Escherichia coli Constructs Expressing Human or Porcine Enterotoxins Induce Identical Diarrheal Diseases in a Piglet Infection Model

ABSTRACT 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.

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Comparison of the Contributions of Heat-Labile Enterotoxin and Heat-Stable Enterotoxin b to the Virulence of Enterotoxigenic Escherichia coli in F4ac Receptor-Positive Young Pigs

Infection and Immunity ◽

10.1128/iai.01743-07 ◽

2008 ◽

Vol 76 (7) ◽

pp. 3141-3149 ◽

Cited By ~ 36

Author(s):

Joseph Erume ◽

Emil M. Berberov ◽

Stephen D. Kachman ◽

Michael A. Scott ◽

You Zhou ◽

...

Keyword(s):

Escherichia Coli ◽

Diarrheal Disease ◽

Bacterial Colonization ◽

Enterotoxigenic Escherichia Coli ◽

Double Deletion ◽

Heat Stable ◽

Heat Labile ◽

Young Pigs ◽

Enterotoxin B ◽

Gnotobiotic Piglets

ABSTRACT In swine, the most common and severe enterotoxigenic Escherichia coli (ETEC) infections are caused by strains that express K88 (F4)+ fimbriae, heat-labile enterotoxin (LT), heat-stable enterotoxin b (STb), and enteroaggregative E. coli heat-stable toxin 1. Previous studies based on a design that involved enterotoxin genes cloned into a nontoxigenic fimbriated strain have suggested that LT but not STb plays an important role in dehydrating diarrheal disease in piglets <1 week old and also enhances bacterial colonization of the intestine. In the present study, we compared these two toxins in terms of importance for piglets >1 week old with a study design that involved construction of isogenic single- and double-deletion mutants and inoculation of 9-day-old F4ac receptor-positive gnotobiotic piglets. Based on the postinoculation percent weight change per h and serum bicarbonate concentrations, the virulence of the STb− mutant (ΔestB) did not significantly differ from that of the parent. However, deletion of the LT genes (ΔeltAB) in the STb− mutant resulted in a complete abrogation of weight loss, dehydration, and metabolic acidosis in inoculated pigs, and LT complementation restored the virulence of this strain. These results support the hypothesis that LT is a more significant contributor than STb to the virulence of F4+ ETEC infections in young F4ac receptor-positive pigs less than 2 weeks old. However, in contrast to previous studies with gnotobiotic piglets, there was no evidence that the expression of LT enhanced the ability of the F4+ ETEC strain to colonize the small intestine.

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Simultaneous Exposure to Escherichia coli Heat-Labile and Heat-Stable Enterotoxins Increases Fluid Secretion and Alters Cyclic Nucleotide and Cytokine Production by Intestinal Epithelial Cells

Infection and Immunity ◽

10.1128/iai.02496-14 ◽

2014 ◽

Vol 82 (12) ◽

pp. 5308-5316 ◽

Cited By ~ 17

Author(s):

Lisa T. Read ◽

Rachel W. Hahn ◽

Carli C. Thompson ◽

David L. Bauer ◽

Elizabeth B. Norton ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Diarrheal Disease ◽

Water Movement ◽

Intestinal Epithelial Cells ◽

Intestinal Lumen ◽

Intestinal Epithelial ◽

Simultaneous Exposure ◽

Heat Stable ◽

Heat Labile

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is a significant cause of diarrheal disease and death, especially in children in developing countries. ETEC causes disease by colonizing the small intestine and producing heat-labile toxin (LT), heat-stable toxin (ST), or both LT and ST (LT+ST). The majority of ETEC strains produce both ST and LT. Despite the prevalence of LT+ST-producing organisms, few studies have examined the physiologic or immunologic consequences of simultaneous exposure to these two potent enterotoxins. In the current report, we demonstrate that when LT and ST are both present, they increase water movement into the intestinal lumen over and above the levels observed with either toxin alone. As expected, cultured intestinal epithelial cells increased their expression of intracellular cyclic GMP (cGMP) when treated with ST and their expression of intracellular cyclic AMP (cAMP) when treated with LT. When both toxins were present, cGMP levels but not cAMP levels were synergistically elevated compared with the levels of expression caused by the corresponding single-toxin treatment. Our data also demonstrate that the levels of inflammatory cytokines produced by intestinal epithelial cells in response to LT are significantly reduced in animals exposed to both enterotoxins. These findings suggest that there may be complex differences between the epithelial cell intoxication and, potentially, secretory outcomes induced by ETEC strains expressing LT+ST compared with strains that express LT or ST only. Our results also reveal a novel mechanism wherein ST production may reduce the hosts' ability to mount an effective innate or adaptive immune response to infecting organisms.

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Importance of Heat-Labile Enterotoxin in Colonization of the Adult Mouse Small Intestine by Human Enterotoxigenic Escherichia coli Strains

Infection and Immunity ◽

10.1128/iai.74.2.869-875.2006 ◽

2006 ◽

Vol 74 (2) ◽

pp. 869-875 ◽

Cited By ~ 86

Author(s):

Kenneth P. Allen ◽

Mildred M. Randolph ◽

James M. Fleckenstein

Keyword(s):

Escherichia Coli ◽

Small Intestine ◽

Diarrheal Disease ◽

Vaccine Development ◽

Small Animal ◽

Enterotoxigenic Escherichia Coli ◽

Colonization Factor ◽

Heat Stable ◽

Heat Labile ◽

Colonization Factor Antigen I

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) infections are a significant cause of diarrheal disease and infant mortality in developing countries. Studies of ETEC pathogenesis relevant to vaccine development have been greatly hampered by the lack of a suitable small-animal model of infection with human ETEC strains. Here, we demonstrate that adult immunocompetent outbred mice can be effectively colonized with the prototypical human ETEC H10407 strain (colonization factor antigen I; heat-labile and heat-stable enterotoxin positive) and that production of heat-labile holotoxin provides a significant advantage in colonization of the small intestine in this model.

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Genetic Fusions of Heat-Labile (LT) and Heat-Stable (ST) Toxoids of Porcine Enterotoxigenic Escherichia coli Elicit Neutralizing Anti-LT and Anti-STa antibodies

Infection and Immunity ◽

10.1128/iai.00497-09 ◽

2009 ◽

Vol 78 (1) ◽

pp. 316-325 ◽

Cited By ~ 59

Author(s):

Weiping Zhang ◽

Chengxian Zhang ◽

David H. Francis ◽

Ying Fang ◽

David Knudsen ◽

...

Keyword(s):

Escherichia Coli ◽

Diarrheal Disease ◽

Rabbit Antiserum ◽

Full Length ◽

Enterotoxigenic Escherichia Coli ◽

Farm Animals ◽

Toxic Activity ◽

Colonization Factor ◽

Heat Stable ◽

Heat Labile

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and farm animals. E. coli fimbriae, or colonization factor antigens (CFAs), and enterotoxins, including heat-labile enterotoxins (LT) and heat-stable enterotoxins (ST), are the key virulence factors in ETEC diarrhea. Unlike fimbriae or LT, STa has not often been included as an antigen in development of vaccines against ETEC diarrhea because of its poor immunogenicity. STa becomes immunogenic only after being coupled with a strongly immunogenic carrier protein. However, native or shorter STa antigens either had to retain toxic activity in order to become antigenic or elicited anti-STa antibodies that were not sufficiently protective. In this study, we genetically mutated the porcine LT (pLT) gene for a pLT192(R→G) toxoid and the porcine STa (pSTa) gene for three full-length pSTa toxoids [STa11(N→K), STa12(P→F), and STa13(A→Q)] and used the full-length pLT192 as an adjuvant to carry the pSTa toxoid for pLT192:pSTa-toxoid fusion antigens. Rabbits immunized with pLT192:pSTa12 or pLT192:pSTa13 fusion protein developed high titers of anti-LT and anti-STa antibodies. Furthermore, rabbit antiserum and antifecal antibodies were able to neutralize purified cholera toxin (CT) and STa toxin. In addition, preliminary data suggested that suckling piglets born by a sow immunized with the pLT192:pSTa13 fusion antigen were protected when challenged with an STa-positive ETEC strain. This study demonstrated that pSTa toxoids are antigenic when fused with a pLT toxoid and that the elicited anti-LT and anti-STa antibodies were protective. This fusion strategy could provide instructive information to develop effective toxoid vaccines against ETEC-associated diarrhea in animals and humans.

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Escherichia coli K88ac Fimbriae Expressing Heat-Labile and Heat-Stable (STa) Toxin Epitopes Elicit Antibodies That Neutralize Cholera Toxin and STa Toxin and Inhibit Adherence of K88ac Fimbrial E. coli

Clinical and Vaccine Immunology ◽

10.1128/cvi.00251-10 ◽

2010 ◽

Vol 17 (12) ◽

pp. 1859-1867 ◽

Cited By ~ 25

Author(s):

Chengxian Zhang ◽

Weiping Zhang

Keyword(s):

Escherichia Coli ◽

Cholera Toxin ◽

Broad Spectrum ◽

Diarrheal Disease ◽

Virulence Determinants ◽

E Coli ◽

B Subunit ◽

Heat Stable ◽

Heat Labile ◽

Bacterial Adhesins

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Bacterial adhesins and heat-labile (LT) and heat-stable (ST) enterotoxins are the virulence determinants in ETEC diarrhea. It is believed that vaccines inducing anti-adhesin immunity to inhibit bacterial adherence and anti-toxin immunity to eliminate toxin activity would provide broad-spectrum protection against ETEC. In this study, an ETEC fimbrial adhesin was used as a platform to express LT and STa for adhesin-toxin fusion antigens to induce anti-toxin and anti-adhesin immunity. An epitope from the B subunit of LT toxin (LTP1, 8LCSEYRNTQIYTIN21) and an STa toxoid epitope (5CCELCCNPQCAGCY18) were embedded in the FaeG major subunit of E. coli K88ac fimbriae. Constructed K88ac-toxin chimeric fimbriae were harvested and used for rabbit immunization. Immunized rabbits developed anti-K88ac, anti-LT, and anti-STa antibodies. Moreover, induced antibodies not only inhibited adherence of K88ac fimbrial E. coli to porcine small intestinal enterocytes but also neutralized cholera toxin and STa toxin. Data from this study demonstrated that K88ac fimbriae expressing LT and STa epitope antigens elicited neutralizing anti-toxin antibodies and anti-adhesin antibodies and suggested that E. coli fimbriae could serve as a platform for the development of broad-spectrum vaccines against ETEC.

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Heat-Labile- and Heat-Stable-Toxoid Fusions (LTR192G-STaP13F) of Human Enterotoxigenic Escherichia coli Elicit Neutralizing Antitoxin Antibodies

Infection and Immunity ◽

10.1128/iai.00165-11 ◽

2011 ◽

Vol 79 (10) ◽

pp. 4002-4009 ◽

Cited By ~ 41

Author(s):

Mei Liu ◽

Xiaosai Ruan ◽

Chengxian Zhang ◽

Steve R. Lawson ◽

David E. Knudsen ◽

...

Keyword(s):

Escherichia Coli ◽

Diarrheal Disease ◽

Full Length ◽

Enterotoxigenic Escherichia Coli ◽

Secretory Iga ◽

Content Type ◽

Human Type ◽

Heat Stable ◽

Heat Labile ◽

Iga Antibodies

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) strains are a major cause of diarrheal disease in humans and animals. Adhesins and enterotoxins, including heat-labile (LT) and heat-stable (STa) toxins, are the key virulence factors. Antigenic adhesin and LT antigens have been used in developing vaccines against ETEC diarrhea. However, STa has not been included because of its poor immunogenicity and potent toxicity. Our recent study showed that porcine-type STa toxoids became immunogenic and elicited neutralizing anti-STa antibodies after being genetically fused to a full-length porcine-type LT toxoid, LTR192G(W. Zhang et al., Infect. Immun. 78:316-325, 2010). In this study, we mutated human-type LT and STa genes, which are highly homologous to porcine-type toxin genes, for a full-length LT toxoid (LTR192G) and a full-length STa toxoid (STaP13F) and genetically fused them to produce LT192-STa13toxoid fusions. Mice immunized with LT192-STa13fusion antigens developed anti-LT and anti-STa IgG (in serum and feces) and IgA antibodies (in feces). Moreover, secretory IgA antibodies from immunized mice were shown to neutralize STa and cholera toxins in T-84 cells. In addition, we fused the STa13toxoid at the N terminus and C terminus, between the A1 and A2 peptides, and between the A and B subunits of LT192to obtain different fusions in order to explore strategies for enhancing STa immunogenicity. This study demonstrated that human-type LT192-STa13fusions induce neutralizing antitoxin antibodies and provided important information for developing toxoid vaccines against human ETEC diarrhea.

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Use of a Multiplex PCR System for the Simultaneous Detection of Heat Labile Toxin I and Heat Stable Toxin II Genes of Enterotoxigenic Escherichia coli in Skim Milk and Porcine Stool

Journal of Food Protection ◽

10.4315/0362-028x-61.2.141 ◽

1998 ◽

Vol 61 (2) ◽

pp. 141-145 ◽

Cited By ~ 12

Author(s):

HAU-YANG TSEN ◽

LIANG-ZHAO JIAN ◽

WAN-RONG CHI

Keyword(s):

Escherichia Coli ◽

Multiplex Pcr ◽

Simultaneous Detection ◽

Skim Milk ◽

Pcr Primers ◽

Enterotoxigenic Escherichia Coli ◽

Farm Animals ◽

Target Cells ◽

Heat Stable ◽

Heat Labile

Enterotoxigenic Escherichia coli (ETEC) strains which produce heat labile and/or heat stable toxins (LT and ST) may cause diarrhea in humans and farm animals. Using PCR primers specific for the LT I and ST II genes, a multiplex PCR system which allows detection of LT I- and ST II-producing ETEC strains was developed. When skim milk was used for a PCR assay, it was found that if target cells in the sample were precultured in MacConkey broth for 8 h prior to PCR as few as 100 cells per ml of the sample could be detected. Without the preculture step, 104 CFU of target cells per 0.2 g of porcine stool specimen were required to generate visible PCR products. The multiplex PCR System can be used for rapid testing of fecal specimens, food and possibly environmental samples for the presence of ETEC strains.

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