Globetrotting strangles: the unbridled national and international transmission of Streptococcus equi between horses.

The equine disease strangles, which is characterized by the formation of abscesses in the lymph nodes of the head and neck, is one of the most frequently diagnosed infectious diseases of horses around the world. The causal agent, Streptococcus equi subspecies equi, establishes a persistent infection in approximately 10 % of animals that recover from the acute disease. Such 'carrier' animals appear healthy and are rarely identified during routine veterinary examinations pre-purchase or transit, but can transmit S. equi to naïve animals initiating new episodes of disease. Here, we report the analysis and visualization of phylogenomic and epidemiological data for 670 isolates of S. equi recovered from 19 different countries using a new core-genome multilocus sequence typing (cgMLST) web bioresource. Genetic relationships among all 670 S. equi isolates were determined at high resolution, revealing national and international transmission events that drive this endemic disease in horse populations throughout the world. Our data argue for the recognition of the international importance of strangles by the Office International des Épizooties to highlight the health, welfare and economic cost of this disease. The Pathogenwatch cgMLST web bioresource described herein is available for tailored genomic analysis of populations of S. equi and its close relative S. equi subspecies zooepidemicus that are recovered from horses and other animals, including humans, throughout the world. This article contains data hosted by Microreact.

SpeS: A Novel Superantigen and Its Potential as a Vaccine Adjuvant against Strangles.

Bacterial superantigens (sAgs) are powerful activators of the immune response that trigger unspecific T cell responses accompanied by the release of proinflammatory cytokines. Streptococcus equi (S. equi) and Streptococcus zooepidemicus (S. zooepidemicus) produce sAgs that play an important role in their ability to cause disease. Strangles, caused by S. equi, is one of the most common infectious diseases of horses worldwide. Here, we report the identification of a new sAg of S. zooepidemicus, SpeS, and show that mutation of the putative T cell receptor (TCR)-binding motif (YAY to IAY) abrogated TCR-binding, whilst maintaining interaction with major histocompatibility complex (MHC) class II molecules. The fusion of SpeS and SpeSY39I to six S. equi surface proteins using two different peptide linkers was conducted to determine if MHC class II-binding properties were maintained. Proliferation assays, qPCR and flow cytometry analysis showed that SpeSY39I and its fusion proteins induced less mitogenic activity and interferon gamma expression when compared to SpeS, whilst retaining Antigen-Presenting Cell (APC)-binding properties. Our data suggest that SpeSY39I-surface protein fusions could be used to direct vaccine antigens towards antigen-presenting cells in vivo with the potential to enhance antigen presentation and improve immune responses.

Intramuscular vaccination with Strangvac is safe and induces protection against equine strangles caused by Streptococcus equi.

The equine disease strangles, caused by Streptococcus equi, remains a major cause of welfare and economic cost to the global horse industry. Here we report the safety, immunogenicity and efficacy of a novel multi-component chimeric fusion protein vaccine, called Strangvac, when administered to ponies via the intramuscular route. Across the four studies, Strangvac was safe and induced robust antibody responses towards the vaccine components in blood serum and the nasopharynx, which were boosted by revaccination up to 12 months after a primary course of 2 vaccinations 4 weeks apart. The vaccine response did not cross-react with a commercial strangles iELISA, which identifies horses that have been exposed to S. equi, demonstrating that it was possible to differentiate infected from vaccinated animals (DIVA). Following challenge with S. equi strain 4047 (Se4047), all 36 control ponies that had received an adjuvant-only placebo vaccine developed clinical signs of strangles. In contrast, intramuscular vaccination with Strangvac protected ponies significantly from challenge with Se4047 at two weeks (5 of 16 ponies protected (31%), P = 0.04) and two months (7 of 12 ponies protected (58%), P = 0.0046 (including pooled control data) after second vaccination. Optimal protection (15 of 16 ponies protected (94%), P < 0.0001) was observed following challenge at two weeks post-third vaccination. Our data demonstrate that Strangvac is safe, has DIVA capability and provides a rapid onset of protective immunity against strangles. We conclude that Strangvac is a valuable tool with which to protect horses from strangles, particularly during high-risk periods, whilst maintaining the mobility of horse populations as required by the global equine industry.

Identification of genes required for the fitness of Streptococcus equi subsp. equi in whole equine blood and hydrogen peroxide.

The availability of next-generation sequencing techniques provides an unprecedented opportunity for the assignment of gene function. Streptococcus equi subspecies equi is the causative agent of strangles in horses, one of the most prevalent and important diseases of equids worldwide. However, the live attenuated vaccines that are utilized to control this disease cause adverse reactions in some animals. Here, we employ transposon-directed insertion-site sequencing (TraDIS) to identify genes that are required for the fitness of S. equi in whole equine blood or in the presence of H2O2 to model selective pressures exerted by the equine immune response during infection. We report the fitness values of 1503 and 1471 genes, representing 94.5 and 92.5 % of non-essential genes in S. equi, following incubation in whole blood and in the presence of H2O2, respectively. Of these genes, 36 and 15 were identified as being important to the fitness of S. equi in whole blood or H2O2, respectively, with 14 genes being important in both conditions. Allelic replacement mutants were generated to validate the fitness results. Our data identify genes that are important for S. equi to resist aspects of the immune response in vitro, which can be exploited for the development of safer live attenuated vaccines to prevent strangles.

The character and distribution of physical contaminants found in soil previously treated with mixed waste organic outputs and garden waste compost.

The re-use of waste materials by application to land is an increasingly common practice around the world, but where municipal solid waste materials are applied, it is almost inevitable that physical contaminants such as glass and plastic will be added to the soil. In many jurisdictions, there are prescribed limits for the amounts of physical contaminants that may be present in these materials, but there is little information on whether these limits safeguard soil functional condition. Here, physical contamination of soil is described after varying rates of a mixed waste organic output (MWOO) and garden waste compost (GWC) were incorporated into field plots. At application rates of 100 and 200 t/ha, both treatments resulted in a coarsening of the topsoil particle size distribution, but only in the MWOO-treated soils were physical contaminants largely responsible for this. The physical contaminant particles present were found only to the depth of cultivation, and included glass, rigid and film plastics, and synthetic fibres. These contaminants were most commonly observed in the gravel and coarse sand-sized fractions, and in those soils treated with the highest rates of MWOO application. Physical contaminant particles acted as both enveloping and nucleating agents for mineral grains and organic matter, and blocked some pores. Although soil physical condition is usually improved by the incorporation of organic matter, the extent of pore blockage evident here suggests that soil physical functions such as water percolation may be affected as the organic matter is broken down and the soil undergoes natural re-consolidation.

Streptococcal sagA activates a proinflammatory response in mast cells by a sublytic mechanism.

Mast cells are implicated in the innate proinflammatory immune defence against bacterial insult, but the mechanisms through which mast cells respond to bacterial encounter are poorly defined. Here, we addressed this issue and show that mast cells respond vividly to wild type Streptococcus equi by up-regulating a panel of proinflammatory genes and by secreting proinflammatory cytokines. However, this response was completely abrogated when the bacteria lacked expression of sagA, whereas the lack of a range of other potential virulence genes (seeH, seeI, seeL, seeM, hasA, seM, aroB, pyrC, and recA) had no effect on the amplitude of the mast cell responses. The sagA gene encodes streptolysin S, a lytic toxin, and we next showed that the wild type strain but not a sagA-deficient mutant induced lysis of mast cells. To investigate whether host cell membrane perturbation per se could play a role in the activation of the proinflammatory response, we evaluated the effects of detergent- and pneumolysin-dependent lysis on mast cells. Indeed, exposure of mast cells to sublytic concentrations of all these agents resulted in cytokine responses of similar amplitudes as those caused by wild type streptococci. This suggests that sublytic membrane perturbation is sufficient to trigger full-blown proinflammatory signalling in mast cells. Subsequent analysis showed that the p38 and Erk1/2 signalling pathways had central roles in the proinflammatory response of mast cells challenged by either sagA-expressing streptococci or detergent. Altogether, these findings suggest that sagA-dependent mast cell membrane perturbation is a mechanism capable of activating the innate immune response upon bacterial challenge.

Strangvac: A recombinant fusion protein vaccine that protects against strangles, caused by Streptococcus equi.

The host-restricted pathogen Streptococcus equi causes strangles in the horse, which is characterised by abscessation of the lymph nodes of the head and neck. The disease is endemic throughout the world causing considerable welfare and economic cost to the horse industry. Here we report the results of three studies where ponies were vaccinated with combinations of recombinant fusion proteins to optimise vaccine production and the level of protection conferred. Optimal protection was conferred by a prototype multicomponent subunit vaccine, Strangvac 4, which contained eight proteins CNE, SclC, SclF, SclI, EAG (fused as CCE), SEQ_402, SEQ_0256 (fused as Eq85) and IdeE. Across the three experiments only three of 16 ponies vaccinated with Strangvac 4 became pyretic compared to all 16 placebo-vaccinated control ponies (P < .001). S. equi was recovered from the lymph nodes of eight Strangvac 4-vaccinated and 15 control ponies (P = .016). None of the ponies vaccinated with Strangvac 4, or the other prototype vaccines developed adverse reactions following vaccination. Our data provide evidence in support of the further clinical development of the Strangvac 4 vaccine.

Genomic Dissection of an Icelandic Epidemic of Respiratory Disease in Horses and Associated Zoonotic Cases.

Iceland is free of the major infectious diseases of horses. However, in 2010 an epidemic of respiratory disease of unknown cause spread through the country's native horse population of 77,000. Microbiological investigations ruled out known viral agents but identified the opportunistic pathogen Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) in diseased animals. We sequenced the genomes of 257 isolates of S. zooepidemicus to differentiate epidemic from endemic strains. We found that although multiple endemic clones of S. zooepidemicus were present, one particular clone, sequence type 209 (ST209), was likely to have been responsible for the epidemic. Concurrent with the epidemic, ST209 was also recovered from a human case of septicemia, highlighting the pathogenic potential of this strain. Epidemiological investigation revealed that the incursion of this strain into one training yard during February 2010 provided a nidus for the infection of multiple horses that then transmitted the strain to farms throughout Iceland. This study represents the first time that whole-genome sequencing has been used to investigate an epidemic on a national scale to identify the likely causative agent and the link to an associated zoonotic infection. Our data highlight the importance of national biosecurity to protect vulnerable populations of animals and also demonstrate the potential impact of S. zooepidemicus transmission to other animals, including humans.IMPORTANCE An epidemic of respiratory disease affected almost the entire native Icelandic horse population of 77,000 animals in 2010, resulting in a self-imposed ban on the export of horses and significant economic costs to associated industries. Although the speed of transmission suggested that a viral pathogen was responsible, only the presence of the opportunistic pathogen Streptococcus zooepidemicus was consistent with the observed clinical signs. We applied genomic sequencing to differentiate epidemic from endemic strains and to shed light on the rapid transmission of the epidemic strain throughout Iceland. We further highlight the ability of epidemic and endemic strains of S. zooepidemicus to infect other animals, including humans. This study represents the first time that whole-genome sequencing has been used to elucidate an outbreak on a national scale and identify the likely causative agent.

Diversity of Streptococcus equi subsp. zooepidemicus strains isolated from the Spanish sheep and goat population and the identification, function and prevalence of a novel arbutin utilisation system.

The zoonotic bacterium Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a diverse, opportunistic pathogen that can cause mastitis in dairy sheep and goats. We used multilocus sequence typing (MLST) to define the genetic diversity of 60 isolates of S. zooepidemicus, which were recovered from sheep and goats in Spain between 2003 and 2010. We identify a novel clonal complex based on sequence type (ST), ST-236, which accounted for 39 of the 60 isolates. A representative ST-236 strain, S. zooepidemicus strain C7 (SzC7), was sequenced and interrogated for the presence of novel nutritional uptake or utilisation systems, the acquisition of which have previously been shown to be important for environmental adaptation in other streptococcal pathogens. A novel phosphoenolpyruvate sugar phosphotransferase system (PTS), which enabled the utilisation of arbutin, was identified. Functionality of the PTS was confirmed following deletion of the PTS from SzC7. Arbutin is found in multiple animal foodstuffs and we propose that the ability to utilise arbutin may have conferred a selective advantage to strains infecting animals, the diet of which contains this sugar.

Investigation of the Fim1 putative pilus locus of Streptococcus equi subspecies equi.

The Gram-positive bacterium Streptococcus equi subspecies equi (S. equi) is the causative agent of strangles, among the most frequently diagnosed infectious diseases of horses worldwide. Genome analysis of S. equi strain 4047 (Se4047) identified a putative operon, Fim1, with similarity to the pilus loci of other Gram-positive bacteria. The Fim1 locus was present in all strains of S. equi and its close relative S. equi subspecies zooepidemicus (S. zooepidemicus) that have been studied to date. In this study we provide evidence that the putative structural pilus proteins, SEQ_0936 and CNE, are produced on the cell surface during in vitro growth and in vivo infection. Although the proteins encoded within the Fim1 locus are not essential for attachment or biofilm formation, over-transcription of SEQ_0936 and CNE enhanced attachment to equine tissue in vitro. Our data suggest that whilst the Fim1 locus does not produce a polymerized pilus structure, the products of the Fim1 locus may fulfil an adhesive function. The putative pilus-associated regulator, tetR, which contains a nonsense mutation in S. equi, was able to regulate transcription of the Fim1 locus following repair and over-transcription, confirming its predicted role in the operon.

Defining the ABC of gene essentiality in streptococci.

BACKGROUND: Utilising next generation sequencing to interrogate saturated bacterial mutant libraries provides unprecedented information for the assignment of genome-wide gene essentiality. Exposure of saturated mutant libraries to specific conditions and subsequent sequencing can be exploited to uncover gene essentiality relevant to the condition. Here we present a barcoded transposon directed insertion-site sequencing (TraDIS) system to define an essential gene list for Streptococcus equi subsp. equi, the causative agent of strangles in horses, for the first time. The gene essentiality data for this group C Streptococcus was compared to that of group A and B streptococci. RESULTS: Six barcoded variants of pGh9:ISS1 were designed and used to generate mutant libraries containing between 33,000-66,000 unique mutants. TraDIS was performed on DNA extracted from each library and data were analysed separately and as a combined master pool. Gene essentiality determined that 19.5% of the S. equi genome was essential. Gene essentialities were compared to those of group A and group B streptococci, identifying concordances of 90.2% and 89.4%, respectively and an overall concordance of 83.7% between the three species. CONCLUSIONS: The use of barcoded pGh9:ISS1 to generate mutant libraries provides a highly useful tool for the assignment of gene function in S. equi and other streptococci. The shared essential gene set of group A, B and C streptococci provides further evidence of the close genetic relationships between these important pathogenic bacteria. Therefore, the ABC of gene essentiality reported here provides a solid foundation towards reporting the functional genome of streptococci.

Transcriptional changes are involved in phenotype switching in Streptococcus equi subspecies equi.

Phenotypic heterogeneity within a population of bacteria, through genetic or transcriptional variation, enables survival and persistence in challenging and changing environments. We report here that a recent clinical isolate of S. equi, strain 1691 (Se1691), yielded a mixture of reduced capsule and mucoid colonies on primary isolation when grown on colistin-oxolinic acid blood agar (COBA) streptococcal selective plates. Passaging colonies of Se1691, with a reduced capsule phenotype maintained this mixed phenotype. In contrast, passaging mucoid colonies fixed the mucoid phenotype, suggesting adaptive genetic or transcriptional changes in response to growth on artificial media. However, despite obvious phenotypic and transcriptional differences, there were no apparent differences in the genome sequences of Se1691 recovered from colonies with a mucoid or reduced capsule phenotype. We identified 105 differentially transcribed genes in the transcriptomes of reduced capsule and mucoid colonies. The reduced capsule phenotype was associated with a significant reduction in transcription of the has locus (SEQ_0269 Q = 0.0015, SEQ_0270 Q = 0.0015, SEQ_0271 Q = 0.0285) and the amount of hyaluronic acid on the surface of S. equi recovered from non-mucoid colonies (P = 0.017). Significant differences in the transcription of 21 surface and secreted proteins were also observed. Our data show that changes in the bacterial transcriptome are linked to the mixed colony phenotype of Se1691.

Characterisation of SEQ0694 (PrsA/PrtM) of Streptococcus equi as a functional peptidyl-prolyl isomerase affecting multiple secreted protein substrates.

Peptidyl-prolyl isomerase (PPIase) lipoproteins have been shown to influence the virulence of a number of Gram-positive bacterial human and animal pathogens, most likely through facilitating the folding of cell envelope and secreted virulence factors. Here, we used a proteomic approach to demonstrate that the Streptococcus equi PPIase SEQ0694 alters the production of multiple secreted proteins, including at least two putative virulence factors (FNE and IdeE2). We demonstrate also that, despite some unusual sequence features, recombinant SEQ0694 and its central parvulin domain are functional PPIases. These data add to our knowledge of the mechanisms by which lipoprotein PPIases contribute to the virulence of streptococcal pathogens.

Genome specialization and decay of the strangles pathogen, Streptococcus equi, is driven by persistent infection.

Strangles, the most frequently diagnosed infectious disease of horses worldwide, is caused by Streptococcus equi. Despite its prevalence, the global diversity and mechanisms underlying the evolution of S. equi as a host-restricted pathogen remain poorly understood. Here, we define the global population structure of this important pathogen and reveal a population replacement in the late 19th or early 20th Century. Our data reveal a dynamic genome that continues to mutate and decay, but also to amplify and acquire genes despite the organism having lost its natural competence and become host-restricted. The lifestyle of S. equi within the horse is defined by short-term acute disease, strangles, followed by long-term infection. Population analysis reveals evidence of convergent evolution in isolates from post-acute disease samples as a result of niche adaptation to persistent infection within a host. Mutations that lead to metabolic streamlining and the loss of virulence determinants are more frequently found in persistent isolates, suggesting that the pathogenic potential of S. equi reduces as a consequence of long-term residency within the horse post-acute disease. An example of this is the deletion of the equibactin siderophore locus that is associated with iron acquisition, which occurs exclusively in persistent isolates, and renders S. equi significantly less able to cause acute disease in the natural host. We identify several loci that may similarly be required for the full virulence of S. equi, directing future research toward the development of new vaccines against this host-restricted pathogen.

PinR mediates the generation of reversible population diversity in Streptococcus zooepidemicus.

Opportunistic pathogens must adapt to and survive in a wide range of complex ecosystems. Streptococcus zooepidemicus is an opportunistic pathogen of horses and many other animals, including humans. The assembly of different surface architecture phenotypes from one genotype is likely to be crucial to the successful exploitation of such an opportunistic lifestyle. Construction of a series of mutants revealed that a serine recombinase, PinR, inverts 114 bp of the promoter of SZO_08560, which is bordered by GTAGACTTTA and TAAAGTCTAC inverted repeats. Inversion acts as a switch, controlling the transcription of this sortase-processed protein, which may enhance the attachment of S. zooepidemicus to equine trachea. The genome of a recently sequenced strain of S. zooepidemicus, 2329 (Sz2329), was found to contain a disruptive internal inversion of 7 kb of the FimIV pilus locus, which is bordered by TAGAAA and TTTCTA inverted repeats. This strain lacks pinR and this inversion may have become irreversible following the loss of this recombinase. Active inversion of FimIV was detected in three strains of S. zooepidemicus, 1770 (Sz1770), B260863 (SzB260863) and H050840501 (SzH050840501), all of which encoded pinR. A deletion mutant of Sz1770 that lacked pinR was no longer capable of inverting its internal region of FimIV. The data highlight redundancy in the PinR sequence recognition motif around a short TAGA consensus and suggest that PinR can reversibly influence the wider surface architecture of S. zooepidemicus, providing this organism with a bet-hedging solution to survival in fluctuating environments.

Vaccination with a live multi-gene deletion strain protects horses against virulent challenge with Streptococcus equi.

Strangles, caused by Streptococcus equi subspecies equi (S. equi) is one of the most frequently diagnosed infectious diseases of horses and there remains a significant need to develop new preventative vaccines. We generated a live vaccine strain of S. equi containing deletions in six genes: sagA, hasA, aroB, pyrC, seM and recA, which was administered to nine Welsh mountain ponies via the intramuscular route. Four vaccinated ponies developed adverse reactions following the first vaccination from which the live vaccine strain was isolated. Two of these ponies were withdrawn from the study and seven ponies received a second vaccination, one of which then developed an adverse reaction. Nine control ponies injected with culture media alone developed no adverse reactions. Following challenge with a virulent strain of S. equi, none of the seven vaccinated ponies had developed clinical signs of strangles eleven days post-challenge, compared to six of nine control ponies over the same period (P=0.0114). A lymph node abscess was identified in one of the seven vaccinated ponies at post-mortem examination, whilst all nine control ponies had at least one lymph node abscess (P=0.0009). Three of the six vaccinated ponies that were protected from strangles had not developed an adverse reaction following vaccination, suggesting that a better understanding of the pro-inflammatory responses to S. equi could lead to the development of a live attenuated vaccine against strangles that is safe for administration via intramuscular injection.

Streptococcus zooepidemicus and Streptococcus equi evolution: the role of CRISPRs.

The host-restricted bacterium Streptococcus equi is the causative agent of equine strangles, the most frequently diagnosed infectious disease of horses worldwide. The disease is characterized by abscessation of the lymph nodes of the head and neck, leading to significant welfare and economic cost. S. equi is believed to have evolved from an ancestral strain of Streptococcus zooepidemicus, an opportunistic pathogen of horses and other animals. Comparison of the genome of S. equi strain 4047 with those of S. zooepidemicus identified examples of gene loss due to mutation and deletion, and gene gain through the acquisition of mobile genetic elements that have probably shaped the pathogenic specialization of S. equi. In particular, deletion of the CRISPR (clustered regularly interspaced short palindromic repeats) locus in the ancestor of S. equi may have predisposed the bacterium to acquire and incorporate new genetic material into its genome. These include four prophages and a novel integrative conjugative element. The virulence cargo carried by these mobile genetic elements is believed to have shaped the ability of S. equi to cause strangles. Further sequencing of S. zooepidemicus has highlighted the diversity of this opportunistic pathogen. Again, CRISPRs are postulated to influence evolution, balancing the need for gene gain over genome stability. Analysis of spacer sequences suggest that these pathogens may be susceptible to a limited range of phages and provide further evidence of cross-species exchange of genetic material among Streptococcus pyogenes, Streptococcus agalactiae and Streptococcus dysgalactiae.

Combining two serological assays optimises sensitivity and specificity for the identification of Streptococcus equi subsp. equi exposure.

The detection of anti-Streptococcus equi antibodies in the blood serum of horses can assist with the identification of apparently healthy persistently infected carriers and the prevention of strangles outbreaks. The aim of the current study was to use genome sequencing data to develop an indirect enzyme linked immunosorbent assay (iELISA) that targets two S. equi-specific protein fragments. The sensitivity and specificity of the antigen A and antigen C iELISAs were compared to an SeM-based iELISA marketed by IDvet - diagnostic Vétérinaire (IDvet). Individually, each assay compromised specificity in order to achieve sufficient sensitivity (SeM iELISA had a sensitivity of 89.9%, but a specificity of only 77.0%) or sensitivity to achieve high specificity. However, combining the results of the antigen A and antigen C iELISAs permitted optimisation of both sensitivity (93.3%) and specificity (99.3%), providing a robust assay for the identification of horses exposed to S. equi.

Detection of Streptococcus equi subspecies equi using a triplex qPCR assay.

Genome sequencing data for Streptococcus equi subspecies equi and zooepidemicus were used to develop a novel diagnostic triplex quantitative PCR (qPCR) assay targeting two genes specific to S. equi (eqbE and SEQ2190) and a unique 100 base pair control DNA sequence (SZIC) inserted into the SZO07770 pseudogene of S. zooepidemicus strain H70. This triplex strangles qPCR assay can provide results within 2h of sample receipt, has an overall sensitivity of 93.9% and specificity of 96.6% relative to the eqbE singlex assay and detects S. equi at levels below the threshold of the culture assay, even in the presence of contaminating bacteria.

Impact of inpatient status and gender on small-bowel capsule endoscopy findings.

BACKGROUND: Video capsule endoscopy (VCE) is most commonly performed in the outpatient setting to evaluate obscure GI bleeding. OBJECTIVE: To determine the impact of gender and inpatient status on VCE findings. DESIGN: Retrospective study. SETTING: Two tertiary medical centers and a VA medical center. PATIENTS: A total of 167 inpatients and 540 outpatients undergoing 707 VCE examinations for obscure GI bleeding. INTERVENTIONS: VCE study. MAIN OUTCOME MEASUREMENTS: Patient age, sex, indication for VCE, gastric and small-bowel transit times, significant VCE findings including detection of blood in the lumen and major lesions outside the small bowel, and presence of comorbid conditions. RESULTS: Significant VCE findings were identified more frequently during inpatient VCE examinations (48% vs 37%, P = .009). Endoscopic placement, nongastric passage, and incomplete studies to the cecum were more common for inpatient VCE examinations. Gastric transit time, but not small-bowel transit time, was longer in inpatient VCE studies. Inpatient VCE examinations were more common in male patients (73% vs 61%, P = .004) and patients with overt bleeding (83% vs 46%, P < .05). The overall diagnostic VCE rate was higher for male patients because of a higher prevalence of angiodysplastic lesions and major findings outside the small bowel. LIMITATIONS: Retrospective study. Lack of information regarding timing of VCE study, most recent episode of obscure bleeding, and comorbidity data for outpatients. CONCLUSION: The overall diagnostic yield was higher for inpatient VCE examinations. Male patients were more likely to demonstrate significant findings on both inpatient and outpatient VCE studies because of a higher prevalence of angiodysplastic lesions and findings outside the small bowel.