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Leafy greens are leading vehicles for Escherichia coli O157:H7 foodborne illness. Pest flies can harbor this pathogen and may disseminate it to produce. We determined the occurrence of E. ...

Different dietary and feed additive strategies have been developed to reduce the liver abscess in feedlot cattle, but liver abscesses are still a major problem in beef production. We have limited knowledge about how rumen microbial communities interact with host epithelial gene expression in healthy and liver-abscessed animals. The objective of this study was to investigate the associations between the rumen content associated and rumen epimural microbiome and epithelial gene expression in liver-abscessed and healthy animals. To this end, we collected the ruminal contents and tissue samples from healthy (N=30; score=0, steers n=19 and heifers n=11) and liver-abscessed (N=30; score=A+, steers n=21 and heifers n=9) feedlot cattle at harvest. The bacterial community compositions in the ruminal contents and papillae were evaluated via 16S rDNA sequencing of the V4 region using the Illumina MiSeq platform. Additionally, total RNA was extracted from rumen epithelial tissues and sequenced using the Illumina NextSeq platform. The permutational analysis (PERMANOVA) on Bray Curtis distances matrices showed the microbial community in the ruminal contents was significantly different (P< 0.001) from the bacterial community observed in rumen papillae. The ruminal contents contained a higher abundance of Bacteroidetes and Proteobacteria while papillae contained higher abundance of Firmicutes. The epimural microbiota was different (P< 0.01) between healthy and liver abscessed animals while ruminal contents microbiome was not different between the two groups. The DeSeq2 algorithm identified differentially expressed genes (221) related to MAPK, NF-kappa B signaling pathway, immune and inflammatory response in liver-abscessed animals. Additionally, a wide range of epimural bacterial taxa were correlated (-0.52 to 0.67) with differentially expressed genes. These data demonstrate the interaction between epimural microbiota and the host and its effect on liver abscesses, and indicate the need to study the epimural microbiome for its impact on liver abscesses in feedlot cattle. USDA is an equal opportunity provider and employer.

Abscess is the highest cause of liver condemnation and is estimated to cost the beef industry US$64 million annually. Fusobacterium necrophorum, commonly found in the bovine rumen, is the primary bacteria associated with liver abscess in cattle. Theoretically, damage to the rumen wall allows F. necrophorum to invade the bloodstream and colonize the liver. The objective of this study was to determine the changes in gene expression in the rumen epithelium and microbial populations adherent to the rumen epithelium and in the rumen contents of beef cattle with liver abscesses compared with those with no liver abscesses. Rumen epithelial tissue and rumen content were collected from 31 steers and heifers with liver abscesses and 30 animals with no liver abscesses. Ribonucleic acid (RNA) sequencing was performed on the rumen epithelium, and a total of 221 genes were identified as differentially expressed in the animals with liver abscesses compared with animals with no abscesses, after removal of genes that were identified as a result of interaction with sex. The nuclear factor kappa-light-chain enhancer of activated B cells signaling and interferon signaling pathways were significantly enriched in the differentially expressed gene (DEG) set. The majority of the genes in these pathways were downregulated in animals with liver abscesses. In addition, RNA translation and protein processing genes were also downregulated, suggesting that protein synthesis may be compromised in animals with liver abscesses. The rumen content bacterial communities were significantly different from the rumen wall epimural bacterial communities. Permutational multivariate analysis of variance (PERMANOVA) analysis did not identify global differences in the microbiome of the rumen contents but did identify differences in the epimural bacterial communities on the rumen wall of animals without and with liver abscesses. In addition, associations between DEG and specific bacterial amplicon sequence variants of epimural bacteria were observed. The DEG and bacterial profile on the rumen papillae identified in this study may serve as a method to monitor animals with existing liver abscesses or to predict those that are more likely to develop liver abscesses.

In light of recent host-microbial association studies, a consensus is evolving that species composition of the gastrointestinal microbiota is a polygenic trait governed by interactions between host genetic factors and the environment. Here, we investigated the effect of host genetic factors in shaping the bacterial species composition in the rumen by performing a genome-wide association study. Using a common set of 61,974 single-nucleotide polymorphisms found in cattle genomes (n = 586) and corresponding rumen bacterial community composition, we identified operational taxonomic units (OTUs), Families and Phyla with high heritability. The top associations (1-Mb windows) were located on 7 chromosomes. These regions were associated with the rumen microbiota in multiple ways; some (chromosome 19; position 3.0–4.0 Mb) are associated with closely related taxa (Prevotellaceae, Paraprevotellaceae, and RF16), some (chromosome 27; position 3.0–4.0 Mb) are associated with distantly related taxa (Prevotellaceae, Fibrobacteraceae, RF16, RFP12, S24-7, Lentisphaerae, and Tenericutes) and others (chromosome 23; position 0.0–1.0) associated with both related and unrelated taxa. The annotated genes associated with identified genomic regions suggest the associations observed are directed toward selective absorption of volatile fatty acids from the rumen to increase energy availability to the host. This study demonstrates that host genetics affects rumen bacterial community composition.

Escherichia coli O157:H7 is a foodborne pathogen commonly associated with cattle feces. Diet, including dietary supplements such as β-agonists, may impact fecal shedding of this pathogen. A series of three experiments were conducted to determine if the β-agonists ractopamine hydrochloride (RAC) or zilpaterol hydrochloride (ZH) would impact the level or prevalence of fecal E. coli O157:H7 shedding. In Experiment 1, dietary RAC did not impact fecal shedding of E. coli O157:H7 based on the level or prevalence, but the addition of dietary soybean meal (SBM) in the study did reduce E. coli O157:H7 shedding. In Experiments 2 and 3, dietary ZH did not affect fecal E. coli O157:H7 shedding as determined by enumeration or prevalence, but in Experiment 2 the addition of 30% (dry matter basis) wet distillers grains with solubles (WDGS) in the diet tended to increase E. coli O157:H7 shedding. Shade is a potential management tool to reduce heat stress in cattle, and in Experiment 3 the presence of shade over the feedlot pens did not affect E. coli O157:H7 shedding. The use of β-agonists in cattle diets did not significantly affect fecal shedding of E. coli O157:H7, and in particular the percentage of animals shedding enumerable levels of the pathogen did not change, indicating that there was not a change in colonization. As has been reported previously and indicated again in this study, the use of WDGS in the diet may increase E. coli O157:H7 shedding. In contrast, the addition of SBM to cattle diets, to increase the dietary crude protein, appeared to reduce E. coli O157:H7 shedding, but this potential dietary intervention needs to be confirmed with additional research.

AIMS: The objective of this study was to determine if the faecal microbiome has an association with Escherichia coli O157:H7 prevalence and enumeration. METHODS AND RESULTS: Pyrosequencing analysis of faecal microbiome was performed from feedlot cattle fed one of three diets: (i) 94 heifers fed low concentrate (LC) diet, (ii) 142 steers fed moderate concentrate (MC) diet, and (iii) 132 steers fed high concentrate (HC) diet. A total of 322 585 OTUs were calculated from 2,411,122 high‐quality sequences obtained from 368 faecal samples. In the LC diet group, OTUs assigned to the orders Clostridiales and RF39 (placed within the class Mollicutes) were positively correlated with both E. coli O157:H7 prevalence and enumeration. In the MC diet group, OTUs assigned to Prevotella copri were positively correlated with both E. coli O157:H7 prevalence and enumeration, whereas OTUs assigned to Prevotella stercorea were negatively correlated with both E. coli O157:H7 prevalence and enumeration. In both the MC diet group and the HC diet group, OTUs assigned to taxa placed within Clostridiales were both positively and negatively correlated with both E. coli O157:H7 prevalence and enumeration. However, all correlations were weak. In both the MC diet group and the HC diet group, stepwise linear regression through backward elimination analyses indicated that these OTUs were significantly correlated (P

The objective of this study was to determine the effect of lysozyme and antibiotics on zoonotic pathogen shedding in faeces from nursery pigs housed without and with an indirect disease challenge.Two replicates of approximately 650 pigs each were weaned and randomly assigned to one of 24 pens in either a nursery room that had been fully disinfected or a nursery room left unclean. Pigs were randomly assigned to control diet (Control), control diet + antibiotics (Antibiotic; chlortetracycline and tiamulin), or control diet + lysozyme (Lysozyme; 100 mg kg(-1) diet). Rectal swab samples were collected on day 0 and 28 of treatment, and enriched and cultured for Campylobacter spp. and shiga-toxigenic Escherichia coli (STEC). Enrichments from rectal swab samples also were analysed for presence of enterohaemorrhagic E. coli (EHEC) virulence genes (hlyA, eae, stx1 and stx2). Room hygiene had little effect on day 28 results. Percentage of samples culture positive for Campylobacter spp. was lowest for lysozyme diets (P0·01), but similar for control and antibiotic diets (43·2, 83·7, and 84·8 respectively). Diet had little effect on the EHEC virulence genes hlyA or eae (P0·1), but there was a tendency for fewer samples positive for stx1/stx2 in antibiotic or lysozyme diet groups (P0·07) compared to control diet (1·2, 2·1 and 5·8% respectively). Salmonella spp. and specific STEC types tested were rarely detected in the study.In nursery swine, room hygiene had little effect on pathogen shedding. Dietary chlortetracycline and tiamulin did not reduce pathogen shedding but dietary lysozyme reduced faecal shedding of Campylobacter.Lysozyme can effectively replace antibiotics in the diet of nursery swine and can be effective for pathogen control.

Feeding corn wet distillers grains with solubles (WDGS) to cattle can increase the load of Escherichia coli O157:H7 in feces and on hides, but the mechanisms are not fully understood. The objective of these experiments was to examine a role for the persistence of E. coli O157:H7 in the feces and feedlot pen surfaces of cattle fed WDGS. In the first study, feces from steers fed 0, 20, 40, or 60% corn WDGS were inoculated with E. coli O157:H7. The E. coli O157:H7 numbers in feces from cattle fed 0% corn WDGS rapidly decreased (P < 0.05), from 6.28 to 2.48 log CFU/g of feces by day 14. In contrast, the E. coli O157:H7 numbers in feces from cattle fed 20, 40, and 60% corn WDGS were 4.21, 5.59, and 6.13 log CFU/g of feces, respectively, on day 14. A second study evaluated the survival of E. coli O157:H7 in feces from cattle fed 0 and 40% corn WDGS. Feces were collected before and 28 days after the dietary corn was switched from high-moisture corn to dry-rolled corn. Within dietary corn source, the pathogen persisted at higher concentrations (P < 0.05) in 40% corn WDGS feces at day 7 than in 0% WDGS. For 40% corn WDGS feces, E. coli O157:H7 persisted at higher concentrations (P < 0.05) at day 7 in feces from cattle fed high-moisture corn (5.36 log CFU/g) than from those fed dry-rolled corn (4.27 log CFU/g). The percentage of WDGS had no effect on the E. coli O157:H7 counts in feces from cattle fed steam-flaked corn-based diets containing 0, 15, and 30% sorghum WDGS. Greater persistence of E. coli O157:H7 on the pen surfaces of animals fed corn WDGS was not demonstrated, although these pens had a higher prevalence of the pathogen in the feedlot surface manure after the cattle were removed. Both or either the greater persistence and higher numbers of E. coli O157:H7 in the environment of cattle fed WDGS may play a part in the increased prevalence of E. coli O157:H7 in cattle by increasing the transmission risk.

Shiga-toxigenic Escherichia coli, such as E. coli O157:H7, are foodborne zoonotic pathogens that can cause severe illness and death in humans. The gastrointestinal tract of ruminant animals has been identified as a primary habitat for E. coli O157:H7 and, in cattle, the hindgut tract appears to be a primary site for colonization. This pathogen has been found in cattle feces, on cattle hides, and in the production environment, and transmission to humans has occurred as a result of consumption of contaminated ground beef, water, and produce. Interventions to reduce the pathogen at beef harvest have significantly reduced the occurrence of the pathogen, but outbreaks and recalls due to the pathogen still occur for beef products. Interventions in the feedyard before harvest have had little success, but critical control points for implementing interventions are limited compared with the beef abattoir. The percentage of animals shedding E. coli O157:H7 in the feces can be highly variable from pen to pen, and the levels in the feces can vary from animal to animal. Animals colonized and shedding E. coli O157:H7 at high levels are a small fraction of animals in a pen but are important source for transferring the pathogen amongst the penmates. Recent research has indicated that diet may greatly influence the shedding of E. coli O157:H7. In addition, diet can influence the microbiota composition of the feces. However, little is known about the interaction between the indigenous microbiota and fecal shedding of E. coli O157:H7. Understanding the influence of indigenous microbiota on the colonization and shedding of E. coli O157:H7 will provide a potential avenue for intervention in the preharvest production environment not yet exploited.

A simple, cow-side test for the presence of drug residues in live animal fluids would provide useful information for tissue drug residue avoidance programs. This work describes adaptation and evaluation of rapid screening tests to detect drug residues in serum and urine. Medicated heifers had urine, serum, and tissue biopsy samples taken while on drug treatment. Samples were tested by rapid methods and high-performance liquid chromatography (HPLC). The adapted microbial inhibition method, kidney inhibition swab test, was useful in detecting sulfadimethoxine in serum, and its response correlated with the prescribed withdrawal time for the drug, 5 to 6 days posttreatment. The lateral flow screening method for flunixin and beta-lactams, adapted for urine, was useful in predicting flunixin in liver detected by HPLC, 96 h posttreatment. The same adapted methods were not useful to detect ceftiofur in serum or urine due to a lack of sensitivity at the levels of interest. These antemortem screening test studies demonstrated that the method selected, and the sampling matrix chosen (urine or serum), will depend on the drug used and should be based on animal treatment history if available. The live animal tests demonstrated the potential for verification that an individual animal is free of drug residues before sale for human consumption.

Foodborne pathogens are a significant cause of disease in humans, and meat-producing animals are reservoirs for a variety of these pathogens. Consumption of contaminated meat or meat products is a major route for human infection, but indirect infection can arise from water and other foods contaminated with animal waste. Hence, reducing colonization and fecal shedding is important for sustaining a safe food chain and environment. Swine are potential reservoirs for several of the foodborne and related pathogens. Piglets can be colonized soon after birth, and the transition to the nursery is a stressful period. At farrowing, Campylobacter is a major pathogenic bacterium observed in feces of piglets, and infection can increase significantly during the nursery phase of production. Moreover, pathogen shedding has been associated with reduced performance. Dietary supplementation of carbodox with copper sulfate or lysozyme from egg whites has been shown to reduce Campylobacter shedding in piglets after weaning, but neither treatment significantly affected potentially pathogenic E. coli shedding. Supplementation with a commercial Lactobacillus acidophilus fermentation product had limited effect on pathogen shedding, whereas tiamulin had no significant effect. Nursery swine are prone to pathogen colonization, and there are dietary supplements shown to be effective for reducing shedding during this important phase of swine production.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains are clinically important foodborne pathogens. Unlike E. coli O157:H7, these foodborne pathogens have no unique biochemical characteristics to readily distinguish them from other E. coli strains growing on plating media. In this study, a chromogenic agar medium was developed in order to differentiate among non-O157 STEC strains of serogroups O26, O45, O103, O111, O121, and O145 on a single agar medium. The ability of this chromogenic agar medium to select and distinguish among these pathogens is based on a combination of utilization of carbohydrates, b -galactosidase activity, and resistance to selective agents. The agar medium in combination with immunomagnetic separation was evaluated and successfully allowed for the detection and isolation of these six serogroups from artificially contaminated fresh beef. The agar medium in combination with immunomagnetic separation also allowed successful detection and isolation of naturally occurring non-O157 STEC strains present in cattle feces. Thirty-five strains of the top six non-O157 STEC serogroups were isolated from 1,897 fecal samples collected from 271 feedlot cattle. This chromogenic agar medium could help significantly in routine screening for the top six non-O157 STEC serogroups from beef cattle and other food.

The incidence of Clostridium difficile infection has recently increased in North American and European countries. This pathogen has been isolated from retail pork, turkey, and beef products and reported associated with human illness. This increase in infections has been attributed to the emergence of a toxigenic strain designated North America pulsed-field gel electrophoresis type 1 (NAP1). The NAP1 strain has been isolated from calves as well as ground meat products, leading to speculation of illness from consumption of contaminated meat products. However, information on C. difficile associated with beef cattle during processing and commercially produced ground beef is limited. To address this data gap, samples from various steps during beef production were collected. Samples from hides (n = 525), preevisceration carcasses (n = 475), postintervention carcasses (n = 471), and 956 commercial ground beef samples were collected from across the United States. The prevalence of C. difficile spores on hides was 3.2%. C. difficile spores were not detected on preevisceration and postintervention carcasses or in commercially produced ground beef. Phenotypic and genetic characterizations were carried out for all 18 isolates collected from hide samples. Twenty-two percent of the isolates were nontoxigenic strains, while 78% of the isolates were toxigenic. Toxinotyping and PCR ribotyping patterns revealed that 6 and 33% of the isolates were identified as NAP1 and NAP7 strains, respectively. This article evidences that the prevalence of C. difficile, specifically pathogenic strains, in the U.S. beef production chain is low.

Aims To determine whether antimicrobials commonly used in swine diets affect zoonotic pathogen shedding in faeces. Methods and Results Barrows (n = 160) were sorted into two treatments at 10 weeks of age (week 0 of the study), and fed growing, grow finishing and finishing diets in 4-week feeding periods. For each feeding phase, diets were prepared without (A−) and with (A+) dietary antimicrobials (chlortetracycline, 0–8 week; bacitracin, 9–12 week) typical of the United States. At week 0, 4, 8, 9, 10 and 12 of the study, faecal swabs or grabs were collected for analyses. Campylobacter spp. was absent at week 0, but prevalence increased over time with most isolates being identified as Campylobacter coli. When chlortetracycline was used in A+ diets (week 4 and 8), prevalence for Campylobacter spp., pathogenic Escherichia coli O26 and stx genes was lower in faeces. On week 12 after the shift to bacitracin, Campylobacter spp. and stx genes were higher in faeces from piglets fed A+ diet. Pathogenic E. coli serogroups O103 and O145 were isolated throughout the study and their prevalence did not differ due to diet. Pathogenic E. coli serogroups O111 and O121 were never found in the piglets, and Salmonella spp. prevalence was low. Conclusions In production swine, growing diets with chlortetracycline may have reduced pathogen shedding compared with the A-growing diets, whereas finishing diets with bacitracin may have increased pathogen shedding compared with the A-finishing diet. Significance and Impact of the Study Inclusion of antimicrobials in the diet can affect zoonotic pathogen shedding in faeces of swine.

Aim: The mammalian intestinal microflora has been shown to impact host physiology. In cattle, intestinal bacteria are also associated with faecal contamination of environmental sources and human illness via foodborne pathogens. Use of wet distillers’ grains with solubles (WDGS) in cattle feed creates a gastrointestinal environment where some bacterial species are enriched. Here, we examine if a diet containing 40% WDGS results in fundamentally different microbial community structures. Methods and Results: The 20 002 16S r-RNA gene sequences from 20 beef cattle were analysed using Sanger sequencing methods. At the genus level, Prevotella (Gram negative) and Anaerobacter (Gram positive) were the most frequently occurring bacteria in our beef cattle faecal samples. Diet-associated differences in prevalence were noted for Prevotella but not Anaerobacter. Conclusions: Diet affects community structure. Faecal communities of co

Feedlot pen soil is a source for transmission of Escherichia coli O157:H7, and therefore a target for preharvest strategies to reduce this pathogen in cattle. The objective of this study was to determine the ability of soil solarization to reduce E. coli O157:H7 in feedlot surface material (FSM). A feedlot pen was identified in which naturally occurring E. coli O157:H7 was prevalent and evenly distributed in the FSM. Forty plots 3 by 3 m were randomly assigned such that five plots of each of the solarization times of 0, 1, 2, 3, 4, 6, 8, and 10 weeks were examined. Temperature loggers were placed 7.5 cm below the surface of each plot, and plots to be solarized were covered with clear 6-mil polyethylene. At each sampling time, five FSM samples were collected from each of five solarized and five unsolarized plots. E. coli concentrations and E. coli O157:H7 presence by immunomagnetic separation and plating were determined for each FSM sample. Initial percentages of E. coli O157:H7–positive samples in control and solarized FSM were 84 and 80%, respectively, and did not differ (P > 0.05). E. coli O157:H7 was no longer detectable by 8 weeks of solarization, but was still detected in unsolarized FSM at 10 weeks. The average initial concentration of E. coli in FSM was 5.56 log CFU/g and did not differ between treatments (P > 0.05). There was a 2.0-log decrease of E. coli after 1 week of solarization, and a >3.0-log reduction of E. coli by week 6 of solarization (P < 0.05). E. coli levels remained unchanged in unsolarized FSM (P > 0.05). Daily peak FSM temperatures were on average 8.7°C higher for solarized FSM compared with unsolarized FSM, and reached temperatures as high as 57°C. Because soil solarization reduces E. coli O157:H7, this technique may be useful for reduction of persistence and transmission of this pathogen in cattle production, in addition to remediation of E. coli O157:H7–contaminated soil used to grow food crops.

Aims: In the United States, carbadox and copper sulfate are growth promoters commonly used in combination in nursery swine diets. Our aim was to determine how selected dietary additives affect selected bacterial populations and pathogens in nursery swine, and compare to larch extract, which contains potential antibacterial activities. Methods and Results: Piglets were weaned and sorted into one of the four treatments: (i) basal diet without antimicrobials; (ii) basal diet with carbadox + copper sulfate; (iii) basal diet + 1000 ppm larch extract; or (iv) basal diet + 2000 ppm larch extract. Diets were fed for a 4-week period after weaning. In both trials, the carbadox + copper sulfate group consumed more feed over the 4-week period relative to the other three diet groups (P

Escherichia coli O157:H7 outbreaks associated with produce consumption have brought attention to livestock manures and manure-based soil amendments as potential sources of pathogens for the contamination of these crops. Procedures for enumeration of E. coli O157:H7 are needed to assess the risks of transmission from these manures and their by-products. A direct plating method employing spiral plating onto CHROMagar O157 was investigated for enumeration of E. coli O157:H7 in feedlot surface material, aged bovine manure, bovine manure compost, and manure-amended soil. In studies utilizing samples spiked with a five-strain cocktail of E. coli O157:H7 at levels ranging from 102 to 10(5) CFU/g of sample, there were strong correlations between the observed and predicted levels of this pathogen. Although the addition of 2.5 mg/liter potassium tellurite and 5 mg/liter novobiocin made the medium more restrictive, these amendments enhanced the ability to identify and enumerate E. coli O157:H7 in feedlot surface material, which contained a higher proportion of fresh feces than did the other three sample types and therefore higher levels of interfering bacterial microflora. The spiral plating method was further assessed to determine its ability to enumerate E. coli O157:H7 in naturally contaminated feedlot surface material. Comparison of E. coli O157:H7 counts in feedlot surface material obtained by the spiral plating method and a most probable number technique were well correlated. We conclude that direct spiral plating onto CHROMagar O157 is effective for estimating E. coli O157:H7 levels in a variety of manures and manure-containing sample types to a lower detection limit of 200 CFU/g. The method has application for determining E. coli O157:H7 concentrations in manures and composts before their sale and use as soil amendments and for measuring the effectiveness of manure treatment processes to reduce or inactivate this pathogen.

A potential source of pathogenic bacteria in ground beef is the lymphatic system, specifically the lymph nodes. Bacteria have been isolated from the lymph nodes of cattle at slaughter; however, most studies have dealt with mesenteric lymph nodes, which are not normally incorporated into ground beef. The objective of the current study was to determine the prevalence and multidrug-resistance status of Salmonella in bovine lymph nodes associated with lean and fat trimmings that might be utilized in ground beef production. Bovine lymph nodes (n = 1,140) were collected from commercial beef processing plants. Half of the lymph nodes sampled were obtained from cull cow and bull processing plants, and the remainder were obtained from fed beef processing plants. Lymph nodes located in chuck and flank adipose tissue were collected for this study. Salmonella prevalence in the lymph node samples was low, with an overall prevalence of 1.6% and a 95% confidence interval of 0.85 to 2.3%. Lymph nodes from cull cattle carcasses had a higher prevalence of Salmonella than did those from fed cattle carcasses. Lymph nodes from the flanks of cow and bull carcasses had the highest prevalence at 3.86%, whereas lymph nodes from the chuck region of fed cattle carcasses had the lowest prevalence at 0.35%. Three of the 18 Salmonella-positive lymph node samples contained multidrug-resistant Salmonella, and all 3 samples were from cull cattle.

Determining the survival of zoonotic pathogens in livestock manure and runoff is critical for understanding the environmental and public health risks associated with these wastes. The occurrence and persistence of the bacterial pathogens Escherichia coli O157:H7 and Campylobacter spp. in a passive beef cattle feedlot runoff control-vegetative treatment system were examined over a 26-mo period. Incidence of the protozoans Cryptosporidium spp. and Giardia spp. was also assessed. The control system utilizes a shallow basin to collect liquid runoff and accumulate eroded solids from the pen surfaces; when an adequate liquid volume is attained, the liquid is discharged from the basin onto a 4.5-ha vegetative treatment area (VTA) of bromegrass which is harvested as hay. Basin discharge transported E. coli O157, Campylobacter spp., and generic E. coli into the VTA soil, but without additional discharge from the basin, the pathogen prevalences decreased over time. Similarly, the VTA soil concentrations of generic E. coli initially decreased rapidly, but lower residual populations persisted. Isolation of Cryptosporidium oocysts and Giardia cysts from VTA samples was infrequent, indicating differences in sedimentation and/or transport in comparison to bacteria. Isolation of generic E. coli from freshly cut hay from VTA regions that received basin discharge (12 of 30 vs. 1 of 30 control samples) provided evidence for the risk of contamination; however, neither E. coli O157 or Campylobacter spp. were recovered from the hay following baling. This work indicates that the runoff control system is effective for reducing environmental risk by containing and removing pathogens from feedlot runoff.

In this study, we conducted a meta-analysis on 16S rRNA gene sequences of bovine fecal origin that are publicly available in the RDP database. A total of 13,663 sequences including 603 isolate sequences were identified in the RDP database (Release 11, Update 1), where 13,447 sequences were assigned to 10 phyla, 17 classes, 28 orders, 59 families, and 110 genera, while the remaining 216 sequences could not be assigned to a known phylum. Firmicutes and Bacteroidetes were the first and the second predominant phyla, respectively. About 41 % of the total sequences could not be assigned to a known genus. The total sequences were assigned to 1252 OTUs at 97 % sequence similarity. A small number of OTUs shared among datasets indicate that fecal bacterial communities of cattle are greatly affected by various factors, specifically diet. This study may guide future studies to further analyze fecal bacterial communities of cattle.

Cattle that were fed an all forage diet had higher numbers of S. bovis than lactobacilli (3×107 versus 4×103). Gradual adaptation of the cattle to 80% cereal grain caused only a modest decline in ruminal pH (always≥5.6), but there was a dramatic decrease in S. bovis and an increase in lactobacilli. The lactobacilli were more resistant to low pH than the S. bovis isolates, but pH alone could not explain the antagonism between ruminal S. bovis and lactobacilli. The ruminal lactobacilli were identified as Lactobacillus fermentum, a bacterium that produces a bacteriocin. Agar overlays, zones of clearing and batch culture growth experiments supported the hypothesis that L. fermentum was producing a bacteriocin that inhibited the growth of S. bovis in the rumen.

Ruminant animals are carriers of Escherichia coli O157:H7, and the transmission of E. coli O157:H7 from cattle to the environment and to humans is a concern. It is unclear if diet can influence the survivability of E. coli O157:H7 in the gastrointestinal system or in feces in the environment. Feces from cattle fed bromegrass hay or corn silage diets were inoculated with E. coli O157:H7, and the survival of this pathogen was analyzed. When animals consumed bromegrass hay for E. coli O157:H7 was not recovered after 28 days postinoculation, but when animals consumed the diet for >1 month, E. coli O157:H7 cells were recovered for >120 days. Viable E. coli O157:H7 cells in feces from animals fed corn silage were detected until day 45 and differed little with the time on the diet. To determine if forage phenolic acids affected the viability of E. coli O157:H7, feces from animals fed corn silage or cracked corn were amended with common forage phenolic acids. When 0.5% trans -cinnamic acid or 0.5% para -coumaric acid was added to feces from silage-fed animals, the E. coli O157:H7 death rate was increased significantly (17-fold and 23-fold, respectively) compared to that with no addition. In feces from animals fed cracked corn, E. coli O157:H7 death rates were increased significantly with the addition of 0.1% and 0.5% trans -cinnamic acid (7- and 13-fold), 0.1% and 0.5% p -coumaric acid (3- and 8-fold), and 0.5% ferulic acid (3-fold). These data suggest that phenolic acids common to forage plants can decrease viable counts of E. coli O157:H7 shed in feces.

The non-metabolizable glucose analog, 2-deoxyglucose (2-DG), decreased the growth rate and optical density of Streptococcus bovis JB1 20%, but it had an even greater effect on stationary phase cultures. Control cultures receiving only glucose (2 mg/ml) lysed very slowly (5% decline in optical density in 48 h), but cultures that had been grown with glucose and 2-DG (2 mg/ml each) lysed much faster (85% decline in optical density in 48 h). Cultures that were treated with inhibitors that decreased intracellular ATP (sodium fluoride, nigericin, and valinomycin or tetrachlorosalicylanilide) or membrane potential (sodium fluoride, nigericin, and valinomycin, tetrachlorosalicylanilide, or phenylmethylsulfonyl fluoride) did not promote lysis. 2-DG had its greatest effect when it was added at inoculation. If 2-DG was added at later times, less lysis was observed, and cells that were given 2-DG just prior to stationary phase were unaffected. Cells that were grown with glucose and 2-DG were more susceptible to cell wall-degrading enzymes (lysozyme and mutanolysin) than cells that had been grown only with glucose, but sublethal doses of penicillin during growth did not promote lysis after the cells had reached stationary phase. The idea that 2-DG might be affecting autolytic activity was supported by the observation that cultures washed and resuspended in fresh medium with or without 2-DG lysed at a slower rate than cultures that were not centrifuged or were resuspended in the culture supernatant.

Reducing Escherichia coli O157:H7 in livestock manures before application to cropland is critical for reducing the risk of foodborne illness associated with produce. Our objective was to determine the fate of naturally occurring E. coli O157:H7 and other pathogens during minimally managed on-farm bovine manure composting processes. Feedlot pen samples were screened to identify E. coli O157:H7-positive manure. Using this manure, four piles of each of three different composting formats were constructed in each of two replicate trials. Composting formats were (i) turned piles of manure plus hay and straw, (ii) static stockpiles of manure, and (iii) static piles of covered manure plus hay and straw. Temperatures in the tops, toes, and centers of the conical piles (ca. 6.0 m(3) each) were monitored. Compost piles that were turned every 2 weeks achieved higher temperatures for longer periods in the tops and centers than did piles that were left static. E. coli O157:H7 was not recovered from top samples of turned piles of manure plus hay and straw at day 28 and beyond, but top samples from static piles were positive for the pathogen up to day 42 (static manure stockpiles) and day 56 (static covered piles of manure plus hay and straw). Salmonella, Campylobacter spp., and Listeria monocytogenes were not found in top or toe samples at the end of the composting period, but E. coli O157:H7 and Listeria spp. were recovered from toe samples at day 84. Our findings indicate that some minimally managed composting processes can reduce E. coli O157:H7 and other pathogens in bovine manure but may be affected by season and/or initial levels of indigenous thermophilic bacteria. Our results also highlight the importance of adequate C:N formulation of initial mixtures for the production of high temperatures and rapid composting, and the need for periodic turning of the piles to increase the likelihood that all parts of the mass are subjected to high temperatures.

Studies using 15N have indicated that as much as 50% of the microbial mass turns over before N passes to the lower gut, and this N recycling significantly decreases the availability of microbial protein. Protozoa digest bacteria and smaller protozoa, but bacterial protein can turn over even if protozoa are not present. Fibrobacter succinogenes cultures lyse even when they are growing, and the lysis rate is independent of growth rate. When extracellular sugar is depleted, F. succinogenes secretes an extracellular proteinase that inactivates the autolysins. This method of autolytic regulation decreases the turnover of stationary cells. Bacteriophage and anaeroplasma can cause lysogeny, but, as yet, there is little proof that these processes are important determinants of bacterial turnover in vivo. Dietary manipulations (e.g., salt feeding and particle size reduction) that increase liquid and solid dilution rates can increase bacterial flow by decreasing bacterial residence time and turnover. Some dead ruminal bacteria are able to maintain their cellular integrity, and the ratio of dead to live cells in ruminal fluid may be as great as 10:1. Bacterial survival appears to be at least partially explained by the method of sugar transport. When bacteria rely solely on mechanisms of ion-coupled sugar symport, an energized membrane is necessary for the reinitiation of growth. If group translocation (phosphotransferase system) is the mechanisms of transport, uptake can be driven by phosphoenolpyruvate, and an energized membrane and the storage of intracellular reserve materials are not an absolute criteria for survival. In some cases, N deprivation accelerates death. When Prevotella ruminicola was limited for N under conditions of excess energy, methylglyoxal production caused a rapid decrease in viability. The impact of bacterial death in the rumen is not clear-cut. If the rate of fermentation is zero-order with respect to cell concentration (substrate-limited), cell death would have little impact on digestion.

Growing cultures of Fibrobacter succinogenes assimilated more ammonia than could be accounted for by cellular protein, RNA, or DNA and released large amounts of nonammonia nitrogen. The difference between net and true growth was most dramatic at low dilution rates, but mathematical derivations indicated that the lysis rate was a growth rate-independent function. The lysis rate was sevenfold greater than the true maintenance rate (0.07 h-1 versus 0.01 h-1). Because slowly growing cells had as much proton motive force and ATP as fast-growing cells, lysis was not a starvation response per se. Stationary-phase cells had a lysis rate that was 10-fold less than that of growing cells. Rapidly growing cells were not susceptible to phenylmethylsulfonyl fluoride, but phenylmethylsulfonyl fluoride increased the lysis rate of the cultures when they reached the stationary phase. This latter result indicated that autolysins of stationary-phase cells were being inactivated by a serine proteinase. When growing cells were treated with the glycolytic inhibitor iodoacetate, the proteinase-dependent transition to the stationary phase was circumvented, and the rate of lysis could be increased by as much as 50-fold.

Prevotella ruminicola B(1)4, TC1-1, TF1-3, and TS1-5 all produced immunologically cross-reacting 88- and 82-kDa carboxymethyl cellulases (CMCases). P. ruminicola 23, 118B, 20-63, and 20-78 had much lower CMCase activities, and Western blots (immunoblots) showed no cross-reaction with the B(1)4 CMCase antiserum. Fibrobacter succinogenes S85 and Selenomonas ruminantium HD4 and D produced CMCase, but these enzymes were smaller and did not cross-react with the B(1)4 CMCase antiserum. The B(1)4 CMCase antiserum inhibited the B(1)4, TC1-1, TF1-3, and TS1-5 CMCase activities and agglutinated these cells, but it had no effect on the other strains or species. On the basis of these results, the B(1)4 CMCase is a strain-specific enzyme that is located on the outside surface of the cells. P. ruminicola B(1)4 cultures, grown on sucrose, did not have significant CMCase activity, but these cells could bind purified 88- and 82-kDa CMCase but not 40.5-kDa CMCase. Because the 40.5-kDa CMCase is a fully active, truncated form of the CMCase, it appears that the N-terminal domain of the 88-kDa B(1)4 CMCase anchors the CMCase to the cells. Cells grown on cellobiose produced at least 10-fold more CMCase than the sucrose-grown cells, and the cellobiose-grown cells could only bind 15% as much CMCase as sucrose-grown cells. Virtually all of the CMCase activity of exponentially growing cultures was cell associated, but CMCase activity was eventually detected in the culture supernatant. On the basis of the observation that the 88-kDa CMCase was gradually converted to the 82-kDa CMCase when cultures reached the stationary phase without a change in specific activity, it appears that the 82-kDa protein is probably a proteolytic degradation product of the 88-kDa CMCase.

The beta-1,4-endoglucanase of the ruminal bacterium, Prevotella ruminicola B14, hydrolysed carboxymethylcellulose and barley glucan but not xylan or mannan. Endoglucanase activity was present in 88- and 82-kDa proteins, and there was at least a 20-fold variation in endoglucanase activity when P. ruminicola B14 was grown on different sugars. The highest activities were observed with mannose, cellobiose or xylose and little activity was observed with sucrose, arabinose or rhamnose, P. ruminicola B14 also had significant xylanase and mannanase activities, but these activities were present in proteins that had lower molecular masses than the endoglucanase and these proteins did not cross-react with antibody made against the endoglucanase. Mannanase activity has a similar pattern of expression to the endoglucanase, while the xylanase was not induced or repressed by the same sugars or combinations of sugars. The xylanase activity was greatest when xylan was the energy source for growth, but xylose was a very poor inducer of xylanase activity.

Mixed ruminal bacteria convert trans-aconitate to tricarballylate, a tricarboxylic acid which chelates blood divalent cations and decreases their availability (J. B. Russell and P. J. Van Soest, Appl. Environ. Microbiol. 47:155-159, 1984). Decreases in blood magnesium in turn cause a potentially fatal disease known as grass tetany. trans-Aconitate was stoichiometrically reduced to tricarballylate by Selenomonas ruminantium, a common ruminal bacterium in grass-fed ruminants (J. B. Russell, Appl. Environ. Microbiol. 49:120-126, 1985). When mixed ruminal bacteria were enriched with trans-aconitate, a trans-aconitate-oxidizing bacterium was also isolated (G. M. Cook, F. A. Rainey, G. Chen, E. Stackebrandt, and J. B. Russell, Int. J. Syst. Bacteriol. 44:576-578, 1994). The trans-aconitate-oxidizing bacterium was identified as Acidaminococcus fermentans, and it converted trans-aconitate to acetate, a nontoxic end product of ruminal fermentation. When S. ruminantium and A. fermentans were cocultured with trans-aconitate and glucose, tricarballylate never accumulated and all the trans-aconitate was converted to acetate. Continuous-culture studies (dilution rate, 0.1 h-1) likewise indicated that A. fermentans could outcompete S. ruminantium for trans-aconitate. When mixed ruminal bacteria were incubated in vitro with 10 mM trans-aconitate for 24 h, 45% of the trans-aconitate was converted to tricarballylate. Tricarballylate production decreased 50% if even small amounts of A. fermentans were added to the incubation mixes (0.01 mg of protein per mg of mixed bacterial protein). When A. fermentans (2 g of bacterial protein) was added directly to the rumen, the subsequent conversion of trans-aconitate to tricarballylate decreased 50%, but this effect did not persist for more than 18 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Cattle fed finishing diets with wet distillers grains with solubles (WDGS) have been shown to harbor increased Escherichia coli O157:H7 populations in the feces and on the hides. To determine if feeding a lower level of WDGS at the end of the feeding period reduces E. coli O157:H7 load at harvest, 608 heifers were sorted into one of five treatments and fed 0, 40, or 70% WDGS (dry matter basis). For three of the treatments, WDGS was reduced midway through the study. Treatment 0W0W heifers (positive control) were fed a corn grain-based diet continuously, and 40W40W heifers (negative control) were fed 40% WDGS continuously. Heifers subjected to treatments 40W0W, 40W15W, and 70W15W were fed either 40 or 70% WDGS for the first 56 days and switched to 0 or 15% WDGS, respectively, for the last 56 days. Prior to the switch in diets, animals fed diets with 40 or 70% had higher prevalence and percent enumerable fecal samples for E. coli O157:H7. After the dietary switch, animals fed 40W0W, 40W15W, and 70W15W diets had fecal prevalence and percent enumerable samples (33.4 and 6.3%, 31.0 and 9.7%, and 34.9 and 8.4%, respectively) similar to those of animals fed 0W0W diets (10.2 and 3.2%, respectively; P0.05), whereas animals fed 40W40W had the highest fecal prevalence and percent enumerable samples (70.1 and 29.2%, respectively; P0.05). Similar relationships between the treatments were observed for hide samples. Time after dietary switch was important, as animals fed lower levels had significantly lower fecal prevalence and percent enumerable samples after 56 days, but not after 28 days. The study indicates that cattle can be switched to lower levels of dietary WDGS (15% or less) 56 days prior to harvest to significantly reduce E. coli O157:H7 in feces and on hides.

Reducing Escherichia coli O157:H7 in cattle and their manure is critical for reducing the risk for human foodborne and waterborne illness. The objective of this study was to evaluate the effects of soil and pond ash surfaces for feedlot pens on the prevalence, levels, and/or persistence of naturally occurring E. coli O157:H7 and total E. coli in cattle (feces and hides) and manure. Cattle (128 beef heifers) were sorted among 16 pens: 8 surfaced with soil and 8 surfaced with pond ash. The prevalence of E. coli O157:H7 in feces decreased (P0.0001) during the study from 57.0% on day 0 to 3.9% on day 84 but did not differ (Por = 0.05) between cattle on soil and on pond ash pens at any sampling period. The prevalence of the pathogen on hides and in feedlot surface material (FSM) also decreased (P0.0001), with no effect of soil or pond ash surface (Por = 0.05). Similarly, levels of E. coli in FSM did not differ (Por = 0.05) at any sampling period, and there were no clear trends for survival differences of E. coli O157:H7 or E. coli in FSM between pond ash and soil surfaces, although E. coli populations survived at 5.0 log CFU/g of FSM on the pen surfaces 6 weeks after the cattle were removed. These results indicate that housing cattle on pens surfaced with pond ash versus pens surfaced with soil does not affect E. coli O157:H7 in cattle or their manure.

Direct-fed microbials (DFM) have been identified as potential preharvest interventions for the reduction of foodborne bacterial pathogens such as Escherichia coli O157:H7. This study evaluated the efficacy of a DFM consisting of Bacillus subtilis strain 166 as an antimicrobial intervention strategy for the reduction of prevalence and load of E. coli O157:H7 in feces and on hides of feedlot cattle. Cattle (n = 526) were divided among 16 feedlot pens. Half of the pens received the DFM, and the other half did not. Hide and fecal samples were collected from each animal on days 28, 63, and 84 of the feeding trial. Over the course of the 84-day feeding period, there were no significant differences observed between treatments for either hide or fecal prevalence of E. coli O157:H7, or for the percentage of animals that were shedding E. coli O157:H7 at high levels (or =200 CFU/g) in their feces or harboring E. coli O157:H7 at high levels (or =40 CFU/cm(2)) on their hides. In addition, there was no significant difference between the average daily gains for the treated and control groups, with both groups averaging 1.3 kg/day. We concluded that the DFM tested would not be an effective preharvest intervention against E. coli O157:H7.

Escherichia coli O157:H7 is a zoonotic pathogen that is an important cause of human foodborne and waterborne disease, with a spectrum of illnesses ranging from asymptomatic carriage and diarrhea to the sometimes fatal hemolytic uremic syndrome. Outbreaks of E. coli O157:H7 disease are often associated with undercooked beef, but there are other sources of transmission, including water, produce, and animal contact, which can often be linked directly or indirectly to cattle. Thus, preharvest control of this pathogen in cattle production should have a large impact on reducing the risk of human foodborne illness. In this review, we will summarize preharvest research on E. coli O157:H7 in cattle and the production environment, focusing on factors that may influence the transmission, prevalence, and levels of this pathogen, such as season, diet, high-level shedders, and animal stress. In addition, we will discuss recent research on the reduction of this pathogen in cattle production, including vaccination, probiotics, bacteriophage, and manure treatments.

The objective of this study was to determine if wet distillers grains with solubles (WDGS) from corn in diets affected Escherichia coli O157:H7 in growing and finishing cattle; steers (n = 603) were randomly assigned to diets with or without WDGS. Hide and fecal samples were collected monthly (October through June) from each animal for enumeration and enrichment of E. coli O157:H7. In the growing phase (0 or 13.9% WDGS diets), fecal prevalence for E. coli O157:H7 in steers fed a diet with WDGS was twice that of the prevalence in control steers (P0.001). In the finishing phase (0 or 40% WDGS diets), the average prevalence in feces (P0.001) and on hides (P0.001) was higher for cattle fed WDGS. The average percentage of fecal E. coli O157:H7 enumerable samples during the finishing phase for cattle fed WDGS was 2.7% compared with 0.1% for control steers (P0.001). The average percentage of E. coli O157:H7 enumerable hide samples was not different between diets, but the cattle fed WDGS had higher levels (P0.05) of the pathogen. Animals fed WDGS had higher levels of E. coli (P0.001), higher pH values (P0.001), and lower concentrations of L-lactate (P0.001) in feces than those values of the control steers. These results indicate that feeding 40% WDGS could increase the level and prevalence of E. coli O157:H7 in and on feedlot cattle when E. coli O157:H7 is seasonally low.

Clinical associations between Crohn's disease in humans and Mycobacterium avium subsp. paratuberculosis (MAP) have been suggested but not confirmed. Cattle could be sources for MAP, but little information on MAP prevalence with beef has been reported. Samples of ileocecal lymph nodes and swabs of hides and carcasses from 343 animals at cull cattle slaughtering facilities and 243 animals at fed cattle slaughtering facilities across the United States were analyzed for the presence of MAP. Amplification of genetic sequences detected MAP DNA predominantly on hides and in lymph nodes of samples taken at both types of processing facilities. More than 34% of the cattle at cull cow slaughtering facilities had ileocecal lymph nodes that tested positive for MAP DNA. From these same cattle, hide prevalence was more than twofold greater than the prevalence in ileocecal lymph nodes, suggesting that cross-contamination could be occurring during transport and lairage. The prevalence of MAP DNA decreased during processing, and less than 11% of the carcasses tested positive after interventions in the cull cow processing facilities. Using standard double-decontamination and culture techniques, less than 1% of the postintervention carcasses tested positive for viable MAP at cull cow facilities. In samples from the facilities processing only fed cattle, MAP prevalence of 1% or less was detected for ileocecal lymph node, hide, and carcass samples, and viable MAP was not detected. Based on this study, fed cattle carcasses are unlikely sources of MAP, and carcasses at cull cow plants have only a slight risk for transmitting viable MAP, due to current interventions.

The reduction of foodborne pathogens in cattle destined for human consumption will require knowledge of the factors that impact the carriage and shedding of these organisms. The objective of this work was to investigate the effects of heat and handling stress levels on the fecal shedding of Escherichia coli O157:H7 and generic E. coli by feedlot cattle. In year 1, 128 feedlot heifers were evaluated for heat tolerance five times per week during the 84-day finishing period from May through August. Heat stress measurements included respiration rate, panting score, and visual assessments. In year 2, panting scores were taken for a group of 256 finishing feedlot heifers on days in July and August for which the temperature humidity index (THI) was predicted to be in the "emergency" category (THIor = 84). For both years, animals were weighed and temperament scored to assess handling stress on a 28-day schedule. At the same time, rectal fecal samples were collected from each animal individually. The presence and concentrations of E. coli O157:H7 and concentrations of generic E. coli in feces were determined. There were no clear trends between the heat stress levels or temperament scores (as an indicator of response to handling) with either fecal generic E. coli concentrations or E. coli O157:H7 concentrations or prevalence in feces, indicating that neither heat nor handling stress contributes to the food safety risk associated with E. coli O157:H7-positive cattle.

Pathogens, ammonia, odor, and greenhouse gas emissions are serious environmental concerns associated with swine production. This study was conducted in two manure pits (33,000 L each) to determine the influence of 1.5 or 3.0 g thymol L(-1) and 80 mg L(-1) urease inhibitor amendments on urea accumulation, coliform bacteria, odor, and methane emission. Each experiment lasted 18 or 19 d, during which time 30 to 36 250-mL samples (six per day) were withdrawn from underneath each pit and analyzed for urea, thymol, volatile fatty acids, coliform bacteria, and Campylobacter. At the end of each experiment, six 50-g samples from each pit were placed in serum bottles, and gas volume and composition were determined periodically for 28 d. Compared with the control pit, volatile fatty acids production was reduced 64 and 100% for the thymol amendments of 1.5 and 3.0 g L(-1), respectively. Viable coliform cells were reduced 4.68 and 5.88 log10 colony-forming units kg(-1) of slurry for the 1.5 and 3.0 g thymol L(-1), respectively, and Escherichia coli were reduced 4.67 and 5.01 log10 colony-forming units kg(-1) of slurry, respectively. Campylobacter was not detected in the pits treated with thymol, in contrast to 63% of the samples being positive for the untreated pit. Urea accumulated in the treated pits from Day 3 to 6. Total gas production from serum bottles was reduced 65 and 76% for thymol amendments of 1.5 and 3.0 g L(-1), respectively, and methane was reduced 78 and 93%, respectively. These results suggest that thymol markedly reduces pathogens, odor, and greenhouse gas emissions from a swine production facility. The urease inhibitor produced a temporary response in conserving urea.

When Escherichia coli XL1-Blue MRA (P2) was infected with lambda DNA containing Prevotella ruminicola B(1)4 chromosomal DNA, only a few plaques produced beta-1,4-endoglucanase activity, and all of these had mannanase activity. Positive phage contained a 17-kb SacI DNA fragment that gave six bands after EcoRI digestion. The EcoRI fragments were ligated into pBluescript and sequenced. The order of the fragments was verified by PCR and by restriction mapping. The DNA sequence contained 6 open reading frames (ORFs). The 4th and 5th ORFs encoded two related beta-1,4-endoglucanases. E. coli clones carrying ORF5 and ORF6 had beta-1,4-endoglucanase and mannanase activities, while a clone carrying only ORF6 hydrolyzed mannan but not carboxymethylcellulose. The 6th ORF had three regions of homology to mannanase A from Pseudomonas fluorescens. Based on these results, ORF6 encoded the mannanase gene. The 3rd ORF had 10 regions of homology with cellulose-binding protein A from Clostridium cellulovorans. The 1st and 2nd ORFs had no significant homology to genes or amino acid sequences in GeneBank or SwissProt. All of the ORFs except 1 encoded a potential signal peptide sequence. The upstream region of ORF1 contained four direct repeats and four inverted repeat elements, but no apparent sigma70 sequence-like promoter was present. The segment of DNA containing the 6 ORFs was preceded and followed by potential transcription termination signals suggesting a single transcriptional unit.