Shiga toxin 2e (Stx2e) has a pivotal role in the colonization and enterotoxicity of F18+Shiga toxin-producing Escherichia coli (STEC), which causes porcine edema disease (ED). In this study, a Stx2eA mutant, which has a Glu167Gln mutation in Stx2eA that inactivates N-glycosidase activity, was genetically engineered to evaluate its potential immunogenicity and protective efficacy. A significant increase in serum IgG1 (a Th2 indicator) was shown in mice immunized with the mutated Stx2eA. However, only 56% of the mice immunized with the toxoid (5 μg) survived following a challenge with a lethal dose 50 (LD50) of a virulent F18+STEC strain (JOL654), while mice immunized with Salmonella ghosts delivering selected antigens of F18+STEC showed an 86% survival rate. The results suggest that sole use of the mutated Stx2eA toxoid may not be an effective preventive strategy for the control of porcine ED.

It has been known that the ability of Shiga toxinproducing Escherichia coli (STEC) to produce Stx2e in culture media plays a role in the diagnosis of edema disease and determination of subunit vaccine candidates in STEC isolates. To examine the efficiency of Stx2e production in several commercial media, a Stx2e-producing strain (KEFS1302) was grown in four different media: ISO-Sensitest broth (ISB), E. coli broth (ECB), trypticase soy broth (TSB), and Mueller Hinton broth (MHB), with or without mitomycin C at 37°C (250 rpm) for 6 h. Toxin production was measured by enzyme-linked immunosorbent assay. In the presence of mitomycin C, ECB was found to be the most suitable medium, reaching a production peak (OD600 = 1.2) at 1 h; Stx2e was mostly produced during the logarithmic phase (within 3 h). On the other hand, toxin production in ISB reached a peak at 3 h after incubation in the absence of mitomycin C. Stx2e was purified by fast protein liquid chromatography (FPLC) using anion-exchange chromatography. The 43 kDa band of Stx2e was confirmed by western blot using the ECB supernatant. Our results showed that ECB and ISB media would be a suitable medium for mass production of Stx2e even if the toxin production is dependent on time.

The emergence of antimicrobial resistant Escherichia (E.) coli is a major problem in pig farms. To tackle this issue, in July 2011, the Korean government banned the use of antimicrobials for growth promotion of animals in farms. Moreover, E. coli encoding the Stx2e gene cause edema disease which results in high mortality and morbidity in pig farms. Therefore, the aim of this study was to investigate the prevalence of antimicrobial resistance among E. coli encoding the Stx2e gene isolated from weaned piglets with diarrhea before and after the ban on antibiotic growth promoters (AGPs) in Korea from 2007 to 2016. In this period, 479 E. coli isolates were obtained from weaned piglets with diarrhea, and of them, 144 E. coli isolates encoding the Stx2e gene were detected by polymerase chain reaction. The susceptibility of the E. coli isolates to antibiotics were tested using the standard Kirby-Bauer disk diffusion method. The most frequently observed resistances in isolates obtained from weaned piglets in the last 10 years were to tetracycline (92.4%) and chloramphenicol (88.9%). The prevalence of resistance to colistin (3.1% to 16.5%) and tetracycline (86.2% to 97.5%) was also observed to have increased over this period. Additionally, multi-drug resistance was also found to have increased (87.7% to 97.5%) after the ban on AGPs. These findings provide useful data for designing prevention and treatment strategies for postweaning diarrhea and edema disease, and can be used in future studies on antimicrobial resistance in Korea.