Search

This study aimed to determine the taxonomic status of a new group of bovine strains of the genus Acinetobacter characterized by the rare ability to grow at temperatures above 44 °C. Initially, 24 strains were isolated from cattle feces collected at 11 farms in Czechia in 2022, representing a tentative new species based on preliminary whole-cell MALDI-TOF MS identification and rpoB gene sequencing. Twelve strains encompassing the within-group diversity were studied in detail, including whole genome de novo sequencing. Core genome-based phylogenetic analysis revealed that the 12 strains form a distinct clade within the genus. The genomic average nucleotide identity based on BLAST (ANIb) values within the clade were ≥ 98.2%, whereas the ANIb values between the clade and the known Acinetobacter species were < 83%. Each of the 12 genomes consisted of a circular chromosome (3.05-3.28 Mb) and 1-6 extrachromosomal elements (1.43-122.5 kb). All strains were non-glucose oxidizing, non-hemolytic, non-proteolytic, and prototrophic, and used acetate, ethanol, and DL-lactate as sole carbon and energy sources. Unrestricted growth at 47 °C was their unique diagnostic characteristic in a genus-wide comparative analysis and their growth kinetics at 37-46 °C differed from that of Acinetobacter baumannii. The studied strains were susceptible to 18 antibiotics except for sporadic resistance to streptomycin, tetracycline, and sulfonamides associated with the presence of strA/strB, tet(Y), and sul2 genes, respectively. We conclude that the studied strains represent a new species, for which we propose the name Acinetobacter thermotolerans sp. nov. The type strain is ANC 7454 T (= CCM 9356 T  = CCUG 77195 T  = CNCTC 8200 T )., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

This study provides an emended description of Acinetobacter faecalis, a species previously described based on a single isolate (YIM 103518 T ) from elephant feces in China. Our emended description is based on 15 novel isolates conspecific with the A. faecalis type strain, obtained from eight cattle farms in the Czech Republic. The A. faecalis strains have relatively small genomes (≈2.5-2.7 Mbp), with a GC content of 36.3-36.7 mol%. Core genome-based phylogenetic analysis showed that the 15 strains, together with the type strain of A. faecalis, form a distinct and internally coherent phylogroup within the genus. Pairwise genomic ANIb values for the 16 A. faecalis strains were 97.32-99.04 %, while ANIb values between the genomes of the 16 strains and those of the other Acinetobacter spp. were ≤ 86.2 %. Analysis of whole-cell MALDI-TOF mass spectra supported the distinctness and cohesiveness of the taxon. The A. faecalis strains could be differentiated from the other validly named Acinetobacter spp. by the absence of hemolytic activity along with their ability to grow at 37 °C and on L-aspartate, ethanol, and L-glutamate but not at 41 °C or on adipate or 2,3-butanediol. Reduced susceptibility to sulfamethoxazole, trimethoprim and/or streptomycin was shown in eight strains, along with the presence of corresponding antibiotic resistance genes. In conclusion, this study provides a comprehensive description of A. faecalis and demonstrates its occurrence in cattle feces. Though the ecological role of A. faecalis remains unknown, our results show its ability to acquire antibiotic resistance genes, likely as an adaptation to antibiotic selection pressure in livestock farms., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Microbiota analyses are key to understanding the bacterial communities within dairy cattle, but the impact of different storage conditions on these analyses remains unclear. This study sought to examine the effects of freezing at -80°C immediately after collection, refrigeration at 4°C for three days and seven days and absolute ethanol preservation on the microbiota diversity of pooled fecal samples from dairy cattle. Examining 16S rRNA gene sequences, alpha (Shannon, Pielou evenness, observed features and Faith PD indices) and beta (Bray-Curtis, βw and Weighted UniFrac) diversity were assessed. The effects of storage conditions on these metrics were evaluated using linear mixed models and PERMANOVA, incorporating the farm as a random effect. Our findings reveal that 7d and E significantly altered the Shannon index, suggesting a change in community composition. Changes in Pielou evenness for 3d and 7d storage when compared to 0d were found, indicating a shift in species evenness. Ethanol preservation impacted both observed features and Faith PD indices. Storage conditions significantly influenced Bray-Curtis, βw, and Weighted UniFrac metrics, indicating changes in community structure. PERMANOVA analysis showed that these storage conditions significantly contributed to microbiota differences compared to immediate freezing. In conclusion, our results demonstrate that while refrigeration for three days had minimal impact, seven days of refrigeration and ethanol preservation significantly altered microbiota analyses. These findings highlight the importance of sample storage considerations in microbiota research., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Jaramillo-Jaramillo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The performance of dairy cows is influenced by the microbial communities hosted within their digestive tract. While the rumen microbiota has long been associated with host phenotypes, the impact of the faecal microbiota remains elusive. In this study, we collected 697 faecal samples from commercial Holstein cows and analysed them with 16S rRNA gene analyses. For each animal, routinely recorded data, i.e., milk yield, fat yield, protein yield, fat content, protein content, and an aggregate production trait (pINEL) based on the French economic dairy index, were available to assess the links between the faecal microbiota and host production. Our findings revealed a strong and significant association between the structure of the bacterial and prokaryote community (β-diversity) and dairy production. In addition, differential abundance analyses identified 48 genera whose abundances were significantly associated with pINEL, milk, fat and protein yield. Among these genera, the increased abundance of Bifidobacterium, and particularly an amplicon sequence variant with a 16S rRNA V3-V4 gene region identical to B. globosum and B. pseudolongum, was found to be the most important for high-yielding animals. Bifidobacterium seemed to be a potential key member of the bovine faecal microbiota that should be further investigated. Conversely, the p-1088-a5 gut group genus was found more abundant in low-productive cows. In conclusion, this study demonstrates significant associations between the faecal microbiota and the performance of dairy cows at the whole lactation scale. A better understanding of the physiology of the gut microbiota could help to improve dairy cow production., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The development and growth of animals coincide with the establishment and maturation of their microbiotas. To evaluate the respiratory and fecal microbiotas of beef calves from birth to weaning, a total of 30 pregnant cows, and their calves at birth, were enrolled in this study. Deep nasal swabs and feces were collected from calves longitudinally, starting on the day of birth and ending on the day of weaning. Nasopharyngeal, vaginal, and fecal samples were also collected from cows, and the microbiotas of all samples were analyzed. The fecal microbiota of calves was enriched with Lactobacillus during the first 8 weeks of life, before being displaced by genera associated with fiber digestion, and then increasing in diversity across time. In contrast, the diversity of calf respiratory microbiota generally decreased with age. At birth, the calf and cow nasal microbiotas were highly similar, indicating colonization from dam contact. This was supported by microbial source-tracking analysis. The structure of the calf nasal microbiota remained similar to that of the cows, until weaning, when it diverged. The changes were driven by a decrease in Lactobacillus and an increase in genera typically associated with bovine respiratory disease, including Mannheimia , Pasteurella , and Mycoplasma . These three genera colonized calves early in life, though Mannheimia was initially transferred from the cow reproductive tract. Path analysis was used to model the interrelationships of calf respiratory and fecal microbiotas. It was observed that respiratory Lactobacillus and fecal Oscillospiraceae UCG-005 negatively affected the abundance of Mannheimia or Pasteurella .IMPORTANCEIn beef cattle production, bovine respiratory disease (BRD) accounts for most of the feedlot morbidities and mortalities. Metaphylaxis is a common management tool to mitigate BRD, however its use has led to increased antimicrobial resistance. Novel methods to mitigate BRD are needed, including microbiota-based strategies. However, information on the respiratory bacteria of beef calves prior to weaning was limited. In this study, it was shown that the microbiota of cows influenced the initial composition of both respiratory and fecal microbiotas in calves. While colonization of the respiratory tract of calves by BRD-associated genera occurred early in life, their relative abundances increased at weaning, and were negatively correlated with respiratory and gut bacteria. Thus, microbiotas of both the respiratory and gastrointestinal tracts have important roles in antagonism of respiratory pathogens and are potential targets for enhancing calf respiratory health. Modulation may be most beneficial, if done prior to weaning, before opportunistic pathogens establish colonization., Competing Interests: The authors declare no conflict of interest.

Researchers have extensively studied the effect of oxygen on the growth and survival of bacteria. However, the impact of oxygen on bacterial community structure, particularly its ability to select for taxa within the context of a complex microbial community, is still unclear. In a 21-day microcosm experiment, we investigated the effect of aerobic exposure on the fecal community structure and succession pattern in broiler, calf, and piglet feces ( n = 10 for each feces type). Bacterial diversity decreased and community structure changed rapidly in the broiler microbiome ( P < 0.001), while the fecal community of calves and piglets, which have higher initial diversity, was stable after initial exposure but decreased in diversity after 3 days ( P < 0.001). The response to aerobic exposure was host animal specific, but in all three animals, the change in community structure was driven by a decrease in anaerobic species, primarily belonging to Firmicutes and Bacteroidetes (except in broilers where Bacteroidetes increased), along with an increase in aerobic species belonging to Proteobacteria and Actinobacteria. Using random forest regression, we identified microbial features that predict aerobic exposure. In all three animals, host-beneficial Prevotella -related ASVs decreased after exposure, while ASVs belonging to Acinetobacter , Corynbacterium, and Tissierella were increased. The decrease of Prevotella was rapid in broilers but delayed in calves and piglets. Knowing when these pathobionts increase in abundance after aerobic exposure could inform farm sanitation practices and could be important in designing animal experiments that modulate the microbiome.IMPORTANCEThe fecal microbial community is contained within a dynamic ecosystem of interacting microbes that varies in biotic and abiotic components across different animal species. Although oxygen affects bacterial growth, its specific impact on the structure of complex communities, such as those found in feces, and how these effects vary between different animal species are poorly understood. In this study, we demonstrate that the effect of aerobic exposure on the fecal microbiota was host-animal-specific, primarily driven by a decrease in Firmicutes and Bacteroidetes, but accompanied by an increase in Actinobacteria, Proteobacteria, and other pathobionts. Interestingly, we observed that more complex communities from pig and cattle exhibited initial resilience, while a less diverse community from broilers displayed a rapid response to aerobic exposure. Our findings offer insights that can inform farm sanitation practices, as well as experimental design, sample collection, and processing protocols for microbiome studies across various animal species., Competing Interests: The authors declare no conflict of interest.

A novel growth-promoting and indole acetic acid-producing strain, designated NEAU-LLB T , was isolated from cow dung collected from Shangzhi, Heilongjiang Province, PR China. Cells of strain NEAU-LLB T were Gram-stain-positive, non-motile, aerobic and non-spore-forming. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain NEAU-LLB T belonged to the genus Microbacterium . Strain NEAU-LLB T had high 16S rRNA sequence similarities of 98.81 and 98.41 % to Microbacterium paludicola DSM 16915 T and Microbacterium marinilacus DSM 18904 T , and less than 98 % to other members of the genus Microbacterium . Chemotaxonomic characteristics showed that MK-11 and MK-12 were detected as the predominant menaquinones. The peptidoglycan contained glutamic acid, aspartic acid, glycine, ornithine and a small amount of alanine, with ornithine as the diagnostic diamino acid. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid. The major fatty acids were identified as anteiso-C 15 : 0 , iso-C 16 : 0 and iso-C 17 : 0 . The genomic DNA G+C content of strain NEAU-LLB T was 70.2 mol%. In addition, the average nucleotide identity values between strain NEAU-LLB T and its reference strains, M. paludicola DSM 16915 T , M. marinilacus DSM 18904 T and M. album SYSU D8007 T , were found to be 81.1, 79.4 and 78.7 %, respectively, and the level of digital DNA-DNA hybridization between them were 23.8, 22.6 and 21.8 %, respectively. Based on the phenotypic, phylogenetic and genotypic data, strain NEAU-LLB T is considered to represent a novel species of the genus Microbacterium , for which the name Microbacterium stercoris sp. nov is proposed, with NEAU-LLB T (=CCTCC AA 2018028 T =JCM 32660 T ) as the type strain.

A Gram-stain-negative, non-spore-forming, yellow-pigmented, aerobic, pleomorphic rod-shaped bacterium, designated ZY171143 T , was isolated from faeces of a cow with diarrhoea in Wenshan, Yunnan Province, south-west China and its taxonomic position was studied. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZY171143 T belonged to the family Weeksellaceae and was most closely related to the only species of the genus Faecalibacter , Faecalibacter macacae CCTCC AB 2016016 T with a sequence similarity of 97.8 %. The genomic OrthoANI and digital DNA-DNA hybridization values between the strain and F. macacae CCTCC AB 2016016 T were 86.2 and 30.5 %, respectively. The genomic G+C content was 31.1 mol%. The predominant fatty acids (>5 %) were C 15 : 0 iso, C 17 : 0 iso 3OH, C 16 : 0 , C 16 : 1  ω5 c and summed feature 3 (C 16 : 1  ω7 c and/or 16 : 1 ω6c). The major polar lipids were phosphatidylethanolamine, triacylglycerol and sulfonolipid. The sole respiratory quinone was MK-6. These chemotaxonomic characterizations also revealed that strain ZY171143 T was a member of the genus Faecalibacter . Based on the phenotypic, chemotaxonomic and genotypic data, strain ZY171143 T represents a novel species within the genus Faecalibacter , for which the name Faecalibacter bovis sp. nov. is proposed. The type strain is ZY171143 T (=CGMCC 1.13663 T =KCTC 62642 T ).

Clostridioides difficile causes severe colitis in people and is a significant enteric pathogen in many species of animals, including swine, horses, and potentially cattle. C. difficile is shed in feces, and transmission occurs horizontally via the fecal-oral route. Livestock has been suggested as a potential reservoir for C. difficile, and while studies have shown that swine and farm workers can be colonized with identical clones of C. difficile, the zoonotic transmission of C. difficile from livestock to people has not been definitively demonstrated. The goal of this study was to determine whether dairy calves and dairy farm workers harbored genetically similar isolates of C. difficile. First, we validated a glove juice protocol for detecting C. difficile on farm workers' hands. We then visited 23 farms and collected 1) fecal samples from 92 dairy calves, 2) hand rinsates from 38 dairy farm workers, and 3) fecal samples from five of the dairy farm workers who were willing to submit them. All samples underwent anaerobic culture and qPCR to detect C. difficile. C. difficile was detected on 15 of the farms (65.2%, 95% confidence interval (CI) 42.7%-83.6%) and in 28 calves (30.4%, 95% CI 21.2-40.9%) but in none of the hand rinsates or human fecal samples. Thus, the zoonotic transmission of C. difficile on dairy farms could not be demonstrated, and dairy farmers did not appear to be at increased risk of acquiring C. difficile via the fecal-oral route., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The aim of this study was to characterize the colostrum and fecal microbiota in calves and to investigate whether fecal microbiota composition was related to colostrum microbiota or factors associated with calf health. Colostrum samples were collected in buckets after hand milking of 76 calving cows from 38 smallholder dairy farms. Fecal samples were taken directly from the rectum of 76 calves at birth and at 14 days age. The bacterial community structure in colostrum and feces was analyzed by terminal restriction fragment length polymorphism for all samples, and the microbial composition was determined by 16S rRNA gene amplicon sequencing for a subset of the samples (8 colostrum, 40 fecal samples). There was a significant difference in fecal microbiota composition between day 0 and day 14 samples, but no associations between the microbiota and average daily gain, birth weight, or transfer of passive immunity. At 14 days of age, Faecalibacterium and Butyricicoccus were prevalent in higher relative abundances in the gut of healthy calves compared to calves with diarrhea that had been treated with antimicrobials. Colostrum showed great variation in composition of microbiota but no association to fecal microbiota. This study provides the first insights into the composition of colostrum and fecal microbiota of young dairy calves in southern Vietnam and can form the basis for future more detailed studies.

Antimicrobial resistance has become a global threat to public health since multidrug-resistant (MDR) bacteria have been reported worldwide carrying different antimicrobial resistance genes (ARGs), and animals have been described as a reservoir of ARGs. The presence of antimicrobial-resistant bacteria and ARGs in the food matrix is a risk to public health. This study aimed to research the presence of clinically relevant ARGs for important antimicrobials and genetic elements in fecal samples from dairy cows and calves on a Brazilian farm. In this study, a total of 21 fecal samples were collected, and then, the DNA of cultivable aerobic bacteria was extracted. Fifty-seven ARGs and twenty-three genetic elements were researched by PCR and confirmed by sequencing. Several ARGs that confer resistance to β-lactams, tetracyclines, fluoroquinolones, sulphonamides, phenicols, aminoglycoside, glycopeptides, and macrolides were detected. A total of 200 amplicons from 23 ARGs (bla CTX-M-Gp2 , bla CMY , bla SHV , tetA, tetB, tetC, qepA, qnrB, qnrS, oqxA, oqxB, vanC1, vanC2/3, aadA, sul1, sul2, sul3, ermB, mefAE, floR, cmlA, aadA, aph(3')-Ia, aac(3')-Ia), and 145 amplicons from 12 genetic elements (IncF, IncFIA, IncFIB, IncI1, IncY, IncU, IncK, IncP, IncR, IncHI1, ColE-like, intI1) were detected. The results presented in this study call attention to the monitoring of antimicrobial resistance in dairy farms worldwide. MDR bacteria and ARGs can spread to different sources, including milk products, which are one of the most consumed products worldwide, representing a potential risk to human health.

The factors that influence the diversity and composition of raw milk and fecal microbiota in healthy commercial dairy herds are not fully understood, partially because the majority of metataxonomic studies involve experimental farms and/or single factors. We analyzed the raw milk and fecal microbiota of 100 healthy cows from 10 commercial alpine farms from the Province of Trento, Italy, using metataxonomics and applied statistical modelling to investigate which extrinsic and intrinsic parameters (e.g. herd, diet and milk characteristics) correlated with microbiota richness and composition in these relatively small traditional farms. We confirmed that Firmicutes, Ruminococcaceae and Lachnospiraceae families dominated the fecal and milk samples of these dairy cows, but in addition, we found an association between the number of observed OTUs and Shannon entropy on each farm that indicates higher microbiota richness is associated with increased microbiota stability. Modelling showed that herd was the most significant factor affecting the variation in both milk and fecal microbiota composition. Furthermore, the most important predictors explaining the variation of microbiota richness were milk characteristics (i.e. percentage fat) and diet for milk and fecal samples, respectively. We discuss how high intra-herd variation could affect the development of treatments based on microbiota manipulation., Competing Interests: The authors have declared that no competing interests exist.

Shiga toxin-producing Escherichia coli (STECs) contamination of produce, as a result of contact with ruminant fecal material, has been associated with serious foodborne illness. Bacteriophages (phages) that infect STECs have primarily been reported to be of cattle origin. However, they likely exist in other environments or in animals that share habitats with cattle, such as goats. To explore the presence and diversity of phages specific to STEC O157 and the top six non-O157 STECs in goat-associated environments, environmental samples consisting of feces (goat and cattle) and soil samples were collected monthly for six months from an organic produce farm. A variety of phages belonging to the Myoviridae, Siphoviridae, and Podoviridae families were isolated from all goat fecal and half of the soil samples. The most commonly isolated phages belonged to Myoviridae and were lytic against STEC O103. The isolated phages had different host ranges, but collectively, showed lytic activity against O157 and the top six non-O157 STEC strains excluding O121. Two non-O157 STECs (O174: H21 and O-antigen-negative: H18) were isolated from soil and cattle feces, respectively. Although prior studies have reported that goats shed STEC into the environment, the findings of the current study suggest that goat feces may also contain lytic STEC-specific phages. The phages of goat origin have the capacity to infect STECs implicated in causing foodborne outbreaks, making them potential candidates for biocontrol pending additional characterization steps. Further work is needed to determine if the addition of goats to the farm environment could potentially reduce the presence of STECs., Competing Interests: The authors have declared that no competing interests exist.

Analysis of shotgun metagenomic data generated from next generation sequencing platforms can be done through a variety of bioinformatic pipelines. These pipelines employ different sets of sophisticated bioinformatics algorithms which may affect the results of this analysis. In this study, we compared two commonly used pipelines for shotgun metagenomic analysis: MG-RAST and Kraken 2, in terms of taxonomic classification, diversity analysis, and usability using their primarily default parameters. Overall, the two pipelines detected similar abundance distributions in the three most abundant taxa Proteobacteria, Firmicutes, and Bacteroidetes. Within bacterial domain, 497 genera were identified by both pipelines, while an additional 694 and 98 genera were solely identified by Kraken 2 and MG-RAST, respectively. 933 species were detected by the two algorithms. Kraken 2 solely detected 3550 species, while MG-RAST identified 557 species uniquely. For archaea, Kraken 2 generated 105 and 236 genera and species, respectively, while MG-RAST detected 60 genera and 88 species. 54 genera and 72 species were commonly detected by the two methods. Kraken 2 had a quicker analysis time (~4 hours) while MG-RAST took approximately 2 days per sample. This study revealed that Kraken 2 and MG-RAST generate comparable results and that a reliable high-level overview of sample is generated irrespective of the pipeline selected. However, Kraken 2 generated a more accurate taxonomic identification given the higher number of "Unclassified" reads in MG-RAST. The observed variations at the genus level show that a main restriction is using different databases for classification of the metagenomic data. The results of this research indicate that a more inclusive and representative classification of microbiomes may be achieved through creation of the combined pipelines., Competing Interests: Francesca Stomeo is currently at The European Molecular Biology Laboratory (EMBL), Heidelberg, Germany., (Copyright © 2020 Felix M. Kibegwa et al.)

Most of the research efforts involving the bovine gastrointestinal microbiota have focused on cattle's forestomach, particularly the rumen, so information concerning the bovine fecal microbiota is more scarce, especially in young beef cattle. The present study was performed to evaluate the ruminal and fecal microbiotas of beef calves as they reached the end of their nursing phase. A total of 18 Angus cow/calf pairs were selected and assigned to one of two treatment groups for the last 92 days of the calves' nursing period, as follows: 1) calves were supplemented with concentrate in a creep feeding system; or 2) control group with no supplementation of calves. After 92 days, ruminal and fecal samples were individually obtained from calves in both groups, and their microbiotas were evaluated using 16S rRNA gene sequencing. Ruminal samples were predominated by Prevotella (18 to 23% of the total bacterial abundance), regardless if calves received supplementation or not; however, in the feces, Prevotella was only the seventh most abundant genus (0.6 to 2.1% of total bacterial abundance). Both the rumen (P = 0.01) and the feces (P = 0.05) of calves that received supplementation had greater abundance of Firmicutes. In addition, calves that were supplemented had lower abundance of Fibrobacteres (P = 0.03) in their rumens. Regardless if the calves were supplemented or not, Faith's Phylogenetic Diversity index (P ≤ 0.007) and total concentration of short chain fatty acids (P < 0.001) were both greater in the rumen than in the feces of calves. In summary, the ruminal and fecal microbiotas of weanling beef calves were considerably distinct. Additionally, supplementation with creep feed caused some significant changes in the composition of the gastrointestinal microbiota of the calves, especially in the rumen, where supplementation caused an increase in Firmicutes and a decrease in abundance of Fibrobacteres., Competing Interests: The authors have declared that no competing interests exist.

The purpose of this study was to determine the effect of intramammary pirlimycin on the fecal microbiome of dairy cattle. Primiparous heifers were enrolled and assigned to a treatment or control group at a ratio of 2:1. In part 1 of the study, treated heifers (T1) were given intramammary pirlimycin into one infected quarter once daily for 2 d at 24-h intervals, according to the label instructions. Control heifers received no treatment. In part 2 of the study, treated heifers (T2) were given intramammary pirlimycin into one infected quarter once daily for 8 d at 24-h intervals, according to the label instructions. All enrolled heifers (T1, T2, and control) had quarter-level milk samples aseptically collected for bacterial culture and fecal samples collected for 16S rRNA gene sequencing on d 0, 2, 7, 14, 21, and 28. Milk samples were plated on Columbia blood agar and incubated at 37°C for 24 h. Bacteria were identified using MALDI-TOF mass spectrometry. The DNA was extracted from feces using PowerFecal kits (Qiagen, Venlo, the Netherlands). The 16S rRNA gene amplicon library construction and sequencing was performed at the University of Missouri DNA Core facility. Testing for differences in fecal community composition was performed via one-way permutational multivariate ANOVA of Bray-Curtis and Jaccard similarities using Past 3.13 (https://folk.uio.no/ohammer/past/). Mean total count of operational taxonomic units and Chao1, Shannon, and Simpson α-diversity indices were determined and compared via t-test or Wilcoxon rank sum test. A treatment-dependent effect was present in the observed and predicted richness of feces from cows in the T1 group at d 2 posttreatment. Additionally, intramammary pirlimycin induced a significant change in the composition of the fecal microbiota by d 2 in the treated groups. Based on calculated intra-subject similarities, intramammary pirlimycin was associated with a significant acute change in the fecal microbiota of dairy heifers and that chance reversed when the antimicrobial exposure was brief, but sustained following longer exposure. Overall, intramammary pirlimycin administration affected the fecal microbiome of lactating dairy heifers. Further work is necessary to determine the effect of these changes on the heifer and the dairy environment as well as if treatment is influencing antimicrobial resistance among enteric and environmental bacteria., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Using multiplex real-time PCR, 960 fecal samples collected from poultry, cattle, and patients with diarrhea in Bangladesh were screened for diarrheagenic Escherichia coli (DEC). The invasion-related gene virB showed the highest prevalence in human patients (41%) and was shown to be positively correlated first with afaB with regards to diffuse adhesion and second with aggR with regards to aggregative adhesion. These three genes were specific to human patients. In contrast, the Shiga toxin genes stx1 (57%) and stx2 (40%) were prevalent in cattle samples. The eae gene, which is associated with attaching and effacing lesion formation, and the elt and est genes, which are associated with enterotoxins, were detected from all three sample sources. Heat map construction and hierarchical clustering assigned the samples into five different clusters, with the patient samples positive for virB and afaB being placed together in one cluster. Although the detection of virulence genes cannot be a direct indication of the distribution of diarrheagenic organisms, their detection suggests that Shigella spp. or enteroinvasive E. coli are the most prevalent diarrheagenic bacteria in Bangladesh and that diffusely adherent E. coli is concomitantly present with these bacteria. eae-possessing organisms in patients may come from cattle and poultry sources. The small number of stx-positive patients could be explained by the small number of animal samples that were positive for both eae and stx.

In sub-Saharan Africa, many families travel to collect water and store it in their homes for daily use, presenting an opportunity for the introduction of fecal contamination. One stored and one source water sample were each collected from 45 households in rural Kenya. All 90 samples were analyzed for fecal indicator bacteria ( E. coli and enterococci) and species-specific contamination using molecular microbial source tracking assays. Human (HF183), avian (GFD), and ruminant (BacR) contamination were detected in 52, two, and four samples, respectively. Stored water samples had elevated enterococci concentrations ( p < 0.01, Wilcoxon matched pairs test) and more frequent BacR detection (89% versus 27%, p < 0.01, McNemar's exact test) relative to source water samples. fsQCA (fuzzy set qualitative comparative analysis) was conducted on the subset of households with no source water BacR contamination to highlight combinations of factors associated with the introduction of BacR contamination to stored water supplies. Three combinations were identified: (i) ruminants in the compound, safe water extraction methods, and long storage time, (ii) ruminants, unsafe water extraction methods, and no soap at the household handwashing station, and (iii) long storage time and no soap. This suggests that multiple pathways contribute to the transmission of ruminant fecal contamination in this context, which would have been missed if data were analyzed using standard regression techniques.

Microbiota of individual cow milk, bulk tank milk, and feces of Jersey cows were examined. Samples were collected from two farms (F1 and F2) in cool (November, Nov) and hot (July, Jul) seasons. Milk yield and milk composition were similar between the two farms and between the two seasons. Prevalent taxa of the fecal microbiota, i.e. Ruminococcaceae, Bacteroidaceae, Lachnospiraceae, Rikenellaceae, and Clostridiaceae, were unaffected by the farm and season. Relative abundance of milk microbiota for Pseudomonadaceae, Enterobacteriaceae, and Streptococcaceae (F1 > F2) and Lactobacillaceae, Bifidobacteriaceae, and Cellulomonadaceae (F1 < F2) were different between the two farms, and those for Staphylococcaceae, Bacillaceae, Ruminococcaceae, and Veillonellaceae (Nov < Jul) and Methylobacteriaceae and Moraxellaceae (Nov > Jul) were different between the two seasons. The microbiota of bulk tank milk was numerically different from that of individual cow milk. Principal coordinate analysis indicated that the milk microbiota was unrelated to the fecal microbiota. The finding that relative abundance of Pseudomonadaceae and Moraxellaceae appeared greater than those reported for Holstein milk suggested that higher protein and fat content may result in a greater abundance of proteolytic and lipolytic taxa in Jersey cow milk., (© 2020 Japanese Society of Animal Science.)

Zinc is one of the micronutrients, required by all types of crops. About 10-100ppm of zinc is present in soil which is generally immobile. The cow dung sustains all life and being practice since aeons. Exploitation of cow dung bacteria can mobilize nutrients besides contributing in sustainable agriculture. Therefore, to examine mobilization of Zn, cow dung is used as a source of bacteria. The objectives of the present study were to isolate an array of bacteria from cow dung and to characterize them for their Zn (ZnO and ZnCO 3 ) mobilization ability in addition to establish the optimum conditions for dissolution of zinc. A total of seventy bacterial isolates have been screened for Zn mobilization. Out of which most potent (CDK15 and CDK25) were selected to study the effect of various parameters viz. pH, temperature and concentration of Zn. These parameters were assessed qualitatively in diverse growth medium and quantitatively using Atomic absorption spectroscopy. Optimum pH and temperature for mobilization was recorded at pH 5 (ZnO) and 37 °C (ZnCO 3 ) by CDK25, whereas, optimum zinc concentration for mobilization was recorded at 0.05% (ZnO) by CDK15. Maximum amount of Zn solubilized was recorded by CDK25 in ZnO (20ppm). Considering the abilities of most potent bacterial isolates with reference to P-mobilization and growth promoting traits, pot culture assay of C. annuum L. was carried out. The findings of which conclude that, bacterium CDK25 (Bacillus megaterium) could be exploited for factors viz. nutrient management of Zn, growth promoting agent, and Zn augmentation in soil.

Tall fescue, the predominant southeastern United States cool-season forage grass, frequently becomes infected with an ergot alkaloid-producing toxic endophyte, Epichloë coenophialum Consumption of endophyte-infected fescue results in fescue toxicosis (FT), a condition that lowers beef cow productivity. Limited data on the influence of ergot alkaloids on rumen fermentation profiles or ruminal bacteria that could degrade the ergot alkaloids are available, but how FT influences the grazing bovine fecal microbiota or what role fecal microbiota might play in FT etiology and associated production losses has yet to be investigated. Here, we used 16S rRNA gene sequencing of fecal samples from weaned Angus steers grazing toxic endophyte-infected (E+; n  = 6) or nontoxic (Max-Q; n  = 6) tall fescue before and 1, 2, 14, and 28 days after pasture assignment. Bacteria in the Firmicutes and Bacteroidetes phyla comprised 90% of the Max-Q and E+ steer fecal microbiota throughout the trial. Early decreases in the Erysipelotrichaceae family and delayed increases of the Ruminococcaceae and Lachnospiraceae families were among the major effects of E+ grazing. E+ also increased abundances within the Planctomycetes , Chloroflexi , and Proteobacteria phyla and the Clostridiaceae family. Multiple operational taxonomic units classified as Ruminococcaceae and Lachnospiraceae were correlated negatively with weight gains (lower in E+) and positively with respiration rates (increased by E+). These data provide insights into how E+ grazing alters the Angus steer microbiota and the relationship of fecal microbiota dynamics with FT. IMPORTANCE Consumption of E+ tall fescue has an estimated annual $1 billion negative impact on the U.S. beef industry, with one driver of these costs being lowered weight gains. As global agricultural demand continues to grow, mitigating production losses resulting from grazing the predominant southeastern United States forage grass is of great value. Our investigation of the effects of E+ grazing on the fecal microbiota furthers our understanding of bovine fescue toxicosis in a real-world grazing production setting and provides a starting point for identifying easy-to-access fecal bacteria that could serve as potential biomarkers of animal productivity and/or FT severity for tall fescue-grazing livestock., (Copyright © 2019 American Society for Microbiology.)

Background: Beef cattle in North America frequently receive an antibiotic injection after feedlot placement to control and manage bovine respiratory disease. The potential collateral effect of these antibiotics on the bovine microbiome is largely unknown. Therefore, we determined the longitudinal impact of two commonly administered veterinary antibiotics, oxytetracycline and tulathromycin, on the fecal and nasopharyngeal (NP) microbiota of beef cattle that were transported to a feedlot. We also report the effect these antibiotics have on several antibiotic resistance determinants in both the fecal and NP microbiome., Results: Oxytetracycline and tulathromycin perturbation of the bovine fecal and NP microbiota was greatest at days 2 and 5. Although the NP microbiota of the tulathromycin-treated cattle had recovered by day 12, the NP microbiota of the oxytetracycline-treated group remained altered through day 34. Overall, the NP microbiota appeared to be more sensitive to antibiotic treatment than the fecal microbiota. Members of the bacterial Microbacteriaceae family were most notably affected by antibiotic administration in the NP microbiota. Both antibiotics protected against Pasteurella spp. in the nasopharynx at days 2 and 5. Despite very similar diets at both locations, the largest shift in the fecal and NP microbiota occurred after transport to the feedlot (P < 0.05). Antibiotic resistance determinants in the NP microbiome were also affected more strongly by antibiotic treatment than those in the fecal microbiome. Oxytetracycline increased the proportion of erm(X), sul2, tet(H), tet(M), and tet(W) in NP samples and tet(M) and tet(W) in fecal samples, at day 12 (P < 0.05). The effect of tulathromycin on the relative abundance of resistance genes in the NP microbiome was greatest at day 34 as erm(X), sul2, and tet(M) were enriched (P < 0.05)., Conclusions: Administration of a single injection of oxytetracycline and tulathromycin resulted in significant changes in the NP and fecal microbiota during the first 5 days after treatment. Antibiotic treatment also increased the relative abundance of several antibiotic resistance determinants in the fecal and NP microbiome at either day 12 or 34.

Objectives: To explore the dynamics of faecal ESBL/AmpC shedding in dairy cattle and farmers, a study was conducted to examine changes in shedding by individual animals, as well as environmental exposure, and to study the association between antimicrobial use (AMU) and ESBL/AmpC shedding., Methods: The study comprised a cross-sectional survey of 20 farms and a 1 year follow-up of 10 farms. Faecal samples were cultured by both direct inoculation on MacConkey agar + 1 mg/L cefotaxime (MC+) and enrichment in LB-broth + 1 mg/L cefotaxime with subsequent inoculation on MC+. Dust samples were collected using electrostatic dustfall collectors (EDCs). Human faecal samples were collected by the farmers. Presence of ESBL/AmpC genes was screened for by PCR and sequencing. Using mixed effects logistic regression, ORs were determined and population-attributable fractions (PAFs) calculated subsequently., Results: In Phase 1, 8/20 farms were positive for ESBL/AmpC and, with 2 negative farms, were selected for Phase 2. Transient shedding of dominant allele variants was observed in the animals. EDCs and human faecal samples did not reflect what was observed in the animals. AMU was related to shedding of ESBLs in the next sampling moment [OR 14.6 (95% CI 3.0-80.0)] and the PAF of AMU was 0.36 (95% CI 0.08-0.77). Calves fed with colostrum from cows on dry-off therapy was not a risk factor [OR 1.7 (95% CI 0.7-4.9, P = 0.28)]., Conclusions: The presence of ESBL/AmpC could only be partly explained by AMU. No link was shown between shedding in cattle and humans or the environment. Interventions should focus on prevention of introduction., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Huge numbers of microorganisms reside in livestock faeces and constitute one of the most complex microbial ecosystems. Here, faecal microbial communities of three typical livestock in Xilingol steppe grassland, i.e. sheep, cattle, and horse, were investigated by Illumina MiSeq sequencing and quantitative real-time polymerase chain reaction (qPCR). Firmicutes and Bacteroidetes comprised the majority of bacterial communities in three livestock faeces. Sordariomycetes, Leotiomycetes, and Dothideomycetes were dominant in fungal communities, as well as Methanobacteria and Methanomicrobia were dominant in archaeal communities in three livestock faeces. Similar fungal community dominated in these samples, with 95.51% of the sequences falling into the overlap of three livestock faeces. In contrast, bacterial communities were quite variable among three different livestock faeces, but a similar community was observed in sheep and cattle faeces. Nearly all the archaea were identified as methanogens, whilst the most diverse and abundant methanogens were detected in cattle faeces. Potential pathogens including Bacteroides spp., Desulfovibrio spp., and Fusarium spp. were also detected in livestock faeces. Overall, this study provides the first detailed microbial comparison of typical livestock faeces dwelling on single grassland, and may be help guide management strategies for livestock grazing and grassland restoration.

There has been a great interest to identify a microbial marker that can be used to predict feed efficiency of beef cattle. Such a marker, specifically one that would allow an early identification of animals with high feed efficiency for future breeding efforts, would facilitate increasing the profitability of cattle operations and simultaneously render them more sustainable by reducing their methane footprint. The work presented here suggests that Prevotella copri might be an ideal microbial marker for identifying beef cattle with high feed efficiency early in their life span and in the production cycle. Developing more refined quantification techniques that allow correlation of P. copri to feed efficiency of beef cattle that can be applied by lay people in the field holds great promise to improve the economy of cattle operations while simultaneously reducing their environmental impact by mitigating methane production from enteric fermentation., (© 2019 Japanese Society of Animal Science.)

The objectives of this study were to describe the frequency of antimicrobial resistance (AMR) in Escherichia coli and Campylobacter spp. isolates in fecal samples from beef cow-calf herds and to examine the associations between herd management practices, reported antimicrobial use, and AMR. Baseline prevalence data are needed to evaluate the effectiveness of antimicrobial stewardship programs. A pooled fecal sample, representing 20 cows, was collected from each of 107 herds during pregnancy testing. In the 305 recovered E. coli isolates (maximum 3 per herd), resistance to ≥ 1 antimicrobial was identified in 12 isolates [4%, 95% confidence interval (CI): 2% to 7%] from 105 herds (11%, 95% CI: 7% to 19%). The most common resistances identified in E. coli isolates were to tetracycline (3%) and to both streptomycin and sulfisoxazole (3%). Only 1 E. coli isolate was resistant to an antimicrobial of very high importance to human health - amoxicillin/clavulanic acid. However, 2 E. coli isolates had intermediate susceptibility to ciprofloxacin. Resistance to 1 antimicrobial was identified in 16 of 87 Campylobacter spp. isolates (18%, 95% CI: 11% to 28%) from 87 herds. Resistance to tetracycline was reported in 15% of Campylobacter spp. isolates and to nalidixic acid in 3.4%. Herds in which cows were treated with florfenicol were more likely to have E. coli resistance to ≥ 2 antimicrobials (OR 7.1, 95% CI: 1.1 to 57, P = 0.03). Herds with calf mortality of > 5% were more likely to have E. coli with resistance to streptomycin and sulfisoxazole [odds ratio (OR): 7.8, P = 0.03]. The results of this study are consistent with previous reports from western Canada and provide a starting point for designing an ongoing antimicrobial surveillance program.

Dehiding during beef cattle processing can introduce fecal contaminants, including Shiga toxin-producing Escherichia coli (STEC), from hides onto carcass surfaces, creating the potential for contaminated beef. Fecal shedding of major STEC serogroups (O26, O45, O103, O111, O121, O145, and O157; STEC-7) may differ among cattle populations, yet no study has been conducted to isolate STEC-7 on hides of multiple cattle types on the same production days at the same processing plant. Our objective was to estimate and compare prevalence and concentrations of STEC-7 on hides of cull dairy, cull beef, and fed beef cattle from the same date and processing plant. Overall, 1,500 cattle hides were sponge sampled from cull dairy ( n = 500), cull beef ( n = 500) and fed beef cattle ( n = 500) over 10 processing days. To determine prevalence, samples were subjected to an immunomagnetic separation culture method, and presumptive STEC isolates were tested by PCR for serogroup and major virulence genes. A spiral plate method was used to enumerate STEC-7 from hide samples. Data were analyzed with linear mixed models. All STEC-7 serogroups except O121 were detected and quantified on cattle hides in this study population. Slightly more fed beef hides (77 of 500; 15.4%) and cull beef hides (76 of 500; 15.2%) were positive for at least one STEC-7 strain compared with cull dairy hides (57 of 500; 11.4%), but cattle type was not significantly associated ( P = 0.19) with STEC-7 prevalence. Fed beef hides had a significantly higher prevalence ( P < 0.05) of STEC O103, O145, and O157 serogroups than did either of the other cattle types. The highest proportions of quantifiable samples were for STEC O145 (32 of 1,500 samples; 2.1%) and O157 (31 of 1,500 samples; 2.1%) serogroups, with the majority of concentrations at 3 to 5 and 2 to 4 log CFU/100 cm 2 of hide, respectively. Results indicate that hide contamination with some major STEC serogroups differs significantly among cattle types at harvest, even within the same day and location.

Aim: The study aimed to isolate and characterize Enterococcus species from apparently healthy waste attendants, cattle and cattle waste in Tanzania. Emphasis was given to antimicrobial resistance and in particular occurrence of vancomycin (VA)-resistant enterococci., Methods and Results: Faecal samples were collected from healthy cattle, cattle waste attendants and cattle house wastes, and isolation of Enterococcus species was performed using Slanetz Bartley agar. Isolates were characterized with regard to species, antimicrobial susceptibility and presence of VA resistance genes. Enterococcus faecalis was the most prevalent species from all sources of isolation (43·5%), followed by Enterococcus faecium (38·4%). Isolates of E. faecium showed a higher number of phenotypic antimicrobial resistance than isolates of E. faecalis. Fifty-eight isolates, which showed resistance or intermediate resistance to VA by disc diffusion test, were analysed for VA-resistant Enterococcus (VRE) by PCR. The vanA gene was detected in 14 isolates of E. faecium and 12 isolates of E. faecalis, while vanB was detected in three isolates. No isolates were found to carry vanC1-gene., Conclusion: VRE was detected in both human and cattle samples, despite no known use of antimicrobial agents that can select for VRE in livestock in Tanzania. Enterococcus faecalis was the most commonly isolated species from cattle and humans., Significance and Impact of the Study: The study provides information on the prevalence of VRE in human and nonhuman samples in Tanzania calling for further studies on the origin of VRE in such isolates, since no selection mechanism in Tanzania are known., (© 2018 The Society for Applied Microbiology.)

High temperature influences rumen and gut health, passage rate, and diet digestibility, with effects on fermentative processes. The main aim of the study was to investigate the effect of hot season on hindgut fermentation, the occurrence of Clostridium tyrobutyricum spores in bovine feces, and on their relationship with metabolic conditions in dairy cows producing milk used for Grana Padano cheese. The study was carried out on 7 dairy farms located in the Po Valley (Italy), involving 1,950 Italian Friesian dairy cows. The study was carried out from November 2013 till the end of July 2014. Temperature and relative humidity were recorded daily by weather stations. Constant management conditions were maintained during the experimental period. Feed and diet characteristics, metabolic conditions, and fecal characteristics were recorded in winter (from late November 2013 to the end of January 2014), spring (from April to May 2014), and summer (July 2014) season. In each season, blood samples were collected from 14 multiparous lactating dairy cows per herd to measure biochemical indices related to energy, protein, and mineral metabolism, as well as markers of inflammation and some enzyme activities. Fecal samples were also collected and measurements of moisture, pH and volatile fatty acids (VFA) were performed. The DNA extracted and purified from fecal samples was used to detect Clostridium tyrobutyricum spores in a quantitative real-time PCR assay. The daily mean temperature-humidity index was 40.7 ± 4.6 (range 25 to 55), 61.2 ± 3.7 (range 39 to 77), and 70.8 ± 3.2 (range 54 to 83) in winter, spring, and summer, respectively. Total VFA concentration in feces progressively decreased from winter to summer. The seasonal changes of acetate and propionate followed the same trend of total VFA; conversely, butyrate did not show any difference between seasons, and its molar proportion was greater in summer compared with winter. A greater occurrence of Cl. tyrobutyricum spores in summer compared with the other seasons was observed. The plasma concentrations of glucose, urea, albumin, Ca, Mg, Cl, Zn, and alkaline phosphatase activity were lower in summer compared with winter, whereas the opposite occurred for bilirubin and Na. Our results show that summer season, through direct and indirect effect of heat stress, affected fecal fermentative parameters and hindgut buffering capacity, and was responsible for the increasing occurrence of Cl. tyrobutyricum spores in feces., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Ten different anaerobic gut fungal strains were isolated from fecal samples of cow and goat in Oklahoma, USA. The isolates displayed polycentric thalli, uniflagellated zoospores, and sausage-shaped hyphal constrictions, all characteristic of the genus Anaeromyces, and lobed or appressorium-like structures on the hyphae, previously reported in Anaeromyces elegans but not in A. mucronatus or A. robustus. Further, the strains exhibited highly coiled and entangled hyphae previously unreported in the Neocallimastigomycota, but resembling fungal traps previously observed in multiple nematophagous fungi. Scanning electron micrographs of isolates grown on switchgrass showed entrapment of plant material by the hyphal coils and the penetration of plant fibers at various sites by the appressorium-like structures. Molecular analysis based on sequences of both partial nuc rDNA ITS1 spacer region and the D1-D2 domains of the nuc28S rDNA confirmed the classification of all isolates in Anaeromyces and confirmed their phylogenetic distance from the available A. mucronatus and A. robustus sequences. On the basis of morphological and phylogenetic distinctions, we conclude that these strains represent a new Anaeromyces species, for which A. contortus is proposed.

The bovine gastrointestinal microbiota is a complex polymicrobial ecosystem that plays an important role in maintaining mucosal health. The role of mucosal microbial populations in the pathogenesis of gastrointestinal diseases has been well established in other species. However, limited information is available about changes in the fecal microbiota that occur under disease conditions, such as hemorrhagic diarrhea in feedlot cattle. The objectives of this study were to characterize the differences in fecal microbiota composition, diversity and functional gene profile between feedlot calves with, and without, hemorrhagic diarrhea. Deep fecal swabs were collected from calves with hemorrhagic diarrhea (n = 5) and from pen matched healthy calves (n = 5). Genomic DNA was extracted, and V1-V3 hypervariable region of 16S rRNA gene was amplified and sequenced using the Illumina MiSeq sequencing. When compared to healthy calves, feedlot cattle with hemorrhagic diarrhea showed significant increases in the relative abundance of Clostridium, Blautia and Escherichia, and significant decreases in the relative abundance of Flavobacterium, Oscillospira, Desulfonauticus, Ruminococcus, Thermodesulfovibrio and Butyricimonas. Linear discriminant analysis effect size (LEfSe) also revealed significant differences in bacterial taxa between healthy calves and hemorrhagic diarrhea calves. This apparent dysbiosis in fecal microbiota was associated with significant differences in the predictive functional metagenome profiles of these microbial communities. In summary, our results revealed a bacterial dysbiosis in fecal samples of calves with hemorrhagic diarrhea, with the diseased calves exhibiting less diversity and fewer observed species compared to healthy controls. Additional studies are warranted in a larger cohort of animals to help elucidate the trajectory of change in fecal microbial communities, and their predictive functional capacity, in calves with other gastrointestinal diseases., (Published by Elsevier Ltd.)

In order to assess the survival of lactic acid bacteria (LAB) in whole crop maize silage in the gut of dairy cows, one representative silage sample and three different feces samples were collected from dairy cows on three dairy farms in Hua Bei, China and three dairy farms in Kyushu, Japan. The composition of the bacterial community was examined by denaturing gradient gel electrophoresis and quantitative polymerase chain reaction. Lactobacillus acetotolerans was detected in all bunker-made maize silage samples, regardless of the dairy farm or sampling region from which they were sourced. A total of eight LAB species were detected in the maize silage samples, of which three (L. acetotolerans, L. pontis and L. casei) appeared to survive digestion. The populations of L. acetotolerans in silage and feces were 10 6-7 and 10 3-4 copies/g, respectively, indicating that, even for the LAB species showing potential survival in the gut, competition in this niche may be harsh and the population may substantially decrease during the digestion process. It may be difficult for silage LAB to survive in the gut of silage-fed dairy cows, because marked decrease in population can take place during the digestion process, even for surviving species., (© 2017 Japanese Society of Animal Science.)

The taxonomic position of four phenotypically closely related strains isolated from faecal samples of yaks (Bos grunniens) collected from the Qinghai-Tibetan plateau, China, was determined by a polyphasic approach. The strains were non-spore-forming, non-motile Gram-stain-positive, ovoid cocci, occurring predominantly in pairs and short chains or in irregular clusters. The 16S rRNA gene of strain MN05 T was related phylogenetically to those of Enterococcushaemoperoxidus, Enterococcusrotai, Enterococcusquebecensis, Enterococcusplantarum, Enterococcuscrotali, Enterococcusmoraviensis, Enterococcussilesiacus, Enterococcuscaccae, Enterococcustermitis, Enterococcusureasiticus and Enterococcusureilyticus, all belonging to the Enterococcusfaecalis species group. The sequence similarities of three selected genes of MN05 T to those of the type strains of phylogenetically related species were measured, with values within the range of 99.2-99.5 % (16S rRNA gene), 90.0-97.3 % (rpoA) and 80.0-85.3 % (pheS), respectively. The genome of MN05 T (3 842 361 bp) contained 4299 genes with a DNA G+C content of 37.5 mol%. A whole-genome phylogenetic tree based on 808 core genes confirmed that MN05 T belongs to a distinct lineage, well separated from all recognized species of the Enterococcusfaecalis species group. DNA-DNA hybridization in silico showed that MN05 T displayed less than 70 % DNA-DNA relatedness with the other 13 species of the Enterococcusfaecalis species group. Moreover, their phenotypic features distinguished the four strains from the other species of the Enterococcusfaecalis species group. Based upon these data obtained from the polyphasic characterization performed in the present study, a novel species of the genus Enterococcus, Enterococcus wangshanyuanii sp. nov., is proposed, with the type strain MN05 T (=DSM 104047 T =CGMCC 1.15942 T ).

Several factors are known to affect the intestinal microbiota of cattle. However, how these changes occur over time is poorly understood. This study aimed to investigate the consequences of entrance into a feedlot and the effects of virginiamycin used as a growth promoter on the bovine fecal microbiota. Two batches of beef cattle (B1, n=50 and B2, n=36) entering a feedlot operation were randomly divided into two pens: one receiving virginiamycin and one group not receiving antibiotic (control group). Fecal samples were collected at arrival, mid feedlot and at exit to slaughter. The V4 region of 16S rRNA gene was amplified and sequenced. Escherichia coli strains isolated in samples from arrival and exit of B2 were also isolated and used as indicators of antimicrobial susceptibility. Marked changes in membership and structure of fecal microbiota occurred following entrance into the feedlot. At mid feedlot, virginiamycin affected bacterial community membership, but not structure, suggesting that the antibiotic had a stronger effect on the rare, but not on the most abundant species. The use of virginiamycin had no demonstrable effect on antibiotic resistance in E. coli. The differences found between batches are suggestive that variations in study conditions are important and can strongly affect the intestinal microbiota., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Metritis is an inflammatory disease of the uterus caused by bacterial infection, particularly Bacteroides, Porphyromonas, and Fusobacterium. Bacteria from the environment, feces, or vagina are believed to be the only sources of uterine contamination. Blood seeps into the uterus after calving; therefore, we hypothesized that blood could also be a seeding source of uterine bacteria. Herein, we compared bacterial communities from blood, feces, and uterine samples from the same cows at 0 and 2 days postpartum using deep sequencing and qPCR. The vaginal microbiome 7 days before calving was also compared., Results: There was a unique structure of bacterial communities by sample type. Principal coordinate analysis revealed two distinct clusters for blood and feces, whereas vaginal and uterine bacterial communities were more scattered, indicating greater variability. Cluster analysis indicated that uterine bacterial communities were more similar to fecal bacterial communities than vaginal and blood bacterial communities. Nonetheless, there were core genera shared by all blood, feces, vaginal, and uterine samples. Major uterine pathogens such as Bacteroides, Porphyromonas, and Fusobacterium were part of the core genera in blood, feces, and vagina. Other uterine pathogens such as Prevotella and Helcococcus were not part of the core genera in vaginal samples. In addition, uterine pathogens showed a strong and significant interaction with each other in the network of blood microbiota, but not in feces or vagina. These microbial interactions in blood may be an important component of disease etiology. The copy number of total bacteria in blood and uterus was correlated; the same did not occur in other sites. Bacteroides heparinolyticus was more abundant in the uterus on day 0, and both B. heparinolyticus and Fusobacterium necrophorum were more abundant in the uterus than in the blood and feces on day 2. This indicates that B. heparinolyticus has a tropism for the uterus, whereas both pathogens thrive in the uterine environment early postpartum., Conclusions: Blood harbored a unique microbiome that contained the main uterine pathogens such as Bacteroides, Porphyromonas, and Fusobacterium. The presence of these pathogens in blood shortly after calving shows the feasibility of hematogenous spread of uterine pathogens in cows.

The microbiota of whole crop corn silage and feces of silage-fed dairy cows were examined. A total of 18 dairy cow feces were collected from six farms in Japan and China, and high-throughput Illumina sequencing of the V4 hypervariable region of 16S rRNA genes was performed. Lactobacillaceae were dominant in all silages, followed by Acetobacteraceae, Bacillaceae, and Enterobacteriaceae. In feces, the predominant families were Ruminococcaceae, Bacteroidaceae, Clostridiaceae, Lachnospiraceae, Rikenellaceae, and Paraprevotellaceae. Therefore, Lactobacillaceae of corn silage appeared to be eliminated in the gastrointestinal tract. Although fecal microbiota composition was similar in most samples, relative abundances of several families, such as Ruminococcaceae, Christensenellaceae, Turicibacteraceae, and Succinivibrionaceae, varied between farms and countries. In addition to the geographical location, differences in feeding management between total mixed ration feeding and separate feeding appeared to be involved in the variations. Moreover, a cow-to-cow variation for concentrate-associated families was demonstrated at the same farm; two cows showed high abundance of Succinivibrionaceae and Prevotellaceae, whereas another had a high abundance of Porphyromonadaceae. There was a negative correlation between forage-associated Ruminococcaceae and concentrate-associated Succinivibrionaceae and Prevotellaceae in 18 feces samples. Succinivibrionaceae, Prevotellaceae, p-2534-18B5, and Spirochaetaceae were regarded as highly variable taxa in this study. These findings help to improve our understanding of variation and similarity of the fecal microbiota of dairy cows with regard to individuals, farms, and countries. Microbiota of naturally fermented corn silage had no influence on the fecal microbiota of dairy cows.

The objective of this study was to determine feedlot- and pen-level fecal prevalence of seven enterohemorrhagic Escherichia coli (EHEC) belonging to serogroups (O26, O45, O103, O111, O121, O145, and O157, or EHEC-7) in feces of feedlot cattle in two feeding areas in the United States. Cattle pens from four commercial feedlots in each of the two major U.S. beef cattle areas were sampled. Up to 16 pen-floor fecal samples were collected from each of 4-6 pens per feedlot, monthly, for a total of three visits per feedlot, from June to August, 2014. Culture procedures including fecal enrichment in E. coli broth, immunomagnetic separation, and plating on selective media, followed by confirmation through polymerase chain reaction (PCR) testing, were conducted. Generalized linear mixed models were fitted to estimate feedlot-, pen-, and sample-level fecal prevalence of EHEC-7 and to evaluate associations between potential demographic and management risk factors with feedlot and within-pen prevalence of EHEC-7. All study feedlots and 31.0% of the study pens had at least one non-O157 EHEC-positive fecal sample, whereas 62.4% of pens tested positive for EHEC O157; sample-level prevalence estimates ranged from 0.0% for EHEC O121 to 18.7% for EHEC O157. Within-pen prevalence of EHEC O157 varied significantly by sampling month; similarly within-pen prevalence of non-O157 EHEC varied significantly by month and by the sex composition of the pen (heifer, steer, or mixed). Feedlot management factors, however, were not significantly associated with fecal prevalence of EHEC-7. Intraclass correlation coefficients for EHEC-7 models indicated that most of the variation occurred between pens, rather than within pens, or between feedlots. Hence, the potential combination of preharvest interventions and pen-level management strategies may have positive food safety impacts downstream along the beef chain.

The aim of this study was to evaluate the ability of bacterial cultures isolated from cattle, poultry or pig faeces and manure to produce rhamnolipids, as well as to investigate the influence of interspecies communication on possible quantitative differences in the production of rhamnolipid congeners. Initial screening methods (oil spreading, drop collapse, haemolytic activity and emulsification activity) showed that approximately 36% of the 51 isolated cultures exhibited the ability to produce biosurfactants. Subsequent studies using a selected culturable mixed culture (which included Enterococcus faecalis, Pseudomonas aeruginosa and Escherichia coli) revealed that only P. aeruginosa was able to produce this biosurfactant. HPLC-MS analysis showed that the surface active compounds were rhamnolipids. Further comparative studies confirmed that the total yield of rhamnolipids was notably higher in the bioreactor inoculated with the selected mixed culture (940.58±1.10mg/L) compared to the bioreactor inoculated with the axenic strain of P. aeruginosa (108.47±0.41mg/L). Twelve rhamnolipid congeners were identified during cultivation of the selected mixed culture, whereas six congeners were detected during cultivation of the sole axenic strain of P. aeruginosa. Furthermore, increased production of rhamnolipids was observed when the concentration of autoinducer molecules (AI-2) responsible for interspecies signaling increased, suggesting the influence of quorum-sensing communication on biosynthesis efficiency. This observation may be of importance for large-scale production of this biosurfactant, as it opens new possible solutions based on the use of mixed cultures or external addition of stimulating autoinducers., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Enterococcus is ubiquitous in nature and is a commensal of both the bovine and human gastrointestinal (GI) tract. It is also associated with clinical infections in humans. Subtherapeutic administration of antibiotics to cattle selects for antibiotic resistant enterococci in the bovine GI tract. Antibiotic resistance genes (ARGs) may be present in enterococci following antibiotic use in cattle. If located on mobile genetic elements (MGEs) their dissemination between Enterococcus species and to pathogenic bacteria may be promoted, reducing the efficacy of antibiotics., Results: We present a comparative genomic analysis of twenty-one Enterococcus spp. isolated from bovine feces including Enterococcus hirae (n = 10), Enterococcus faecium (n = 3), Enterococcus villorum (n = 2), Enterococcus casseliflavus (n = 2), Enterococcus faecalis (n = 1), Enterococcus durans (n = 1), Enterococcus gallinarum (n = 1) and Enterococcus thailandicus (n = 1). The analysis revealed E. faecium and E. faecalis from bovine feces share features with human clinical isolates, including virulence factors. The Tn917 transposon conferring macrolide-lincosamide-streptogramin B resistance was identified in both E. faecium and E. hirae, suggesting dissemination of ARGs on MGEs may occur in the bovine GI tract. An E. faecium isolate was also identified with two integrative conjugative elements (ICEs) belonging to the Tn916 family of ICE, Tn916 and Tn5801, both conferring tetracycline resistance., Conclusions: This study confirms the presence of enterococci in the bovine GI tract possessing ARGs on MGEs, but the predominant species in cattle, E. hirae is not commonly associated with infections in humans. Analysis using additional complete genomes of E. faecium from the NCBI database demonstrated differential clustering of commensal and clinical isolates, suggesting that these strains may be specifically adapted to their respective environments.

The phylum Neocallimastigomycota contains eight genera (about 20 species) of strictly anaerobic fungi. The evolutionary relationships of these genera are uncertain due to insufficient sequence data to infer their phylogenies. Based on morphology and molecular phylogeny, thirteen isolates obtained from yak faeces and rumen digesta in China were assigned to Neocallimastix frontalis (nine isolates), Orpinomyces joyonii (two isolates) and Caecomyces sp. (two isolates), respectively. The phylogenetic relationships of the eight genera were evaluated using complete ITS and partial LSU sequences, compared to the ITS1 region which has been widely used in this phylum in the past. Five monophyletic lineages corresponding to six of the eight genera were statistically supported. Isolates of Caecomyces and Cyllamyces were present in a single lineage and could not be separated properly. Members of Neocallimastigomycota with uniflagellate zoospores represented by Piromyces were polyphyletic. The Piromyces-like genus Oontomyces was consistently closely related to the traditional Anaeromyces, and separated the latter genus into two clades. The phylogenetic position of the Piromyces-like genus Buwchfawromyces remained unresolved. Orpinomyces and Neocallimastix, sharing polyflagellate zoospores, were supported as sister genera in the LSU phylogeny. Apparently ITS, specifically ITS1 alone, is not a good marker to resolve the generic affinities of the studied fungi. The LSU sequences are easier to align and appear to work well to resolve generic relationships. This study provides a comparative phylogenetic revision of Neocallimastigomycota isolates known from culture and sequence data.

Clinical outcomes of Shiga toxin (stx)-producing Escherichia coli infection are largely determined by virulence gene subtypes. This study used a polymerase chain reaction (PCR)-pyrosequencing assay to analyze single-nucleotide polymorphisms for subtyping three major virulence genes (stx 1 , stx 2 , eae) of pathogenic E. coli (O157, O26, O111, and O103) isolated from cattle over a 2-year interval (n = 465) and human clinical cases (n = 42) in western Canada. Most bovine isolates were PCR positive for at least one target virulence gene (367/465), whereas 100% of human isolates harbored eae in combination with at least one stx gene. Four Shiga toxin (1a, 2a, 2c, and 2e) and four eae (λ/γ1-eae, ɛ-eae, θ/γ2-eae, and β-eae) subtypes were identified in over 25 distinct virulence genotypes. Among cattle isolates, every serogroup, but O103, presented a dominant genotype (O157: stx 1a +stx 2a +λ/γ1-eae, O26: β-eae alone, and O111: stx 1a +θ/γ2-eae). Similar patterns were found in human isolates, although it was not possible to establish a clear genotypic association between the two sources. Many O157 and non-O157 cattle isolates lacked stx genes; the absence was greater in non-O157 (75/258) and O157:non-H7 (19/40) than in O157:H7 strains (1/164). In addition, there was a greater diversity of virulence genotypes of E. coli isolated from cattle than those of human diseases, which could be due to sample characteristics (e.g., source and clinical condition). However, the majority of cattle strains had virulence profiles identical to those of clinical cases. Consequently, determining the presence of certain stx (stx 1a and stx 2a ) and eae (λ/γ1-eae) subtypes known to cause human disease would be a valuable tool for risk assessment and prediction of disease outcome along the farm-to-fork continuum.

Storm water runoff is a major source of pollution, and understanding the components of storm water discharge is essential to remediation efforts and proper assessment of risks to human and ecosystem health. In this study, culturable Escherichia coli and ampicillin-resistant E. coli levels were quantified and microbial source tracking (MST) markers (including markers for general Bacteroidales spp., human, ruminant/cow, gull, and dog) were detected in storm water outfalls and sites along the Humber River in Toronto, Ontario, Canada, and enumerated via endpoint PCR and quantitative PCR (qPCR). Additionally, chemical source tracking (CST) markers specific for human wastewater (caffeine, carbamazepine, codeine, cotinine, acetaminophen, and acesulfame) were quantified. Human and gull fecal sources were detected at all sites, although concentrations of the human fecal marker were higher, particularly in outfalls (mean outfall concentrations of 4.22 log 10 copies, expressed as copy numbers [CN]/100 milliliters for human and 0.46 log 10 CN/100 milliliters for gull). Higher concentrations of caffeine, acetaminophen, acesulfame, E. coli, and the human fecal marker were indicative of greater raw sewage contamination at several sites (maximum concentrations of 34,800 ng/liter, 5,120 ng/liter, 9,720 ng/liter, 5.26 log 10 CFU/100 ml, and 7.65 log 10 CN/100 ml, respectively). These results indicate pervasive sewage contamination at storm water outfalls and throughout the Humber River, with multiple lines of evidence identifying Black Creek and two storm water outfalls with prominent sewage cross-connection problems requiring remediation. Limited data are available on specific sources of pollution in storm water, though our results indicate the value of using both MST and CST methodologies to more reliably assess sewage contamination in impacted watersheds., Importance: Storm water runoff is one of the most prominent non-point sources of biological and chemical contaminants which can potentially degrade water quality and pose risks to human and ecosystem health. Therefore, identifying fecal contamination in storm water runoff and outfalls is essential for remediation efforts to reduce risks to public health. This study employed multiple methods of identifying levels and sources of fecal contamination in both river and storm water outfall sites, evaluating the efficacy of using culture-based enumeration of E. coli, molecular methods of determining the source(s) of contamination, and CST markers as indicators of fecal contamination. The results identified pervasive human sewage contamination in storm water outfalls and throughout an urban watershed and highlight the utility of using both MST and CST to identify raw sewage contamination., (© Crown copyright 2016.)

We examined the effects of encapsulated lactic acid bacteria administrated orally to lactating cattle on the intestinal flora. A dose of 3 X 10¹¹ colony forming unit (cfu) of freeze-dried Lactobacillus coryniformis subsp. torquens (JCM1099) encapsulated in an enteric capsule capable of bypassing the rumen was administered for seven days. DNA was extracted from feces 0 and 24 hr after daily administration. Metagenomic analysis showed an increasing trend of the alpha diversity, an index of the species diversity. Furthermore, principal component analysis of intestinal flora revealed that cattle could be differentiated by JCM1099 capsule and suspension administration via principal components 1, 2, and 3. We conclude that administration of encapsulated JCM1099 can alter the intestinal bacterial flora of cattle.

Pooled feces collected over two years from 1749 transport trailers hauling western-Canadian slaughter cattle were analysed by PCR for detection of Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157. Sequential immunomagnetic separation was then used to collect bacterial isolates (n = 1035) from feces positive for target serogroups. Isolated bacteria were tested by PCR to confirm serogroup and the presence of eae, ehxA, stx1, and stx2 virulence genes. Based on PCR screening, serogroup prevalence in feces ranged from 7.0% (O145) to 94.4% (O103) with at least 3 serogroups present in 79.5% of samples. Origin of cattle affected serogroup PCR prevalence and O157 was most prevalent in feces from south-west Alberta (P < 0.001). All serogroups demonstrated seasonal variations in PCR prevalence, with O26, O45, O103, O121, and O157 least prevalent (P < 0.001) in cooler winter months, while uncommon serogroups O111 and O145 increased in prevalence during winter (P < 0.001). However, isolates collected during winter were predominantly from serogroups O103 and O45. No seasonal variation was noted in proportion of isolates which were Shiga toxin containing E. coli (STEC; P = 0.18) or positive for Shiga toxin and eae (enterohemorrhagic E. coli; EHEC; P = 0.29). Isolates of serogroups O111, O145, and O157 were more frequently EHEC than were others, although 37.6-54.3% of isolates from other serogroups were also EHEC. Shiga-toxin genes present also varied by geographic origin of cattle (P < 0.05) in all serogroups except O157. As cattle within feedlots are sourced from multiple regions, locational differences in serogroup prevalence and virulence genes imply existence of selection pressures for E. coli and their virulence in western-Canadian cattle. Factors which reduce carriage or expression of virulence genes, particularly in non-O157 serogroups, should be investigated.

The aim of this study was to determine possible differences in the faecal microbiota of dairy cows infected with Mycobacterium avium subsp. paratuberculosis (Johne's disease) in comparison with noninfected cows from the same herds. Faecal samples from cows in 4 herds were tested for M. avium subsp. paratuberculosis by real-time PCR, and faecal bacterial populations were analysed by 454 pyrosequencing of the 16S rRNA gene. The most notable differences between shedding and nonshedding cows were an increase in the genus Psychrobacter and a decrease in the genera Oscillospira, Ruminococcus, and Bifidobacterium in cows infected with M. avium subsp. paratuberculosis. The present study is the first to report the faecal microbial composition in dairy cows infected with M. avium subsp. paratuberculosis.

Escherichia coli O104:H4, an hybrid pathotype of Shiga toxigenic and enteroaggregative E. coli, involved in a major foodborne outbreak in Germany in 2011, has not been detected in cattle feces. Serogroup O104 with H type other than H4 has been reported to cause human illnesses, but their prevalence and characteristics in cattle have not been reported. Our objectives were to determine the prevalence of E. coli O104 in feces of feedlot cattle, by culture and PCR detection methods, and characterize the isolated strains. Rectal fecal samples from a total of 757 cattle originating from 29 feedlots were collected at a Midwest commercial slaughter plant. Fecal samples, enriched in E. coli broth, were subjected to culture and PCR methods of detection. The culture method involved immunomagnetic separation with O104-specific beads and plating on a selective chromogenic medium, followed by serogroup confirmation of pooled colonies by PCR. If pooled colonies were positive for the wzxO104 gene, then colonies were tested individually to identify wzxO104-positive serogroup and associated genes of the hybrid strains. Extracted DNA from feces were also tested by a multiplex PCR to detect wzxO104-positive serogroup and associated major genes of the O104 hybrid pathotype. Because wzxO104 has been shown to be present in E. coli O8/O9/O9a, wzxO104-positive isolates and extracted DNA from fecal samples were also tested by a PCR targeting wbdDO8/O9/O9a, a gene specific for E. coli O8/O9/O9a serogroups. Model-adjusted prevalence estimates of E. coli O104 (positive for wzxO104 and negative for wbdDO8/O9/O9a) at the feedlot level were 5.7% and 21.2%, and at the sample level were 0.5% and 25.9% by culture and PCR, respectively. The McNemar's test indicated that there was a significant difference (P < 0.01) between the proportions of samples that tested positive for wzxO104 and samples that were positive for wzxO104, but negative for wbdDO8/O9/O9a by PCR and culture methods. A total of 143 isolates, positive for the wzxO104, were obtained in pure culture from 146 positive fecal samples. Ninety-two of the 143 isolates (64.3%) also tested positive for the wbdDO8/O9/O9a, indicating that only 51 (35.7%) isolates truly belonged to the O104 serogroup (positive for wzxO104 and negative for wbdDO8/O9/O9a). All 51 isolates tested negative for eae, and 16 tested positive for stx1 gene of the subtype 1c. Thirteen of the 16 stx1-positive O104 isolates were from one feedlot. The predominant serotype was O104:H7. Pulsed-field gel electrophoresis analysis indicated that stx1-positive O104:H7 isolates had 62.4% homology to the German outbreak strain and 67.9% to 77.5% homology to human diarrheagenic O104:H7 strains. The 13 isolates obtained from the same feedlot were of the same PFGE subtype with 100% Dice similarity. Although cattle do not harbor the O104:H4 pathotype, they do harbor and shed Shiga toxigenic O104 in the feces and the predominant serotype was O104:H7.

Antimicrobial resistant determinants (ARDs) can be transmitted from livestock systems through meat products or environmental effluents. The public health risk posed by these two routes is not well understood, particularly in non-pathogenic bacteria. We collected pooled samples from 8 groups of 1741 commercial cattle as they moved through the process of beef production from feedlot entry through slaughter. We recorded antimicrobial drug exposures and interrogated the resistome at points in production when management procedures could potentially influence ARD abundance and/or transmission. Over 300 unique ARDs were identified. Resistome diversity decreased while cattle were in the feedlot, indicating selective pressure. ARDs were not identified in beef products, suggesting that slaughter interventions may reduce the risk of transmission of ARDs to beef consumers. This report highlights the utility and limitations of metagenomics for assessing public health risks regarding antimicrobial resistance, and demonstrates that environmental pathways may represent a greater risk than the food supply., Competing Interests: The authors declare that no competing interests exist.

This study aims to describe in detail the temporal dynamics of E. coli O157 shedding and risk factors for shedding in a grass-fed beef herd. During a 9-month period, 23 beef cows were sampled twice a week (58 sampling points) and E. coli O157 was enumerated from faecal samples. Isolates were screened by PCR for presence of rfbE, stx 1 and stx 2 . The prevalence per sampling day ranged from 0% to 57%. This study demonstrates that many members of the herd were concurrently shedding E. coli O157. Occurrence of rainfall (P < 0·01), feeding silage (P < 0·01) and lactating (P < 0·01) were found to be predictors of shedding. Moving cattle to a new paddock had a negative effect on shedding. This approach, based on short-interval sampling, confirms the known variability of shedding within a herd and highlights that high shedding events are rare.

The United States Department of Agriculture Food Safety and Inspection Service has declared seven Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, O145, and O157) as adulterants in raw, nonintact beef products. The objective of this study was to determine the prevalence of these seven serogroups and the associated virulence genes (Shiga toxin [stx1, stx2], and intimin [eae]) in cattle feces during summer (June-August 2013) and winter (January-March 2014) months. Twenty-four pen floor fecal samples were collected from each of 24 cattle pens, in both summer and winter months, at a commercial feedlot in the United States. Samples were subjected to culture-based detection methods that included enrichment, serogroup-specific immunomagnetic separation and plating on selective media, followed by a multiplex polymerase chain reaction for serogroup confirmation and virulence gene detection. A sample was considered STEC positive if a recovered isolate harbored an O gene, stx1, and/or stx2, and eae genes. All O serogroups of interest were detected in summer months, and model-adjusted prevalence estimates are as follows: O26 (17.8%), O45 (14.6%), O103 (59.9%), O111 (0.2%), O121 (2.0%), O145 (2.7%), and O157 (41.6%); however, most non-O157 isolates did not harbor virulence genes. The cumulative model-adjusted sample-level prevalence estimates of STEC O26, O103, O145, and O157 during summer (n=576) were 1.0, 1.6, 0.8, and 41.4%, respectively; STEC O45, O111, and O121 were not detected during summer months. In winter, serogroups O26 (0.9%), O45 (1.5%), O103 (40.2%), and O121 (0.2%) were isolated; however, no virulence genes were detected in isolates from cattle feces collected during winter (n=576). Statistically significant seasonal differences in prevalence were identified for STEC O103 and O157 (p<0.05), but data on other STEC were sparse. The results of this study indicate that although non-O157 serogroups were present, non-O157 STEC were rarely detected in feces from the feedlot cattle populations tested in summer and winter months.

A 3-year (2009 to 2011) grazing study was conducted to assess the effects of purple prairie clover (PPC; Dalea purpurea Vent) on fecal shedding of total Escherichia coli in cattle. Three pasture types were used in the experiment: bromegrass (Check), mixed cool season grasses with PPC (Simple), and mixed cool and warm grasses with PPC (Complex). Pastures were rotationally grazed during a summer and fall grazing period. PPC was grazed in summer at the vegetative or early flower stage and at the flower or early seed stage during the fall. Fecal samples were collected for enumeration of E. coli and chemical analyses. Forage samples were collected throughout grazing for analysis. Condensed tannins (CT) were only detected in Simple and Complex pastures that contained PPC, with higher concentrations found in the fall than in the summer. Fecal counts of E. coli in cattle grazing Simple and Complex pastures linearly decreased (P < 0.05) over summer to fall in all 3 years, an outcome not observed in cattle grazing the Check pasture. Across the three grazing seasons, fecal E. coli was lower (P < 0.05) in cattle grazing Simple and Complex pastures than in those grazing the Check pasture during the fall. During the fall, feces collected from cattle grazing the Check pasture had higher (P < 0.05) values for pH, N, NH3-N, total volatile fatty acids, and branched-chain volatile fatty acids, but a lower (P < 0.05) acetate:propionate ratio than feces collected from cattle grazing Simple or Complex pastures. In a second experiment, two strains of E. coli were cultured in M9 medium containing 25 to 200 μg/ml of PPC CT. Growth of E. coli was linearly (P < 0.01) reduced by increasing levels of PPC CT. Scanning electron micrographs showed electron-dense filamentous material associated with the outer membrane of E. coli cells exposed to CT. Incorporation of PPC into forage reduced the fecal shedding of E. coli from grazing cattle, likely due to the anti-E. coli properties of PPC CT.