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

1998 ◽  
Vol 61 (2) ◽  
pp. 141-145 ◽  
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.

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

2009 ◽  
Vol 78 (1) ◽  
pp. 316-325 ◽  
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.

Multiplex Real-Time PCR Detection of Heat-Labile and Heat-Stable Toxin Genes in Enterotoxigenic Escherichia coli †

2006 ◽  
Vol 69 (2) ◽  
pp. 412-416 ◽  
Author(s):  
MICHAEL A. GRANT ◽  
JINXIN HU ◽  
KAREN C. JINNEMAN
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Membrane Filtration ◽  
Enterotoxigenic Escherichia Coli ◽  
Pcr Analysis ◽  
E Coli ◽  
Toxin Genes ◽  
Heat Stable ◽  
Heat Labile

A multiplex real-time PCR method was developed for detection of heat-labile and heat-stable toxin genes in enterotoxigenic Escherichia coli. Approximately 10 CFU per reaction mixture could be detected in rinsates from produce samples. Several foods representative of varieties previously shown to have caused enterotoxigenic E. coli outbreaks were spiked and enriched for 4 or 6 h. Both heat-labile and heat-stable toxin genes could be detected in the foods tested, with the exception of hot sauce, with threshold cycle values ranging from 25.2 to 41.1. A procedure using membrane filtration which would allow enumeration of the enterotoxigenic E. coli population in a food sample in less than 28 h by real-time PCR analysis of colonies picked from media highly selective for E. coli was also developed.

scholarly journals A Tripartite Fusion, FaeG-FedF-LT192A2:B, of Enterotoxigenic Escherichia coli (ETEC) Elicits Antibodies That Neutralize Cholera Toxin, Inhibit Adherence of K88 (F4) and F18 Fimbriae, and Protect Pigs against K88ac/Heat-Labile Toxin Infection

2011 ◽  
Vol 18 (10) ◽  
pp. 1593-1599 ◽  
Author(s):  
Xiaosai Ruan ◽  
Mei Liu ◽  
Thomas A. Casey ◽  
Weiping Zhang
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Enterotoxigenic Escherichia Coli ◽  
Content Type ◽  
Etec Strain ◽  
Severe Diarrhea ◽  
Heat Stable ◽  
Heat Labile ◽  
Young Pigs ◽  
Gnotobiotic Piglet

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) strains expressing K88 (F4) or F18 fimbriae and heat-labile (LT) and/or heat-stable (ST) toxins are the major cause of diarrhea in young pigs. Effective vaccines inducing antiadhesin (anti-K88 and anti-F18) and antitoxin (anti-LT and anti-ST) immunity would provide broad protection to young pigs against ETEC. In this study, we genetically fused nucleotides coding for peptides from K88ac major subunit FaeG, F18 minor subunit FedF, and LT toxoid (LT192) A2 and B subunits for a tripartite adhesin-adhesin-toxoid fusion (FaeG-FedF-LT192A2:B). This fusion was used for immunizations in mice and pigs to assess the induction of antiadhesin and antitoxin antibodies. In addition, protection by the elicited antiadhesin and antitoxin antibodies against a porcine ETEC strain was evaluated in a gnotobiotic piglet challenge model. The data showed that this FaeG-FedF-LT192A2:B fusion elicited anti-K88, anti-F18, and anti-LT antibodies in immunized mice and pigs. In addition, the anti-porcine antibodies elicited neutralized cholera toxin and inhibited adherence against both K88 and F18 fimbriae. Moreover, immunized piglets were protected when challenged with ETEC strain 30302 (K88ac/LT/STb) and did not develop clinical disease. In contrast, all control nonvaccinated piglets developed severe diarrhea and dehydration after being challenged with the same ETEC strain. This study clearly demonstrated that this FaeG-FedF-LT192A2:B fusion antigen elicited antibodies that neutralized LT toxin and inhibited the adherence of K88 and F18 fimbrialE. colistrains and that this fusion could serve as an antigen for vaccines against porcine ETEC diarrhea. In addition, the adhesin-toxoid fusion approach used in this study may provide important information for developing effective vaccines against human ETEC diarrhea.

scholarly journals Use of colony pools for diagnosis of enterotoxigenic Escherichia coli diarrhea

1979 ◽  
Vol 9 (4) ◽  
pp. 493-497
Author(s):  
M H Merson ◽  
R B Sack ◽  
A K Kibriya ◽  
A Al-Mahmood ◽  
Q S Adamed ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enterotoxigenic Escherichia Coli ◽  
Adult Males ◽  
E Coli ◽  
Heat Stable ◽  
Heat Labile ◽  
Stool Specimens ◽  
Stool Cultures ◽  
Made In

Diagnosis of enterotoxigenic Escherichia coli diarrhea was made in 109 adult males with an acute dehydrating cholera-like syndrome in Dacca, Bangladesh, by testing 10 colonies isolated from admission stool specimens for production of heat-labile and heat-stable toxins. Toxin testing of one colony yielded a diagnosis in 92% of the cases, testing of two colonies yielded a diagnosis in 95% of the cases, testing of a pool of 5 colonies yielded a diagnosis in 95% of the cases, and testing of a pool of 10 colonies yielded a diagnosis in 96% of the cases. From stool cultures obtained on subsequent days, toxin testing of individual colonies and pools revealed diminished efficacy of pooling with decreasing numbers of enterotoxin-positive isolates in the pool. To detect the presence of enterotoxigenic E. coli in stools, toxin testing of 5 individual isolates and a pool of 10 colonies was found to be almost as effective as the testing of 10 individual isolates.

scholarly journals Heat-Stable Enterotoxins of Enterotoxigenic Escherichia coli and Their Impact on Host Immunity

Toxins ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 24 ◽  
Author(s):  
Haixiu Wang ◽  
Zifu Zhong ◽  
Yu Luo ◽  
Eric Cox ◽  
Bert Devriendt
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Enterotoxigenic Escherichia Coli ◽  
Farm Animals ◽  
Future Research ◽  
Intestinal Epithelial ◽  
Heat Stable ◽  
Intestinal Immune System ◽  
Global Threat ◽  
The Impact

Enterotoxigenic Escherichia coli (ETEC) are an important diarrhea-causing pathogen and are regarded as a global threat for humans and farm animals. ETEC possess several virulence factors to infect its host, including colonization factors and enterotoxins. Production of heat-stable enterotoxins (STs) by most ETEC plays an essential role in triggering diarrhea and ETEC pathogenesis. In this review, we summarize the heat-stable enterotoxins of ETEC strains from different species as well as the molecular mechanisms used by these heat-stable enterotoxins to trigger diarrhea. As recently described, intestinal epithelial cells are important modulators of the intestinal immune system. Thus, we also discuss the impact of the heat-stable enterotoxins on this role of the intestinal epithelium and how these enterotoxins might affect intestinal immune cells. Finally, the latest developments in vaccination strategies to protect against infections with ST secreting ETEC strains are discussed. This review might inform and guide future research on heat-stable enterotoxins to further unravel their molecular pathogenesis, as well as to accelerate vaccine design.

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

2006 ◽  
Vol 74 (2) ◽  
pp. 869-875 ◽  
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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