Sample Type and Processing Plant Differences in the Proportion of Enterohemorrhagic Escherichia coli O157 and Non-O157 Serogroups in Feces and on Hides of Cull Dairy Cattle at Slaughter.

The study was conducted to determine the proportion and concentration of enterohemorrhagic Escherichia coli (EHEC) O157 and six non-O157 (O26, O45, O103, O111, O121, and O145) serogroups and identify seasonal and processing plant differences in feces and on hides of cull dairy cattle processed in commercial slaughterhouses in the United States. Approximately 60 rectal and 60 hide-on samples from matched carcasses were collected in each of three processing plants, in two periods; summer of 2017 and spring of 2018. Samples before enrichment were spiral plated to quantify EHEC, and postenriched samples underwent culture methods that included immuno-magnetic separation, plating on selective media, and PCR assays for identification and serogroup confirmation of putative isolates. An isolate was considered EHEC O157 positive if it harbored serogroup-specific (rfbE), Shiga toxin (stx1 and/or stx2), and intimin (eae) genes and EHEC non-O157 positive if at least one of the non-O157 serogroup-specific, stx1 and/or stx2, and eae genes was identified. Generalized linear mixed models were fitted to estimate overall proportion of positives for EHEC O157 and non-O157 EHEC serogroups, as well as seasonal and processing plant differences in fecal and hide-on proportion of positives. The fecal EHEC proportion at the sample level was 1.8% (95% CI = 0.0-92.2%) and 4.2% (95% CI = 0.0-100.0%) for EHEC O157 and EHEC non-O157, respectively. Hide sample level proportion of positives was 3.0% (95% CI = 0.0-99.9%) for EHEC O157 and 1.6% (95% CI = 0.0-100.0%) for EHEC non-O157. The proportion of EHEC O157 and non-O157 significantly differed by processing plant and sample type (hide vs. feces), but not by season. The association between proportion of EHEC serogroups in feces with the proportion on hides collected from matched cattle was 7.8% (95% CI = 0.6-53.3%) and 3.8% (95% CI = 0.3-30.8%) for EHEC O157 and non-O157, respectively. Taken together, our findings provide evidence of a low proportion of EHEC serogroups in the feces and on hides of cull dairy cattle and that their proportion varies across processing plants.

Whole-genome sequencing analysis of uncommon Shiga toxin-producing Escherichia coli from cattle: Virulence gene profiles, antimicrobial resistance predictions, and identification of novel O-serogroups.

Shiga toxin-producing E. coli (STEC) are major foodborne pathogens. While many studies have focused on the "top-7 STEC", little is known for minor serogroups. A total of 284 non-top-7 STEC strains isolated from cattle feces were subjected to whole-genome sequencing (WGS) to determine the serotypes, the presence of virulence genes and antimicrobial resistance (AMR) determinants. Nineteen typeable and three non-typeable serotypes with novel O-antigen loci were identified. Twenty-one AMR genes and point mutations in another six genes that conferred resistance to 10 antimicrobial classes were detected, as well as 46 virulence genes. The distribution of 33 virulence genes and 15 AMR determinants exhibited significant differences among serotypes (p < 0.05). Among all strains, 81.7% (n = 232) and 14.1% (n = 40) carried stx2 and stx1 only, respectively; only 4.2% (n = 12) carried both. Subtypes stx1a, stx1c, stx2a, stx2c, stx2d, and stx2g were identified. Forty-six strains carried eae and stx2a and therefore had the potential cause severe diseases; 47 strains were genetically related to human clinical strains inferred from a pan-genome phylogenetic tree. We were able to demonstrate the utility of WGS as a surveillance tool to characterize the novel serotypes, as well as AMR and virulence profiles of uncommon STEC that could potentially cause human illness.

Effectiveness of a Direct-Fed Microbial Product Containing Lactobacillus acidophilus and Lactobacillus casei in Reducing Fecal Shedding of Escherichia coli O157:H7 in Commercial Feedlot Cattle.

The objective of this study was to evaluate the effectiveness of a direct-fed microbial (DFM) product in reducing fecal shedding of Escherichia coli O157:H7 in finishing commercial feedlot cattle in Kansas (KS) and Nebraska (NE). Utilizing a randomized complete block design within the feedlot (KS, n = 1; NE, n = 1), cattle were randomly allocated to 20 pens, grouped in blocks of two based on allocation date, and then, within the block, randomly assigned to a treatment group (DFM or negative control). The DFM product was included in the diet at a targeted daily dose of 1 × 109 colony-forming units (CFU) of the Lactobacillus acidophilus and Lactobacillus casei combination per animal for at least 60 d before sampling. Feedlots were sampled for four consecutive weeks; weekly sampling consisted of collecting 20 pen floor fecal samples per pen. Fecal samples were subjected to culture-based methods for detection and isolation of E. coli O157, and positive samples were quantified using real-time polymerase chain reaction. Primary outcomes of interest were fecal prevalence of E. coli O157:H7 and E. coli O157 supershedding (≥104 CFU/g of feces) prevalence. Data for each feedlot were analyzed at the pen level using mixed models accounting for the study design features. Model-adjusted mean E. coli O157:H7 fecal prevalence estimates (standard error of the mean [SEM]) for DFM and control groups were 8.2% (SEM = 2.2%) and 9.9% (SEM = 2.5%) in KS and 14.6% (SEM = 2.8%) versus 14.3% (SEM = 2.6%) in NE; prevalence did not differ significantly between treatment groups at either site (KS, p = 0.51; NE, p = 0.92). Mean E. coli O157 supershedding prevalence estimates for DFM and control groups were 2.2% (SEM = 0.7%) versus 1.8% (SEM = 0.7%) in KS (p = 0.66) and 6.7% (SEM = 1.5%) versus 3.2% (SEM = 1.0%) in NE (p = 0.04). In conclusion, administering the DFM product in the finishing diet of feedlot cattle did not significantly reduce E. coli O157:H7 fecal prevalence or supershedding prevalence in study pens at either commercial feedlot.

Leukotoxin production by Fusobacterium necrophorum strains in relation to severity of liver abscesses in cattle.

Fusobacterium necrophorum, a Gram-negative anaerobe, is the primary etiologic agent of liver abscesses of beef cattle. The bacterium, a member of the microbial community of the rumen, travels to the liver via portal circulation to cause abscesses. The severity of liver abscesses vary from mild with one or two small abscesses to severe with medium to large multiple abscesses. Leukotoxin, a secreted protein, is the critical virulence factor involved in the infection. Our objective was to compare leukotoxin production between strains of F. necrophorum isolated from mild and severe liver abscesses collected from slaughtered cattle. The quantification of leukotoxin was based on assays to measure cytotoxicity and protein antigen concentration. One-hundred strains, 50 from mild and 50 from severe abscesses, were utilized in the study. Cell-free supernatants were prepared from cultures grown in anaerobic broth at 9 and 24 h incubations. The leukotoxic activity was quantified by measuring cytotoxicity based on the release of lactic dehydrogenase from bovine lymphocyte cells, BL3, treated with the culture supernatant. Leukotoxin protein concentration was quantified by a sandwich ELISA assay with a leukotoxin-specific monoclonal antibody as the capture antibody. The leukotoxin activity and concentration were highly variable among the strains within each severity of liver abscesses. Although the leukotoxic activity was unaffected by incubation time, leukotoxin protein concentration was consistently higher at 24 h compared to 9 h incubation. Strains from severe liver abscesses had significantly higher leukotoxic activity and higher protein concentration compared to strains from mild liver abscesses (P < 0.0001) at both 9 and 24 h culture supernatants. Across all strains, the correlation coefficients between leukotoxic activity and leukotoxin concentration at 9 and 24 h were 0.14 (P = 0.17) and 0.47 (P < 0.0001), respectively. In conclusion, strains isolated from severe liver abscesses had significantly higher leukotoxic activities and leukotoxin protein concentrations compared to strains isolated from mild liver abscesses.

Single-Cell-Based Digital PCR Detection and Association of Shiga Toxin-Producing Escherichia coli Serogroups and Major Virulence Genes.

Escherichia coli serogroups O157, O26, O45, O103, O111, O121, and O145, when carrying major virulence genes, the Shiga toxin genes stx1 and stx2 and the intimin gene eae, are important foodborne pathogens. They are referred to as the "top 7" Shiga toxin-producing E. coli (STEC) serogroups and were declared by the USDA as adulterants to human health. Since top 7 serogroup-positive cattle feces and ground beef can also contain nonadulterant E. coli strains, regular PCR cannot confirm whether the virulence genes are carried by adulterant or nonadulterant E. coli serogroups. Thus, traditional gold-standard STEC detection requires bacterial isolation and characterization, which are not compatible with high-throughput settings and often take a week to obtain a definitive result. In this study, we demonstrated that the partition-based multichannel digital PCR (dPCR) system can be used to detect and associate the E. coli serogroup-specific gene with major virulence genes and developed a single-cell-based dPCR approach for rapid (within 1 day) and accurate detection and confirmation of major STEC serogroups in high-throughput settings. Major virulence genes carried by each of the top 7 STEC serogroups were detected by dPCR with appropriately diluted intact bacterial cells from pure cultures, culture-spiked cattle feces, and culture-spiked ground beef. Furthermore, from 100 randomly collected, naturally shed cattle fecal samples, 3 O103 strains carrying eae and 2 O45 strains carrying stx1 were identified by this dPCR assay and verified by the traditional isolation method. This novel and rapid dPCR assay is a culture-independent, high-throughput, accurate, and sensitive method for STEC detection and confirmation.

Potential risk-factors affecting Salmonella sp. and Escherichia coli occurrence and distribution in Midwestern United States swine feed mills.

AIM: This study aimed to evaluate the patterns and potential risk factors associated with the occurrence of Salmonella sp. and Escherichia coli in selected United States swine feed mills. METHODS AND RESULTS: A total of 405 samples were collected during fall 2018, spring and summer 2019 from selected sites including floors, equipment, shoes and feed in six feed mills in the US Midwest region. Each sample was analysed for the presence of Salmonella and E. coli with culture methods and confirmed by PCR. A survey regarding production volumes, hygiene practices and microbial testing capabilities was conducted in each facility All mills had at least one sampling site positive for either Salmonella or E. coli. Of the 405 samples, 4·7, and 14·1% were positive for Salmonella sp., and E. coli respectively. Sites with higher percentages of positive samples were the receiving, manufacturing, and control area floors. The survey responses indicated that the age of the mill might be a risk factor for bacterial contamination: the older the facility, the higher the number of positive samples. Other risk factors evaluated, such as the production capacity, did not appear to relate to bacterial prevalence. CONCLUSION: The data documents the presence of E. coli and Salmonella in selected US swine feed mills, and an association between E. coli occurrence and number of ingredient suppliers to feed mill. SIGNIFICANCE AND IMPACT OF THE STUDY: This information could be used to understand risk factors affecting the occurrence of Salmonella sp. and E. coli in feed mills and help implement monitoring and mitigation strategies for public health.

Leukotoxic activity of Fusobacterium necrophorum of cattle origin.

Fusobacterium necrophorum is a Gram negative, rod-shaped and aero tolerant anaerobe. In animals, it is an opportunistic pathogen frequently associated with necrotic infections, generally called necrobacillosis, such as calf diphtheria, foot rot and liver abscesses in cattle. Two subspecies exist: subsp. necrophorum and subsp. funduliforme. Among several virulence factors, leukotoxin (Lkt) is considered to be a major factor and a protective antigen. The objective of the study was to utilize BL3 cells and measure the release of lactic dehydrogenase to quantify Lkt activity of F. necrophorum. The assay was used to examine the effects of storage and handling conditions, growth media, polymyxin B addition on the cytotoxicity and evaluate Lkt activities of F. necrophorum strains isolated from bovine liver abscesses and foot rot. The Lkt activity peaked at 9 h of incubation. There was a significant decrease in the cytotoxicity measured in the samples after each freeze and thaw cycle. No difference was observed in the cytotoxicity for the samples handled aerobically versus anaerobically. Lkt activities of strains grown in anaerobic Brain-Heart Infusion broth were higher compared to Vegitone broth. A small reduction in the cytotoxicity activity was observed after the addition of polymyxin. The Lkt activity was consistently higher in strains of subsp. necrophorum than subsp. funduliforme of liver abscess origin. Among the strains isolated from cattle foot rot, Lkt activities of subsp. necrophorum strains appear to be much more variable. Use of BL3 cells in combination of lactic acid dehydrogenase assay appears to be a simple and valid assay to measure Lkt activity of F. necrophorum.

Campylobacter Prevalence and Quinolone Susceptibility in Feces of Preharvest Feedlot Cattle Exposed to Enrofloxacin for the Treatment of Bovine Respiratory Disease.

Campylobacter spp. can be pathogenic to humans and often harbor antimicrobial resistance genes. Data on resistance in relation to fluoroquinolone use in beef cattle are scarce. This cross-sectional study of preharvest cattle evaluated Campylobacter prevalence and susceptibility to nalidixic acid and ciprofloxacin in feedlots that previously administered a fluoroquinolone as primary treatment for bovine respiratory disease. Twenty fresh fecal samples were collected from each of 10 pens, in each of five feedlots, 1-2 weeks before harvest. Feces were cultured for Campylobacter using selective enrichment and isolation methods. Genus and species were confirmed via PCR. Minimum inhibitory concentrations (MICs) of ciprofloxacin and nalidixic acid were determined using a micro-broth dilution method and human breakpoints. Antimicrobial use within each pen was recorded. Data were analyzed using generalized linear mixed-models (prevalence) and survival analysis (MICs). Overall, sample-level prevalence of Campylobacter was 27.2% (272/1000) and differed significantly among feedlots (p < 0.01). Campylobacter coli was the most common species (55.1%; 150/272), followed by Campylobacter hyointestinalis (42.6%; 116/272). Within-pen prevalence was not significantly associated with the number of fluoroquinolone treatments, sex, body weight, or metaphylaxis use, but was associated with the number of days cattle were in the feedlot (p = 0.03). The MICs for the majority of Campylobacter isolates were above the breakpoints for nalidixic acid (68.4%; 175/256) and for ciprofloxacin (65.6%; 168/256). Distributions of MICs for nalidixic acid (p ≤ 0.01) and ciprofloxacin (p ≤ 0.05) were significantly different among feedlots, and by Campylobacter species. However, fluoroquinolone treatments, sex, body weight, days on feed, and metaphylaxis were not significantly associated with MIC distributions within pens. We found no evidence that the number of fluoroquinolone treatments within feedlot pens significantly affected the within-pen fecal prevalence or quinolone susceptibilies of Campylobacter in feedlots that used a fluoroquinolone as primary treatment for bovine respiratory disease.

A Randomized Trial to Assess the Effect of Fluoroquinolone Metaphylaxis on the Fecal Prevalence and Quinolone Susceptibilities of Salmonella and Campylobacter in Feedlot Cattle.

The study objective was to determine effects of fluoroquinolone metaphylaxis on fecal prevalence of Salmonella and Campylobacter and fecal prevalence of quinolone-resistant Salmonella and Campylobacter in feedlot cattle. On Day 0, cattle (n = 288) at risk for bovine respiratory disease (BRD) were randomly assigned to either a nontreated control pen (12 pens) or a fluoroquinolone-treated (enrofloxacin; Baytril® 100) pen (12 pens). Rectal fecal samples were collected from cattle on days 0, 7, 14, 21, and 28. Feces were cultured for Salmonella enterica and Campylobacter spp. using enrichment and selective isolation methods, and confirmed by serology and PCR. Susceptibilities to nalidixic acid and ciprofloxacin were determined using microbroth dilution methods. Data analyses were performed using linear mixed models. Overall, Salmonella sp. and Campylobacter spp. were recovered from 10.2% (139/1,364) and 12.4% (170/1,364) of the fecal samples, respectively. Campylobacter species included hyointestinalis, jejuni, and coli. Neither Salmonella sp. nor Campylobacter spp. prevalence was significantly impacted by fluoroquinolone treatment (p = 0.80, p = 0.61, respectively). However, Salmonella prevalence differed between study weeks (p < 0.01) with prevalence decreasing over time. Before treatment, 98.9% (91/92) of Salmonella isolates were susceptible to nalidixic acid and ciprofloxacin. All Salmonella recovered posttreatment (n = 43) were susceptible to both antimicrobials. The majority of Campylobacter spp. recovered before treatment were resistant to nalidixic acid (23/35; 65.7%) and ciprofloxacin (21/35; 60.0%). There was no significant treatment by week interaction (p = 0.85) or treatment effects (p = 0.61) on the posttreatment prevalence of Campylobacter resistance. There was, however, a significant week effect (p = 0.05), with Campylobacter resistance prevalence decreasing over time. In this 28-day study, we found no evidence that a fluoroquinolone used for metaphylaxis significantly impacts fecal prevalence of Salmonella sp. or Campylobacter spp. or the fecal prevalence of nalidixic acid or ciprofloxacin resistance.

Virulence Gene Profiles and Clonal Relationships of Escherichia coli O26:H11 Isolates from Feedlot Cattle as Determined by Whole-Genome Sequencing.

UNLABELLED: Escherichia coli O26 is the second most important enterohemorrhagic E. coli (EHEC) serogroup worldwide. Serogroup O26 strains are categorized mainly into two groups: enteropathogenic (EPEC) O26, carrying a locus of enterocyte effacement (LEE) and mostly causing mild diarrhea, and Shiga-toxigenic (STEC) O26, which carries the Shiga toxin (STX) gene (stx), responsible for more severe outcomes. stx-negative O26 strains can be further split into two groups. One O26 group differs significantly from O26 EHEC, while the other O26 EHEC-like group shows all the characteristics of EHEC O26 except production of STX. In order to determine the different populations of O26 E. coli present in U.S. cattle, we sequenced 42 O26:H11 strains isolated from feedlot cattle and compared them to 37 O26:H11 genomes available in GenBank. Phylogenetic analysis by whole-genome multilocus sequence typing (wgMLST) showed that O26:H11/H(-) strains in U.S. cattle were highly diverse. Most strains were sequence type 29 (ST29). By wgMLST, two clear lineages could be distinguished among cattle strains. Lineage 1 consisted of O26:H11 EHEC-like strains (ST29) (4 strains) and O26:H11 EHEC strains (ST21) (2 strains), and lineage 2 (36 strains) consisted of O26:H11 EPEC strains (ST29). Overall, our analysis showed U.S. cattle carried pathogenic (ST21; stx1 (+) ehxA(+) toxB(+)) and also potentially pathogenic (ST29; ehxA(+) toxB(+)) O26:H11 E. coli strains. Furthermore, in silico analysis showed that 70% of the cattle strains carried at least one antimicrobial resistance gene. Our results showed that whole-genome sequence analysis is a robust and valid approach to identify and genetically characterize E. coli O26:H11, which is of importance for food safety and public health. IMPORTANCE: Escherichia coli O26 is the second most important type of enterohemorrhagic E. coli (EHEC) worldwide. Serogroup O26 strains are categorized into two groups: enteropathogenic (EPEC) carrying LEE, causing mild diarrhea, and Shiga toxigenic (STEC) carrying the stx gene, responsible for more severe outcomes. However, there are currently problems in distinguishing one group from the other. Furthermore, several O26 stx-negative strains are consistently misidentified as either EHEC-like or EPEC. The use of whole-genome sequence (WGS) analysis of O26 strains from cattle in the United States (i) allowed identification of O26 strains present in U.S. cattle, (ii) determined O26 strain diversity, (iii) solved the misidentification problem, and (iv) screened for the presence of antimicrobial resistance and virulence genes in the strains. This study provided a framework showing how to easily and rapidly use WGS information to identify and genetically characterize E. coli O26:H11, which is important for food safety and public health.

Effects of in-feed copper and tylosin supplementations on copper and antimicrobial resistance in faecal enterococci of feedlot cattle.

AIMS: The objective was to investigate whether in-feed supplementation of copper, at elevated level, co-selects for macrolide resistance in faecal enterococci. METHODS AND RESULTS: The study was conducted in cattle (n = 80) with a 2 × 2 factorial design of copper (10 or 100 mg kg(-1) of feed) and tylosin (0 or 10 mg kg(-1) of feed). Thirty-seven isolates (4·6%; 37/800) of faecal enterococci were positive for the tcrB and all were Enterococcus faecium. The prevalence was higher among cattle fed diets with copper and tylosin (8·5%) compared to control (2·0%), copper (4·5%) and tylosin (3·5%) alone. All tcrB-positive isolates were positive for erm(B) and tet(M) genes. Median copper minimum inhibitory concentrations (MICs) for tcrB-positive and tcrB-negative enterococci were 20 and 4 mmol l(-1) , respectively. CONCLUSIONS: Feeding of elevated dietary copper and tylosin alone or in combination resulted in an increased prevalence of tcrB and erm(B)-mediated copper and tylosin-resistant faecal enterococci in feedlot cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: In-feed supplementation of elevated dietary copper has the potential to co-select for macrolide resistance. Further studies are warranted to investigate the factors involved in maintenance and dissemination of the resistance determinants and their co-selection mechanism in relation to feed-grade antimicrobials' usage in feedlot cattle.

Evaluation of a Postbiotic on Salmonella enterica Prevalence, Serotype Diversity, and Antimicrobial Resistance in the Subiliac Lymph Nodes of Cull Dairy Cattle.

This study aimed to determine whether the farm-level use of a Saccharomyces cerevisiae-based postbiotic was associated with Salmonella prevalence and concentration, serotype diversity, and antimicrobial resistance in the subiliac lymph nodes (LN) of cull dairy cows. In collaboration with two commercial processing plants in the Southwestern (SW) and Northeastern (NE) regions of the U.S., cull dairy cattle lots processed in the same week from dairy farms that fed or not the postbiotic were sampled. Up to 20 LN were collected from dairy cattle from each supplier farm at least once every season. Samples were analyzed for Salmonella by culture and quantitative PCR methods, and isolates were subjected to serotype identification and antimicrobial susceptibility testing. Although a numerically lower prevalence was observed, the preharvest administration of the postbiotic was not significantly associated with Salmonella prevalence in cull dairy cattle. However, Salmonella prevalence significantly varied by region; the SW region showed a higher prevalence than the NE region. Whereas dominant Salmonella serotypes included Montevideo, Mbandaka, Muenster, Cerro, Meleagridis, and Anatum, the probability of isolating a dominant serotype did not significantly vary by feed additive status (FAS) or region, but varied by season. Up to 34 isolates (out of 391) exhibited resistance to each antimicrobial, with the highest number of isolates exhibiting resistance to streptomycin and ciprofloxacin; however, this did not significantly vary by FAS, season, or region. The precise reasons for the lack of effectiveness of the postbiotic in reducing Salmonella burden are unknown; however, varied administration durations of the postbiotic due to culling at different lactation cycle stages or during dry periods may have hindered its impact, especially if dairy cows were culled early. Other factors may include dietary components, farm management practices, and external environmental influences.

Temporal dynamics of the fecal microbiome in female pigs from early life through estrus, parturition, and weaning of the first litter of piglets.

BACKGROUND: Age-associated changes in the gastrointestinal microbiome of young pigs have been robustly described; however, the temporal dynamics of the fecal microbiome of the female pig from early life to first parity are not well understood. Our objective was to describe microbiome and antimicrobial resistance dynamics of the fecal microbiome of breeding sows from early life through estrus, parturition and weaning of the first litter of piglets (i.e., from 3 to 53 weeks of age). RESULTS: Our analysis revealed that fecal bacterial populations in developing gilts undergo changes consistent with major maturation milestones. As the pigs progressed towards first estrus, the fecal bacteriome shifted from Rikenellaceae RC9 gut group- and UCG-002-dominated enterotypes to Treponema- and Clostridium sensu stricto 1-dominated enterotypes. After first estrus, the fecal bacteriome stabilized, with minimal changes in enterotype transition and associated microbial diversity from estrus to parturition and subsequent weaning of first litter piglets. Unlike bacterial communities, fecal fungal communities exhibited low diversity with high inter- and intra-pig variability and an increased relative abundance of certain taxa at parturition, including Candida spp. Counts of resistant fecal bacteria also fluctuated over time, and were highest in early life and subsequently abated as the pigs progressed to adulthood. CONCLUSIONS: This study provides insights into how the fecal microbial community and antimicrobial resistance in female pigs change from three weeks of age throughout their first breeding lifetime. The fecal bacteriome enterotypes and diversity are found to be age-driven and established by the time of first estrus, with minimal changes observed during subsequent physiological stages, such as parturition and lactation, when compared to the earlier age-related shifts. The use of pigs as a model for humans is well-established, however, further studies are needed to understand how our results compare to the human microbiome dynamics. Our findings suggest that the fecal microbiome exhibited consistent changes across individual pigs and became more diverse with age, which is a beneficial characteristic for an animal model system.

Detection of Escherichia coli O104 in the feces of feedlot cattle by a multiplex PCR assay designed to target major genetic traits of the virulent hybrid strain responsible for the 2011 German outbreak.

A multiplex PCR was designed to detect Escherichia coli O104:H4, a hybrid pathotype of Shiga toxigenic and enteroaggregative E. coli, in cattle feces. A total of 248 fecal samples were tested, and 20.6% were positive for serogroup O104. The O104 isolates did not carry genes characteristic of the virulent hybrid strain.

Occurrence of the transferable copper resistance gene tcrB among fecal enterococci of U.S. feedlot cattle fed copper-supplemented diets.

Copper, an essential micronutrient, is supplemented in the diet at elevated levels to reduce morbidity and mortality and to promote growth in feedlot cattle. Gut bacteria exposed to copper can acquire resistance, which among enterococci is conferred by a transferable copper resistance gene (tcrB) borne on a plasmid. The present study was undertaken to investigate whether the feeding of copper at levels sufficient to promote growth increases the prevalence of the tcrB gene among the fecal enterococci of feedlot cattle. The study was performed with 261 crossbred yearling heifers housed in 24 pens, with pens assigned randomly to a 2×2 factorial arrangement of treatments consisting of dietary copper and a commercial linseed meal-based energy protein supplement. A total of 22 isolates, each identified as Enterococcus faecium, were positive for tcrB with an overall prevalence of 3.8% (22/576). The prevalence was higher among the cattle fed diets supplemented with copper (6.9%) compared to normal copper levels (0.7%). The tcrB-positive isolates always contained both erm(B) and tet(M) genes. Median copper MICs for tcrB-positive and tcrB-negative enterococci were 22 and 4 mM, respectively. The transferability of the tcrB gene was demonstrated via a filter-mating assay. Multilocus variable number tandem repeat analysis revealed a genetically diverse population of enterococci. The finding of a strong association between the copper resistance gene and other antibiotic (tetracycline and tylosin) resistance determinants is significant because enterococci remain potential pathogens and have the propensity to transfer resistance genes to other bacteria in the gut.

In vitro degradation of lysine by ruminal fluid-based fermentations and by Fusobacterium necrophorum.

The objective of these studies was to characterize some factors affecting lysine degradation by mixed ruminal bacteria and by ruminal Fusobacterium necrophorum. Mixed ruminal bacteria degraded lysine, and addition of pure cultures of F. necrophorum did not increase lysine degradation. Addition of acetic or propionic acid strikingly reduced NH(3) production from lysine by mixed ruminal bacteria at pH 6, but not at pH 7. Although typical ruminal environments with acidic pH and normal concentrations of volatile fatty acids might inhibit lysine degradation by F. necrophorum, ruminal fluid contained enough bacteria with a lysine-degrading capacity to ferment 50 mM lysine in vitro. Of 7 strains of ruminal F. necrophorum tested, all grew on both lactate and lysine as the primary energy source. Both subspecies of ruminal F. necrophorum (necrophorum and funduliforme) used lysine as a primary C and energy source. Lysine and glutamic acid were effectively fermented by F. necrophorum, but alanine and tryptophan were not, and histidine and methionine were fermented only to a minor extent. The end products of lactate fermentation by F. necrophorum were propionate and acetate, and those of lysine degradation were butyrate and acetate. Fermentation of glutamic acid by F. necrophorum yielded acetate and butyrate in a ratio near to 2:1. The minimum inhibitory concentration of tylosin for F. necrophorum was not dependent on whether bacteria were grown with lactate or lysine, but F. necrophorum was more susceptible to monensin when grown on lysine than on lactate. Although F. necrophorum is generally resistant to monensin, the ionophore may reduce lysine degradation by F. necrophorum in the rumen. The essential oil components limonene, at 20 or 100 µg/mL, and thymol, at 100 µg/mL, inhibited F. necrophorum growth, whereas eugenol, guaiacol, and vanillin had no effect. Our findings may lead to ways to minimize ruminal lysine degradation and thus increase its availability to the animal.

Metabolome of purulent materials of liver abscesses from crossbred cattle and Holstein steers fed finishing diets with or without in-feed tylosin.

Liver abscesses in feedlot cattle are a polymicrobial infection with Fusobacterium necrophorum and Trueperella pyogenes as the primary and secondary etiologic agents, respectively. Cattle with liver abscesses do not exhibit clinical signs and the abscesses are detected only at slaughter. The objective was to conduct metabolomics analysis of purulent materials of liver abscesses to identify biochemicals. Liver abscesses from crossbred cattle (n = 24) and Holstein steers (n = 24), each fed high-grain finishing diet with tylosin (n = 12) or no tylosin (n = 12), were included in the study. Abscess purulent materials were analyzed by ultrahigh-performance liquid chromatography-tandem mass spectroscopy. A total of 759 biochemicals were identified and were broadly categorized into carbohydrates, energy metabolism pathways intermediates, peptides, amino acids and their metabolites, lipids and their metabolites, nucleotides, vitamins and cofactors, xenobiotics, and partially characterized molecules. The top 50 biochemicals identified included amino acids, lipids, nucleotides, xenobiotics, peptides, and carbohydrates and their metabolites. Among the 15 amino acid metabolites in the top 50 biochemicals, four were tryptophan metabolites, indoleacrylate, indolepropionate, tryptamine, and anthranilate. The 3-phenylpropionate, a product of phenylalanine metabolism, was the predominant metabolite in purulent materials. Between the four treatment groups, a two-way ANOVA analysis identified biochemicals that exhibited significant main effects for cattle type and in-feed tylosin use and their interactions. A total of 59 and 85 biochemicals were different (P < 0.05) between the cattle type (crossbred vs. Holstein steers) and in-feed tylosin use (tylosin vs. no tylosin), respectively. Succinate, an intermediate of lactate fermentation by some bacterial species, was one of the top 30 biochemicals that differentiated the four treatment groups. A number of lysophospholipids, indicative of bacterial and host cell membrane lyses, were identified in the purulent materials. In conclusion, to our knowledge this is the first report on the metabolome of liver abscess purulent materials and several biochemicals identified were related to metabolic activities of the bacterial community, particularly F. necrophorum and T. pyogenes. Biochemicals unique to liver abscesses that appear in the blood may serve as biomarkers and be of diagnostic value to detect liver abscesses of cattle before slaughter.

Liver abscesses in feedlot cattle, a consequence of feeding a diet of high-grain and low-roughage, are a mixed bacterial infection with Fusobacterium necrophorum, a ruminal bacterium as the primary causative agent. Cattle with liver abscesses do not exhibit clinical signs and the abscesses are detected only at slaughter. The study analyzed purulent materials of liver abscesses of feedlot cattle collected at slaughter for biochemical molecules. A total of 759 biochemicals were identified and a majority belonged to biochemical classes of lipids and amino acids and their metabolites. Biochemicals unique to liver abscesses that enter blood circulation may have the potential to be used as biomarkers in cattle with liver abscess before slaughter.

Age influences the temporal dynamics of microbiome and antimicrobial resistance genes among fecal bacteria in a cohort of production pigs.

BACKGROUND: The pig gastrointestinal tract hosts a diverse microbiome, which can serve to select and maintain a reservoir of antimicrobial resistance genes (ARG). Studies suggest that the types and quantities of antimicrobial resistance (AMR) in fecal bacteria change as the animal host ages, yet the temporal dynamics of AMR within communities of bacteria in pigs during a full production cycle remains largely unstudied. RESULTS: A longitudinal study was performed to evaluate the dynamics of fecal microbiome and AMR in a cohort of pigs during a production cycle; from birth to market age. Our data showed that piglet fecal microbial communities assemble rapidly after birth and become more diverse with age. Individual piglet fecal microbiomes progressed along similar trajectories with age-specific community types/enterotypes and showed a clear shift from E. coli/Shigella-, Fusobacteria-, Bacteroides-dominant enterotypes to Prevotella-, Megaspheara-, and Lactobacillus-dominated enterotypes with aging. Even when the fecal microbiome was the least diverse, the richness of ARGs, quantities of AMR gene copies, and counts of AMR fecal bacteria were highest in piglets at 2 days of age; subsequently, these declined over time, likely due to age-related competitive changes in the underlying microbiome. ARGs conferring resistance to metals and multi-compound/biocides were detected predominately at the earliest sampled ages. CONCLUSIONS: The fecal microbiome and resistome-along with evaluated descriptors of phenotypic antimicrobial susceptibility of fecal bacteria-among a cohort of pigs, demonstrated opposing trajectories in diversity primarily driven by the aging of pigs.

Evaluation of a Lactococcus lactis-based dried fermentation product administered through drinking water on nursery pig growth performance, fecal Escherichia coli virulence genes and pathotypes, antibiotic usage, and mortality.

A total of 34,749 pigs were used in two experiments to evaluate the effects of a postbiotic dried fermentation product (DFP) administered through drinking water on nursery pig growth performance, antibiotic injection frequency, morbidity, mortality, fecal consistency, and characterization of fecal Escherichia coli. The DFP is composed of bioactive molecules derived from Lactococcus lactis. In Exp. 1, 350 barrows (DNA Line 200 × 400; initial body weight [BW] 6.1 ± 0.01 kg) were used in a 42-d study with five pigs per pen and 35 pens per treatment. The DFP was supplied for 14 d at a target dosage of 24 mg/kg BW using a water medicator at a 1:128 dilution. On days 7 and 14, fecal samples were collected for dry matter (DM) and to determine, by a multiplex polymerase chain reaction (PCR) assay, prevalence of 11 virulence genes characteristic of E. coli pathotypes. There was no evidence (P > 0.10) for differences for growth, incidence of diarrhea, number of antibiotic injections, removals, or fecal DM. On both fecal collection days, E. coli virulence genes were present with day 7 samples positive for genes that encode for hemolysins (hlyA, exhA), intimin (eae), and enteroaggregative heat-stable enterotoxin (astA). Prevalence of enterotoxin genes (elt, estA, estB, astA) increased on day 14, but DFP had no effects on the prevalence of any of the virulence genes. A total of 32 out of 72 E. coli isolates were identified as enterotoxigenic pathotype and all except one were from day 14 fecal samples. Fourteen isolates were positive for F4 fimbria and one isolate was positive for F4 and F18 fimbriae. In Exp. 2, 34,399 nursery pigs (initially 5.6 kg) were used in 20 nursery barns with 10 barns per treatment (control or DFP). The target dosage of the DFP for the first 14 d was 35 mg/kg BW. Following the 14-d supplementation period, pigs continued to be monitored for approximately 31 d. There was no evidence (P > 0.05) for the DFP to influence the overall percentage of pigs that died or growth performance. From days 0 to 14, providing the DFP reduced (P < 0.05) the percentage of pigs that were euthanized. However, providing the DFP increased (P < 0.05) the overall percentage of pigs that were euthanized and total mortality. For the number of antibiotic injections (treatment interventions), providing the DFP reduced the number of injections for the common period (P < 0.001) and overall (P = 0.002). These results indicate that the DFP did not influence growth performance but providing the DFP in Exp. 2 led to increased total nursery pig mortality.