Shiga toxin 2eB-transgenic lettuce vaccine: N-glycosylation is important for protecting against porcine edema disease.

Porcine edema disease (ED) is a life-threatening toxemia caused by enteric infection with Shiga toxin 2e (Stx2e)-producing Escherichia coli (STEC) in weaned piglets. We previously reported that the stx2eB-transgenic lettuce 2BH strain shows potential for use as an oral vaccine candidate against ED. However, the 2BH strain expressed a hemagglutinin (HA)-tag together with Stx2eB and contained non-canonical N-glycosylation. Therefore, we developed two Stx2eB-lettuce strains, the 3 (G+) strain in which the HA-tag was removed from 2BH, and the 3 (G-) lettuce strain, in which the 73rd Asn was replaced with Ser to prevent non-canonical N-glycosylation of Stx2eB from the 3 (G+) strain. We examined the protective effect of these newly developed two strains compared with the previous 2BH strain against ED using a colostrum-deprived piglet STEC infection model. We found that the N-glycosylated 2BH and 3 (G+) strains relieved the pathogenic symptoms of ED in STEC-challenged piglets, whereas the non-glycosylated 3 (G-) strain did not. N-Glycosylation of the Stx2eB product in lettuce may be involved in the immune response in piglets.

Shiga toxin 2eB-transgenic lettuce vaccine is effective in protecting weaned piglets from edema disease caused by Shiga toxin-producing Escherichia coli infection.

Porcine edema disease (ED) is a toxemia that is caused by enteric infection with Shiga toxin 2e (Stx2e)-producing Escherichia coli (STEC) and is associated with high mortality. Since ED occurs most frequently during the weaning period, preweaning vaccination of newborn piglets is required. We developed stx2eB-transgenic lettuce as an oral vaccine candidate against ED and examined its protective efficacy using a piglet STEC infection model. Two serially developed Stx2eB-lettuce strains, 2BN containing ingredient Stx2eB constituting a concentration level of 0.53 mg Stx2eB/g of powdered lettuce dry weight (DW) and 2BH containing ingredient Stx2eB constituting a concentration level of 2.3 mg of Stx2eB/g of powdered lettuce DW, were evaluated in three sequential experiments. Taken the results together, oral administration of Stx2eB-lettuce vaccine was suggested to relieve the pathogenic symptoms of ED in piglets challenged with virulent STEC strain. Our data suggested that Stx2eB-lettuce is a promising first oral vaccine candidate against ED.

Improved porcine model for Shiga toxin-producing Escherichia coli infection by deprivation of colostrum feeding in newborn piglets.

Porcine edema disease (ED) is a toxemia caused by enteric infection with Shiga toxin 2e (Stx2e)-producing Escherichia coli (STEC). ED occurs most frequently during the weaning period and is manifested as emaciation associated with high mortality. In our experimental infection with a specific STEC strain, we failed to cause the suppression of weight gain in piglets, which is a typical symptom of ED, in two consecutive experiments. Therefore, we examined the effects of deprivation of colostrum on the sensitivity of newborn piglets to STEC infection. Neonatal pigs were categorized into two groups: one fed artificial milk instead of colostrum in the first 24 h after birth and then returned to the care of their mother, the other breastfed by a surrogate mother until weaning. The oral challenge with 1011  colony-forming units of virulent STEC strain on days 25, 26 and 27 caused suppression of weight gain and other ED symptoms in both groups, suggesting that colostrum deprivation from piglets was effective in enhancing susceptibility to STEC. Two successive STEC infection experiments using colostrum-deprived piglets reproduced this result, leading us to conclude that this improved ED piglet model is more sensitive to STEC infection than the previously established models.

Comparison of gene expression profiles of T cells in porcine colostrum and peripheral blood.

OBJECTIVE To compare gene expression patterns of T cells in porcine colostrum and peripheral blood. ANIMALS 10 multiparous sows. PROCEDURES Cytotoxic and CD4-CD8 double-positive T cells were separated from porcine colostrum and peripheral blood. Total RNA was extracted. The cDNA prepared from RNA was amplified, labeled, fragmented, and competitively hybridized to DNA microarray slides. The DNA microarray data were validated by use of a real-time reverse-transcription PCR assay, and expression of the genes FOS, NFKBI, IFNG, CXCR6, CCR5, ITGB2, CCR7, and SELL was assessed. Finally, DNA microarray data were validated at the protein level by use of flow cytometry via expression of c-Fos and integrin ß-2. RESULTS Evaluation of gene expression profiles indicated that in contrast to results for peripheral blood, numerous cell-signaling pathways might be activated in colostrum. Profile analysis also revealed that FOS and NFKBI (genes of transcription factors) were involved in most cell-signaling pathways and that expression of these genes was significantly higher in colostral T cells than in peripheral blood T cells. Furthermore, CCR7 and SELL (genes of T-cell differentiation markers) in colostral T cells had expression patterns extremely similar to those found in effector or effector memory T cells. CONCLUSIONS AND CLINICAL RELEVANCE All or most of the T cells in colostrum had an effector-like phenotype and thus were more activated than those in peripheral blood. This gene expression profile would enable T cells to migrate to mammary glands, be secreted in colostrum, and likely contribute to passive immunity provided by sows to newborn pigs.

The effect of colostrum ingestion during the first 24 hours of life on early postnatal development of piglet immune systems.

It has been suggested that colostrum is important not only for direct protection from pathogens but also for proper development of immune systems in piglets. In this study, we focused on the effect of colostrum ingestion during the first 24 h of life on early postnatal development of piglet immune systems. Thirty-six piglets from five litters were divided into colostrum-fed (CoF) and colostrum-deprived (CoD) groups. The former group was allowed to suckle normally while formula milk was fed to the latter group during the first 24 h of life. At the weaning period, the concentrations of fecal immunoglobulin (Ig) A and plasma IgG as well as the number of blood leukocyte subsets were analyzed. Fecal IgA and plasma IgG concentrations in the CoF group were more than twice as high as those in the CoD group (P < 0.01). In addition, the number of blood B cells was significantly higher in the CoF group than that in the CoD group (P < 0.05). This study demonstrates that colostrum ingestion during the first 24 h plays a significant role in early postnatal development of both mucosal and systemic immunity of piglets.

Evaluation of secretion volume and immunoglobulin A and G concentrations in sow colostrum from anterior to posterior teats.

Among domestic animals, teat order is only observed in the pig. In order to achieve the healthy growth and weaning of piglets, it is important to elucidate if volume of colostrum secretion and immunoglobulin A (IgA) and IgG concentrations differ among the teats of a sow. Nine sows were used to evaluate the difference in colostrum secretion volume (CSV) and four of these sows were assessed for IgA and IgG concentrations from each teat. Samples were collected five times during 21 h following parturition. Teats were assigned anatomical locations of teat (1 to 7) from anterior to posterior. The CSV of anterior (locations 1 and 2) and middle teats (locations 3-5) was significantly higher than those of posterior teats (locations 6 and 7) throughout the experiment except for 18 h post-parturition (P < 0.05). The CSV of the teats at location 1 was significantly higher at most collection times than those at locations 6 and 7. A positive correlation of CSV was observed with IgA and IgG concentrations from 12 h and 6 h post-parturition, respectively (P < 0.05). The results suggest that anterior teats secrete greater volumes of colostrum and that these tend to contain higher IgA and IgG than posteriors teats.

Shotgun proteomic analysis of porcine colostrum and mature milk.

The epitheliochorial nature of the porcine placenta prevents the transfer of maternal immunity. Therefore, ingestion of the colostrum immediately after birth is crucial for neonatal piglets to acquire passive immunity from the sow. We performed a shotgun proteomic analysis of porcine milk to reveal in detail the protein composition of porcine milk. On the basis of the Swiss-Prot database, 113 and 118 proteins were identified in the porcine colostrum and mature milk, respectively, and 50 of these proteins were common to both samples. Some immune-related proteins, including interleukin-18 (IL-18), were unique to the colostrum. The IL-18 concentration in the colostrum and mature milk of four sows was measured to validate the proteomic analysis, and IL-18 was only detected in the colostrum (191.0 ± 53.9 pg/mL) and not in mature milk. In addition, some proteins involved in primary defense, such as azurocidin, which has never been detected in any other mammal's milk, were also identified in the colostrum.

Interaction between optineurin and the bZIP transcription factor NRL.

Although the gene encoding optineurin (OPTN) is a causative gene for glaucoma and amyotrophic lateral sclerosis, it is ubiquitously expressed in all body tissues, including the retina. To study the function of OPTN in retinal ganglion cells as well as the whole retina, we previously isolated OPTN-interacting proteins and identified the gene encoding the bZIP transcription factor neural retina leucine zipper (NRL), which is a causative gene for retinitis pigmentosa. Herein, we investigated the binding between OPTN and NRL proteins in HeLaS3 cells. Co-expression of HA-tagged NRL and FLAG-tagged OPTN in HeLaS3 cells followed by immunoprecipitation and Western blotting with anti-tag antibodies demonstrated the binding of these proteins in HeLaS3 cells, which was confirmed by proximity ligation assay. NRL is the first OPTN-binding protein to show eye-specific expression. A series of partial-deletion OPTN plasmids demonstrated that the tail region (423-577 amino acids [aa]) of OPTN was necessary for binding with NRL. Immunostaining showed that Optn (rat homologue of OPTN) was expressed in rat photoreceptors and localised in the cytoplasm of photoreceptor cells. This is a novel demonstration of Optn expression in photoreceptor cells. OPTN was not detected in photoreceptor nuclei under our experimental conditions. Further analyses are necessary to elucidate the function of OPTN and the significance of its possible binding with NRL in photoreceptor cells.

The preventive effect of Bacillus subtilus strain DB9011 against experimental infection with enterotoxcemic Escherichia coli in weaning piglets.

Porcine edema disease (ED) is caused by Shiga toxin 2e-producing Escherichia coli (STEC). Post-weaned piglets often suffer from ED as a result of intestinal infection with STEC, which causes impaired growth performance and high mortality. Antimicrobial therapy is a curative treatment for piglets infected with STEC, but the emergence of antimicrobial-resistant STEC has become a serious problem for Japanese pig farmers. Therefore, an alternative strategy other than antimicrobial therapy is needed for the prevention or treatment of ED. In this study, we evaluated the effect of oral administration of Bacillus subtilis DB9011 (DB9011) to prevent the experimental infection of STEC in weaning piglets. Eight 21-day-old piglets were divided into two groups: STEC challenge with the basal diet, and STEC challenge with DB9011 supplemented diet. The challenge was carried out when the animals were 25, 26 and 27 days old using STEC contained in capsules resistant against gastric digestion. All pigs were euthanized at 36 days of age. DB9011 improved the symptoms of ED and decreased the number of STEC in the ileal digesta and feces. Accordingly, oral administration of DB9011 in weaned piglets prevents ED through the suppression of the growth of STEC in the ileum.

Correlation between villous height and the disaccharidase activity in the small intestine of piglets from nursing to growing.

Early weaning induces villous atrophy in the small intestine. Reduction in villous height in the small intestine after weaning is associated with reductions in brush-border enzyme activity. Body weight gain after weaning is, therefore, correlated with villous height. This evidence suggested that the maintenance of small intestinal structure and function after weaning is important for the growth of young pigs. On the other hand, the relationship between villous height and the activity of the digestive enzymes in the small intestine has not been studied with piglets from the suckling to the growing period. Five suckling piglets, four piglets in the proximal stage of weaning, four pigs in the distal stage of weaning and four growing pigs were used. The activities of lactase (LA), sucrase (SA) and maltase (MA) were determined. LA showed a positive correlation with villous height in weaning. SA and MA were positively correlated with villous height from suckling to growing. In a previous study, non-infectious dyspeptic diarrhea was frequently observed in growing piglets on Japanese swine farms. The maintenance of villous height to retain disaccharidase activity may prevent dyspepsic diarrhea in this stage.

Interference of kallikrein 1b26 (klk1b26) translation by microRNA specifically expressed in female mouse submandibular glands: an additional mechanism for sexual dimorphism of klk1b26 protein in the glands.

BACKGROUND: Mouse kallikrein 1b26 (klk1b26) protein is more abundant in male submandibular glands (SMGs) than in female ones. This sexual dimorphism has been thought to be due to increased mRNA synthesis stimulated by androgen. However, the klk1b26 protein level in female SMG is far less than that expected from the mRNA level, suggesting an additional mechanism for down-regulation of klk1b26 expression in female SMGs. METHODS: We examined the effects of small non-coding RNAs in mouse SMGs on in vitro translation of klk1b26 using a reticulocyte lysate system and reverse transcription (RT)-PCR for klk1b26 mRNA. Statistical analyses were performed with a computer package (Microsoft Excel). RESULTS: The microRNA (miRNA) preparation from female SMGs, but not male SMGs, interfered with the in vitro translation of the klk1b26 protein and inhibited the RT-PCR for klk1b26 mRNA with forward primers targeting its 5'-terminal region (between the 15th and 40th nucleotide from the 5'-terminal). The miRNA preparation from castrated mouse SMGs showed the inhibitory effect on the klk1b26 translation, but that from a 5α-dihydrotestosterone-treated female mouse SMGs did not. Synthetic miRNAs (miR-325 and miR-1497a), which have partial complementarity with klk1b26 mRNA at its 5'-terminal region (15th to 40th nucleotide position from the 5'-terminal), also interfered with the in vitro klk1b26 translation. When the female miRNA preparation was incubated with a 30-nucleotide-long single-strand oligoDNA (named [15th-44th]ssDNA, whose sequence corresponded to the 15th to 44th position from the 5'-terminal of klk1b26 mRNA) prior to the addition into the in vitro translation system, the inhibitory effect of the miRNA preparation on klk1b26 translation disappeared, while [15th-44th]ssDNA itself had no effect on the translation. Preincubation of the miRNA preparation with another single-strand DNA ([169th-198th]ssDNA, whose sequence corresponded with 169th to 198th position of klk1b26 mRNA) did not show the inhibitory effect. CONCLUSIONS: The small non-coding RNA, most probably miRNA, specifically expressed in female mouse SMGs interfered with klk1b26 protein synthesis in the in vitro translation system. Therefore sexual dimorphism observed in klk1b26 expression in mouse SMGs is due at least in part to the female-specific small non-coding RNA in SMGs.

Differential modulation of immunostimulant-triggered NO production by endoplasmic reticulum stress inducers in vascular smooth muscle cells.

We investigated the effects of endoplasmic reticulum (ER) stress inducers thapsigargin (TG) and tunicamycin (Tm) on immunostimulant lipopolysaccharide/interferon (LPS/IFN)-induced expression of isoform of nitric oxide synthase (iNOS) and nitric oxide (NO) production in vascular smooth muscle cells. LPS/IFN-induced iNOS mRNA expression was markedly enhanced by TG, whereas iNOS mRNA expression was strongly attenuated by Tm. Similarly, production of iNOS protein was markedly upregulated by TG but virtually eliminated by Tm. LPS/IFN-induced guanosine triphosphate cyclohydrolase I mRNA expression was slightly reduced by TG and markedly inhibited by Tm. Similarly, LPS/IFN-mediated induction of cellular biopterin was modestly reduced by TG and markedly inhibited by Tm. TG modestly enhanced LPS/IFN-induced activation of NF-κB, whereas Tm had no effect on it. Cellular respiration was reduced by TG and Tm in a concentration-dependent manner, which was confirmed by apoptosis assay. Thus, TG and Tm-induced ER stress and differently modulated NO production through alterations in iNOS expression and activity independently of NF-κB activation and caused a similar degree of ER stress-induced apoptosis.

YPEL5 protein of the YPEL gene family is involved in the cell cycle progression by interacting with two distinct proteins RanBPM and RanBP10.

YPEL5 is a member of the YPEL gene family that is highly conserved in the eukaryotic species and apparently involved in a certain cell division-related function. In this study, we examined the functional and phylogenetic aspects of YPEL5 protein in more detail. During cell cycle, YPEL5 protein was detected at different subcellular localizations; at interphase, it was located in the nucleus and centrosome, then it changed location sequentially to spindle poles, mitotic spindle, and spindle midzone during mitosis, and finally transferred to midbody at cytokinesis. Knockdown of YPEL5 function by siRNA or anti-sense morpholino oligonucleotide inhibited the growth of cultured COS-7 cells and early development of medaka fish embryos, indicating its involvement in cell cycle progression. Interestingly, RanBPM (Ran Binding Protein in the Microtubule organizing center, encoded by RANBP9) was identified as a YPEL5-binding protein by yeast two-hybrid method. A paralog of RanBPM, namely RanBP10 (encoded by RANBP10), was found to be another YPEL5-binding protein, and these two protein genes are highly conserved each other. Comparative genomic analysis allowed us to define a new gene family consisting of RanBPM and RanBP10, named Scorpin, providing a basis to better understand how they interact with YPEL5.

An epidemiological survey on pigs showing symptoms of infectious enteric diseases and dyspepsia in Japan.

Diarrhea in pigs has the potential to have a serious economic impact on the swine industry. Previously, we suggested that the likely cause of the presence of non-infectious diarrhea in pigs characterized by lactate accumulation was dyspepsia. In this experiment, the prevalence of enteropathogens and hyper-lactate accumulation in feces of piglets in 4 distinct growth stages was examined. The feces were collected when veterinarian experts recognized abnormalities in sporadic outbreaks. Prevalence of enteropathogens in diarrheal feces was 100% in fattening pigs (FP), 75% in weaning pigs (WP), 50% in suckling pigs (SP), and 42% in growing pigs (GP). Prevalence of enteropathogens in loose feces was 53% in WP, 50% in SP, 40% in FP, and 28% in GP. Prevalence of hyper-lactate accumulation in diarrheal feces was 33% in GP, 33% in SP, 25% in WP, and 25% in FP. Prevalence of hyper-lactate accumulation in loose feces was 40% in GP, 0% in SP, 7% in WP, and 5% in FP. Accordingly, non-infectious dyspepsia is frequent in growing pigs. In this period, pigs are potentially exposed to needless antimicrobial therapeutic treatments in sporadic cases.

Decreasing traits of fecal immunoglobulin A in neonatal and weaning piglets.

The concentration of fecal secretory immunoglobulin A (sIgA) in neonate and weaning piglets was measured daily from 1 day after birth to 50 days of age. The concentration of fecal sIgA started from the level of 10(4) microg/g wet feces 1 day after birth and then increased to a maximal value of up to 10(5) microg/g within a few days of birth. The values constantly declined to between 10(1) and 10(2) microg/g for the next 10 days and were relatively constant until weaning. The level of sIgA in the feces remained very low until at least 50 days of age. The vulnerability of pre- or post-weaning piglets can be explained, at least in part, by this low level of sIgA in the intestine.

Systematic evaluation of nitric oxide, tetrahydrobiopterin, and anandamide levels in a porcine model of endotoxemia.

PURPOSE: Using a lipopolysaccharide (LPS)-treated porcine model, we examined: (1) whether nitric oxide (NO), anandamide, and tetrahydrobiopterin (BH4) increased or not in early endotoxic shock; and (2) the location of the major site of production of these molecules, by comparing their concentrations in arteries and the portal and hepatic veins. METHODS: Ten pigs received an infusion of LPS at 1.7 microg x kg(-1)x h(-1) via the portal vein for 240 min. Consecutive changes in systemic hemodynamics, hepatosplanchnic circulation, and oxygen delivery were measured. Furthermore, the variable changes in the concentrations of nitrite and nitrate (NOx), anandamide, and BH4 were measured. To access the effects of surgery, anesthesia, and fluid management on BH4, an experiment without LPS infusion was performed in two other animals. RESULTS: Mean arterial pressure and cardiac index started to decrease at 60 min after LPS infusion. However, systemic vascular resistance remained unchanged. Total hepatic blood flow and hepatic oxygen delivery also decreased significantly. NOx and anandamide did not change during LPS infusion. BH4 values did not change without LPS infusion. However, BH4 values increased significantly in the arterial, portal, and hepatic circulation during LPS infusion, especially in the hepatic vein (from 136.8 +/- 27.5 to 281.3 +/- 123.2 mol/ml; P < 0.01). CONCLUSION: Our data suggest that the BH4 values were significantly increased in several organs, especially in the liver during endotoxic shock. Impaired cardiac output and decreased blood pressure appeared in the early phase of porcine endotoxemia. Longer-term observation of these parameters after LPS treatment should be performed as the next step in future studies.

Novel RDH5 mutation in family with mother having fundus albipunctatus and three children with retinitis pigmentosa.

PURPOSE: To identify mutations in the RDH5 gene in a family with a mother having fundus albipunctatus (FA) and 3 children with retinitis pigmentosa (RP). METHODS: Ophthalmological examinations were performed to diagnose FA and RP. Mutational analysis of RDH5 was performed. RESULTS/CONCLUSIONS: The mother was diagnosed with FA, and 3 children were diagnosed with RP. The proband's mother, brother, and sister had a novel mutation c.689_690CT > GG in RDH5. The proband and mother had a previously reported mutation c.928delCinsGAAG. Consequently, the mother's FA was caused by compound heterozygous mutations. Further studies will be needed to determine the gene responsible for children's RP.

Cilostazol activates AMP-activated protein kinase and restores endothelial function in diabetes.

BACKGROUND: Endothelial dysfunction plays a key role in atherogenesis. We investigated whether AMP-activated protein kinase (AMPK) activity is a downstream mediator of the beneficial effects of cilostazol on vascular endothelial cells and whether cilostazol might reverse endothelial dysfunction in diabetic rats. METHODS AND RESULTS: Treatment of human umbilical vein endothelial cells (HUVECs) with cilostazol resulted in time-dependent activation of AMPK, as monitored by phosphorylation of AMPK and its down-stream target, acetyl-CoA carboxylase (ACC). Activation of AMPK by cilostazol was through signaling pathway independent of cyclic AMP and caused phosphorylation of endothelial nitric oxide synthase (eNOS), leading to increased production of nitric oxide (NO), while inhibiting cytokine-induced nuclear factor-kappaB (NF-kappaB) activation, leading to suppression of VCAM-1 gene expression. Significantly reduced eNOS activity and NO production in response to cilostazol and attenuation of cilostazol-induced inhibition of NF-kappaB activation were observed in cells treated with AMPK siRNA. We also demonstrated that administration of cilostazol to diabetic rats significantly restored endothelium-dependent vasodilation. Furthermore, treatment of diabetic rats with cilostazol increased tetrahydrobiopterin (BH4) levels in the aorta. Thus, recovery of BH4 following administration of cilostazol might also contribute to restoration of endothelial function in diabetic rats. CONCLUSIONS: Our findings suggest that the beneficial effects of cilostazol on endothelial function may be due to AMPK activation. Restoration of endothelial dysfunction in diabetic rats by cilostazol is at least partly attributed to amelioration of biopterin metabolism in the aorta.

Expression of Na(+)/K(+)-ATPase alpha subunit isoforms in rat salivary glands: occurrence of sense and antisense RNAs of the alpha3 isoform in the sublingual gland.

We examined the expression of Na(+)/K(+)-ATPase alpha-subunit isoforms in rat salivary glands by RT-PCR. Isoform alpha1 was expressed strongly in all three major salivary glands. The alpha2 isoform was expressed in both submandibular gland (SMG) and sublingual gland (SLG) but faintly in the parotid gland (PG). The alpha3 was detected only in the SLG, and the alpha3 mRNA in the SLG was 1/8 of its level in the brain. Na(+)/K(+)-ATPase alpha3 isoform in the SLG, was localized predominantly on the basolateral plasma membranes in serous cells by immunohistochemical method. We also found the presence of natural antisense RNA of the alpha3 isoform in rat SLG: the 1st-strand cDNA prepared with gene-specific forward primers targeted to the CDS region and to the promoter region of the alpha3 gene in place of oligo(dT) or gene-specific reverse primers produced reasonable PCR products corresponding to the alpha3 cDNA sequence by the subsequent PCR reaction. Synthesis of the 1st-strand cDNA with the gene-specific forward primers was prevented by RNase digestion of the total RNA preparation, indicating that the PCR products in the RT-PCR system were not due to the contaminated genomic DNA, if any. The alpha3 protein level in rat SLG increased with aging, and levels of both alpha3 mRNA (sense RNA) and alpha3 antisense RNA were higher in SLGs of aged rats than in those of young rats, respectively.

Evaluation of the low dose level of a heat-killed and dried cell preparation of Enterococcus faecalis to prevent porcine edema disease using experimental infection model with enterotoxcemic Escherichia coli in weaning pigs.

Porcine edema disease (ED) is caused by Shiga toxin 2e-producing Escherichia coli (STEC). ED has become frequent in pig farms, and the use of antimicrobials has resulted in the development of antimicrobial-resistant STEC. Accordingly, the use of materials other than antimicrobials is requested for the prevention of ED. Oral administration of a heat-killed and dried cell preparation of Enterococcus faecalis strain EC-12 (EC-12) to weaning pigs was previously demonstrated to decrease animal mortality in a STEC-contaminated farm at 0.05% (w/w) dose level. In this study, pigs experimentally infected with STEC were used as a model for ED to evaluate the low dose level of EC-12 to prevent ED. Fifteen 21-day-old pigs were divided into 5 groups: STEC challenge with the basal diet, STEC challenge with EC-12 supplemented at 0.005, 0.01, or 0.05% (w/w) to the basal diet, and no STEC challenge with the basal diet. The challenge was carried out when the animals were 25, 26, and 27 days old using STEC contained in capsules resistant against gastric digestion. All pigs were euthanized at 32 days of age. The daily weight gain, feed conversion ratio, and palpebral edema were improved by supplementation with 0.05% EC-12, but not by the low dose levels. Accordingly, 0.05% level of supplementation was needed for EC-12 to improve clinical symptoms in weaning piglets infected by STEC.