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 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.

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.

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.

Growth of infectious and non-infectious B. burgdorferi at different salt concentrations.

Borrelia burgdorferi, the causative agent of Lyme disease, grows in vitro in modified Barbour-Stoenner-Kelly (BSK-H) medium. We have studied the effect of increased osmotic strength of culture media on growth of infectious and non-infectious B. burgdorferi strains B31 and N40. Relatively small increases in the NaCl concentration of the medium significantly inhibited growth in infectious as well as non-infectious strains. Growth of low passage, infectious clone B31-4a was more sensitive to increased NaCl concentrations than high passage, non-infectious clone B31-a. Growth of two infectious N40 strains, one low passage (N40-Lp) and one high passage (N40-P31) was more resistant to increased NaCl concentration than growth of infectious B31-4a. Osmotic strength is an important physical parameter for growth of B. burgdorferi in vitro and could influence its ability to adapt and to establish an infection within ticks and mammals.

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.

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.

Detection of antibodies and delayed hypersensitivity with Rotofor preparative IEF fractions of Blastomyces dermatitidis yeast phase lysate antigen.

Blastomyces dermatitidis (dog isolate T-58) yeast phase lysate antigen was concentrated and separated by Rotofor preparative isoelectric focusing cell (Bio-Rad). The pH values of the fractions were determined and equilibrated to pH 7.2 and then analysed by enzyme-linked immunosorbent assay using horseradish peroxidase enzyme system against serum specimens from dogs with blastomycosis, histoplasmosis, aspergillosis, and coccidioidomycosis. The results showed a peak absorbance at pH 3.89-4.31 (fractions 4 and 5) with the blastomycosis serum specimens. This was a single sharp peak while the rest of the fractions were lower. In contrast the sera from dogs with histoplasmosis showed a peak absorbance at pH 5.54-5.97 (fractions 9 and 10), while the other mycoses showed patterns that did not resemble the blastomycosis or histoplasmosis specimens. Serum specimens from dogs with blastomycosis being treated with itraconazole were also assayed (pre-treatment and 1, 2, 3, and 12 months post-treatment sera). The characteristic peak for blastomycosis was observed and a decrease in the peak was seen as the treatment progressed. Fractions 3-12 were also used to detect delayed dermal hypersensitivity in hyperimmunized hairless guinea-pigs. Fraction 5 (pH 4.31) elicited the optimal response in B. dermatitidis-immunized animals, while no cross-reactivity was observed in guinea-pigs sensitized with Histoplasma capsulatum killed cells.

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.

Archaeal-type lysyl-tRNA synthetase in the Lyme disease spirochete Borrelia burgdorferi.

Lysyl-tRNAs are essential for protein biosynthesis by ribosomal mRNA translation in all organisms. They are synthesized by lysyl-tRNA synthetases (EC 6.1.1.6), a group of enzymes composed of two unrelated families. In bacteria and eukarya, all known lysyl-tRNA synthetases are subclass IIc-type aminoacyl-tRNA synthetases, whereas some archaea have been shown to contain an unrelated class I-type lysyl-tRNA synthetase. Examination of the preliminary genomic sequence of the bacterial pathogen Borrelia burgdorferi, the causative agent of Lyme disease, indicated the presence of an open reading frame with over 55% similarity at the amino acid level to archaeal class I-type lysyl-tRNA synthetases. In contrast, no coding region with significant similarity to any class II-type lysyl-tRNA synthetase could be detected. Heterologous expression of this open reading frame in Escherichia coli led to the production of a protein with canonical lysyl-tRNA synthetase activity in vitro. Analysis of B. burgdorferi mRNA showed that the lysyl-tRNA synthetase-encoding gene is highly expressed, confirming that B. burgdorferi contains a functional class I-type lysyl-tRNA synthetase. The detection of an archaeal-type lysyl-tRNA synthetase in B. burgdorferi and other pathogenic spirochetes, but not to date elsewhere in bacteria or eukarya, indicates that the gene that encodes this enzyme has a common origin with its orthologue from the archaeal kingdom. This difference between the lysyl-tRNA synthetases of spirochetes and their hosts may be readily exploitable for the development of anti-spirochete therapeutics.

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.

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.

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.

Borrelia burgdorferi erp proteins are immunogenic in mammals infected by tick bite, and their synthesis is inducible in cultured bacteria.

Borrelia burgdorferi, the causative agent of Lyme disease, can contain multiple genes encoding different members of the Erp lipoprotein family. Some arthropod-borne bacteria increase the synthesis of proteins required for transmission or mammalian infection when cultures are shifted from cool, ambient air temperature to a warmer, blood temperature. We found that all of the erp genes known to be encoded by infectious isolate B31 were differentially expressed in culture after a change in temperature, with greater amounts of message being produced by bacteria shifted from 23 to 35 degrees C than in those maintained at 23 degrees C. Mice infected with B31 by tick bite produced antibodies that recognized each of the Erp proteins within 4 weeks of infection, suggesting that the Erp proteins are produced by the bacteria during the early stages of mammalian infection and may play roles in transmission from ticks to mammals. Several of the B31 Erp proteins were also recognized by antibodies from patients with Lyme disease and may prove to be useful antigens for diagnostic testing or as components of a protective vaccine.

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.

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.

Sensitivity and specificity of an isoelectric focusing fraction of Blastomyces dermatitidis yeast lysate antigen for the detection of canine blastomycosis.

Blastomyces dermatitidis yeast lysate antigen (T-58, dog isolate) fractions prepared using the Rotofor preparative isoelectric focusing (IEF) cell (Bio-Rad) were compared with B. dermatitidis yeast lysate and filtrate reagents with respect to the detection of antibodies in sera from dogs with blastomycosis, histoplasmosis, coccidioidomycosis, cryptococcosis and aspergillosis. A horseradish peroxidase enzyme immunoassay with Turbo TMB substrate was used in the study. One particular IEF fraction (pH 4.3) was optimal in the assay, and it exhibited greater sensitivity (100%) and specificity (93%) than the lysate or filtrate preparations. The highest degree of cross-reactivity was encountered with the histoplasmosis and coccidioidomycosis specimens and considerably less with the cryptococcosis and aspergillosis sera. Studies are in progress to purify further the optimal IEF fraction.

The Borrelia burgdorferi circular plasmid cp26: conservation of plasmid structure and targeted inactivation of the ospC gene.

The 26 to 28kb circular plasmid of B. burgdorferi sensu lato (cp26) is ubiquitous among bacteria of this group and contains loci implicated in the mouse-tick transmission cycle. Restriction mapping and Southern hybridization indicated that the structure of cp26 is conserved among isolates from different origins and culture passage histories. The cp26 ospC gene encodes an outer surface protein whose synthesis within infected ticks increases when the ticks feed, and whose synthesis in culture increases after a temperature upshift. Previous studies of ospC coding sequences showed them to have stretches of sequence apparently derived from the ospC genes of distantly related isolates by homologous recombination after DNA transfer. We found conservation of the promoter regions of the ospC and guaA genes, which are divergently transcribed. We also demonstrated that the increase in OspC protein after a temperature upshift parallels increases in mRNA levels, as expected if regulatory regions adjoin the conserved sequences in the promoter regions. Finally, we used directed insertion to inactivate the ospC gene of a non-infectious isolate. This first example of directed gene inactivation in B. burgdorferi shows that the OspC protein is not required for stable maintenance of cp26 or growth in culture.