Determination of gastrointestinal tract colonization sites from feedlot cattle transiently shedding or super-shedding Escherichia coli O157:H7 at harvest.

AIMS: The objective of the study was to determine levels of Escherichia coli O157:H7 colonization in the gastrointestinal tract (GIT) of naturally shedding cattle shedding the pathogen at low- or super-shedder levels. METHODS AND RESULTS: Over 2 years, feedlot cattle were sampled multiple times for faecal shedding of E. coli O157:H7. Just prior to harvest (1-2 days), animals that were super-shedders (≥104  CFU per gram of faeces) were specifically identified, and based on the longer term screening data, pen cohorts that were low-shedders (years 1 and 2) or chronic-shedders (year 1) were also identified. At harvest, samples were collected from throughout the GIT, including the rectoanal junction (RAJ) for enumeration and enrichment of E. coli O157:H7. The mouth samples exhibited the greatest prevalence for the pathogen, and the abomasum and rumen exhibited the lowest prevalence (P < 0·05). Super-shedders had significantly greater prevalence for all GIT locations except the mouth and abomasum compared to low-shedders, but the super-shedders were the only animals with positive abomasum samples. Samples from the super-shedders were enumerable for most GIT locations, and the rectum and RAJ locations were the only locations that were significantly greater than other locations (P < 0·05). CONCLUSIONS: Across all animals naturally exposed to E. coli O157:H7, the risk of ingestion is high, but rumen and abomasum are potential barriers to passage. In super-shedders, the passage through the GIT was greater, allowing colonization in the rectum and at the RAJ. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli O157:H7 low-shedding cattle had lower pathogen levels throughout the GIT, indicating intrinsic GIT factors to these cattle may reduce pathogen passage through the GIT, including the abomasum, and minimize risk of RAJ colonization.

The effects of monensin in diets fed to finishing beef steers and heifers on growth performance and fecal shedding of Escherichia coli O157:H7.

Two experiments were conducted to study the effects of monensin dose on growth performance and O157:H7 shedding in finishing beef cattle. In Exp. 1, 198 heifers (298 ± 1.1 kg BW) were allocated to 1 of 2 treatments consisting of 1) 200 mg/heifer daily of monensin and 2) 400 mg/heifer daily of monensin and fed for 151 d. In Exp. 2, 199 steers (430 ± 1.9 kg BW) were stratified by BW and allocated to 1 of 2 treatments consisting of 1) 0 mg/steer daily of monensin and 2) 400 mg/steer daily of monensin and fed for 128 d. For both experiments, there were 4 pen replicates per treatment. For Exp. 1 and Exp. 2, the model included the fixed effect of treatment for growth performance measures and the fixed effects of treatment, time, and treatment × time interaction, respectively, for O157:H7 shedding. In Exp. 1, final BW was 1.9% greater for heifers fed 400 mg/d monensin than for heifers fed 200 mg/d monensin ( = 0.05). Furthermore, ADG was 4.9% greater ( = 0.05) and G:F was 3.1% greater ( = 0.04) when the heifers were fed 400 mg/d monensin vs. 200 mg/d monensin. Pen prevalence for O157:H7 ( = 0.96) and the percentage of animals in the pen shedding O157:H7 at enumerable levels ( = 0.82) did not differ between heifers fed 200 mg/d monensin and heifers fed 400 mg/d monensin over the 4 sampling periods. For Exp. 2, steers fed the supplement containing monensin had a 1.9% greater final BW ( = 0.04) and a 5.2% greater ADG ( = 0.02) than steers fed a control supplement without monensin. No differences in DMI or G:F were noted across the treatments ( ≥ 0.14). O157:H7 percentage of enumerable cattle within the pen was greater for the steers fed monensin than the control steers not fed monensin than the control steers not fed monensin ( = 0.02) over the 4 sampling periods. However, the percentage of animals in the pen shedding O157:H7 (prevalence positive) did not differ between treatments ( = 0.18), nor did the average fecal counts ( = 0.45). In conclusion, feeding a higher dose (400 mg/d) of monensin improved final BW and ADG compared with a low dose of monensin or a no-monensin control in steers and heifers across multiple years. The percentage of animals shedding O157:H7 at enumerable levels was greater for steers fed the monensin supplement than for steers fed the control supplement, yet the presence of monensin, irrespective of the dose, did not affect the percentage of animals in the pen shedding O157:H7.

The impact of the bovine faecal microbiome on Escherichia coli O157:H7 prevalence and enumeration in naturally infected cattle.

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 < 0·001) with prevalence or enumeration, explaining as much as 50% of variability in E. coli O157:H7 prevalence or enumeration. CONCLUSIONS: Individual colonic bacterial species have little impact on E. coli O157:H7 shedding but collectively groups of bacteria were strongly associated with pathogen shedding. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial groups in the bovine colon may impact faecal shedding of the zoonotic pathogen E. coli O157:H7, and manipulation of the intestinal microbiota to alter these bacteria may reduce shedding of this pathogen and foodborne illnesses.

Meat Science and Muscle Biology Symposium: Escherichia coli O157:H7, diet, and fecal microbiome in beef cattle.

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.

Investigation of bacterial diversity in the feces of cattle fed different diets.

The objective of this study is to investigate individual animal variation of bovine fecal microbiota including as affected by diets. Fecal samples were collected from 426 cattle fed 1 of 3 diets typically fed to feedlot cattle: 1) 143 steers fed finishing diet (83% dry-rolled corn, 13% corn silage, and 4% supplement), 2) 147 steers fed late growing diet (66% dry-rolled corn, 26% corn silage, and 8% supplement), and 3) 136 heifers fed early growing diet (70% corn silage and 30% alfalfa haylage). Bacterial 16S rRNA gene amplicons were determined from individual fecal samples using next-generation pyrosequencing technology. A total of 2,149,008 16S rRNA gene sequences from 333 cattle with at least 2,000 sequences were analyzed. Firmicutes and Bacteroidetes were dominant phyla in all fecal samples. At the genus level, Oscillibacter, Turicibacter, Roseburia, Fecalibacterium, Coprococcus, Clostridium, Prevotella, and Succinivibrio were represented by more than 1% of total sequences. However, numerous sequences could not be assigned to a known genus. Dominant unclassified groups were unclassified Ruminococcaceae and unclassified Lachnospiraceae that could be classified to a family but not to a genus. These dominant genera and unclassified groups differed (P < 0.001) with diets. A total of 176,692 operational taxonomic units (OTU) were identified in combination across all the 333 cattle. Only 2,359 OTU were shared across 3 diet groups. UniFrac analysis showed that bacterial communities in cattle feces were greatly affected by dietary differences. This study indicates that the community structure of fecal microbiota in cattle is greatly affected by diet, particularly between forage- and concentrate-based diets.

Proposed model for the high rate of rearrangement and rapid migration observed in some IncA/C plasmid lineages.

IncA/C plasmids are a class of plasmids from the Enterobacteriaceae that are relatively large (49 to >180 kbp), that are readily transferred by conjugation, and that carry multiple antimicrobial resistance genes. Reconstruction of the phylogeny of these plasmids has been difficult because of the high rate of remodeling by recombination-mediated horizontal gene transfer (HGT). We hypothesized that evaluation of nucleotide polymorphisms relative to the rate of HGT would help to develop a clock to show whether anthropic practices have had significant influences on the lineages of the plasmid. A system was developed to rapidly sequence up to 191 known open reading frames from each of 39 recently isolated IncA/C plasmids from a diverse panel of Salmonella enterica and Escherichia coli strains. With these data plus sequences from GenBank, we were able to distinguish six distinct lineages that had extremely low numbers of polymorphisms within each lineage, especially among the largest group designated as group 1. Two regions, each about half the plasmid in size, could be distinguished with a separate lineal pattern. The distribution of group 1 showed that it has migrated extremely rapidly with fewer polymorphisms than can be expected in 2,000 years. Remodeling by frequent HGT was evident, with a pattern that appeared to have the highest rate just upstream of the putative conjugation origin of transfer (oriT). It seems likely that when an IncA/C plasmid is transferred by conjugation there is an opportunity for plasmid remodeling adjacent to the oriT, which was also adjacent to a multiple antimicrobial resistance gene cassette.

Comparison of bacterial communities in faeces of beef cattle fed diets containing corn and wet distillers' grain with solubles.

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-housed beef cattle are not identical. SIGNIFICANCE AND IMPACT OF THE STUDY: It is known that a diet of 40% corn-based WDGS increases the generic Escherichia coli in the faeces and enriches E. coli O157:H7. The results from the current study suggest that in addition to previously observed changes in E. coli, the entire bacterial community structure is different for animals fed 40% corn-based WDGS compared to a traditional corn-finishing diet.

Interferon type I response in porcine reproductive and respiratory syndrome virus-infected MARC-145 cells.

Infection by porcine reproductive and respiratory syndrome virus (PRRSV) results in a weak induction of the innate immune response. There are many genes that collectively comprise this response and the extent to which each gene responds to PRRSV infection is unclear and warrants further investigation. To this end, we have utilized real-time PCR using SYBR Green I dye-based detection to quantify transcript abundance of the type I interferons (IFN-alpha and -beta) and IFN-beta transcriptional enhanceasome genes. In MARC-145 cells, both IFN-alpha and -beta transcript abundance were unaffected by PRRSV infection. However, stimulation of MARC-145 cells by exogenous double-stranded RNA, resulted in significant increases in transcript abundance of both IFN-alpha and -beta as well as IFN-beta enhanceasome components, indicating that a type I IFN response could be induced in these cells. The double-stranded RNA induction of type I IFN transcription was significantly inhibited by dual-exposure with PRRSV. These results suggest that PRRSV infection directly interferes with type I IFN transcriptional activation early in its pathway, at the level of IFN-beta gene transcription.

Genetics and regulation of chitobiose utilization in Borrelia burgdorferi.

Borrelia burgdorferi spends a significant proportion of its life cycle within an ixodid tick, which has a cuticle containing chitin, a polymer of N-acetylglucosamine (GlcNAc). The B. burgdorferi celA, celB, and celC genes encode products homologous to transporters for cellobiose and chitobiose (the dimer subunit of chitin) in other bacteria, which could be useful for bacterial nutrient acquisition during growth within ticks. We found that chitobiose efficiently substituted for GlcNAc during bacterial growth in culture medium. We inactivated the celB gene, which encodes the putative membrane-spanning component of the transporter, and compared growth of the mutant in various media to that of its isogenic parent. The mutant was no longer able to utilize chitobiose, while neither the mutant nor the wild type can utilize cellobiose. We propose renaming the three genes chbA, chbB, and chbC, since they probably encode a chitobiose transporter. We also found that the chbC gene was regulated in response to growth temperature and during growth in medium lacking GlcNAc.

Transduction by phiBB-1, a bacteriophage of Borrelia burgdorferi.

We previously described a bacteriophage of the Lyme disease agent Borrelia burgdorferi designated phiBB-1. This phage packages the host complement of the 32-kb circular plasmids (cp32s), a group of homologous molecules found throughout the genus Borrelia. To demonstrate the ability of phiBB-1 to package and transduce DNA, a kanamycin resistance cassette was inserted into a cloned fragment of phage DNA, and the resulting construct was transformed into B. burgdorferi CA-11.2A cells. The kan cassette recombined into a resident cp32 and was stably maintained. The cp32 containing the kan cassette was packaged by phiBB-1 released from this B. burgdorferi strain. phiBB-1 has been used to transduce this antibiotic resistance marker into naive CA-11.2A cells, as well as two other strains of B. burgdorferi. This is the first direct evidence of a mechanism for lateral gene transfer in B. burgdorferi.

Telomere resolution in the Lyme disease spirochete.

The genus Borrelia includes the causative agents of Lyme disease and relapsing fever. An unusual feature of these bacteria is a genome that includes linear DNA molecules with covalently closed hairpin ends referred to as telomeres. We have investigated the mechanism by which the hairpin telomeres are processed during replication. A synthetic 140 bp sequence having the predicted structure of a replicated telomere was shown to function as a viable substrate for telomere resolution in vivo, and was sufficient to convert a circular replicon to a linear form. Our results suggest that the final step in the replication of linear Borrelia replicons is a site-specific DNA breakage and reunion event to regenerate covalently closed hairpin ends. The telomere substrate described here will be valuable both for in vivo manipulation of linear DNA in Borrelia and for in vitro studies to identify and characterize the telomere resolvase.

Isolation of a circular plasmid region sufficient for autonomous replication and transformation of infectious Borrelia burgdorferi.

Borrelia burgdorferi contains abundant circular and linear plasmids, but the mechanism of replication of these extrachromosomal elements is unknown. A B. burgdorferi 9 kb circular plasmid (cp9) was amplified in its entirety by the polymerase chain reaction and used to construct a shuttle vector that replicates in Escherichia coli and B. burgdorferi. A 3.3 kb region of cp9 containing three open reading frames was used to construct a smaller shuttle vector, designated pBSV2. This vector was stably maintained in B. burgdorferi, indicating that all elements necessary for autonomous replication are probably located on this 3.3 kb fragment. A non-infectious B. burgdorferi strain was efficiently transformed by pBSV2. Additionally, infectious B. burgdorferi was also successfully transformed by pBSV2, indicating that infectious strains of this important human pathogen can now be genetically manipulated.

Genetic diversity and transcriptional analysis of the bys1 gene from Blastomyces dermatitidis.

Blastomyces dermatitidis, a pathogenic fungal organism, is able to exist in two different morphologies, a multicellular mycelium or a unicellular yeast, according to temperature, 25 degrees C and 37 degrees C respectively. The switching between morphologies must be accompanied by a cascade of signaling events in which expression of genes responsible for the change of morphology is increased or decreased. bys1, a gene from B. dermatitidis isolate #58, is expressed at high levels in the unicellular yeast, but gradually diminishes as the temperature is lowered and the organism converts to the mycelial phase where there is no transcription of bys1. We explored if bys1 homologs are found in other B. dermatitidis isolates and if the transcription of the homologs were regulated by temperature. bys1 was identified in all B. dermatitidis isolates tested and could be grouped into two classes by Southern blot, PCR, and DNA sequence. Although the bys1 transcripts of both classes were regulated by temperature, transcription rates varied between the three isolates tested.

DNA exchange and insertional inactivation in spirochetes.

Spirochetes have complex life cycles and are associated with a number of diseases in humans and animals. Despite their significance as pathogens, spirochete genetics are in their early stages. However, gene inactivation has been achieved in Borrelia burgdorferi, Brachyspira hyodysenteriae, and Treponema denticola. Here, we review methods that have been used in spirochetes for gene inactivation and DNA exchange, with a primary focus on B. burgdorferi. We also describe factors influencing electrotransformation in B. burgdorferi. In summary, optimal transformation frequencies are obtained with log phase bacteria, large amounts of DNA (up to 50 microg per transformation), and high field strength (12.5-37.5 kV/cm). Infectious B. burgdorferi isolates transform with frequencies 100-fold lower than those found for high passage, non-infectious strains. Surface characteristics of the bacteria, which often correlate with infectivity, are among the obstacles to effective transformation by electroporation.

A second allele of eppA in Borrelia burgdorferi strain B31 is located on the previously undetected circular plasmid cp9-2.

Although sequence analysis of Borrelia burgdorferi isolate B31 was recently declared "complete," we found that cultures of this strain can contain a novel 9-kb circular plasmid, cp9-2. The newly described plasmid contains both sequence similarities with and differences from the previously identified B31 plasmid cp9-1 (formerly cp9). cp9-1 and cp9-2 each encode a unique allele of EppA, a putative membrane protein synthesized by B. burgdorferi during mammalian infection.

Borrelia burgdorferi periplasmic flagella have both skeletal and motility functions.

Bacterial shape usually is dictated by the peptidoglycan layer of the cell wall. In this paper, we show that the morphology of the Lyme disease spirochete Borrelia burgdorferi is the result of a complex interaction between the cell cylinder and the internal periplasmic flagella. B. burgdorferi has a bundle of 7-11 helically shaped periplasmic flagella attached at each end of the cell cylinder and has a flat-wave cell morphology. Backward moving, propagating waves enable these bacteria to swim in both low viscosity media and highly viscous gel-like media. Using targeted mutagenesis, we inactivated the gene encoding the major periplasmic flagellar filament protein FlaB. The resulting flaB mutants not only were nonmotile, but were rod-shaped. Western blot analysis indicated that FlaB was no longer synthesized, and electron microscopy revealed that the mutants were completely deficient in periplasmic flagella. Wild-type cells poisoned with the protonophore carbonyl cyanide-m-chlorophenylhydrazone retained their flat-wave morphology, indicating that the periplasmic flagella do not need to be energized for the cell to maintain this shape. Our results indicate that the periplasmic flagella of B. burgdorferi have a skeletal function. These organelles dynamically interact with the rod-shaped cell cylinder to enable the cell to swim, and to confer in part its flat-wave morphology.

Expression and immunological analysis of the plasmid-borne mlp genes of Borrelia burgdorferi strain B31.

A lipoprotein gene family first identified in Borrelia burgdorferi strain 297, designated 2.9 LP and recently renamed mlp, was found on circular and linear plasmids in the genome sequence of B. burgdorferi strain B31-M1. Sequence analyses of the B31 mlp genes and physically linked variant gene families indicated that mlp gene heterogeneity is unique and unrelated to location or linkage to divergent sequences. Evidence of recombination between B31 mlp alleles was also detected. Northern blot analysis of cultured strain B31 indicated that the mlp genes were not expressed at a temperature (23 degrees C) characteristic of that of ticks in the environment. In striking contrast, expression of many mlp genes increased substantially when strain B31 was shifted to 35 degrees C, a temperature change mimicking that occurring in the natural transmission cycle of the spirochete from tick to mammal. Primer extension analysis of the mlp mRNA transcripts suggested that sigma 70-like promoters are involved in mlp expression during temperature shift conditions. Antibodies were made against strain B31 Mlp proteins within the first 4 weeks after experimental mouse infection. Importantly, Lyme disease patients also had serum antibodies reactive with purified recombinant Mlp proteins from strain B31, a result indicating that humans are exposed to Mlp proteins during infection. Taken together, the data indicate that strain B31 mlp genes encode a diverse array of lipoproteins which may participate in early infection processes in the mammalian host.

Altered stationary-phase response in a Borrelia burgdorferi rpoS mutant.

The homolog of the chromosomally encoded stationary-phase sigma factor RpoS in Borrelia burgdorferi was inactivated using gyrB(r) as a selectable marker. Two-dimensional nonequilibrium pH gradient electrophoresis of stationary-phase cell lysates identified at least 11 differences between the protein profiles of the rpoS mutant and wild-type organisms. Wild-type B. burgdorferi had a growth phase-dependent resistance to 1 N NaCl, similar to the stationary-phase response reported for other bacteria. The B. burgdorferi rpoS mutant strain was less resistant to osmotic stress in stationary phase than the isogenic rpoS wild-type organism. The results indicate that the B. burgdorferi rpoS homolog influences protein composition and participates in stationary-phase-dependent osmotic resistance. This rpoS mutant will be useful for studying regulation of gene expression in response to changing environmental conditions.

Efficient targeted mutagenesis in Borrelia burgdorferi.

Genetic studies in Borrelia burgdorferi have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is gyrB(r), a mutated form of the chromosomal gyrB gene that encodes the B subunit of DNA gyrase and confers resistance to the antibiotic coumermycin A(1). The utility of the coumermycin-resistant gyrB(r) gene for targeted gene disruption is limited by a high frequency of recombination with the endogenous gyrB gene. A kanamycin resistance gene (kan) was introduced into B. burgdorferi, and its use as a selectable marker was explored in an effort to improve the genetic manipulation of this pathogen. B. burgdorferi transformants with the kan gene expressed from its native promoter were susceptible to kanamycin. In striking contrast, transformants with the kan gene expressed from either the B. burgdorferi flaB or flgB promoter were resistant to high levels of kanamycin. The kanamycin resistance marker allows efficient direct selection of mutants in B. burgdorferi and hence is a significant improvement in the ability to construct isogenic mutant strains in this pathogen.

Transformation of the Lyme disease spirochete Borrelia burgdorferi with heterologous DNA.

Studies of the spirochete Borrelia burgdorferi have been hindered by the scarcity of genetic tools that can be used in these bacteria. For the first time, a method has been developed by which heterologous DNA (DNA without a naturally occurring B. burgdorferi homolog) can be introduced into and persistently maintained by B. burgdorferi. This technique uses integration of circular DNA into the bacterial genome via a single-crossover event. The ability to transform B. burgdorferi with heterologous DNA will now permit a wide range of experiments on the biology of these bacteria and their involvement in the many facets of Lyme disease.