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The aim of this study was to elucidate the biological phenotypes of 16S rRNA methylase RmtB in Escherichia coli and the impact of RmtB acquisition on the fitness of the target bacterium. An rmtB in-frame deletion mutant in E. coli was constructed using a suicide vector (pDMS197)-based double crossover allelic exchange, and its corresponding complemented strain was established. Combined studies of microdilution susceptibility testing, conjugation experiments, growth kinetics assays, competitive experiments, biofilm formation tests and motility assays were performed to study the rmtB-mediated fitness among the prototype E. coli strain, its isogenic mutant and the corresponding complemented strain. The minimum inhibitory concentrations (MICs) of 4,6-disubstituted 2-deoxystreptamines for the rmtB wild-type strain, its isogenic mutant and the complemented strain were ≥1024, ≤2 and ≥1024mg/L, respectively. Both the growth rates and the competitive abilities of the wild-type and complemented strains were relatively inferior to the ΔrmtB mutant. There was no significant difference in biofilm formation and motility among the three strains. In conclusion, the data presented here suggest that acquisition of the 16S rRNA methylase gene rmtB in E. coli can exact a fitness cost on the bacteria, subsequently reducing the growth rate slightly and decreasing the competitive capacity of the bacterium, whereas it does not affect biofilm formation or motility., (Copyright © 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Virulence of enterotoxigenic Escherichia coli (ETEC) is associated with fimbrial adhesins and enterotoxins such as heat-labile (LT) and/or heat-stable (ST) enterotoxins. Previous studies using a cell culture model suggest that exclusion of ETEC from attachment to epithelial cells requires expression of both an adhesin such as K88 (F4) fimbriae, and LT. To test the ability of non-pathogenic E. coli constructs to exclude virulent ETEC sufficiently to prevent clinical disease, we utilized a piglet ETEC challenge model. Thirty-nine 5-day-old piglets were inoculated with a placebo (control), or with either of the three K88(+)E. coli strains isogenic with regard to modified LT expression: 8017 (pBR322 plasmid vector control), non-toxigenic mutant 8221 (LT(R192G)) in pBR322, or 8488, with the LT gene fused to the STb gene in pBR322 (LT(R192G)-STb). Piglets were challenged with virulent ETEC Strain 3030-2 (K88(+)/LT/STb) 24h post-inoculation. K88ac receptor-positive piglets in the control group developed diarrhea and became dehydrated 12-24h post-challenge. Piglets inoculated with 8221 or 8488 did not exhibit clinical signs of ETEC disease; most piglets inoculated with 8017 showed diarrhea. Control pigs exhibited significant weight loss, increased blood total protein, and higher numbers of colony-forming units of 3030-2 E. coli in washed ileum and jejunum than treated pigs. This study shows for the first time that pre-inoculation with an avirulent strain expressing adhesive fimbriae and a non-toxic form of LT provides significant short term protection from challenge with a virulent ETEC strain that expresses the same fimbrial adhesion and enterotoxin., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Tetracyclines and bacitracin are used extensively as a growth promotant and a therapeutic, respectively in livestock. In this study, we test whether there is an interaction between bacitracin and tetracycline that can select for an increase in microbial tetracycline sensitivity, using Escherichia coli and Salmonella as a model. We found via in vitro studies that bacitracin at sublethal concentrations preferentially selects for outgrowth of tetracycline-sensitive bacteria from among a population of tetracycline-resistant and tetracycline-sensitive E. coli and Salmonella strains. Most of the bacterial strains employed in our study were shown by PCR to possess the tetracycline resistance gene tet(A) or tet(C), either of whose activity is associated with tetracycline efflux. Bacitracin could be substituted for the lipophylic chelator, fusaric acid, used as a positive selective agent for tetracycline sensitivity. Together, these observations suggest that bacitracin-mediated selection for tetracycline sensitivity alters the function of tetracycline efflux proteins. Using bacitracin and tetracycline simultaneously may improve the effectiveness of the antibiotics, while decreasing the risk of selecting for a population of tetracycline-resistant microorganisms.

Enterotoxigenic Escherichia coli (ETEC)-associated post-weaning diarrhea (PWD) is economically one of the most important diseases for the swine industry. Porcine ETEC strains typically express K88 or F18 fimbria and heat-labile (LT) and/or heat-stable (STa, STb) enterotoxins. However, recent studies indicate that EAST1 toxin, adhesin involved in diffuse adherence (AIDA-I) and porcine attaching and effacing-associated factor (paa) may also be expressed by ETEC strains associated with diarrhea. To better understand the virulence factors of E. coli strains that cause PWD, we applied PCR to screen for K88, F18, F41, 987P and K99 fimbrial genes; LT, STa, STb, Stx2e and EAST1 toxic genes; and AIDA-I, paa and EAE adhesin genes in E. coli strains recently isolated from young pigs with PWD in the US. Of 304 E. coli isolates from diarrheic pigs submitted for testing, 175 (57.6%) strains possessed fimbrial genes: K88 (64.6%), F18 (34.3%), F41 (0.57%), K99 (0.57%), 987P (0); toxin genes: LT (57.7%), STb (72.6%), STa (27.4%), STx2e (17.4%), EAST1 (35%); and adhesin genes: AIDA-I (26.9%), paa (60%), EAE (1.1%). All toxin genes except the EAST1 toxin gene, were almost exclusively associated with K88+ or F18+ isolates, and most of these isolates carried multiple toxin genes. The non-fimbrial adhesin paa was found present in over half of the K88+ isolates. A total of 129 (42%) isolates carried no fimbrial genes, including 66 (21.7%) isolates that did not have any of the above virulence genes. These results suggest a broad array of virulence genes associated with PWD in pigs.

Enterotoxigenic Escherichia coli (ETEC) strains that produce multiple enterotoxins are important causes of severe dehydrating diarrhea in human beings and animals, but the relative importance of these enterotoxins in the pathogenesis is poorly understood. Gnotobiotic piglets were used to study the importance of heat-labile enterotoxin (LT) in infection with an ETEC strain that produces multiple enterotoxins. LT(-) (DeltaeltAB) and complemented mutants of an F4(+) LT(+) STb(+) EAST1(+) ETEC strain were constructed, and the virulence of these strains was compared in gnotobiotic piglets expressing receptors for F4(+) fimbria. Sixty percent of the piglets inoculated with the LT(-) mutant developed severe dehydrating diarrhea and septicemia compared to 100% of those inoculated with the nalidixic acid-resistant (Nal(r)) parent and 100% of those inoculated with the complemented mutant strain. Compared to piglets inoculated with the Nal(r) parent, the mean rate of weight loss (percent per hour) of those inoculated with the LT(-) mutant was 67% lower (P < 0.05) and that of those inoculated with the complemented strain was 36% higher (P < 0.001). Similarly, piglets inoculated with the LT(-) mutant had significant reductions in the mean bacterial colony count (CFU per gram) in the ileum; bacterial colonization scores (square millimeters) in the jejunum and ileum; and clinical pathology parameters of dehydration, electrolyte imbalance, and metabolic acidosis (P < 0.05). These results indicate the significance of LT to the development of severe dehydrating diarrhea and postdiarrheal septicemia in ETEC infections of swine and demonstrate that EAST1, LT, and STb may be concurrently expressed by porcine ETEC strains.

Cesarean-derived piglets were reared for 5 wk under germfree conditions or monoassociated with a benign Escherichia coli (G58-1) or a enterohemorrhagic strain (933D) derived from O157:H7, and immunized i.p. with the T-dependent (TD) Ags fluorescein-labeled (FL) keyhole limpet hemocyanin or trinitrophenylated (TNP) keyhole limpet hemocyanin and the type 2 T-independent Ags TNP-Ficoll or FL-Ficoll. Only colonized piglets showed an increase in serum IgG, IgA, and IgM and had serum Abs to FL, TNP, and colonizing bacteria. While serum Abs to FL or TNP appeared following colonization alone, secondary responses were restricted to piglets immunized using TD carriers. While animals colonized with 933D had significantly higher total serum IgG and IgM levels and specific IgG Abs than those colonized with G58-1, no differences were seen in serum IgA levels, B cell diversification in the ileal Peyer's patches, and specific activity (ELISA activity per micrograms of Ig) of pre-boost serum IgG and IgM anti-TNP and anti-FL Abs. Serum IgA Abs to TNP, FL, or bacteria were not detected. Ag-driven responses, as measured by an increase in specific Ab activity, were only observed in secondary responses to TD Ags and to colonizing, pathogenic E. coli. We propose that germline-encoded, isotype-switched B cells in newborn piglets differentiate to Ab-secreting cells 1) after stimulation by bacteria-activated APCs or 2) through direct stimulation by bacterial products. We further propose that Ag-driven systemic responses require both bacterial colonization and TD Ags translocated to the peritoneum.

An experiment was conducted to evaluate the effects of including canola meal (CM) in diets for weaning pigs challenged with a F18 strain of Escherichia coli on growth performance and gut health. A total of 36 individually housed weaned pigs (initial body weight [BW] = 6.22 kg) were randomly allotted to one of the three diets (12 pigs/diet). The three diets were corn– soybean meal (SBM)-based basal diet (control diet) and the basal diet with 0.3% zinc oxide, 0.2% chlortetracycline, and 0.2% tiamulin (antibiotic diet) or with 20% CM diet. The diets were fed in two phases: Phase 1: days 0 to 7 and Phase 2: days 7 to 20. All pigs were given an oral dose of 2 × 109 CFU of F18 strain of E. coli on day 7. Fecal score was assessed daily throughout the trial. Dietary antibiotics increased (P < 0.05) overall average daily gain (ADG) and average daily feed intake (ADFI) compared by 48% and 47%, respectively. Dietary CM increased (P < 0.05) overall ADG and ADFI by 22% and 23%, respectively; but the ADG and ADFI values for CM-containing diet did not reach those for the antibiotics-containing diet. Dietary antibiotics reduced (P < 0.05) fecal score; however, dietary CM unaffected fecal score. Dietary antibiotics decreased (P < 0.05) liver weight per unit live BW by 16% at day 20, whereas dietary CM did not affect liver weight per unit live BW (29.2 vs. 28.6). Also, dietary antibiotics increased (P < 0.05) serum triiodothyronine and tetraiodothyronine levels for day 14, whereas dietary CM did not affect the serum level of these hormones. Dietary antibiotics reduced (P < 0.05) the number white blood cells and neutrophils by 38% and 43% at day 20, respectively, whereas dietary CM tended to reduce (P = 0.09) the number white blood cells by 19% at day 20. The number white blood cells for CM diet tended to be greater (P < 0.10) than that for antibiotics diet. The dietary antibiotics decreased (P < 0.05) the concentration of individual volatile fatty acids and hence of total volatile fatty acid in cecum by 61% at day 20, whereas dietary CM decreased (P < 0.05) cecal butyric acid concentration by 61% and tended to reduce (P < 0.10) total volatile fatty acid concentration by 30% at day 20. In conclusion, the dietary inclusion of 20% CM improved ADG and tended to reduce white blood cell counts. Thus, inclusion of CM in antibiotics-free corn-SBM-based diets for weaned pigs that are challenged with F18 strain of E. coli can result in their improved performance partly through a reduction of the inflammatory response. [ABSTRACT FROM AUTHOR]

This study was conducted to investigate the effects of a direct-fed microbial (DFM) product (Bacillus subtilis strain DSM 32540) in weaned pigs challenged with K88 strain of Escherichia coli on growth performance and indicators of gut health. A total of 21 weaned pigs [initial body weight (BW) = 8.19 kg] were housed individually in pens and fed three diets (seven replicates per diet) for 21 d in a completely randomized design. The three diets were a corn-soybean meal-based basal diet without feed additives, a basal diet with 0.25% antibiotics (neo-Oxy 10-10; neomycin + oxytetracycline), or a basal diet with 0.05% DFM. All pigs were orally challenged with a subclinical dose (6.7 × 108 CFU/mL) of K88 strain of E. coli on day 3 of the study (3 d after weaning). Feed intake and BW data were collected on days 0, 3, 7, 14, and 21. Fecal scores were recorded daily. On day 21, pigs were sacrificed to determine various indicators of gut health. Supplementation of the basal diet with antibiotics or DFM did not affect the overall (days 0–21) growth performance of pigs. However, antibiotics or DFM supplementation increased (P = 0.010) gain:feed (G:F) of pigs during the post- E. coli challenge period (days 3–21) by 23% and 24%, respectively. The G:F for the DFM-supplemented diet did not differ from that for the antibiotics-supplemented diet. The frequency of diarrhea for pigs fed a diet with antibiotics or DFM tended to be lower (P = 0.071) than that of pigs fed the basal diet. The jejunal villous height (VH) and the VH to crypt depth ratio (VH:CD) were increased (P < 0.001) by 33% and 35%, respectively, due to the inclusion of antibiotics in the basal diet and by 43% and 41%, respectively due to the inclusion of DFM in the basal diet. The VH and VH:CD for the DFM-supplemented diet were greater (P < 0.05) than those for the antibiotics-supplemented diet. Ileal VH was increased (P < 0.05) by 46% due to the inclusion of DFM in the basal diet. The empty weight of small intestine, cecum, or colon relative to live BW was unaffected by dietary antibiotics or DFM supplementation. In conclusion, the addition of DFM to the basal diet improved the feed efficiency of E. coli -challenged weaned pigs to a value similar to that of the antibiotics-supplemented diet and increased jejunal VH and VH:CD ratio to values greater than those for the antibiotics-supplemented diet. Thus, under E. coli challenge, the test DFM product may replace the use of antibiotics as a growth promoter in diets for weaned pigs to improve feed efficiency and gut integrity. [ABSTRACT FROM AUTHOR]

Infection with F4 enterotoxigenic Escherichia coli (ETEC) responsible for diarrhea in neonatal and post-weaned piglets leads to great economic losses in the swine industry. These pathogenic bacteria express either of three fimbrial variants F4ab, F4ac, and F4ad, which have long been known for their importance in host infection and initiating protective immune responses. The initial step in infection for the bacterium is to adhere to host enterocytes through fimbriae-mediated recognition of receptors on the host cell surface. A number of receptors for ETEC F4 have now been described and characterized, but their functions are still poorly understood. The current review summarizes the latest research addressing the characteristics of F4 fimbriae receptors and the interactions of F4 fimbriae and their receptors on host cells. These include observations that as follows: (1) FaeG mediates the binding activities of F4 and is an essential component of the F4 fimbriae, (2) the F4 fimbrial receptor gene is located in a region of chromosome 13, (3) the biochemical properties of F4 fimbrial receptors that form the binding site of the bacterium are now recognized, and (4) specific receptors confer susceptibility/resistance to ETEC F4 infection in pigs. Characterizing the host-pathogen interaction will be crucial to understand the pathogenicity of the bacteria, provide insights into receptor activation of the innate immune system, and develop therapeutic strategies to prevent this illness. [ABSTRACT FROM AUTHOR]

Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system (T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had the highest LT secretion level all porcine-origin WT strains tested (P<0.05). The level of LT secretion (concentration in supernatants at 6-h culture) explained 92% of the variation in time-to-a-moribund-condition (R2 = 0.92, P<0.0001) in gnotobiotic piglets inoculated with either strain 3030-2, or an ETEC strain of lesser virulence (2534-86), or a non-enterotoxigenic WT strain (G58-1). All 16 porcine ETEC strains were positive by PCR analysis for the T2SS genes, gspD and gspK, and bioinformatic analysis of 4 porcine-origin strains for which complete genomic sequences were available revealed a T2SS with a high degree of homology to that of H10407. Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18. Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml. This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and between the latter and virulence. [ABSTRACT FROM AUTHOR]

Effects of dietary canola meal (CM) on growth performance, fecal score, and blood parameters of E. colichallenged nursery pigs were investigated. Thirty-six pigs (initial body weight = 6.22 kg) weaned at 21 d of age were housed individually and fed 3 diets (12 replicates/diet) in randomized complete block design. The diets were corn-soybean meal-based diet without or with antibiotics (0.2% chlortetracycline and 0.2% tiamulin) or with CM (20%). The diets were fed in 2 phases; Phase 1: d 0 to 7, and Phase 2: d 7 to 21. Pigs were orally challenged with F18 strain of E. coli on d 7. Feed intake and body weight were measured on d 7, 14, and 21. Blood samples for measuring thyroid hormones were collected from each pig on d 7, 14, and 21. Blood samples for measuring white blood cells, immunoglobulins, and cytokine were collected on d 21. Fecal score was assessed daily. Dietary antibiotics increased (P < 0.05) ADG by 50%. Dietary SECM also increased (P < 0.05) ADG by 23%. Week 1 fecal score was unaffected by diet. However, during weeks 2 and 3, fecal score for diet with antibiotics was less (P < 0.05) than that for basal diet or diet with CM, which did not differ in fecal score. During weeks 1 and 2, serum tetraiodothyronine level for diet with antibiotics was less (P < 0.05) than that for basal diet or diet with CM, which did not differ in serum tetraiodothyronine level; week 3 serum tetraiodothyronine level was unaffected by diet. White blood cell count was reduced (P < 0.05) by dietary antibiotics, and tended to be reduced (P = 0.087) by dietary CM. In conclusion, dietary CM increased ADG and tended to reduce white blood cell count. Thus, dietary CM at 20% may improve the performance of pigs challenged with E. coli F18 partly by decreasing immune response. [ABSTRACT FROM AUTHOR]

Enterotoxigenic Escherichia coli (ETEC) infection is of global importance because of its high morbidity and mortality rate in humans and animals. ETEC has been the principal agent causing the traveler's diarrhea in humans and post-weaning diarrhea in pigs. Intestinal innate immunity is the first line of defense in enteric bacterial infections. The pathogenicity of ETEC is due to the fimbriae and heat labile (LT) and heat stable (ST) toxins. LT has two subunits A and B. Porcine intestinal epithelial cells (IPEC-J2) are a suitable in vitro model for studying ETEC infections. This study was conducted to analyze the changes in the gene expression of proinflammatory cytokines and chemokines upon stimulation of IPEC-J2 cells with different strains of ETEC and LT toxins for 3 hrs. We used wild type ETEC strains with K88ac fimbriae 3030-2 (O157:K87 LT+, STb+), 2534-86 (O8:K87: NM: LT-I+, STb+), 1836-2 (LT_, ST-, astA+), and G58-1, a non pathogenic, non-fimbriated, wild-type (O101:K28: NM, LT_), at multiplicity of infection 10:1. LT and LTb were used at 100 ng/ml. G58-1 and cells with media alone served as negative controls. The changes in the gene expression of proinflammatory cytokines such as IL-1α, IL-1β, TNF-α, IL-6 and chemokine, IL-8, were quantified relative to porcine specific cyclophilin-A. We found that the 3030-2 strain decreased IL-6 and increased TNF-α expression at 3h. Strain 1836-2 increased IL-8 and LTb decreased IL-8 gene expressions. Further investigations will help in understanding more about enteric mucosal innate immune responses to ETEC, which may potentially contribute to its prevention and treatment. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies in piglets indicate that heat labile enterotoxin (LT) expression enhances intestinal colonization by K88 adhesin-producing enterotoxigenic Escherichia coli (ETEC) as wild-type ETEC adhered to intestinal epithelium in substantially greater numbers than did non-toxigenic constructs. Enzymatic activity of the toxin was also shown to contribute to the adhesion of ETEC and non-ETEC bacteria to epithelial cells in culture. To further characterize the contribution of LT to host cell adhesion, a nontoxigenic, K88-producing E. coli was transformed with either the gene encoding for LT holotoxin, a catalytically-attenuated form of the toxin [LT(R192G)], or LTB subunits, and resultant changes in bacterial adherence to IPEC-J2 porcine intestinal epithelial cells were measured. Strains expressing LT holotoxin or mutants were able to adhere in significantly higher numbers to IPEC-J2 cells than was an isogenic, toxin-negative construct. LT+ strains were also able to significantly block binding of a wild-type LT+ ETEC strain to IPEC-J2 cells. Adherence of isogenic strains to IPEC-J2 cells was unaltered by cycloheximide treatment, suggesting that LT enhances ETEC adherence to IPEC-J2 cells independent of host cell protein synthesis. However, pretreating IPEC-J2 cells with LT promoted adherence of negatively charged latex beads (a surrogate for bacteria which carry a negative change), which adherence was inhibited by cycloheximide, suggesting LT may induce a change in epithelial cell membrane potential. Overall, these data suggest that LT may enhance ETEC adherence by promoting an association between LTB and epithelial cells, and by altering the surface charge of the host plasma membrane to promote non-specific adherence. [Copyright &y& Elsevier]

Abstract: Heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) increases bacterial adherence to porcine enterocytes in vitro and enhances small intestinal colonization in swine. Heat-stable enterotoxin-b (STb) is not known to affect colonization; however, through an induction of net fluid accumulation it might reduce bacterial adherence. The relationship between fluid accumulation and bacterial adherence in jejunal loops inoculated with ETEC strains that produce LT, STb, both, or neither toxin was studied. Ligated jejunal loops were constructed in weaned Yorkshire pigs in two independent experiments (Exp. 1, n =5, 8-week-old; Exp. 2, n =6, 6–8-week-old). Each pig was inoculated with six F4ac+ E. coli strains: (1) LT+, STb+ parent (WAM2317); (2) STb− (ΔestB) mutant (MUN297); (3) MUN297 complemented with STb (MUN298); (4) LT− STb− (ΔeltAB ΔestB) mutant (MUN300); (5) MUN300 complemented with LT (MUN301); and (6) 1836-2 (non-enterotoxigenic, wild-type). Pigs were confirmed to be K88 (F4)ab/ac receptor-positive in Exp. 2 by testing for intestinal mucin-type glycoproteins and inferred to be receptor-positive in both Exp. 1 and 2 based on histopathologic evidence of bacterial adherence. Strains that produced STb induced marked fluid accumulation with the response (ml/cm) to WAM2317 and MUN298 significantly greater than that to the other strains (P <0.0001). Conversely, bacterial adherence scores based on immunohistochemistry and CFU/g of washed mucosa were both lowest in the strains that expressed STb and highest in those that did not. For the two experiments combined, the Pearson correlation coefficient (R) between fluid volume (ml/cm) and log CFU per gram was −0.57021 (P <0.0001); R 2 =0.3521 (n =197). These results support the hypothesis that enterotoxin-induced fluid accumulation flushes progeny organisms into the lumen of the bowel, thereby increasing the likelihood of fecal shedding and transmission of the pathogen to new hosts. [Copyright &y& Elsevier]

To develop a piglet model for studying diarrheal disease and developing vaccines, we challenged gnotobiotic piglets with isogenic Escherichia coli strains constructed to express porcine 987P(F6) fimbriae and a heat-labile or a heat-stable enterotoxin to examine clinical outcomes. Piglets developed identical diarrheal diseases when inoculated with constructs expressing human or porcine enterotoxins. [ABSTRACT FROM AUTHOR]

Escherichia coli O157:H7 causes life-threatening outbreaks of diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans and significant economic loss in agriculture and could be a potential agent of bioterrorism. Although the prevalence of E. coli O157:117 in cattle and other species with which humans have frequent contact is high, human infections are relatively uncommon, despite a low infectious dose. A plausible explanation for the low disease incidence is the possibility that not all strains are virulent in humans. If there are substantial differences in virulence among strains in nature, then human disease may select for high virulence. We used a gnotobiotic piglet model to investigate the virulence of isolates from healthy cattle and from humans in disease outbreaks and to determine the correlation between production of Shiga toxin 1 (Stx1) and Stx2 and virulence. Overall, E. coli O157:117 strains isolated from healthy cattle were less virulent in gnotobiotic piglets than strains isolated from humans during disease outbreaks. The amount of Stx2 produced by E. coli O157:H7 strains correlated with strain virulence as measured by a reduction in piglet survival and signs of central nervous system disease due to brain infarction. The amount of Stx1 produced in culture was not correlated with the length of time of piglet survival or with signs of central nervous system disease. We suggest that disease outbreaks select for producers of high levels of Stx2 among E. coli O157:H7 strains shed by animals and further suggest that Stx1 expression is unlikely to be significant in human outbreaks. [ABSTRACT FROM AUTHOR]