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Aims: To isolate and identify cellulolytic bacteria from yak rumen and further evaluate the effects of the isolates on the silage quality, structural carbohydrates degradation and cellulose convertibility of Napier grass silage., Methods and Results: Two out of 218 strains were selected based on their most extensive transparent zone and the highest filter paper disintegration rate. The two isolates (JFL12 and JF85) could grow normally at 15-55°C, pH 3·0-7·0 and NaCl (3·0, 6·5%), and were identified as Enterococcus casseliflavus and Enterococcus faecalis by 16S rDNA sequence analysis, respectively. Napier grass was ensiled with no additive control (C), Lactobacillus plantarum (Lp), JFL12, JF85, JFL12 + Lp and JF85 + Lp for 3, 5, 7, 14, 30 and 60 days. All inoculated silages had higher lactic acid content, lower pH, ammonia nitrogen (NH 3 -N) and lignocellulose contents than the control silage. Silages treated with JFL12 + Lp and JF85 + Lp had the lowest pH and NH 3 -N contents, the highest lactic acid content and lignocellulose degradation among all treatments. The isolates with or without Lp significantly (P < 0·01) increased water soluble carbohydrates (WSC), glucose, fructose and sucrose contents as compared with the control silage. Silages treated with JFL12 + Lp and JF85 + Lp had higher glucose yield and cellulose convertibility than the other silages., Conclusions: Therefore, the application of isolates (JFL12 and JF85) with Lp had synergistic effects on accelerating the degradation of structural carbohydrates and improving the silage quality., Significance and Impact of the Study: Napier grass presents difficulty to ensiling due to its low WSC and high structural carbohydrates contents. The screened cellulolytic bacteria could be a candidate strain in improving fermentation quality and structural carbohydrates degradability of ensiled forages., (© 2020 The Society for Applied Microbiology.)

Aims: The effect of increasing dietary cation-anion difference (DCAD) on rumen fermentation and ruminal microbial community in dairy cows under heat stress (HS) conditions were evaluated., Methods and Results: This study was performed as a two-period cross-over design during the summer season, with eight lactating dairy cows randomly distributed to either a control DCAD diet (CON: 33·5 mEq/100 g DM) or high DCAD diet (HDCAD: 50·8 mEq/100 g DM). Throughout the present study, the temperature and humidity index (THI; 80·2 ± 4·29) was generally elevated above the threshold (THI = 72) that is reported to cause HS in lactating dairy cows. Rumen liquid samples were collected on 15 and 21 d during each 21 d-period. The absolute concentration of ruminal total volatile fatty acid (TVFA) in HDCAD treatment was significantly (P < 0·05) higher than those in the control, whilst the ruminal pH, NH 3 -N, and VFA molar percentages were unaffected through increasing DCAD. Furthermore, the copy numbers of the cellulolytic bacteria Ruminococcus albus and Ruminococcus flavefaciens in rumen fluid significantly (P < 0·05) rose along with the increment of DCAD. Although the Alpha diversity indexes and the bacterial microbiota structure were unaffected, increasing DCAD significantly (P < 0·05) enriched the phylum Fibrobacteres and genus Fibrobacter in the microflora of rumen fluid, whilst the genera Flexilinea and Dubosiella were the most differentially abundant taxa in the control., Conclusions: Increasing DCAD under HS conditions resulted in a greater concentration of total VFA without affecting rumen bacteria diversity or structure, although the enrichment of some cellulolytic/hemicellulolytic bacteria was observed., Significance and Impact of the Study: The present study provides information on the modulation of rumen fermentation and microbial community through the increment of DCAD in Holstein dairy cows under HS conditions., (© 2020 The Society for Applied Microbiology.)

Manipulation of the rumen microorganisms during early life has emerged as a promising strategy for persistent improvement of nutrient utilisation and lowering of enteric methanogenesis. However, limited understanding of the dynamics of rumen microbial colonisation has prevented the identification of the optimum timeframe for such interventions. The present study used DNA amplicon sequencing of the 16S rRNA gene to assess bacterial and archaeal dynamics in the rumen digesta of beef calves raised on two farms from birth through to post-weaning. The colonisation patterns of both communities were influenced by age (P < 0.05) and farm of origin (P < 0.05). The bacterial community exhibited an age-wise progression during the first month of life which appeared to be partly related to diet, and settled by day 21, indicating that this may mark the boundary of a timeframe for intervention. The archaeal community appeared less sensitive to age/diet than bacteria in the first month of life but was more sensitive to farm environment. These data show that ruminal microbial composition during early life is driven by calf age, diet and local environment, and provide important fundamental information concerning the ontogeny of the rumen microbiota from birth., (© FEMS 2020.)

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.)

The bovine gastrointestinal (GI) tract microbiota has important influences on animal health and production. Presently, a large number of studies have used high-throughput sequencing of the archaeal and bacteria 16S rRNA gene to characterize these microbiota under various experimental parameters. By aggregating publically available archaeal and bacterial 16S rRNA gene datasets from 52 studies we were able to determine taxa that are common to nearly all microbiota samples from the bovine GI tract as well as taxa that are strongly linked to either the rumen or feces. The methanogenic genera Methanobrevibacter and Methanosphaera were identified in nearly all fecal and rumen samples (> 99.1%), as were the bacterial genera Prevotella and Ruminococcus (≥ 92.9%). Bacterial genera such as Alistipes, Bacteroides, Clostridium, Faecalibacterium and Escherichia/Shigella were associated with feces and Fibrobacter, Prevotella, Ruminococcus and Succiniclasticum with the rumen. As expected, individual study strongly affected the bacterial community structure, however, fecal and rumen samples did appear separated from each other. This meta-analysis provides the first comparison of high-throughput sequencing 16S rRNA gene datasets generated from the bovine GI tract by multiple studies and may serve as a foundation for improving future microbial community research with cattle., (© Her Majesty the Queen in Right of Canada 2019. Reproduced with the permission of the Minister of Agriculture and Agri-Food.)

Objectives: To investigate the prevalence and molecular characteristics of ESBL-producing Escherichia coli (ESBL-EC) in faecal samples from dairy cows in China., Methods: In total, 651 faecal samples were collected from cows distributed among the 10 provinces of China. Potential ESBL-EC isolates were cultured on selective medium. The clonal relatedness of the ESBL-EC isolates was assessed using MLST. WGS was conducted on 3 mcr-positive isolates and 14 additional randomly selected ESBL-EC isolates. Southern blot, S1-PFGE and conjugation were performed for mcr-1-carrying isolates. The genetic environment of the pMCR-JLF4 plasmid was also analysed., Results: In total, 290 unique ESBL-EC isolates were detected from 284 cows (43.6%). Alleles of CTX-M were observed in 94.1% (273/290) of all isolates. The most prevalent genotypes observed in this study were blaCTX-M-14, blaCTX-M-15, blaCTX-M-17 and blaCTX-M-55. Differentiation of 79 STs with a polyclonal structure was accomplished using MLST. Clonal complex 10 was the most prevalent major complex detected here. Furthermore, the mcr-1 gene was detected in three isolates. The complete sequence of the mcr-1-containing pMCR-JLF4 was determined. The plasmid was 66.7 kb in length, with a genetic structure of nikA-nikB-mcr-1-pap2. Conjugation analysis confirmed that the mcr-1 gene in pMCR-JLF4 was transferable without the assistance of the ISApl1 gene., Conclusions: The data presented here suggest high prevalence of ESBL-EC in Chinese cow farms. Furthermore, it was clearly demonstrated that commensal E. coli strains can be reservoirs of blaCTX-M genes, potentially contributing to the dissemination and transfer of the mcr-1 gene to pathogenic bacteria among cows.

This study evaluated the effect of feeding non-heated and heated colostrum on the mucosa- and digesta-associated microbiota in the colon of dairy calves during the first 12 h of life. Thirty-two neonatal Holstein male calves were fed: no colostrum (NC, n = 8), non-heated colostrum (FC, n = 12) and heated colostrum (HC (60 °C, 60 min), n = 12) immediately after birth. The abundances of mucosa- and digesta-associated total bacteria were higher in the colon of FC fed calves compared to those fed no colostrum (NC) at 12 h of life. Compare to NC calves, a higher proportion of mucosa- and digesta-associated Clostridium cluster XIVa and Bifidobacterium, and a lower abundance of mucosa and digesta-associated E. coli were detected in the colon of FC and HC fed calves, as well as a tentatively lower relative abundance of Escherichia-Shigella genus in colon mucosa of HC fed calves. In addition, HC calves had lower abundances of E. coli and higher abundances of Bifidobacterium in mucosa-associated microbiota than FC fed calves. Our results suggest that feeding non-heated colostrum immediately after birth benefit neonatal calves with increased Bifidobacterium and decreased opportunistic pathogenic E. coli and Escherichia-Shigella genus in the colon, and feeding heated colostrum can fortify such effects.

Objectives: To sequence the genomes and determine the genetic mechanisms for linezolid resistance identified in three strains of Enterococcus isolated from cattle and swine caecal contents as part of the US National Antimicrobial Resistance Monitoring System (NARMS) surveillance programme., Methods: Broth microdilution was used for in vitro antimicrobial susceptibility testing to assess linezolid resistance. Resistance mechanisms and plasmid types were identified from data generated by WGS on Illumina® and PacBio® platforms. Conjugation experiments were performed to determine whether identified mechanisms were transmissible., Results: Linezolid resistance plasmids containing optrA were identified in two Enterococcus faecalis isolates and one Enterococcus faecium. The E. faecium isolate also carried the linezolid resistance gene cfr on the same plasmid as optrA. The linezolid resistance plasmids had various combinations of additional resistance genes conferring resistance to phenicols (fexA), aminoglycosides [spc and aph(3')-III] and macrolides [erm(A) and erm(B)]. One of the plasmids was confirmed to be transmissible by conjugation, resulting in linezolid resistance in the transconjugant., Conclusions: To the best of our knowledge, this is the first identification of linezolid resistance in the USA in bacteria isolated from food animals. The oxazolidinone class of antibiotics is not used in food animals in the USA, but the genes responsible for resistance were identified on plasmids with other resistance markers, indicating that there may be co-selection for these plasmids due to the use of different antimicrobials. The transmissibility of one of the plasmids demonstrated the potential for linezolid resistance to spread horizontally. Additional surveillance is necessary to determine whether similar plasmids are present in human strains of Enterococcus.

Objectives: To elucidate the phylogenetic relationships among ST9-MRSA-XII isolates from different sources and their genetic features in colonization of different hosts., Methods: We obtained whole-genome sequences of two ST9-MRSA-XII isolates from nasal swabs associated with live pigs in China, and compared them with 135 previously sequenced genomes of 78 human-associated, 39 bovine and 18 porcine Staphylococcus aureus consisting of 11 MRSA of SCCmecXII, 62 MRSA of other SCCmec types and 62 MSSA. The distribution of diverse mobile genetic elements (MGEs), resistance genes and virulence determinants was investigated in relation to isolate phylogeny. Comparisons of SNPs and small insertion/deletions (indels) were conducted to examine genome-level variation between porcine and bovine ST9-MRSA-XII., Results: Phylogenetic analysis revealed that both of our porcine ST9-MRSA-XII isolates clustered with porcine, bovine and human-associated ST9-MRSA-XII. All of these isolates possessed a novel type V pathogenicity island, νSaα, carrying the von Willebrand-binding protein gene vwb, the immune evasion complex gene scn, the aminoglycoside resistance gene aadE, staphylococcal superantigen-like genes (ssl1-ssl11) and lpl tandem genes. Compared with bovine ST9-MRSA-XII BA01611, our porcine isolates contain non-synonymous nucleotide substitutions in genes encoding adhesins and an indel located in a phosphonate ABC transporter pseudogene., Conclusions: The data suggest transmission of ST9-MRSA-XII among swine, cattle and humans. The extraordinary success of the ST9-MRSA-XII group in colonization of various hosts is likely due to acquisition of many MGEs harbouring functional antimicrobial resistance and virulence genes. Transmission of ST9-MRSA-XII between porcine and bovine hosts was accompanied by changes in binding profile and function in genes involved in metabolism.

Dairy farming involves frequent contact among animals, workers and farm environments. To explore the Staphylococcus spp. diversity that occurs on dairy farms, a pilot study sampled dairy workers, cows and the farm environments from five farms, two organic and three conventional farms, in Washington State. Samples were taken from the nares and hands of consenting workers (n = 24), udders and nares of selected cows (n = 25) and representative environmental surfaces (n = 96) from each farm. To increase diversity of the Staphylococcus spp. characterized, five distinct colonies were selected from each sample for identification with 16S analysis. A total of 198 staphylococci were characterized representing 19 different Staphylococcus spp. The diversity of species ranged from 9-15 Staphylococcus spp./farm with no difference between conventional and organic farms. S. haemolyticus [n = 60 isolates] was the most common species and was isolated from all farms and from cows, humans and environmental samples. Whole genome sequencing of selected S. haemolyticus found no genetically related isolates among human, animal and environmental samples within the same farm. S. epidermidis, S. saprophyticus, S. sciuri and S. xylosus were also found in ≥1 farms from human, animal and environmental samples.

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.)

This study aimed to characterize mucosa- and digesta-associated microbiota in the hindgut (cecum, colon and rectum) of newborn (NB, n = 6), day 7 (n = 6), day 21 (n = 6) and day 42 (n = 6) Holstein bull calves using amplicon sequencing. The hindgut microbiota was diverse at birth, and mucosa-attached microbial community had higher individual variation than that of digesta-associated community. In total, 16 phyla were identified with Firmicutes, Bacteroidetes and Proteobacteria being the dominant microbial taxa in the hindgut. Quantitative real-time PCR analysis showed a significant age effect on the proportion of mucosa-attached Escherichia coli, Bifidobacterium, Clostridium cluster XIVa and Faecalibacterium prausnitzii. Especially, high abundance of mucosa-associated Escherichia was detected during the first week of life, suggesting higher chance of the pathogenic infection during this stage. The relative abundances of predicted microbial genes involved in amino acid metabolism, carbohydrate metabolism and energy metabolism were enriched, indicating the importance of hindgut microbiota in fermentation during the pre-weaned period. Moreover, the significant correlation between short-chain fatty acid concentration and mucosa-attached carbohydrate utilizing (Coprococcus 1, Blautia, Lachnospiraceae NC2004 group, etc.) and health-related bacteria (Escherichia-Shigella and Salmonella) suggests the importance of hindgut microbiota in the fermentation and health of dairy calves during pre-weaned period.

Aims: The probiotic Bacillus amyloliquefaciens H57 increased weight gain, increased nitrogen retention and increased feed intake in ruminants when administered to the diet. This study aims to develop a better understanding of this probiotic effect by analysing changes in the rumen prokaryotic community., Methods and Results: Sequencing the 16S rRNA gene PCR amplicons of the rumen microbiome, revealed that ewes fed H57 had a significantly different rumen microbial community structure to Control sheep. In contrast, dairy calves showed no significant differences in rumen community structure between treatment groups. In both instances, H57 was below detection in the rumen community profile and was only present at low relative abundance as determined by qPCR., Conclusions: The altered rumen microbial community in sheep likely contributes to increased weight gain through more efficient digestion of plant material. As no change occurred in the rumen community of dairy calves it is suggested that increased weight gain may be due to changes in community function rather than structure. The low relative abundance of H57 as determined by qPCR, suggests that weight gain was not directly mediated by the probiotic, but rather by influencing animal behaviour (feed consumption) and/or altering the native rumen community structure or function., Significance and Impact of the Study: This study provides a novel look at the rumen prokaryotic community in both sheep and dairy calves when fed H57. These findings improve our understanding for the potential rumen community involvement in H57-enabled weight gain. The study reveals that the probiotic B. amyloliquefaciens H57 is capable of benefiting ruminants without colonizing the rumen, suggesting an indirect mechanism of action., (© 2018 The Society for Applied Microbiology.)

Aims: The effects of partial replacement of a barley-based concentrate with flaxseed-based products on the rumen bacterial population of lactating Holstein dairy cows were evaluated., Methods and Results: Treatments fed were CONT, a normal diet that included barley silage, alfalfa hay and a barley-based concentrate that contained no flaxseed or faba beans; FLAX, inclusion of a nonextruded flaxseed-based product containing 55·0% flaxseed, 37·8% field peas and 6·9% alfalfa; EXT, similar to FLAX, but the product was extruded and EXTT, similar to FLAX, but product was extruded and field peas were replaced by high-tannin faba beans. The rumen bacterial population was evaluated by utilizing 16S rRNA gene sequencing. Most abundant phyla, families and genera were unaffected. However, some taxa were affected; for example, unsaturated fatty acid content was negatively correlated with Clostridiaceae, and tannin content was negatively correlated with BS11 and Paraprevotellaceae., Conclusions: Predominant rumen bacterial taxa were not affected, but the abundance of some taxa found in lower proportions shifted, possibly due to sensitivity to unsaturated fatty acids or tannins., Significance and Impact of the Study: Flaxseed-based products were effective for partially replacing barley-based concentrate in rations of lactating dairy cows. No negative effects of these products were observed on the abundance of predominant rumen bacterial taxa, with only minor shifts in less abundant bacteria., (© 2017 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Aims: The effect of feeding dried distillers grains with solubles (DDGS) or reduced-fat DDGS (RFDG) on ruminal methanogenesis and the rumen bacterial community of dairy cattle was evaluated., Methods and Results: Treatments were CONT, a diet with no distillers grains; DG, inclusion of 20% DDGS; rfDG, inclusion of 20% RFDG; and MIX, inclusion of 10% DDGS and 10% RFDG. Methane emission was measured; rumen bacterial community was evaluated by sequencing the V4 region of the 16S rRNA gene. Total methane production remained unaffected. However, feeding distillers grains tended to reduce methanogenesis per unit of feed intake, decreased the abundance of the phylum Bacteroidetes and tended to increase Firmicutes. The abundance of Prevotellaceae positively correlated with feed intake; methane emission was positively correlated with the abundance of Prevotellaceae and was negatively correlated with the abundance of Succinivibrionaceae., Conclusions: DDGS or RFDG may reduce methanogenesis per unit of feed intake; shifts in the abundance of predominant ruminal bacterial families may influence methane formation, likely because of their role on hydrogen liberation and utilization pathways., Significance and Impact of the Study: Replacing corn and soybean meal with DDGS or RFDG in dairy rations may reduce the proportion of dietary energy wasted as methane, without detrimental effects on the overall bacterial population., (© 2017 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of The Society for Applied Microbiology.)

Twenty-six lactose non-fermenting, oxidase, urease and citrate-positive Gram-negative rods, isolated from broiler chickens, pigs and cattle at slaughter, were subjected to the matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and 16S rDNA sequencing for identification. Susceptibility to 14 antimicrobials was determined by the disc diffusion method. Ochrobactrum isolates resistant to third-generation cephalosporins were PCR-screened for the presence of the Ochrobactrum anthropi ampC gene (blaOCH). A 547-bp internal segment of blaOCH in the Ochrobactrum spp isolates was amplified with a newly designed primer set, and a phylogenetic reconstruction based on the complete amino acid sequence of blaOCH obtained from nine Ochrobactrum strains in our collection and 20 O. anthropi available in the GenBank was undertaken. All the Ochrobactrum isolates were resistant to the expanded-spectrum beta-lactams and streptomycin. None of the isolates was resistant to imipenem while 41.7% to 50.0% of them were resistant to fluoroquinolones. The blaOCH gene was detected in 16 (66.7%) and 20 (83.3%) of the 24 Ochrobactrum isolates (O. intermedium/O. tritici species), using primers designed for O. anthropi and the newly designed primer set, respectively. Six blaOCH variants grouped into two divergent clusters were identified. This is the first report of the complete nucleotide sequence of the blaOCH gene in non-antropi Ochrobactrum species., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: To identify and characterize a novel cfr variant that recently emerged and confers multidrug resistance in Campylobacter , a major foodborne pathogen., Methods: WGS was initially used to identify the cfr (C) gene in Campylobacter isolates and its function was further verified by cloning into an antibiotic-susceptible Campylobacter jejuni strain. Distribution of cfr (C) in various Campylobacter isolates was determined by PCR analysis. Genotyping of cfr (C)-positive strains was done by PFGE and MLST., Results: The cfr (C) gene is predicted to encode a protein that shares 55.1% and 54.9% identity with Cfr and Cfr(B), respectively. cfr (C) was located on a conjugative plasmid of ∼48 kb. Cloning of cfr (C) into C. jejuni NCTC 11168 and conjugative transfer of the cfr (C)-containing plasmid confirmed its role in conferring resistance to phenicols, lincosamides, pleuromutilins and oxazolidinones, and resulted in an 8-256-fold increase in their MICs in both C. jejuni and Campylobacter coli . The cfr (C) gene was detected in multiple C. coli (34 of 344; 10%) isolates derived from different cattle farms in different states, and molecular typing of the cfr (C)-positive C. coli isolates revealed its spread mainly via clonal expansion., Conclusions: These results identify cfr (C) as a new multidrug resistance mechanism in Campylobacter and suggest the potential transmission of this mechanism via the foodborne route, warranting enhanced efforts to monitor its spread in Campylobacter and other foodborne pathogens., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Campylobacter sputorum is a nonthermotolerant campylobacter that is primarily isolated from food animals such as cattle and sheep. C. sputorum is also infrequently associated with human illness. Based on catalase and urease activity, three biovars are currently recognized within C. sputorum: bv. sputorum (catalase negative, urease negative), bv. fecalis (catalase positive, urease negative), and bv. paraureolyticus (catalase negative, urease positive). A multi-locus sequence typing (MLST) method was recently constructed for C. sputorum. MLST typing of several cattle-associated C. sputorum isolates suggested that they are members of a divergent C. sputorum clade. Although catalase positive, and thus technically bv. fecalis, the taxonomic position of these strains could not be determined solely by MLST. To further characterize C. sputorum, the genomes of four strains, representing all three biovars and the divergent clade, were sequenced to completion. Here we present a comparative genomic analysis of the four C. sputorum genomes. This analysis indicates that the three biovars and the cattle-associated strains are highly related at the genome level with similarities in gene content. Furthermore, the four genomes are strongly syntenic with one or two minor inversions. However, substantial differences in gene content were observed among the three biovars. Finally, although the strain representing the cattle-associated isolates was shown to be C. sputorum, it is possible that this strain is a member of a novel C. sputorum subspecies; thus, these cattle-associated strains may form a second taxon within C. sputorum., (Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2017. This work is written by US Government employees and is in the public domain in the US.)

Aims: The objective was to determine the effect of biochanin A (BCA), an isoflavone produced by red clover (Trifolium pratense L.), on corn fermentation by rumen micro-organisms., Methods and Results: When bovine rumen bacterial cell suspensions (n = 3) were incubated (24 h, 39°C) with ground corn, amylolytic bacteria including group D Gram-positive cocci (GPC; Streptococcus bovis; enterococci) proliferated, cellulolytic bacteria were inhibited, lactate accumulated and pH declined. Addition of BCA (30 μg ml -1 ) inhibited lactate production, and pH decline. BCA had no effect on total amylolytics, but increased lactobacilli and decreased GPC. The initial rate and total starch disappearance was decreased by BCA addition. BCA with added Strep. bovis HC5 supernatant (containing bacteriocins) inhibited the amylolytic bacteria tested (Strep. bovis JB1; Strep. bovis HC5; Lactobacillus reuteri, Selenemonas ruminatium) to a greater extent than either addition alone. BCA increased cellulolytics and dry matter digestibility of hay with corn starch., Conclusions: These results indicate that BCA mitigates changes associated with corn fermentation by bovine rumen bacteria ex vivo., Significance and Impact of the Study: BCA could serve as an effective mitigation strategy for rumen acidosis. Future research is needed to evaluate the effect of BCA on mitigating rumen acidosis in vivo., (Published 2017. This article is a U.S. Government work and is in the public domain in the USA.)

Aim: The occurrence of virulence markers, serotypes and invasive ability were investigated in Shiga toxin-producing Escherichia coli (STEC) isolated from faecal samples of healthy dairy cattle at Rio de Janeiro State, Brazil., Methods and Results: From 1562 stx-positive faecal samples, 105 STEC strains were isolated by immuno-magnetic separation (IMS) or plating onto MacConkey agar (MC) followed by colony hybridisation. Fifty (47·6%) strains belonged to nine serotypes (O8:H19, O22:H8, O22:H16, O74:H42, O113:H21, O141:H21, O157:H7, O171:H2 and ONT:H21). The prevalent serotypes were O157:H7 (12·4%), O113:H21 (6·7%) and O8:H19 (5·7%). Virulence genes were identified by polymerase chain reaction (PCR). E-hlyA (77·1%) was the more prevalent virulence marker, followed by espP (64·8%), saa (39%), eae (24·8%) and astA (21·9%). All O157:H7 strains carried the γ (gamma) variant of the locus of enterocyte effacement (LEE) genes and the stx2c gene, while the stx1/stx2 genotype prevailed among the eae-negative strains. None of the eae-positive STEC produced the localized adherence (LA) phenotype in HEp-2 or Caco-2 cells. However, intimate attachment (judged by the fluorescent actin staining test) was detected in some eae-positive strains, both in HEp-2 (23·1%) and in Caco-2 cells (11·5%). Most strains (87·5%) showed 'peripheral association' (PA) adherence phenotype to undifferentiated Caco-2 cells. Twenty-five (92·6%) of 27 strains invaded Caco-2 cells. The highest average value of invasion (9·6%) was observed among the eae-negative bovine strains from serotypes described in human disease., Conclusion: Healthy dairy cattle is a reservoir of STEC carrying virulence genes and properties associated with human disease., Significance and Impact of the Study: Although reports of human disease associated with STEC are scarce in Brazil, the colonization of the animal reservoir by potentially pathogenic strains offers a significant risk to our population., (© 2016 The Society for Applied Microbiology.)

Aims: This study was performed in a well-established in vitro model to investigate whether the application of a glyphosate-containing herbicide might affect the bacterial communities and some biochemical parameters in a cow's rumen., Methods and Results: The test item was applied in two concentrations (high and low) for 5 days. In a second trial, fermentation vessels were inoculated with Clostridium sporogenes before the high dose was applied. Effluents were analysed by biochemical, microbiological and genetic methods. A marginal increase in short-chain fatty acid production and a reduction in NH3 -N were observed. There were minor and rather equivocal changes in the composition of ruminal bacteria but no indications of a shift towards a more frequent abundance of pathogenic Clostridia species. Clostridium sporogenes counts declined consistently., Conclusions: No adverse effects of the herbicide on ruminal metabolism or composition of the bacterial communities could be detected. In particular, there was no evidence of a suspected stimulation of Clostridia growth., Significance and Impact of the Study: Antibiotic activity of glyphosate resulting in microbial imbalances has been postulated. In this exploratory study, however, intraruminal application of concentrations reflecting potential exposure of dairy cows or beef cattle did not exhibit significant effects on bacterial communities in a complex in vitro system. The low number of replicates (n = 3/dose) may leave some uncertainty., (© 2016 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Rumen bacteria form a dynamic, complex, symbiotic relationship with their host, degrading forages to provide volatile fatty acids (VFA) and other substrates as energy to the animal. The objectives were to characterize rumen bacteria in three genetic lines of primiparous dairy cattle, Holstein (HO, n = 7), Jersey (JE, n = 8), and HO × JE crossbreeds (CB, n = 7) across a lactation [3, 93, 183 and 273 days in milk (DIM)] and correlate these factors with VFA, bacterial cell membrane fatty acids (FA), and animal production (i.e. milk yield). This study employed Illumina MiSeq (v. 3) to investigate rumen bacterial communities and gas-liquid chromatography/mass spectroscopy to identify bacterial membrane FA. Lactation stage had a prominent effect on rumen bacterial communities, whereas genetics had a lesser effect on rumen bacteria. The FA composition of bacterial cell membranes was affected by both lactation stage and genetics. Few correlations existed between VFA and bacterial communities; however, moderate correlations occurred between milk yield, protein percentage, fat yield and rumen bacterial communities. Positive correlations were found between branched-chain FA (BCFA) in bacterial cell membranes and bacterial genera. In conclusion, bacterial communities and their FA compositions are more affected by stage of lactation than by genetics of dairy cow., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: Recent studies have demonstrated RAMP, a complete starter feed, to have beneficial effects for animal performance. However, how RAMP may elicit such responses is unknown. To understand if RAMP adaptation results in changes in the rumen bacterial community that can potentially affect animal performance, we investigated the dynamics of rumen bacterial community composition in corn-adapted and RAMP-adapted cattle., Methods and Results: During gradual acclimation of the rumen bacterial communities, we compared the bacterial community dynamics in corn and RAMP-adapted using 16S rRNA gene amplicon sequencing. Significant shifts in bacterial populations across diets were identified. The shift in corn-adapted animals occurred between adaptation step3 and step4, whereas in RAMP-adapted cattle, the shift occurred between step2 and step3. As the adaptation program progressed, the abundance of OTUs associated with family Prevotellaceae and S24-7 changed in corn-adapted animals. In RAMP-adapted animals, OTUs belonging to family Ruminococcaceae and Lachnospiraceae changed in abundance., Conclusions: Rumen bacteria can be acclimated faster to high concentrate diets, such as RAMP, than traditional adaptation programs and the speed of bacterial community acclimation depends on substrate composition., Significance and Impact of the Study: These findings may have implications for beef producers to reduce feedlot costs, as less time adapting animals would result in lower feed costs. However, animal feeding behavior patterns and other factors must be considered., (© 2016 The Authors published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Aim: To obtain co-cultures of anaerobic fungi and their indigenously associated methanogens from the rumen of yaks grazing on the Qinghai-Tibetan Plateau and investigate their morphology features and ability to degrade lignocellulose., Methods and Results: Twenty fungus-methanogen co-cultures were obtained by Hungate roll-tube technique. The fungi were identified as Orpinomyces, Neocallimastix and Piromyces genera based on the morphological characteristics and internal transcribed spacer 1 sequences analysis. All methanogens were identified as Methanobrevibacter sp. by 16S rRNA gene sequencing. There were four types of co-cultures: Neocallimastix with Methanobrevibacter ruminantium, Orpinomyces with M. ruminantium, Orpinomyces with Methanobrevibacter millerae and Piromyces with M. ruminantium among 20 co-cultures. In vitro studies with wheat straw as substrate showed that the Neocallimastix with M. ruminantium co-cultures and Piromyces with M. ruminantium co-cultures exhibited higher xylanase, filter paper cellulase (FPase), ferulic acid esterase, acetyl esterase activities, in vitro dry matter digestibility, gas, CH4 , acetate production, ferulic acid and p-coumaric acid releases. The Neocallimastix frontalis Yak16 with M. ruminantium co-culture presented the strongest lignocellulose degradation ability among 20 co-cultures., Conclusions: Twenty fungus-methanogen co-cultures were obtained from the rumen of grazing yaks. The N. frontalis with M. ruminantium co-cultures were highly effective combination for developing a fermentative system that bioconverts lignocellulose to high activity fibre-degrading enzyme, CH4 and acetate., Significance and Impact of the Study: The N. frontalis with M. ruminantium co-cultures from yaks grazing on the Qinghai-Tibetan Plateau present great potential in lignocellulose biodegradation industry., (© 2015 The Society for Applied Microbiology.)

Molecular information suggests that there is a broad diversity of acetogens in the rumen, distinct from any currently isolated acetogens. We combined molecular analysis with enrichment culture techniques to investigate this diversity further. Methane-inhibited, hydrogenotrophic enrichment cultures produced acetate as the dominant end product. Acetyl-CoA synthase gene analysis revealed putative acetogens in the cultures affiliated with the Lachnospiraceae and Ruminococcaceae as has been found in other rumen studies. No formyltetrahydrofolate synthetase genes affiliating with acetogens or with 'homoacetogen similarity' scores >90% were identified. To further investigate the hydrogenotrophic populations in these cultures and link functional gene information with 16S rRNA gene identity, cultures were subcultured quickly, twice, through medium without exogenous hydrogen, followed by incubation without exogenous hydrogen. Comparison of cultures lacking hydrogen and their parent cultures revealed novel Lachnospiraceae and Ruminococcaceae that diminished in the absence of hydrogen, supporting the hypothesis that they were likely the predominant acetogens in the enrichments. Interestingly, a range of Bacteroidetes rrs sequences that demonstrated <86% identity to any named isolate also diminished in cultures lacking hydrogen. Acetogens or sulphate reducers from the Bacteroidetes have not been reported previously; therefore this observation requires further investigation., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2015 The Society for Applied Microbiology.)

Aims: Microcin MccPDI-producing Escherichia coli have a fitness advantage in dairy calves. For this project, we determined whether MccPDI is responsible for the in vivo fitness advantage, which is a necessary condition before MccPDI strains can be considered viable candidates for inhibiting pathogenic serovars of E. coli., Methods and Results: Neonatal calves were coinoculated with either MccPDI-producing E. coli or MccPDI-knockout mutants in conjunction with a susceptible strain. After 6 days, the MccPDI-producing E. coli-25 strain clearly dominated the E. coli-186 susceptible strain in the inoculated calves (P = 0·003). MccPDI-producing E. coli composed a higher log percentage of the total population of lactose-fermenting bacteria in the faeces (5·51 log CFU per 8·03 log CFU) compared with the knockout strain (2·6 log CFU per 8·23 log CFU) (P = 0·01), and it was more consistently recovered from the lower gastrointestinal tract at the time of necropsy (P = 0·01)., Conclusion: Our findings support the hypothesis that MccPDI is functional in vivo and it is most likely responsible for a fitness advantage in vivo., Significance and Impact of the Study: MccPDI-producing E. coli strongly inhibit pathogenic E. coli strains in vitro. We show herein that MccPDI functions in vivo, and thus, these strains may be candidate probiotics against pathogenic strains of E. coli., (© 2014 The Society for Applied Microbiology.)

Unlabelled: A total of 300 isolates of Enterococcus, from different sources including faeces of poultry, cow and sheep, raw milk, ricotta cheese and water, in Tunisia, were screened for their antibacterial activity. Amongst them, 59 bacteriocin-producing strains were detected and identified by molecular methods. Genes encoding for entA, entP, entB, entL50A/B, AS-48 and bac31 bacteriocins were targeted by PCR. The bacteriocin-producing strains were assigned to the species Enterococcus faecium, Enterococcus faecalis, Enterococcus hirae, Enterococcus mundtii and Enterococcus durans, respectively, 34, 19, 3, 2 and 1 isolates. Antimicrobial activity was specifically observed against different spoilage and pathogenic micro-organisms, such as Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Escherichia coli, Ent. faecalis, Staphylococcus aureus, Salmonella enterica serovar Enteritidis and Paenibacillus larvae. The inhibitory activity was totally lost after proteinase K treatment, thereby revealing the proteinaceous nature of the antimicrobial compound. Only three bacteriocin genes, namely entP, entA and entL50A/B were detected in the isolates included in this study. Enterocins A and P were the most frequent genes and they were found in 55 (93.2%) and 39 isolates (66.1%), respectively, followed by enterocin L50A/B present in 27 isolates (45.7%). These newly identified bacteriocin-producing enterococci have the potential to be used in bio-preservation of food as well as biological control of foulbrood disease., Significance and Impact of the Study: Enterococci possess interesting properties not only for the food industry, but also for animal and human health. The antimicrobial potential of these bacteria includes principally bacteriocin-like molecules. With the aim of identifying bacteriocinogenic strains, a collection of 300 enterococci isolated from different origins were screened and their spectrum of action, as well as the gene encoding the bacteriocin, was determined. Fifty-nine bacteriocin-producing Enterococcus showed high activity against Listeria monocytogenes and Paenibacillus larvae, the causative agent of American foulbrood. Enterocins A, P and L50A/B were found in various combinations. The most important finding of this study is the growth inhibition of P. larvae due to bacteriocin-producing Enterococcus, which opens up the possibility to use these strains to control the disease in honeybees., (© 2014 The Society for Applied Microbiology.)

Unlabelled: The aim of our study was to determine the effect of diets with different types of roughage on the ruminal microbial metabolite profile in dairy cows. Holstein dairy cows were fed a diet containing either corn stover (CS group) or a mixture of alfalfa hay, Leymus chinensis hay and corn silage (MF group) at 0700 and 1900 h daily. Rumen fluid was sampled from each cow through a ruminal cannula at 0630 and 1030 h, and the mixed ruminal fluid from 3 day in each cow was analysed using nuclear magnetic resonance (NMR) spectroscopy. A multivariate analysis revealed a significant difference between the ruminal metabolome of the CS and MF groups at both time points. The MF group had higher levels of acetate, valerate, hydrocinnamate and methylamine and lower levels of glucose, glycine, propionate and isovalerate than those in the CS group. Our results showed that different types of roughages can significantly influence the ruminal microbial metabolome, especially with regard to organic acids, amines and amino acids., Significance and Impact of the Study: The microbial metabolites in the rumen provide nutritional precursors that are critical for general health and milk production in dairy cows. However, studies of the effect of diet on ruminal microbial metabolism are scant. In our current study, we analysed the ruminal microbial metabolite profile of cows fed different types of roughage. We found that the ruminal microbial metabolite profile of cows fed a mixed-roughage diet differed significantly from that of cows fed a single type of roughage. Certain metabolites, such as acetate, hydrocinnamate and methylamine, were closely correlated with specific types of roughage. Our findings provide insight into the effects of different roughages on ruminal microbial fermentation in dairy cows., (© 2014 The Society for Applied Microbiology.)

Unlabelled: The aim of this study was to determine concentrations of culturable faecal indicator organisms (FIOs) in freshly excreted dairy faeces and assess seasonal, within-herd and year-on-year variability in counts. Such values are essential in order to provide input parameters and associated uncertainty bounds for empirical models designed to determine the burden of FIOs on pasture. A longitudinal faecal analysis survey (n = 80) was conducted at a conventional dairy farm in central Scotland over a 2-year period. The analysis quantified counts of Escherichia coli and other nonE. coli coliforms and compared the concentrations of these FIO groups across contrasting seasons. The overall mean concentration of E. coli was 6.63 and 6.58 log10 CFU g(-1) dry weight in 2012 and 2013, respectively. However, concentrations of E. coli in faecal pats on each seasonal sampling event were highly variable and spanned several orders of magnitude on all occasions. Concentrations of E. coli in faeces excreted in winter were found to be lower than those excreted in all other seasons in 2012, though patterns of seasonal shedding were not consistent in observations the following year highlighting additional sources of uncertainty in FIO loading to land from dairy herds., Significance and Impact of the Study: This study provides a comprehensive temporal data set of faecal indicator organism (FIO) counts (both E. coli and other coliforms) in fresh dairy faeces for Scotland. Such faecal audits for the UK are scarce which is surprising given that livestock constitute one of the largest agricultural sources of diffuse microbial pollution of surface waters and contributors to poor bathing water quality. Such FIO concentration data (and evaluation of variability across seasonal, within-herd and year-on-year counts) in fresh faeces is a fundamental precursor to the robust parameterization of models that aim to predict the fate and transfer of both FIOs and pathogens in agricultural catchments., (© 2014 The Society for Applied Microbiology.)

Aim: To determine the faecal excretion of Campylobacter jejuni by dairy cows that used housing in combination with outdoor grazing., Methods and Results: Campylobacter jejuni prevalence and concentration were measured in a total of 990 cow faecal samples collected from seven herd home farms (HH), seven stand-off pad farms (SOP) and seven pasture farms (P) over a 2-year period. On all the farms, cows had access to pasture but were restricted to narrow grazing strips in winter. The overall Camp. jejuni prevalence was 55, 49 and 54% on HH, SOP and P farms, respectively. The Camp. jejuni concentration ranged from 0 to 6·7 log10 g(-1) faeces and was not statistically different among the farm systems. However, Camp. jejuni prevalence (P = 0·014) and concentration (P = 0·0001) were significantly greater in winter and early spring after intensive use of HH, SOP and strip-grazing. Typing of 30 Camp. jejuni isolates revealed a dominance of ruminant types (MLST CC-61, CC-21, CC-42 and CC-48), which are associated with human disease., Conclusion: No overall difference was observed among systems, but seasonal management practices that force cows close together increased the prevalence and concentration of Camp. jejuni in faeces., Significance and Impact of the Study: These findings are important when identifying farm practices that reduce Camp. jejuni excretion and the associated risk to human health., (© 2013 The Society for Applied Microbiology.)

Treponema saccharophilum is a pectinolytic bacterium isolated from the bovine rumen. The abundance of this bacterium has not been well determined, reflecting the lack of a reliable and accurate detection method. To develop a rapid method for monitoring T. saccharophilum, we performed pyrosequencing of genomic DNA isolated from rumen microbiota to explore the 16S rRNA gene sequences of T. saccharophilum candidates. Species-specific primers were designed based on fifteen sequences of partial 16S rRNA genes generated through pyrosequencing with 97% or higher similarity with T. saccharophilum DSM2985 along with sequence from type strain. The relative abundance of T. saccharophilum was quantified in both in vitro and in vivo rumen systems with varied pectin-containing forages using real-time PCR. There was a clear association of T. saccharophilum with alfalfa hay, which contains more pectin than Chinese wild rye hay or corn stover. The relative abundance of T. saccharophilum was as high as 0.58% in vivo, comparable with the population density of other common rumen bacteria. It is recognized that T. saccharophilum plays an important role in pectin digestion in the rumen., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Unlabelled: We proposed in this study that during intramammary infection, biofilm formation may facilitate adherence and colonization of Enterococcus faecalis to mammary gland epithelium. This was established by comparing six different Ent. faecalis isolates with different biofilm-forming profiles for their adhesive, invasive and survival capabilities to bovine mammary epithelial cell line (MAC-T). Our results showed increased ability of the biofilm-producer Ent. faecalis strains to adhere, invade and survive inside MAC-T cells rather than nonbiofilm-producer strains. We showed that growth of bacteria in bovine milk significantly augmented the adherence and invasion of all tested strains, and this feature was abolished again when strains were subcultured in brain heart infusion broth. Moreover, growth in bovine milk significantly increased biofilm formation by all tested strains. These results indicated that biofilm formation by Ent. faecalis, especially after expressing milk-dependent induction, may have special relevance in the pathogenesis of Ent. faecalis mastitis during intramammary infection by enhancing bovine mammary epithelial adhesion and colonization., Significance and Impact of the Study: Results obtained from current work highlighted the role of biofilm in the pathogenesis of Enterococcus faecalis mastitis. Those biofilm-forming strains might be substantial as useful antigens in diagnostic assays and as future vaccine candidates to control Ent. faecalis mastitis., (© 2013 The Society for Applied Microbiology.)

Aims: The objectives of this study were to investigate the correlations between Shiga toxin-producing Escherichia coli (STEC) shedding and faecal microflora in beef cattle and to identify functional species that might be used for STEC control., Methods and Results: Faecal samples were collected from 110 calves and 92 dams. The number and prevalence of STEC were determined using CHROMagar™ STEC; denaturing gradient gel electrophoresis (DGGE) was employed to analyse faecal bacterial composition. Six-month-old calves had the highest STEC shedding levels (3.03 ± 1.41 Log CFU g(-1)) and prevalence (95.5%). Both the number and prevalence decreased significantly as the calf age increased (P < 0.05). The DGGE analysis showed that faecal bacterial diversity increased, while cattle ages increased and STEC shedding levels decreased. Significant correlations between STEC shedding, cattle age and bacterial compositions were observed by redundancy analysis (P < 0.05). T-value biplots and sequencing results indicated that butyrate-producing bacteria (BPB) negatively correlated with STEC shedding., Conclusions: Higher STEC shedding levels and prevalence were associated with younger cattle age, lower faecal bacterial diversity and lower BPB levels., Significance and Impact of the Study: Butyrate-producing bacteria in GI tract might serve as an option for the future development of STEC shedding control strategy., (© 2013 The Society for Applied Microbiology.)

Unlabelled: Ruminal organic acid production, especially lactic acid, can be modified by feeding cattle highly concentrated diets, which have been shown to adversely affect dairy cattle health. Therefore, the use of lactic acid-utilizing organisms is considered to be a potential method for controlling lactic acid levels. This study was conducted to isolate and identify lactic acid-utilizing yeasts from the ruminal fluid of dairy cattle and to determine the specific growth rate and generation time when using lactic acid as a carbon source instead of glucose. Seventeen yeast isolates were examined in this study. Yeasts isolated from dairy cattle that were fed a high cassava pulp diet (HCP) had higher specific growth rates and shorter generation times than yeasts isolated from dairy cattle that were fed a high-concentrate diet (HC) and a mixed diet (M). The three most effective yeasts in terms of specific growth rate and generation time were Pichia kudriavzevii, Candida rugosa and Kodamaea ohmeri, with 99, 100 and 99% nucleotide identities, respectively. These three isolates could be used as potential probiotics in dairy cattle diets., Significance and Impact of the Study: This study demonstrates that yeasts isolated from the ruminal fluid of dairy cattle can utilize lactic acid as a carbon and energy source for growth. The isolated yeasts can be used as probiotic supplements for dairy cattle that are fed highly concentrated diets to reduce ruminal lactic acid production., (© 2013 The Society for Applied Microbiology.)

Aims: To determine the herd prevalence of Enterobacteriaceae producing CTX-M-type extended-spectrum β-lactamases (ESBLs) among 381 dairy farms in Japan., Methods and Results: Between 2007 and 2009, we screened 897 faecal samples using BTB lactose agar plates containing cefotaxime (2 μg ml(-1)). Positive isolates were tested using ESBL confirmatory tests, PCR and sequencing for CTX-M, AmpC, TEM and SHV. The incidence of Enterobacteriaceae producing CTX-M-15 (n = 7), CTX-M-2 (n = 12), CTX-M-14 (n = 3), CMY-2 (n = 2) or CTX-M-15/2/14 and CMY-2 (n = 4) in bovine faeces was 28/897 (3·1%) faecal samples. These genes had spread to Escherichia coli (n = 23) and three genera of Enterobacteriaceae (n = 5). Herd prevalence was found to be 20/381 (5·2%) dairy farms. The 23 E. coli isolates showed clonal diversity, as assessed by multilocus sequence typing and pulsed-field gel electrophoresis. The pandemic E. coli strain ST131 producing CTX-M-15 or CTX-M-27 was not detected., Conclusions: Three clusters of CTX-M (CTX-M-15, CTX-M-2, CTX-M-14) had spread among Japanese dairy farms., Significance and Impact of the Study: This is the first report on the prevalence of multidrug-resistant CTX-M-15-producing E. coli among Japanese dairy farms., (Journal of Applied Microbiology © 2013 The Society for Applied Microbiology.)

Aims: To characterize a psychrotrophic bacterium, designated TC1, previously isolated from a cattle hide in Ireland, and to investigate the ability of this strain to cause 'blown pack' spoilage (BPS) of vacuum-packaged beef primals., Methods and Results: TC1 was characterized using a combination of phenotypic, chemotaxonomic and genotypic analyses and was assessed for its ability to spoil vacuum-packaged beef at refrigerated temperatures. TC1 was Gram-positive and formed elliptical subterminal endospores. The strain was able to grow between 0 and 33 °C, with optimal growth between 23 and 24 °C. TC1 could be differentiated from its phylogenetically closest neighbour (Clostridium lituseburense DSM 797(T)) by 16S rRNA gene sequencing, pulsed-field gel electrophoresis and cellular fatty acid composition. TC1 spoiled (BPS) beef within 42 days when inoculated in cold-stored (1 °C) vacuum-packed beef., Conclusions: The phenotypic, chemotaxonomic and genotypic characterization indicated that TC1 may represent a potentially novel, cold-tolerant, gas-producing bacterium of considerable economic significance to the beef industry., Significance and Impact of the Study: This study reports and characterizes an emerging BPS bacterium, which should be considered in future activities designed to minimize the psychrophilic and psychrotrophic spoilage of vacuum-packaged beef., (© 2012 The Society for Applied Microbiology.)

Aims: Anaerobic rumen fungi (Neocallimastigales) play important roles in the breakdown of complex, cellulose-rich material. Subsequent decomposition products are utilized by other microbes, including methanogens. The aim of this study was to determine the effects of dietary changes on anaerobic rumen fungi diversity., Methods and Results: Altered diets through increasing concentrate/forage (50 : 50 vs 90 : 10) ratios and/or the addition of 6% soya oil were offered to steers and the Neocallimastigales community was assessed by PCR-based fingerprinting with specific primers within the barcode region. Both a decrease in fibre content and the addition of 6% soya oil affected Neocallimastigales diversity within solid and liquid rumen phases. The addition of 6% soya oil decreased species richness. Assemblages were strongly affected by the addition of 6% soya oil, whereas unexpectedly, the fibre decrease had less effect. Differences in volatile fatty acid contents (acetate, propionate and butyrate) were significantly associated with changes in Neocallimastigales assemblages between the treatments., Conclusions: Diet clearly influences Neocallimastigales assemblages. The data are interpreted in terms of interactions with other microbial groups involved in fermentation processes within the rumen., Significance and Impact of the Study: Knowledge on the influence of diet on anaerobic fungi is necessary to understand changes in microbial processes occurring within the rumen as this may impact on other rumen processes such as methane production., (© 2012 The Society for Applied Microbiology.)

Aims: The objective of this study was to examine the prevalence of enteropathogenic Escherichia coli (EPEC) on beef and dairy farms and in beef abattoirs and to characterize the isolates in terms of serogroup and virulence markers., Methods and Results: Bovine faecal samples (n = 1200), farm soil samples (n = 600), hide samples (n = 450) and carcass samples (n = 450) were collected from 20 farms and three abattoirs throughout Ireland over a 12-month period. After selective enrichment, samples testing positive for the intimin gene (eae) using PCR screening were cultured, and colonies were examined for the presence of the eae, vt(1) and vt(2) genes. Colonies that were positive for the intimin gene and negative for the verotoxin genes were further screened using PCR for a range of virulence factors including tir, espA, espB katP, espP, etpD, saa, sab, toxB, iha, lpfA(O157/OI-141) , lpfA(O113) and lpfA(O157/OI-154) . PCR screening was also used to screen for variations in the intimin gene (eae). Of the 2700 source samples analysed, 3.9% (47 of 1200) of faecal, 2% (12 of 600) of soil, 6.4% (29 of 450) of hide and 0.7% (3 of 450) of carcass samples were PCR positive (for the presence of the eae gene). All 140 isolates obtained were atypical EPEC (aEPEC), while θ and β intimin types were common. The virulence factors hlyA, tir, lpfA (O113) , lpfA (O157/OI-154) , and iha were frequently detected, while lpfA(O157/OI-141) , saa, espA, espB and toxB were also present but to a lesser extent., Conclusions: It was concluded that cattle are a source of aEPEC, many of which have the virulence machinery necessary to be pathogenic to humans., Significance and Impact of the Study: These findings suggest the need for increased research on aEPEC with particular emphasis on food safety and public health risk., (© 2012 The Society for Applied Microbiology.)

Aims: To assess the prevalence of Shiga toxin-producing Escherichia coli (STEC) on a cluster of twelve beef farms in the north-east of Ireland., Methods and Results: Samples were screened for stx1 and stx2 using PCR. Positive samples were enriched in mTSB and STEC O157 isolated using immunomagnetic separation. Enrichment cultures were plated onto TBX agar to isolate non-O157 STEC. All isolates were serotyped and examined for a range of virulence genes and their antibiotic resistance phenotype determined. Eighty-four isolates of 33 different serotypes were cultured from the 13·7% of samples that were stx positive. The most prevalent serotype was O157:H7, the most common Shiga toxin was stx(2) , and a variety of virulence factor combinations was observed. O-:H-, O26:H11, O76:H34, O157:H7, O157:H16 and OX18:H+ also carried eaeA and hlyA genes. Twenty-nine per cent of strains were resistant to at least one antibiotic, 48% of which had multiple drug resistance (MDR) with O2:H32 displaying resistance to five antibiotics., Conclusions: The ubiquitous nature of STEC on beef farms, the detection of stx(+) eaeA(+) hlyA(+) in the serotypes O-:H-, O157:H16 and OX18:H+ in addition to O157:H7 and O26:H11 and the widespread distribution of antibiotic resistance are of public health concern as new virulent STEC strains are emerging., Significance and Impact of the Study: This study found no relationship between serotype and antibiotic resistance, therefore negating efforts to isolate serotypes using specific antibiotic supplemented media. The data presented provide further evidence of the emergence of new STEC virulotypes of potential public health significance., (© 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.)

Aims: To investigate, using culture-independent techniques, the presence and diversity of methanogenic archaea in the foregut of kangaroos., Methods and Results: DNA was extracted from forestomach contents of 42 kangaroos (three species), three sheep and three cattle. Four qualitative and quantitative PCR assays targeting the archaeal domain (16S rRNA gene) or the functional methanogenesis gene, mcrA, were used to determine the presence and population density of archaea in kangaroos and whether they were likely to be methanogens. All ruminal samples were positive for archaea, produced PCR product of expected size, contained high numbers of archaea and high numbers of cells with mcrA genes. Kangaroos were much more diverse and contradictory. Fourteen kangaroos had detectable archaea with numbers 10- to 1000-fold fewer than sheep and cattle. Many kangaroos that did not possess archaea were positive for the mcrA gene and had detectable numbers of cells with this gene and vice versa. DNA sequence analysis of kangaroos' archaeal 16S rRNA gene clones show that many methanogens were related to Methanosphaera stadmanae. Other sequences were related to non-methanogenic archaea (Thermoplasma sp.), and a number of kangaroos had mcrA gene sequences related to methane oxidising archaea (ANME)., Conclusions: Discrepancies between qualitative and quantitative PCR assays for archaea and the mcrA gene suggest that the archaeal communities are very diverse and it is possible that novel species exist., Significance and Impact of the Study: Archaea (in general) were below detectable limits in many kangaroos, especially Red kangaroos; when present they are in lower numbers than in ruminants, and the archaea are not necessarily methanogenic. The determination of why this is the case in the kangaroo foregut could assist in reducing emissions from other ecosystems in the future., (© 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.)

Aims: The focus of this work was to compare the survival of Escherichia coli introduced into streambed sediments from goose, deer and bovine faeces vs indigenous E. coli., Methods and Results: The survival experiments were conducted in flow-through chambers for 32days using two sediments (mineral and organic) obtained from a first-order creek in Maryland. Bovine, goose and deer faeces were collected fresh and diluted or enriched so that added E. coli and indigenous populations were equivalent. Escherichia coli and total coliforms were enumerated using the Colilert-18 Quanti-Tray system. Patterns of E. coli survival and inactivation rates were virtually identical for indigenous strains in both mineral and organic sediments. The addition of E. coli strains from bovine, goose or deer faeces had relatively little impact on final E. coli concentrations, with the exception of deer-borne E. coli populations in the organic sediment., Conclusion: These results indicate that indigenous sediment-borne E. coli strains are generally, or more, persistent than those deposited into sediments, including wildlife., Significance and Impact of the Study: This is the first study on the survival of E. coli originating from wildlife faeces, in sediments, as opposed to bovine faeces or laboratory-cultured strains. As wildlife are likely to be the primary source of E. coli in most non agricultural watersheds, an understanding of the persistence of these strains is important to understanding microbial water quality., (© 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.)

Aims: To evaluate the antimicrobial susceptibility and genetic relatedness of 11 Stenotrophomonas maltophilia isolates from an outbreak of bovine clinical mastitis in one herd and two isolates from two separate mastitis cases in two other herds., Methods and Results: Thirteen S. maltophilia isolates were obtained from milk samples from 11 cows from three dairy herds in Japan during 2008. We tested their susceptibility to 14 antimicrobials by broth microdilution and identified their genotypes by enterobacterial repetitive intergenic consensus 2 (ERIC2)-PCR. Every cow had acute mild mastitis (slightly watery foremilk with flakes) without systemic symptoms and all resolved within 3-5 weeks of diagnosis. Eleven of the 13 isolates derived from nine cows in one herd over a 7-month period exhibited a closely related ERIC2 type (A). The remaining two isolates derived from two cows from two other herds exhibited two distinct ERIC2 types (B and C). Most of the 13 isolates exhibited susceptibility to trimethoprim-sulfamethoxazole, chloramphenicol, minocycline and levofloxacin; however, they were resistant to four β-lactams, kanamycin, gentamicin and oxytetracycline. They were intermediate to enrofloxacin., Conclusions: Eleven closely related S. maltophilia isolates were involved in a herd outbreak of mastitis to some extent. Bovine S. maltophilia isolates exhibited resistance to many classes of antimicrobials., Significance and Impact of Study: This is a rare report of a herd outbreak of bovine mastitis involving closely related S. maltophilia isolates., (© 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.)

Aims: To determine the effects of the removal of forage in high-concentrate diets on rumen fermentation conditions and rumen bacterial populations using culture-independent methods., Methods and Results: Detectable bacteria and fermentation parameters were measured in the solid and liquid fractions of digesta from cattle fed two dietary treatments, high concentrate (HC) and high concentrate without forage (HCNF). Comparison of rumen fermentation conditions showed that duration of time spent below pH 5·2 and rumen osmolality were higher in the HCNF treatment. Simpson's index of 16S PCR-DGGE images showed a greater diversity of dominant species in the HCNF treatment. Real-time qPCR showed populations of Fibrobacter succinogenes (P = 0·01) were lower in HCNF than HC diets. Ruminococcus spp., F. succinogenes and Selenomonas ruminantium were at higher (P ≤ 0·05) concentrations in the solid vs the liquid fraction of digesta regardless of diet., Conclusions: The detectable bacterial community structure in the rumen is highly diverse. Reducing diet complexity by removing forage increased bacterial diversity despite the associated reduction in ruminal pH being less conducive for fibrolytic bacterial populations. Quantitative PCR showed that removal of forage from the diet resulted in a decline in the density of some, but not all fibrolytic bacterial species examined., Significance and Impact of the Study: Molecular techniques such as DGGE and qPCR provide an increased understanding of the impacts of dietary changes on the nature of rumen bacterial populations, and conclusions derived using these techniques may not match those previously derived using traditional laboratory culturing techniques., (© 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.)

Aims: To investigate the relationship between ruminal methanogen community and host enteric methane (CH(4) ) production in lactating dairy cows fed diets supplemented with an exogenous fibrolytic enzyme additive., Methods and Results: Ecology of ruminal methanogens from dairy cows fed with or without exogenous fibrolytic enzymes was examined using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analyses and quantitative real-time PCR (qRT-PCR). The density of methanogens was not affected by the enzyme additive or sampling times, and no relationship was observed between the total methanogen population and CH(4) yield (as g per head per day or g kg(-1) DMI). The PCR-DGGE profiles consisted of 26 distinctive bands, with two bands similar to Methanogenic archaeon CH1270 negatively correlated, and one band similar to Methanobrevibacter gottschalkii strain HO positively correlated, with CH(4) yield. Three bands similar to Methanogenic archaeon CH1270 or Methanobrevibacter smithii ATCC 35061 appeared after enzyme was added., Conclusions: Supplementing a dairy cow diet with an exogenous fibrolytic enzyme additive increased CH(4) yield and altered the composition of the rumen methanogen community, but not the overall density of methanogens., Significance and Impact of the Study: This is the first study to identify the correlation between methanogen ecology and host CH(4) yield from lactating dairy cows., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

Aims: Campylobacter jejuni isolates from various sources in Iceland were genotyped with the aim of assessing the genetic diversity, population structure, source distribution and campylobacter transmission routes to humans., Methods and Results: A collection of 584 Campylobacter isolates were collected from clinical cases, food, animals and environment in Iceland in 1999-2002, during a period of national Campylobacter epidemic in Iceland. All isolates were characterized by pulse field gel electrophoresis (PFGE), and selected subset of 52 isolates representing the diversity of the identified PFGE types was further genotyped using multilocus sequence typing (MLST) and fla-SVR sequencing to gain better insight into the population structure., Conclusions: The results show a substantial diversity within the Icelandic Campylobacter population. Majority of the human Campylobacter infections originated from domestic chicken and cattle isolates. MLST showed the isolates to be distributed among previously reported and common sequence type complexes in the MLST database., Significance and Impact of the Study: The genotyping of Campylobacter from various sources has not previously been reported from Iceland, and the results of the study gave a valuable insight into the population structure of Camp. jejuni in Iceland, source distribution and transmission routes to humans. The geographical isolation of Iceland in the north Atlantic provides new information on Campylobacter population dynamics on a global scale., (Journal of Applied Microbiology © 2011 The Society for Applied Microbiology No claim to Icelandic Government works.)

Aims: To provide molecular epidemiological evidence of avian transmission of Escherichia coli O157:H7 between dairy farms in Ohio, this study was designed to identify genetic relatedness between isolates originating from bovine faecal samples and intestinal contents of European starlings captured on these farms., Methods and Results: During a three-year period (2007-2009), cattle (n = 9000) and starlings (n = 430) on 150 different dairy farms in northern Ohio were sampled for the presence of E. coli O157:H7. Isolates were subjected to multiple-locus variable-nucleotide tandem repeat analysis (MLVA). Distinct allelic groups were identified on most farms; however, isolates clustering into three MLVA groups originated from both cattle and birds on different farms., Conclusions: Sharing of indistinguishable epidemiologically linked E. coli O157 MLVA subtypes between starlings and cattle on different farms supports the hypothesis that these birds contribute to the transmission of E. coli O157:H7 between dairy farms., Significance and Impact of Study: A continued need exists to identify and to improve preharvest measures for controlling E. coli O157:H7. Controlling wildlife intrusion, particularly European starlings, on livestock operations, may be an important strategy for reducing dissemination of E. coli O157:H7 between farms and thereby potentially decreasing the on-farm prevalence of E. coli O157:H7 and enhancing the safety of the food supply., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

Aims: To determine the effects of sodium bisulfate (SBS) on the bacterial populations in cattle waste., Methods and Results: We applied SBS at 0, 60, 70 or 100 kg week(-1) to cattle waste as it accumulated on the floors of four cattle pens, housing eight cattle each. We observed significant pH decreases in all of the treated wastes on day one; however, the 60 kg week(-1) treatment returned to control levels by day four, while the others remained significantly lower. Heterotrophic plate counts of the waste revealed that all treatments reduced the bacterial populations in the wastes on day one; however, all returned to control levels by day four. The 16S rRNA gene libraries derived from the wastes revealed significant reductions in sequences associated with the phyla Bacteroidetes and Firmicutes and increases in the Proteobacteria, Actinobacteria and Spirochaetes on day one, but resembled the control by day seven. Sequences associated with Escherichia coli increased significantly after SBS application, but became undetectable by day seven., Conclusions: SBS application significantly alters the bacterial population structure of waste during the first few days of application, but the populations return to almost normal after 7 days., Significance and Impact of the Study: Application of SBS to animal waste can reduce emissions; however, biosecurity precautions must be rigorously maintained during the initial application to ensure that pathogenic E. coli is not released into the environment., (Journal of Applied Microbiology © 2011 The Society for Applied Microbiology. No claim to Brazilian Government works.)