• Journal of the Korean Society of Food Science and Nutrition
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• v.20 no.6
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• pp.621-628
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• 1991

Heat-stable enterotoxin(ST) from enterotoxigenic E. coli eKT-53($ST^{+}\;LT^{-}$, transformant from isolate KM-7) that was produced in succinate salts medium. The culture supernatant(crude ST) was purifed by mulitpled steps and investigated some characterization of the ST. The heatstability of purified ST activity was completely lost by treating at $100^{\circ}C$ for 30minutes. ST activity was lost by treatment at pH 1 and 12 conditions, while the activity was not reduced by treatment at pH 2~10, and then the ${\alpha}-amylase$ and pepsin was not decreased activity but disulfide reducing agnets was lost the activity. The molecular weight of the purified ST was approximately 4,200, the isoelectric point was about 4.0.

• Journal of Microbiology and Biotechnology
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• v.31 no.11
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• pp.1552-1558
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• 2021

The diverse microbial communities in kimchi are dependent on fermentation period and temperature. Here, we investigated the effect of enterotoxigenic Escherichia coli (ETEC) during the fermentation of kimchi at two temperatures using high-throughput sequencing. There were no differences in pH between the control group, samples not inoculated with ETEC, and the ETEC group, samples inoculated with ETEC MFDS 1009477. The pH of the two groups, which were fermented at 10 and 25℃, decreased rapidly at the beginning of fermentation and then reached pH 3.96 and pH 3.62. In both groups, the genera Lactobacillus, Leuconostoc, and Weissella were predominant. Our result suggests that microbial communities during kimchi fermentation may be affected by the fermentation parameters, such as temperature and period, and not enterotoxigenic E. coli (ETEC).

• Journal of Microbiology
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• v.43 no.4
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• pp.354-360
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• 2005

Heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli (ETEC) is both a strong mucosal adjuvant and immunogen. It is a subunit vaccine candidate to be used against ETEC-induced diarrhea. It has already been expressed in several bacterial and plant systems. In order to construct yeast expressing vector for the LTB protein, the eltB gene encoding LTB was amplified from a human origin enterotoxigenic E. coli DNA by PCR. The expression plasmid pLTB83 was constructed by inserting the eltB gene into the pYES2 shuttle vector immediately downstream of the GAL1 promoter. The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The LTB protein was detected in the total soluble protein of the yeast by SDS-PAGE analysis. Quantitative ELISA showed that the maximum amount of LTB protein expressed in the yeast was approximately $1.9\%$ of the total soluble protein. Immunoblotting analysis showed the yeast-derived LTB protein was antigenically indistinguishable from bacterial LTB protein. Since the whole-recombinant yeast has been introduced as a new vaccine formulation the expression of LTB in S. cerevisiae can offer an inexpensive yet effective strategy to protect against ETEC, especially in developing countries where it is needed most.

• The Journal of the Korean Society for Microbiology
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• v.21 no.1
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• pp.47-52
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• 1986

A heat-labile enterotoxin and no heat-stable enterotoxin producing($LT^+ST^-$) plasmid (110 kilobases in size) was isolated from an enterotoxigenic Escherichia coli of human strain O15:H11 and used for analysis of the $LT^+$ deoxyrionucleic acid region using recombinant DNA technology. A DNA segment containing the $LT^+$ DNA region which was one restriction endonuclease BamHl fragment(6.2 kb in size) was joind to a small multicopy plasmid, pUC9. E. coli K-12 strain, JM103 harboring the chimeric plasmid produced greater amounts of LT than did the enterotoxigenic E. coli O15:H11 strain. The BamHl fragment was further digested with various restriction endonucleases and contained no HindIll restriction site which is an essential in $LT^+ST^+$ plasmid. The detailed DNA sequencing of this $LT^+ST^-$ plasmid is required.

• Korean Journal of Veterinary Research
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• v.38 no.4
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• pp.829-836
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• 1998

Immunogenicity of Escherichia coli pilus and LT were evaluated in 20-week-old hens. The antigens were consisted of K88, K99, 987p pilus and heat labile toxin purified from enterotoxigenic Escherichia coli. The durations of antibody titers in sera and egg yolk were investigated by an enzyme-linked immunosorbent assay(ELISA). After first inoculation, antibody titers in sera reached at peak 2 weeks postinoculation. However, peak antibody titers in egg yolk were detected 4 weeks postinoculation, indicating that transfer of immunoglobulin from serum to egg yolk took about two weeks period. Although there were slight reduction in titers, the specific antibodies in egg yolk lasted up to 3 months. Immune responses against monovalent and combined antigens were showed as almost same patterns. The transfer rate of antibodies from serum to egg yolk didn't show any significant differences among three pilus antigens in this study. Considering the concentrations of antigens in each inoculated group, multivalent antigens containing heat labile toxin of E coli were found to be more immunogenic than monovalent antigen in producing specific antibodies. From this experiment, it was demonstrated that multivalent antigens containing three pilus and heat labile toxin could be a promising candidate for the production of egg yolk antibodies for prophylactic use in preventing swine colibacillosis in future.

• Korean Journal of Veterinary Service
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• v.20 no.4
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• pp.359-370
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• 1997

This study was carried out to Investigate the biochemical characteristics, antibiotic susceptibility, serogroups and pili producibility test of enterotoxigenic Escherichia coli(ETEC) isolated from piglets with diarrhea in Kangwon province from March to October 1996. 1. Sixty eight E coli strains were isolated from 72 piglets with diarrhea and the biochemical and cultural reaction were compared with the classification criteria of Edwards and Ewing. 2. The serogroups of 26 isolates were classified as 08 : K87 6(8.8%), O20 : K1O1 4(5.9%), O141 : K85 4(5.9%), 09 : K103 : P987 3(4.4%), O45 : K 2(2.9%) 0139 : K82 2(2.9%), O64 : $K^{-}$2(2.9%), O149 : K91 1(1.5%), O157 : K88ac 1(1.5%) and O115 : $K^{-}$1(1.5%), respectively. 3. In antibiotic susceptibility test, the isolates showed high susceptible to Ak, Eno, Na, Gm, Am and Km, whereas resistance to Tc, Sm and Cf. 4. Sixty one strains(89.7%) of 68 I coli Isolates were resistant to one or more drugs. The isolates resistant to 2 and 3 or more drugs were 60.3% and 19.1%, respectively. Amog the 16 multiple resistant patterns, Sm Tc(11.5% ), Cf Sm Tc(11.5% ), Cf Cp Sm Su Tc(9.8% ) and Cf Cp Sm Su Tc(8.2%) patterns were frequently observed. 5. MRHA of guinea pig erythrocytes was detected in 9 out of 26 OK serotype and 9 out of 42 unidentified serotypes. MRHA titers of serotypes showed from 16 to 32 in O141 : K85 and no titers in O139 : K82. 6. By the GM1 ganglioside ELISA, $\beta$-, $\alpha$-, and $\gamma$-hemolysin producing strains was detected as 36, 6, and 5 from heat labile enterotoxin(LT) of 47 ETEC, respectively. The distribution of LT toxin from 112 isolates was showed $\beta$- hemolysin, 2 isolates $\alpha$-hemolysin and 3 isolates $\gamma$-hemol-ysin from 26 OK serotypes.

• The Journal of the Korean Society for Microbiology
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• v.22 no.4
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• pp.377-392
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• 1987

Monoclonal antibody to the Escherichia coli heat-stable enterotoxin(ST) was produced to develop a rapid and convenient diagnostic method to the ST. The toxin was purified from culture supernatant of enterotoxigenic E. coli O148H28($ST^+/LT^+$) and conjugated to bovine serum albumin(BSA). The ST-BSA conjugate was used to immunize BALB/c mice and the immune spleen cells from these mice were fused with $P3{\times}63$ Ag8.V653 plasmacytoma cells. Hybridomas were screened by ELISA and positive hybridomas were cloned by limiting dilution. Finally, one stable clone (AS36) specific to ST was selected for further growth and characterization. Antibody titers of culture supernatant and ascitic fluid from BALB/c mice were 1:1,024 and 1:20,480 respectively in ELISA. The isotype and subclass of monoclonal antibody was IgG1 in sandwich ELISA. To test the neutralizing effect of monoclonal antibody on toxin activity of ST, mixture of ascitic fluid and ST was assayed by infant mouse assay and this monoclonel antibody was proved to be a neutralizing antibody. The titer of ascitic fluid which completely neutralized biological activity of 4 units of ST was 1:4. Purified ST was quantitatively measured by competitive ELISA and minimum amount of ST detectable by this assay was 250pg, which was an amount six-fold smaller than that detectable by infant mouse assay. Four reference strains of enterotoxigenic E. coli from WHO were detected by competitive ELISA and highly specific, sensitive and reproducible result was obtained.

• Korean Journal of Agricultural Science
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• v.43 no.5
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• pp.788-793
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• 2016

Post-weaning diarrhea (PWD), mostly caused by enterotoxigenic Escherichia coli (ETEC), remains to be a major source of economic loss in swine industry. The use of the ETEC-oral challenge model is often applied to mimic unsanitary commercial swine farm conditions where pathogens and unknown complex microbes exist and can cause severe infections in pigs. The purpose of this study was (1) to estimate ETEC density using spectrophotometric computation, (2) to determine survivability of ETEC after storing at $-20^{\circ}C$ for 7 days, and (3) to evaluate survivability of ETEC after blending with diluted sweeteners (0, 5, 10, 20, and 40% sucrose in phosphate buffered saline [PBS]). Cell density was quantified using UV-VIS spectrophotometer and counting ETEC colony forming units (cfu) at 0, 30, 60, 90, 120, 150, 180, 210, and 240 min. The established linear equation ($y=0.0031x^2-0.0079x+0.0043$ and $y=0.0046x^2-0.0151x+0.0113$) was used for robust quantification of each ETEC cell density. ETEC stored at $-20^{\circ}C$ showed 108 cfu/mL after thawing and incubation. When ETEC was blended with sweeteners (20 and 40%), survival of ETEC was decreased by 58 and 54% in 5 min post blending. However, addition of 20% of sweetener resulted in a higher survivability than those with other media concentrations. Therefore, the use of ETEC-oral challenge model would be possible as a stable method if we could confirm the appropriate medium that increases survivability of ETEC in weaner pigs.

• Journal of Animal Science and Technology
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• v.57 no.1
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• pp.4.1-4.5
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• 2015

The study was performed to investigate the effect of dietary supplementation of a lipid-encapsulated Zinc oxide on growth parameters and intestinal mucosal morphology piglets born to Duroc-sired Landrace ${\times}$ Yorkshire dams. Twenty-four 30-day-old piglets weaned at 25 days of age were orally challenged with $5{\times}10^8$ colony forming units of enterotoxigenic Escherichia coli (ETEC) K88 and fed one of the four diets for 7 days: (i) a nursery basal diet containing 100-ppm ZnO (referred to as BASAL), (ii) BASAL supplemented with 120-ppm apramycin (referred to as ANTIBIO), (iii) BASAL with 2,400-ppm ZnO (referred to as HIGH), and BASAL containing 100-ppm lipid-encapsulated ZnO (referred to as LE). All piglets were killed at the end of the experiment for histological examination on the intestine. The results showed that the average daily gain (ADG), the villus height: crypt depth (CD) ratio in the ileum, and the goblet cell density of the villus and crypt in the duodenum, jejunum, and colon were greater in the LE-fed group that those of the BASAL (p < 0.05). Fecal consistency score (FCS) and the CD ratio in the ileum were less in the LE-fed group, compared to the BASAL-fed one (p < 0.05). The effects observed in the LE-fed group were almost equal to those of the HIGH-fed group as well as even superior to those of the ANTIBIO-fed group. Taken together, our results imply that dietary supplementation of 100-ppm lipid-encapsulated ZnO is as effective as that of 2,400-ppm ZnO for promoting growth diarrhea and intestinal morphology caused by ETEC infection.

• Korean Journal of Veterinary Service
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• v.40 no.4
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• pp.235-244
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• 2017

Enterotoxigenic Escherichia coli (ETEC) strains producing each F4, F5, F6 and F41 fimbriae were lysed by GI24 peptide. The lysate cells were used as ETEC vaccine candidate. This study was carried out to examine whether intramuscular (im) immunization of pregnant sows with the novel vaccine candidate could effectively protect their neonatal piglets against ETEC colibacillosis. All pregnant sows were primed at 11 weeks and were boosted at 14 weeks of pregnancy. Group A sows were im inoculated with PBS. Group B sows were im immunized with $2{\times}10^9$ the mixture. Seral IgG, colostral IgA and IgG titers from group B sows, and seral IgG and IgA levels in group B piglets were significantly higher than those of group A sows and piglets, respectively. After challenge with wild-type ETEC, diarrhea and mortality was not observed in group B piglets. However, diarrhea was observed in 66.7% of group A piglets, and 33.3% mortality was observed. These findings indicate that im immunization of sows with the mixture of the novel vaccine candidate can effectively protect their offspring from ETEC colibacillosis.