other isolated strain (C20) presented a biphasic inactivation model with a shoulder. Whole genome sequencing determined that unlike C20, C23 carried yehW, which may have led to Weibull inactivation. Possibly, very rapid interaction of C20 with the QAC favored increased survival of C23 and overall persistence of the T18 mixture. Consequently, our results indicate that individual E. coli with low-QAC resistance can synergistically interfere with QAC inactivation.

Stress Resistance and Virulence Gene Profiles Associated with Phylogeny and Phenotypes of Escherichia coli from Cattle.

Seven serogroups of E. coli (Top seven E. coli) are frequently implicated in foodborne outbreaks in North America, largely due to their carriage of Shiga toxin genes (stx). This study aimed to profile resistance genes and virulence factors (VF), and their potential association with phylogeny and phenotypes of Top seven E. coli originating from cattle in Canada. 155 Top seven E. coli isolates previously characterized for heat and acid resistance and biofilm-forming ability were whole-genome sequenced and analyzed for phylogeny, VF, and stress resistance genes. The 155 E. coli strains belonged to six phylogroups: A (n = 32), B1 (n = 93), C (n = 3), D (n = 11), E (n = 15), and G (n = 1). Different phylogroups were clearly separated on the core genome tree, with strains of the same serotype closely clustered. The carriage of stx and the transmissible locus of stress tolerance (tLST), the extreme heat resistance marker, was mutually exclusive, in 33 and 15 genomes, respectively. A novel O84:H2 strain carrying stx1a was also identified. In total, 70, 41, and 32 VF, stress resistance genes and antibiotic resistance genes were identified. The stress resistance genes included those for metal (n = 29), biocides/acid (n = 4), and heat (n = 8) resistance. All heat resistance genes and most metal-resistance genes that were differentially distributed among the phylogroups were exclusively in phylogroup A. VF were least and most present in phylogroups A and D, respectively. No specific genes associated with acid resistance or biofilm formation phenotypes were identified. VF were more abundant (P < 0.05) in the non-biofilm-forming population and acid-resistant population.

Association of resistance to quaternary ammonium compounds and organic acids with genetic markers and their relationship to Escherichia coli serogroup.

Sanitizer resistance is being extensively investigated due to the potential for bacterial survival and cross-resistance with other antimicrobials. Similarly, organic acids are being used due to their microbial inactivation potential as well as being generally recognized as safe (GRAS). However, little is known about associations of genetic and phenotypic factors in Escherichia coli related to resistance to sanitizers and organic acids as well as differences between "Top 7" serogroups. Therefore, we investigated 746 E. coli isolates for resistance to lactic acid and two commercial sanitizers based on quaternary ammonium and peracetic acid. Furthermore, we correlated resistance to several genetic markers and investigated 44 isolates using Whole Genome Sequencing. Results indicate that factors related to motility, biofilm formation, and Locus of Heat Resistance played a role in resistance to sanitizers and lactic acid. In addition, Top 7 serogroups significantly differed in sanitizer and acid resistance, with O157 being the most consistently resistant to all treatments. Finally, mutations in rpoA, rpoC, and rpoS genes were observed, in addition to presence of a Gad gene with alpha-toxin formation in all O121 and O145 isolates, which may be related to increased resistance of these serogroups to the acids used in the present study.

Genomic features and heat resistance profiles of Escherichia coli isolated from Brazilian beef.

AIMS: Characterize Escherichia coli and E. coli -producing (STEC) isolates from Brazilian beef to determine heat resistance and the presence of the transmissible locus of stress tolerance (tLST). METHODS AND RESULTS: Twenty-two STEC previously isolated from beef and characterized as STEC by PCR were subjected to different heat survival challenges (60°C and 71°C). Furthermore, the three tLST-positive isolates and one tLST-negative isolate by PCR were selected for WGS analysis. Phenotypic results indicated that 3/22 (13.64%) were heat resistant, 12/22 (54.54%) were moderately resistant, and 7/22 (31.82%) were sensitive to heat treatments. WGS analyses showed that three isolates with heat resistance showed tLST with up to 80% and 42% of similarity by BLAST analysis, with the major tLST genes being responsible for the homeostasis module. However, WGS showed the absence of stx genes associated with tLST-positive isolates, albeit with virulence and resistance genes found in extraintestinal pathogenic E. coli (ExPEC). CONCLUSION: Our findings demonstrate the presence of heat-resistant E. coli as well as confirm some tLST genes in E. coli isolated from Brazilian beef.

Lactic Acid Resistance and Population Structure of Escherichia coli from Meat Processing Environment.

To explore the effect of beef processing on Escherichia coli populations in relation to lactic acid resistance, this study investigated the links among acid response, phylogenetic structure, genome diversity, and genotypes associated with acid resistance of meat plant E. coli. Generic E. coli isolates (n = 700) were from carcasses, fabrication equipment, and beef products. Acid treatment was carried out in Luria-Bertani broth containing 5.5% lactic acid (pH 2.9). Log reductions of E. coli ranged from <0.5 to >5 log CFU/mL (median: 1.37 log). No difference in lactic acid resistance was observed between E. coli populations recovered before and after a processing step or antimicrobial interventions. E. coli from the preintervention carcasses were slightly more resistant than E. coli isolated from equipment, differing by <0.5 log unit. Acid-resistant E. coli (log reduction <1, n = 45) had a higher prevalence of genes related to energy metabolism (ydj, xap, ato) and oxidative stress (fec, ymjC) than the less resistant E. coli (log reduction >1, n = 133). The ydj and ato operons were abundant in E. coli from preintervention carcasses. In contrast, fec genes were abundant in E. coli from equipment surfaces. The preintervention E. coli contained phylogroups A and B1 in relatively equal proportions. Phylogroup B1 predominated (95%) in the population from equipment. Of note, E. coli collected after sanitation shared either the antigens of O8 or H21. Additionally, genome diversity decreased after chilling and equipment sanitation. Overall, beef processing did not select for E. coli resistant to lactic acid but shaped the population structure. IMPORTANCE Antimicrobial interventions have significantly reduced the microbial loads on carcasses/meat products; however, the wide use of chemical and physical biocides has raised concerns over their potential for selecting resistant populations in the beef processing environment. Phenotyping of acid resistance and whole-genome analysis described in this study demonstrated beef processing practices led to differences in acid resistance, genotype, and population structure between carcass- and equipment-associated E. coli but did not select for the acid-resistant population. Results indicate that genes coding for the metabolism of long-chain sugar acids (ydj) and short-chain fatty acids (ato) were more prevalent in carcass-associated than equipment-associated E. coli. These results suggest E. coli from carcasses and equipment surfaces have been exposed to different selective pressures. The findings improve our understanding of the microbial ecology of E. coli in food processing environments and in general.

Effects of Heating, Pelleting, and Feed Matrix on Apparent Concentrations of Cereal Ergot Alkaloids in Relation to Growth Performance and Welfare Parameters of Backgrounding Beef Steers.

As the contamination of cereal grains with ergot has been increasing in Western Canada, studies were undertaken to evaluate the impacts of heating (60, 80, 120, or 190 °C) alone or in combination with pelleting on concentrations of ergot alkaloids. Fifteen samples of ergot-contaminated grain from Alberta and Saskatchewan were assayed for R and S epimers of six alkaloids (ergocryptine, ergocristine, ergocornine, ergometrine, ergosine, and ergotamine) using HPLC MS/MS. Five samples with distinct alkaloid profiles were then selected for heating and pelleting studies. Heating resulted in a linear increase (p < 0.05) of total R and total S epimers with increasing temperature, although some individual R epimers were stable (ergometrine, ergosine, ergotamine). Pelleting also increased (p < 0.05) concentrations of total R and total S epimers detected, although ergometrine concentration decreased (p < 0.05) after pelleting. A feeding study arranged in a 2 × 2 factorial structure used 48 backgrounding Angus-cross steers fed four different diets: (1) Control Mash (CM, no added ergot), (2) Control Pellet (CP), (3) Ergot Mash (EM), or (4) Ergot Pellet (EP). Pelleting heated the ergot to 90−100 °C under 4 bars pressure, but the ergot used in the feeding study was not otherwise heated. Alkaloid concentrations of EM and EP varied by up to 1.1 mg/kg depending on the feed matrix assayed. No differences among treatments were noted for growth performance, feed intake, feed conversion, concentrations of serum prolactin and haptoglobin, hair cortisol, or in temperatures of extremities measured by infrared thermography. The only negative impacts of ergot alkaloids were on blood parameters indicative of reduced immune function or chronic inflammation. Pelleting did not heighten the negative clinical outcomes of ergot, although alkaloid concentrations of pelleted feed increased depending on the matrix assayed. It was hypothesized that the heat and pressure associated with pelleting may enhance the recovery of alkaloids from pelleted feed.

Genomic Analysis of Shiga Toxin-Producing E. coli O157 Cattle and Clinical Isolates from Alberta, Canada.

Shiga toxin (stx) is the principal virulence factor of the foodborne pathogen, Shiga toxin-producing Escherichia coli (STEC) O157:H7 and is associated with various lambdoid bacterio (phages). A comparative genomic analysis was performed on STEC O157 isolates from cattle (n = 125) and clinical (n = 127) samples to characterize virulence genes, stx-phage insertion sites and antimicrobial resistance genes that may segregate strains circulating in the same geographic region. In silico analyses revealed that O157 isolates harboured the toxin subtypes stx1a and stx2a. Most cattle (76.0%) and clinical (76.4%) isolates carried the virulence gene combination of stx1, stx2, eae and hlyA. Characterization of stx1 and stx2-carrying phages in assembled contigs revealed that they were associated with mlrA and wrbA insertion sites, respectively. In cattle isolates, mlrA and wrbA insertion sites were occupied more often (77% and 79% isolates respectively) than in clinical isolates (38% and 1.6% isolates, respectively). Profiling of antimicrobial resistance genes (ARGs) in the assembled contigs revealed that 8.8% of cattle (11/125) and 8.7% of clinical (11/127) isolates harboured ARGs. Eight antimicrobial resistance genes cassettes (ARCs) were identified in 14 isolates (cattle, n = 8 and clinical, n = 6) with streptomycin (aadA1, aadA2, ant(3'')-Ia and aph(3'')-Ib) being the most prevalent gene in ARCs. The profound disparity between the cattle and clinical strains in occupancy of the wrbA locus suggests that this trait may serve to differentiate cattle from human clinical STEC O157:H7. These findings are important for stx screening and stx-phage insertion site genotyping as well as monitoring ARGs in isolates from cattle and clinical samples.

Effect of ergot alkaloids and a mycotoxin deactivating product on in vitro ruminal fermentation using the Rumen simulation technique (RUSITEC).

The rumen simulation technique (RUSITEC) was used to investigate the effect of ergot alkaloids (EA) and a mycotoxin deactivating product (Biomin AA; MDP) on nutrient digestion, ruminal fermentation parameters, total gas, methane, and microbial nitrogen production. Ruminal fermentation vessels received a feedlot finishing diet of 90:10 concentrate:barley silage (DM basis). Using a randomized complete block design, treatments were assigned (n = 4 vessels/treatment) within two RUSITEC apparatuses in a 2 × 2 factorial arrangement. Treatments included: (1) control (CON) diet (no EA and no MDP); (2) CON diet + 1 g/d MDP; (3) CON diet + 20 mg/kg EA; and (4) CON diet + 20 mg/kg EA + 1 g/d MDP. The study was conducted over 14 d with 7 d of adaptation and 7 d of sample collection. Data were analyzed in SAS using PROC MIXED including fixed effects of EA, MDP, and the EA×MDP interaction. Random effects included RUSITEC apparatus and cow rumen inoculum (n = 4). Ergot alkaloids decreased dry matter (DMD) (P = 0.01; 87.9 vs. 87.2%) and organic matter disappearance (OMD) (P = 0.02; 88.8 vs. 88.4%). Inclusion of MDP increased OMD (P = 0.01; 88.3 vs. 88.9%). Neutral detergent fiber disappearance (NDFD) was improved with MDP; however, an EA×MDP interaction was observed with MDP increasing (P < 0.001) NDFD more with EA diet compared to CON. Acetate proportion decreased (P = 0.01) and isovalerate increased (P = 0.03) with EA. Consequently, acetate:propionate was reduced (P = 0.03) with EA. Inclusion of MDP increased total volatile fatty acid (VFA) production (P < 0.001), and proportions of acetate (P = 0.03) and propionate (P = 0.03), and decreased valerate (P < 0.001), isovalerate (P = 0.04), and caproate (P = 0.002). Treatments did not affect (P ≥ 0.17) ammonia, total gas, or methane production (mg/d or mg/g of organic matter fermented). The inclusion of MDP reduced (P < 0.001) microbial nitrogen (MN) production in the effluent and increased (P = 0.01) feed particle-bound MN. Consequently, total MN decreased (P = 0.001) with MDP. In all treatments, the dominant microbial phyla were Firmicutes, Bacteroidota, and Proteobacteria, and the major microbial genus was Prevotella. Inclusion of MDP further increased the abundance of Bacteroidota (P = 0.04) as it increased both Prevotella (P = 0.04) and Prevotellaceae_UCG-003 (P = 0.001). In conclusion, EA reduced OMD and acetate production due to impaired rumen function, these responses were successfully reversed by the addition of MDP.

Ergot formed from a parasitic fungus (Claviceps purpurea) affects various types of grains (rye, wheat, or oats) and may contain several toxic ergot alkaloids (EA). Individual EA may impact the rumen microorganisms, and cattle feed intake, digestibility, health, and overall performance. A common method to alleviate toxicity in mycotoxin-contaminated feed is through the addition of mycotoxin binders (MDP); however, their efficacy against EA is unknown. To better understand the effect of EA in cattle, we performed an in vitro experiment to examine the impact of EA on the ruminal microbial populations and fermentation of a finishing feedlot diet using an artificial rumen (RUSITEC). Additionally, an MDP was added to test if it could reduce the detrimental effects of EA on rumen fermentation. MDP increased total volatile fatty acids (VFA) and reduced total microbial protein synthesis. Furthermore, EA reduced microbial diversity and the acetate:propionate ratio. Although EA reduced organic matter digestibility and acetate production, these negative effects were reversed by the addition of the MDP.

Formation and Transfer of Multi-Species Biofilms Containing E. coli O103:H2 on Food Contact Surfaces to Beef.

Interactions of Shiga toxin-producing E. coli (STEC; O103:H2) with lactic acid bacteria (LAB) or spoilage bacteria (SP) multispecies biofilms on polyurethane (TPU) and stainless-steel (SS) were assessed at 10 and 25°C under wet and dry conditions after 6, 30, and 60 days of storage. One LAB T1: Carnobacterium piscicola + Lactobacillus bulgaricus, and two SP T2: Comamonas koreensis + Raoultella terrigena; T3: Pseudomonas aeruginosa + C. koreensis were assessed for their ability to form multispecies biofilms with O103:H2. O103:H2 single-species biofilms served as a control positive (T4). Coupons were stored dry (20-50% relative humidity; RH) or moist (60-90% RH) for up to 60 days, at which point O103:H2 transfer to beef and survival was evaluated. At 25°C, T3 decreased beef contamination with O103:H2 by 2.54 log10 CFU/g (P < 0.001). Overall, at 25°C contamination of beef with O103:H2 decreased (P < 0.001) from 3.17 log10 CFU/g on Day 6 to 0.62 log10 CFU/g on Day 60. With 60 days dry biofilms on TPU, an antagonistic interaction was observed among O103:H2 and multispecies biofilm T1 and T3. E. coli O103:H2 was not recovered from T1 and T3 after 60 days but it was recovered (33%) from T2 and T4 dry biofilms. At 10°C, contamination of beef with O103:H2 decreased (P < 0.001) from 1.38 log10 CFU/g after 6 days to 0.47 log10 CFU/g after 60 days. At 10°C, recovery of O103:H2 from 60 days dry biofilms could only be detected after enrichment and was always higher for T2 than T4 biofilms. Regardless of temperature, the transfer of O103:H2 to beef from the biofilm on TPU was greater (P < 0.001) than SS. Moist biofilms also resulted in greater (P < 0.001) cell transfer to beef than dry biofilms at 10 and 25°C. Development of SP or LAB multispecies biofilms with O103:H2 can either increase or diminish the likelihood of beef contamination. Environmental conditions such as humidity, contact surface type, as well as biofilm aging all can influence the risk of beef being contaminated by STEC within multi-species biofilms attached to food contact surfaces.

Genomic Profiling of Non-O157 Shiga Toxigenic Escherichia coli-Infecting Bacteriophages from South Africa.

Background: Non-O157 Shiga toxigenic Escherichia coli (STEC) are one of the most important food and waterborne pathogens worldwide. Although bacteriophages (phages) have been used for the biocontrol of these pathogens, a comprehensive understanding of the genetic characteristics and lifestyle of potentially effective candidate phages is lacking. Materials and Methods: In this study, 10 non-O157-infecting phages previously isolated from feedlot cattle and dairy farms in the North-West province of South Africa were sequenced, and their genomes were analyzed. Results: Comparative genomics and proteomics revealed that the phages were closely related to other E. coli-infecting Tunaviruses, Seuratviruses, Carltongylesviruses, Tequatroviruses, and Mosigviruses from the National Center for Biotechnology Information GenBank database. Phages lacked integrases associated with a lysogenic cycle and genes associated with antibiotic resistance and Shiga toxins. Conclusions: Comparative genomic analysis identified a diversity of unique non-O157-infecting phages, which could be used to mitigate the abundance of various non-O157 STEC serogroups without safety concerns.

Single- and Dual-Species Biofilm Formation by Shiga Toxin-Producing Escherichia coli and Salmonella, and Their Susceptibility to an Engineered Peptide WK2.

Shiga toxin-producing Escherichia coli (STEC) and Salmonella enterica are important foodborne pathogens capable of forming both single- and multi-species biofilms. In this study, the mono- and dual-species biofilms were formed by STEC O113:H21 and Salmonella enterica serovar Choleraesuis 10708 on stainless steel in the presence of beef juice over 5 d at 22 °C. The dual-species biofilm mass was substantially (p < 0.05) greater than that produced by STEC O113:H21 or S. Choleraesuis 10708 alone. However, numbers (CFU/mL) of S. Choleraesuis 10708 or STEC O113:H21 cells in the dual-species biofilm were (p < 0.05) lower than their respective counts in single-species biofilms. In multi-species biofilms, the sensitivity of S. Choleraesuis 10708 to the antimicrobial peptide WK2 was reduced, but it was increased for STEC O113:H21. Visualization of the temporal and spatial development of dual-species biofilms using florescent protein labeling confirmed that WK2 reduced cell numbers within biofilms. Collectively, our results highlight the potential risk of cross-contamination by multi-species biofilms to food safety and suggest that WK2 may be developed as a novel antimicrobial or sanitizer for the control of biofilms on stainless steel.

Biofilm-Forming Capacity of Escherichia coli Isolated from Cattle and Beef Packing Plants: Relation to Virulence Attributes, Stage of Processing, Antimicrobial Interventions, and Heat Tolerance.

Despite the importance of biofilm formation in the contamination of meat by pathogenic Escherichia coli at slaughter plants, drivers for biofilm remain unclear. To identify selection pressures for biofilm, we evaluated 745 isolates from cattle and 700 generic E. coli isolates from two beef slaughter plants for motility, the expression of curli and cellulose, and biofilm-forming potential. Cattle isolates were also screened for serogroup, stx1, stx2, eae, and rpoS. Generic E. coli isolates were compared by source (hide of carcass, hide-off carcass, and processing equipment) before and after the implementation of antimicrobial hurdles. The proportion of E. coli isolates capable of forming biofilms was lowest (7.1%; P < 0.05) for cattle isolates and highest (87.3%; P < 0.05) from equipment. Only one enterohemorrhagic E. coli (EHEC) isolate was an extremely strong biofilm former, in contrast to 73.4% of E. coli isolates from equipment. Isolates from equipment after sanitation had a greater biofilm-forming capacity (P < 0.001) than those before sanitation. Most cattle isolates were motile and expressed curli, although these traits along with the expression of cellulose and the detection of rpoS were not necessary for biofilm formation. In contrast, isolates capable of forming biofilms on equipment were almost exclusively motile and able to express curli. The results of the present study indicate that cattle rarely carry EHEC capable of making strong biofilms in slaughter plants. However, if biofilm-forming EHEC contaminates equipment, current sanitation procedures may not eliminate the most robust biofilm-forming strains. Accordingly, new and effective antibiofilm hurdles for meat-processing equipment are required to reduce future instances of foodborne disease. IMPORTANCE As the majority of enterohemorrhagic E. coli (EHEC) isolates are not capable of forming biofilms, sources were undetermined for biofilm-forming EHEC isolated from "high-event periods" in beef slaughter plants. This study demonstrated that sanitation procedures used on beef-processing equipment may inadvertently lead to the survival of robust biofilm-forming strains of E. coli. Cattle only rarely carry EHEC capable of forming strong biofilms (1/745 isolates evaluated), but isolates with greater biofilm-forming capacity were more likely (P < 0.001) to survive equipment sanitation. In contrast, chilling carcasses for 3 days at 0°C reduced (P < 0.05) the proportion of biofilm-forming E. coli. Consequently, an additional antibiofilm hurdle for meat-processing equipment, perhaps involving cold exposure, is necessary to further reduce the risk of foodborne disease.