• Korean Journal of Veterinary Research
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• v.26 no.1
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• pp.103-108
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• 1986

The oil emulsion and alhydrogel pilli vaccines were prepared from a strain(O9: K35, K99, F41) of enterotoxigenic Escherichia coli isolated from calves with diarrhea and their immunogenicity was tested in guinea-pigs, pregnant goats and cows. Serum antibody responses to K99 and F41 antigens in guinea-pigs given experimental oil and gel vaccines peaked at 4 and 6 weeks after vaccinations. At that time, the mean hemagglutination inhibition titers to K99 and F41 antigens in guinea-pigs given oil vaccine were 1:25 and 1:1, 218 and those given gel vaccine were 1:54 and 1:724 respectively. Agglutinin titers in pregnant goats given the oil vaccine were significantly higher(mean 1:2,347) compared to those of control group(mean 1:160). Less than 12.5% of goatlings from vaccinated goats developed scours compared to nearly 100% in control group after oral challenge with enterotoxigenic Escherichia coil within 24 hours after birth. The highest agglutinin titers of cow serum and colostrum and of the serum of calves 48 hours after birth from cows given oil vaccine were 1:256, 1:512 and 1:64 respectively. On the other hand, those titers of serum and colostrum and of the serum of nursing calves from nonvaccinated cows were 1:8, 1:16 and 1:20 respectively. The protective efficacy of the oil emulsion vaccine was 72.1% under field conditions. These results strongly indicated that the vaccine could be applied for protection of diarrhea caused by enterotoxigenic Escherichia coli in calves.

• Korean Journal of Veterinary Research
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• v.26 no.1
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• pp.97-102
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• 1986

The prevalence of diarrhea caused by enterotoxigenic Escherichia coli was surveyed on 445 calves in 6 farms which were located in the central part of Korea. The incidence of enterotoxigenic Escherichia coli in calves with diarrhea was investigated by detecting the K99 and F41 antigens from the isolated strains of Escherichia coli The incidence of colibacillosis in calves was 23.3%. Of 238 strains of Escherichia coli isolated from calves with diarrhea, 73 strains(30.6%) were proved possessing the K99 antigen by mannose-resistant hemagglutination(MRHA) using horse red blood cells and 79(33.1%) possessing F41 antigens by MRHA using guinea-pig red blood cells. The minca medium, nutrient broth, tryptic soy broth and brain heart infusion were tested for yield of K99 and F41 pili. The production of pili was greatest in minea medium. The best detachment method of the K99 and F41 pili from the cells was heat treatment for 20 minutes at $60^{\circ}C$ and concentration by precipitation with ammonium sulfate. The purified antigens of K99 and F41 were polypeptides with molecular weights of 18,500 and 29,500, respectively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

• Biomedical Science Letters
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• v.18 no.4
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• pp.362-368
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• 2012

Enterotoxigenic Bacteroides fragilis (ETBF) is an intestinal commensal bacterium implicated as a risk factor for colon cancer. The key virulence factor is a secreted toxin called B. fragilis toxin (BFT). In this study we used an in vitro bioassay to examine the prevalence of ETBF in colonic washings from patients with colorectal polyps and normal control patients. We found that 9.3% of polyp patients and 10.9% of non-polyp patients harbored ETBF, respectively. A total of nine ETBF clinical isolates were isolated and confirmed to be positive for the BFT gene by PCR analysis and the ability to induce IL-8 secretion in the colonic epithelial cell line HT29/c1. Two of the ETBF clinical strains were characterized further in vitro and in vivo. We found that the two ETBF clinical isolates induced E-cadherin cleavage in HT29/c1 cells and promoted colonic inflammation in C57BL/6 mice. Our results indicate that the prevalence of ETBF in polyp patients were similar in non-polyp patients suggesting that ETBF carriage does not positively correlate to polyp incidence.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.8
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• pp.1308-1314
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• 2018

Objective: Gut health improvements were monitored with respect to growth performance, diarrhea incidence, fecal bacterial population and intestinal morphology of suckling pigs orally supplemented with live Lactobacillus salivarius (L. salivarius) oral suspensions and challenged with $F4^+$ enterotoxigenic Escherichia coli (ETEC). Methods: Two groups of newborn pigs from 18 multiparous sows were randomly designated as non-supplemented (control: n = 114 piglets) and L. salivarius supplemented groups (treatment: n = 87 piglets). Treatment pigs were orally administered with 2 mL of $10^9$ colony-forming unit (CFU)/mL L. salivarius on days 1 to 3, then they were orally administered with 5 mL of $10^9CFU/mL$ L. salivarius on days 4 to 10, while those in control group received an equal amount of phosphate buffered saline solution. On day 24 (2 weeks post supplementation), one pig per replicate of both groups was orally administered with $10^8CFU/mL$ $F4^+$ ETEC, then they were euthanized on day 29 of experiment. Results: Results revealed that pigs in treatment group had a statistically significant increase in average daily gain, body weight and weight gain, and tended to lower diarrhea throughout the study. Numbers of Lactobacillus population in feces of treatment pigs were higher than control pigs, especially on day 10 of study. Numbers of total bacteria in intestinal contents of control pigs were also increased, but not Coliform and Lactobacillus populations. Histological examination revealed statistically significant improvements of villous height and villous/crypt ratio of duodenum, proximal jejunum and distal jejunum parts of treatment pigs compared with controls. Duodenal pH of treatment group was significantly decreased. Conclusion: Oral supplementation of live L. salivarius during the first 10 days of suckling pig promoted growth performance and gut health, reduced diarrhea incidence, increased fecal Lactobacillus populations and improved intestinal morphology.

• Food Science of Animal Resources
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• v.40 no.5
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• pp.746-757
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• 2020

Enterotoxigenic Escherichia coli (ETEC) is the major pathogenic E. coli that causes diarrhea and edema in post-weaning piglets. In this study, we describe the morphology and characteristics of ØCJ19, a bacteriophage that infects ETEC, and performed genetic analysis. Phage ØCJ19 belongs to the family Myoviridae. One-step growth curve showed a latent phase of 5 min and burst size of approximately 20 phage particles/infected cell. Phage infectivity was stable for 2 h between 4℃ and 55℃, and the phage was stable between pH 3 and 11. Genetic analysis revealed that phage ØCJ19 has a total of 49,567 bases and 79 open reading frames (ORFs). The full genomic sequence of phage ØCJ19 showed the most similarity to an Escherichia phage, vB_EcoS_ESCO41. There were no genes encoding lysogeny, toxins, virulence factors, or antibiotic resistance in this phage, suggesting that this phage can be used safely as a biological agent to control ETEC. Comparative genomic analysis in terms of the tail fiber proteins could provide genetic insight into host recognition and the relationship with other coliphages. These results showed the possibility to improve food safety by applying phage ØCJ19 to foods of animal origin contaminated with ETEC and suggests that it could be the basis for establishing a safety management system in the animal husbandry.

• 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.

• Journal of Veterinary Science
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• v.23 no.1
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• pp.7.1-7.14
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• 2022

Background: Enterotoxigenic Escherichia coli (ETEC) infection is a primary cause of livestock diarrhea. Therefore, effective vaccines are needed to reduce the incidence of ETEC infection. Objectives: Our study aimed to develop a multivalent ETEC vaccine targeting major virulence factors of ETEC, including enterotoxins and fimbriae. Methods: SLS (STa-LTB-STb) recombinant enterotoxin and fimbriae proteins (F4, F5, F6, F18, and F41) were prepared to develop a multivalent vaccine. A total of 65 mice were immunized subcutaneously by vaccines and phosphate-buffered saline (PBS). The levels of specific immunoglobulin G (IgG) and pro-inflammatory cytokines were determined at 0, 7, 14 and 21 days post-vaccination (dpv). A challenge test with a lethal dose of ETEC was performed, and the survival rate of the mice in each group was recorded. Feces and intestine washes were collected to measure the concentrations of secretory immunoglobulin A (sIgA). Results: Anti-SLS and anti-fimbriae-specific IgG in serums of antigen-vaccinated mice were significantly higher than those of the control group. Immunization with the SLS enterotoxin and multivalent vaccine increased interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) concentrations. Compared to diarrheal symptoms and 100% death of mice in the control group, mice inoculated with the multivalent vaccine showed an 80% survival rate without any symptom of diarrhea, while SLS and fimbriae vaccinated groups showed 60 and 70% survival rates, respectively. Conclusions: Both SLS and fimbriae proteins can serve as vaccine antigens, and the combination of these two antigens can elicit stronger immune responses. The results suggest that the multivalent vaccine can be successfully used for preventing ETEC in important livestock.

• Journal of Food Hygiene and Safety
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• v.6 no.3
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• pp.147-155
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• 1991

Enterotoxigenic E. coli is one of the major causative agents of the infantile diarrhea and traveler's diarrhea. The heat-stable enterotoxin(ST) is thought to be a virulence factor in the pathogenesis of the diarrhea and to be a maker for identification of the enterotoxingeic E. coli from non pathogenic E. coli. The isolate of enterotoxigenlc E. coli was isolated from swine during 1989 year(from 5 to 10 month) in the Kyong-gi and Chung-Cheong provinces, and three strains(KM-4, KM-7 and KM-12) was selected from 189 isolates of ST producing E. coli. The detection of a ST produced of the isolated E. coli was performed by the infant mouse assay(IMA). This study was designed to know optimal conditions for the production of the ST and the molecular properties of plasmids of the enterotoxigenic E. coli. Amount of ST produced were the most at initial pH 8.5~9.0 of succinate salts medium culture. The cultural time of the same medium was accumulated the highest level of ST was at the 14 to 16 hours, and then stationary phase was at the 20 hours. From this experiment the KM-7 strain was selected among ST producing strains by IMA. Partial plasmid-curing experiment was done to select plasmid encoding for ST among other plasmids and then comparing the plasmid pattern of ST producing strain(KM-7) with those of other ST non-producing strains, it is found that ST gene exists on the about 80 Kbp plasmid. Each fragment of this plasmid digested with EcoRl was ligated to vector pBR 322 and transformed into E. coli K-12. A clone producing ST(eKT 53) was selected by IMA. The EcoRl digestion pattern of the isolated plasmid(pKD 37) from the ST producing clone it is indicated that the size of the inserted fragment in eKT 53 strain is 16 Kbp. The cultured supernatant of eKT 53 strain was positive result of ST production in IMA.