RESUMO
Enterotoxigenic Escherichia coli (ETEC) strains produce at least one of two types of enterotoxins: the heat-labile (LT) and heat-stable (ST) toxins, which are responsible for the watery secretory diarrhoea that is a hallmark of the human ETEC infection. One regulatory system that controls the transcription of virulence genes in pathogenic bacteria is the CpxRA two-component system (TCS). We reported that the eltAB bicistronic operon, which encodes for the A and B subunits of LT, was repressed for the CpxRA TCS by direct binding of CpxR-P from -12 to +6 bp with respect to the transcription start site of eltAB. Moreover, the Cpx-response activation down-regulated the transcription of eltAB genes, and this negative effect was CpxRA-dependent. Our data show that CpxRA TCS is a negative regulator of the LT, one of the main virulence determinants of ETEC.
Assuntos
Toxinas Bacterianas , Escherichia coli Enterotoxigênica , Infecções por Escherichia coli , Proteínas de Escherichia coli , Humanos , Escherichia coli Enterotoxigênica/genética , Escherichia coli Enterotoxigênica/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Temperatura Alta , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Enterotoxinas/genética , Enterotoxinas/metabolismo , Infecções por Escherichia coli/microbiologia , Diarreia/microbiologia , Expressão GênicaRESUMO
Enterotoxigenic Escherichia coli (ETEC) is one of the main bacterial pathotypes in- volved in diarrhea, mainly affecting children under 5 years old and travelers to areas where this pathogen is endemic. Heat-labile type I toxin (LT-I), one of the main virulence factors of this pathotype, is associated with diarrhea in humans and is described as a potent mucosal and systemic immune adjuvant. Early diagnosis, therapeutic approaches, and in vitro and in vivo models are the main concerns in diarrhea caused by ETEC. Different in vitro and in vivo models have already been described, however, its use does not always allow screening of biomolecules and more systemic studies with the LT toxin. In addition, the generation of specific and high-affinity recombinant antibodies for diagnosis and therapy are of great importance. Thus, the present work aimed to establish strategies for the generation of recombinant antibodies such as scFv and Fab and evaluate and validate Caco-2 cells and zebrafish as models for studies with LT-I. Caco-2 cells were seeded in the 62nd passage and at different cells density per microplate well. The internalization of LT-I conjugated to FITC was visualized in Caco-2 cells at a density of 3x104 cells per well, both associated with the cell membrane and with the nucleus, thus demonstrating its retrograde transport and validating the use of this model. In a zebrafish, the fish embryo test revealed the sensitivity of embryos to different concentrations of LT-I, as well as evident malformation phenotypes, mainly in the pericardial and yolk region. Systemically, migration of the FITC labeled toxin was observed in the cardiac region, yolk, and intestine, suggesting the observed phenotypes of cardiac edema (100%), absence of swim bladder (100%), yolk edema (80%), in addition to growth delay in larvae, were caused by the toxin. None of these phenotypes were observed in the control group of animals. There was also a decrease in heart rate during the larvae' survival kinetics, showing the cardiotoxic effect of LT-I. In ELISA assays the scFv-LT obtained in the present study was reactive against the purified antigen as well to LT-I producing strains supernatant, but not to LT-I-non-producing strains. For the Fab-LT, a synthetic library was constructed, but no LT-I-reactive clones were generated. Therefore, herein we established in vitro and in vivo models that allowed us to validate and demonstrate unknown characteristics of the LT-I concerning its relationship with the host. Moreover, the generation of scFv-LT will allows us to employ recombinant antibodies for ETEC diagnosis avoiding animals immunization.
Escherichia coli enterotoxigênica (ETEC), é um dos principais patotipos bacterianos causadores de diarreia, afetando principalmente crianças menores de cinco anos e viajantes em áreas onde esse patógeno é endêmico. A toxina termolábil do tipo I (LT-I) é um dos principais fatores de virulência deste patotipo e está associada à diarreia em humanos; é também descrita como potente adjuvante de mucosa e sistêmico. O diagnóstico precoce, a abordagem terapêutica e os modelos experimentais são importantes aspectos que merecem atenção em relação à diarreia causada por ETEC. Diferentes modelos in vitro e in vivo já foram descritos, no entanto, seu uso nem sem- pre permite a triagem de biomoléculas e estudos mais sistêmicos com a toxina LT. Além disso, a geração de anticorpos específicos e de alta afinidade para uso no diagnóstico e terapia são de grande importância. Assim, o presente trabalho propôs estabelecer estratégias para geração de fragmentos de anticorpos do tipo scFv e Fab; bem como avaliar e validar as células Caco-2 e o zebrafish como modelos para estudos da LT-I. As células Caco-2 foram utilizadas na 62a passagem e em diferentes densidades de célula por poço da microplaca de cultivo. A internalização da LT-I conjugada ao FITC foi visualizada com 3x104 células Caco-2, tanto associada à membrana celular, quanto ao núcleo, evidenciando seu transporte retrógrado e validando o uso deste modelo. Em zebrafish, pelo teste de sensibilidade em embriões, foi observada sensibilidade dos embriões frente a diferentes concentrações de LT-I e fenótipos de malformações, principalmente na região pericárdica e no vitelo. Por via sistêmica, a migração da LT-I foi observada na região cardíaca, vitelo e intestino, sugerindo que os fenótipos observados de edema cardíaco (100%), ausência de bexiga natatória (100%), edema de vitelo (80%), além de retardo no crescimento nas larvas, tenham sido ocasionados pela toxina. Nenhum desses efeitos foi encontrado no grupo controle de animais. Houve também diminuição dos batimentos cardíacos durante a cinética de sobrevivência, mostrando o efeito cardiotóxico da LT-I. Em testes por técnica de ELISA o scFv-LT obtido no presente estudo mostrou-se reativo contra a toxina LT purificada, reconheceu as cepas produtoras de LT-I e não reconheceu cepas não produtoras da toxina. Para o Fab-LT foi estabelecida a biblioteca sintética, mas não foram gerados clones reativos contra a toxina LT-I. Assim, podemos afirmar que os modelos in vitro e in vivo utilizados permitiram-nos validar e demonstrar características até então não exploradas da LT-I na sua relação com o hospedeiro. A geração do scFv-LT nos permitirá utilizar anticorpos recombinantes no diagnóstico de ETEC sem depender da imunização de animais.
RESUMO
Heat-labile toxin I (LT-I), produced by strains of enterotoxigenic Escherichia coli (ETEC), causes profuse watery diarrhea in humans. Different in vitro and in vivo models have already elucidated the mechanism of action of this toxin; however, their use does not always allow for more specific studies on how the LT-I toxin acts in systemic tracts and intestinal cell lines. In the present work, zebrafish (Danio rerio) and human intestinal cells (Caco-2) were used as models to study the toxin LT-I. Caco-2 cells were used, in the 62nd passage, at different cell concentrations. LT-I was conjugated to FITC to visualize its transport in cells, as well as microinjected into the caudal vein of zebrafish larvae, in order to investigate its effects on survival, systemic traffic, and morphological formation. The internalization of LT-I was visualized in 3 × 104 Caco-2 cells, being associated with the cell membrane and nucleus. The systemic traffic of LT-I in zebrafish larvae showed its presence in the cardiac cavity, yolk, and regions of the intestine, as demonstrated by cardiac edema (100%), the absence of a swimming bladder (100%), and yolk edema (80%), in addition to growth limitation in the larvae, compared to the control group. There was a reduction in heart rate during the assessment of larval survival kinetics, demonstrating the cardiotoxic effect of LT-I. Thus, in this study, we provide essential new depictions of the features of LT-I.
Assuntos
Toxinas Bacterianas/toxicidade , Escherichia coli Enterotoxigênica , Enterotoxinas/toxicidade , Proteínas de Escherichia coli/toxicidade , Animais , Toxinas Bacterianas/farmacocinética , Células CACO-2 , Edema/induzido quimicamente , Embrião não Mamífero/anormalidades , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Enterotoxinas/farmacocinética , Proteínas de Escherichia coli/farmacocinética , Cardiopatias Congênitas/induzido quimicamente , Frequência Cardíaca/efeitos dos fármacos , Humanos , Intestinos/metabolismo , Miocárdio/metabolismo , Saco Vitelino/efeitos dos fármacos , Peixe-Zebra/anormalidades , Peixe-Zebra/metabolismoRESUMO
Heat-labile toxin I (LT-I), produced by strains of enterotoxigenic Escherichia coli (ETEC), causes profuse watery diarrhea in humans. Different in vitro and in vivo models have already elucidated the mechanism of action of this toxin; however, their use does not always allow for more specific studies on how the LT-I toxin acts in systemic tracts and intestinal cell lines. In the present work, zebrafish (Danio rerio) and human intestinal cells (Caco-2) were used as models to study the toxin LT-I. Caco-2 cells were used, in the 62nd passage, at different cell concentrations. LT-I was conjugated to FITC to visualize its transport in cells, as well as microinjected into the caudal vein of zebrafish larvae, in order to investigate its effects on survival, systemic traffic, and morphological formation. The internalization of LT-I was visualized in 3 × 104 Caco-2 cells, being associated with the cell membrane and nucleus. The systemic traffic of LT-I in zebrafish larvae showed its presence in the cardiac cavity, yolk, and regions of the intestine, as demonstrated by cardiac edema (100%), the absence of a swimming bladder (100%), and yolk edema (80%), in addition to growth limitation in the larvae, compared to the control group. There was a reduction in heart rate during the assessment of larval survival kinetics, demonstrating the cardiotoxic effect of LT-I. Thus, in this study, we provide essential new depictions of the features of LT-I.
RESUMO
In the present study, we evaluated the immunological responses induced by dengue vaccines under experimental conditions after delivery via a transcutaneous (TC) route. Vaccines against type 2 Dengue virus particles (DENV2 New Guinea C (NGC) strain) combined with enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) were administered to BALB/c mice in a three-dose immunization regimen via the TC route. As a control for the parenteral administration route, other mouse groups were immunized with the same vaccine formulation via the intradermic (ID) route. Our results showed that mice vaccinated either via the TC or ID routes developed similar protective immunity, as measured after lethal challenges with the DENV2 NGC strain. Notably, the vaccine delivered through the TC route induced lower serum antibody (IgG) responses with regard to ID-immunized mice, particularly after the third dose. The protective immunity elicited in TC-immunized mice was attributed to different antigen-specific antibody properties, such as epitope specificity and IgG subclass responses, and cellular immune responses, as determined by cytokine secretion profiles. Altogether, the results of the present study demonstrate the immunogenicity and protective properties of a dengue vaccine delivered through the TC route and offer perspectives for future clinical applications.
Assuntos
Vacinas contra Dengue/administração & dosagem , Vírus da Dengue/imunologia , Dengue/prevenção & controle , Administração Cutânea , Animais , Anticorpos Antivirais/sangue , Dengue/sangue , Dengue/imunologia , Dengue/virologia , Vacinas contra Dengue/genética , Vacinas contra Dengue/imunologia , Vírus da Dengue/genética , Humanos , Imunização , Imunoglobulina G/sangue , Injeções Intradérmicas , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Enterotoxigenic Escherichia coli (ETEC) represents one of the most important etiological agents of diarrhea in developing countries and characteristically produces at least one of two enterotoxins: heat-labile toxin (LT) and heat-stable toxin (ST). It has been previously shown that the production and release of LT by human-derived ETEC strains are variable. Although the natural genetic polymorphisms of regulatory sequences of LT-encoding (eltAB) genes may explain the variable production of LT, the knowledge of the transcriptional and posttranscriptional aspects affecting LT expression among ETEC strains is not clear. To further understand the factors affecting LT expression, we evaluated the impact of the natural polymorphism in noncoding regulatory sequences of eltAB among clinically derived ETEC strains. Sequence analyses of seven clinically derived strains and the reference strain H10407 revealed polymorphic sites at both the promoter and upstream regions of the eltAB operon. Operon fusion assays with GFP revealed that specific nucleotide changes in the Pribnow box reduce eltAB transcription. Nonetheless, the total amounts of LT produced by the tested ETEC strains did not strictly correspond to the detected LT-specific mRNA levels. Indeed, the stability of LT varied according to the tested strain, indicating the presence of posttranscriptional mechanisms affecting LT expression. Taken together, our results indicate that the production of LT is a strain-specific process and involves transcriptional and posttranscriptional mechanisms that regulate the final amount of toxin produced and released by specific strains.
Assuntos
Toxinas Bacterianas/genética , Escherichia coli Enterotoxigênica/genética , Enterotoxinas/genética , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , Proteínas de Escherichia coli/genética , Óperon , Polimorfismo Genético , TemperaturaRESUMO
We standardized an immunochromatographic test (IC) for heat-labile toxin I (LT-I) detection using LT-I antibodies and a specific platform containing the apparatus for application, assembly and cutting. IC detected as little as 62.5ng/mL of purified LT-I toxin and presented 91% sensitivity, 99.5% specificity and 96.0% accuracy, thereby proving to be an excellent point-of-care test for the diagnosis of enterotoxigenic E. coli infection in low-income countries.
Assuntos
Toxinas Bacterianas/isolamento & purificação , Testes Diagnósticos de Rotina/métodos , Diarreia/diagnóstico , Escherichia coli Enterotoxigênica/isolamento & purificação , Enterotoxinas/isolamento & purificação , Infecções por Escherichia coli/diagnóstico , Proteínas de Escherichia coli/isolamento & purificação , Imunoensaio/métodos , Anticorpos Antibacterianos/imunologia , Toxinas Bacterianas/imunologia , Testes Diagnósticos de Rotina/instrumentação , Diarreia/microbiologia , Escherichia coli Enterotoxigênica/metabolismo , Escherichia coli Enterotoxigênica/patogenicidade , Enterotoxinas/imunologia , Proteínas de Escherichia coli/imunologia , Temperatura Alta , Humanos , Imunoensaio/instrumentação , Sensibilidade e EspecificidadeRESUMO
We standardized an immunochromatographic test (IC) for heat-labile toxin I (LT-I) detection using LT-I antibodies and a specific platform containing the apparatus for application, assembly and cutting. IC detected as little as 62.5 ng/mL of purified LT-I toxin and presented 91% sensitivity, 99.5% specificity and 96.0% accuracy, thereby proving to be an excellent point-of-care test for the diagnosis of enterotoxigenic E. coli infection in low-income countries.
RESUMO
In Part II we discuss the following bacterial pathogens: Shigella, Salmonella (non-typhoidal), diarrheogenic E. coli (enterotoxigenic and enterohemorragic) and Campylobacter jejuni. In contrast to the enteric viruses and Vibrio cholerae discussed in Part I of this series, for the bacterial pathogens described here there is only one licensed vaccine, developed primarily for Vibrio cholerae and which provides moderate protection against enterotoxigenic E. coli (ETEC) (Dukoral(®)), as well as a few additional candidates in advanced stages of development for ETEC and one candidate for Shigella spp. Numerous vaccine candidates in earlier stages of development are discussed.
Assuntos
Vacinas Bacterianas/imunologia , Campylobacter jejuni/imunologia , Diarreia/prevenção & controle , Escherichia coli/imunologia , Gastroenterite/prevenção & controle , Salmonella/imunologia , Shigella/imunologia , Ensaios Clínicos como Assunto , Diarreia/epidemiologia , Diarreia/microbiologia , Diarreia/virologia , Aprovação de Drogas , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Gastroenterite/epidemiologia , Gastroenterite/microbiologia , Gastroenterite/parasitologia , Gastroenterite/virologia , HumanosRESUMO
E. coli O111 strains are responsible for outbreaks of blood diarrhea and hemolytic uremic syndrome throughout the world. Because of their phenotypic variability, the development of a vaccine against these strains which targets an antigen that is common to all of them is quite a challenge. Previous results have indicated, however, that O111 LPS is such a candidate, but its toxicity makes LPS forbidden for human use. To overcome this problem, O111 polysaccharides were conjugated either to cytochrome C or to EtxB (a recombinant B subunit of LT) as carrier proteins. The O111-cytochrome C conjugate was incorporated in silica SBA-15 nanoparticles and administered subcutaneously in rabbits, while the O111-EtxB conjugate was incorporated in Vaxcine(TM), an oil-based delivery system, and administered orally in mice. The results showed that one year post-vaccination, the conjugate incorporated in silica SBA-15 generated antibodies in rabbits able to inhibit the adhesion of all categories of O111 E. coli to epithelial cells. Importantly, mice immunized orally with the O111-EtxB conjugate in Vaxcine(TM) generated systemic and mucosal humoral responses against all categories of O111 E. coli as well as antibodies able to inhibit the toxic effect of LT in vitro. In summary, the results obtained by using 2 different approaches indicate that a vaccine that targets the O111 antigen has the potential to prevent diarrhea induced by O111 E. coli strains regardless their mechanism of virulence. They also suggest that a conjugated vaccine that uses EtxB as a carrier protein has potential to combat diarrhea induced by ETEC.
Assuntos
Anticorpos Antibacterianos/sangue , Portadores de Fármacos/uso terapêutico , Infecções por Escherichia coli/prevenção & controle , Escherichia coli/imunologia , Polissacarídeos Bacterianos/imunologia , Vacinas Conjugadas/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Aderência Bacteriana/imunologia , Toxinas Bacterianas/química , Toxinas Bacterianas/imunologia , Linhagem Celular , Citocromos c/química , Citocromos c/imunologia , Endotoxinas/imunologia , Enterotoxinas/química , Enterotoxinas/imunologia , Escherichia coli/classificação , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/imunologia , Feminino , Síndrome Hemolítico-Urêmica/imunologia , Síndrome Hemolítico-Urêmica/microbiologia , Síndrome Hemolítico-Urêmica/prevenção & controle , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/uso terapêutico , Coelhos , Dióxido de Silício/química , Vacinas Conjugadas/uso terapêuticoRESUMO
Native type I heat-labile toxins (LTs) produced by enterotoxigenic Escherichia coli (ETEC) strains exert strong adjuvant effects on both antibody and T cell responses to soluble and particulate antigens following co-administration via mucosal routes. However, inherent enterotoxicity and neurotoxicity (following intra-nasal delivery) had reduced the interest in the use of these toxins as mucosal adjuvants. LTs can also behave as powerful and safe adjuvants following delivery via parenteral routes, particularly for activation of cytotoxic lymphocytes. In the present study, we evaluated the adjuvant effects of a new natural LT polymorphic form (LT2), after delivery via intradermal (i.d.) and subcutaneous (s.c.) routes, with regard to both antibody and T cell responses. A recombinant HIV-1 p24 protein was employed as a model antigen for determination of antigen-specific immune responses while the reference LT (LT1), produced by the ETEC H10407 strain, and a non-toxigenic LT form (LTK63) were employed as previously characterized LT types. LT-treated mice submitted to a four dose-base immunization regimen elicited similar p24-specific serum IgG responses and CD4(+) T cell activation. Nonetheless, mice immunized with LT1 or LT2 induced higher numbers of antigen-specific CD8(+) T cells and in vivo cytotoxic responses compared to mice immunized with the non-toxic LT derivative. These effects were correlated with stronger activation of local dendritic cell populations. In addition, mice immunized with LT1 and LT2, but not with LTK63, via s.c. or i.d. routes developed local inflammatory reactions. Altogether, the present results confirmed that the two most prevalent natural polymorphic LT variants (LT1 or LT2) display similar and strong adjuvant effects for subunit vaccines administered via i.d. or s.c. routes.
RESUMO
Aeromonas species are autochtonous in the aquatic environment and some of them have been associated with health effects like wound infections, septicemia and diarrhoeal illness. In this study, the occurrence of Aeromonas spp. and microbial indicators in raw drinking water from wells, springs, fountains and mineral waters was evaluated. A total of 126 water samples was analyzed for Aeromonas spp. by the membrane filtration technique using ADA media and by P/A test. Typical colonies of Aeromonas spp. were submitted to biochemical tests for species differentiation. Toxin production was tested using Y-1 mouse adrenal cells. Coliforms and heterotrophic bacteria were enumerated by membrane filtration and pour plate techniques, respectively. P. aeruginosa, C. perfringens and fecal streptococci were determined by P/A method. Aeromonas spp. were isolated in 36.5% of the samples, whereas total and thermotolerant coliforms were detected in 51.2% and in 23.8% of the samples, respectively. C. perfringens, fecal streptococci and P. aeruginosa were present in 16.5%, 20.4% and 3.8% of the samples, respectively. The concentrations of heterotrophic bacteria were higher than 1,0x10³ CFU/mL in 52.5% of the samples. A. hydrophila was the most frequent species, followed by A. allosaccharophila,A. jandaei,A.sobria and HG2. A heat label toxin was detected in 13 from the 58 strains tested. These data show that the drinking water sources analyzed can represent a risk for human health. It is important to consider that wells and springs are used as drinking water supply in poor areas and rural regions, where undernourished people more susceptible to infections by these microorganisms predominate.
Bactérias do gênero Aeromonas são naturais no ambiente aquático e algumas espécies podem causar infecções em humanos como feridas, septicemia e diarréia. Este trabalho objetivou avaliar a ocorrência de Aeromonas sp. em 126 amostras de água de poços, nascentes, fontes e água mineral, e associar sua presença com indicadores microbianos de contaminação. Foi utilizada a técnica de membrana filtrante com o meio ADA e o teste P/A. Colônias típicas de Aeromonas sp. foram submetidas a testes bioquímicos para identificação da espécie. A produção de toxina foi avaliada utilizando-se células Y-1 de adrenal de camundongo. Coliformes e bactérias heterotróficas foram analisados através de filtração em membrana e pela técnica de inoculação em profundidade, respectivamente. P. aeruginosa, C. pefringens e os estreptococos fecais foram determinados pelo teste P/A. Aeromonas sp. foi isolada em 36,5% das amostras, enquanto que os coliformes totais e termotolerantes estavam presentes em 51,2% e 23,8% das amostras, respectivamente. C. perfringens, estreptococos fecais e P. aeruginosa foram detectados em 16,5%, 20,4% e 3,8% das amostras respectivamente. Concentrações de bactérias heterotróficas superiores a 1,0x10³ UFC/mL ocorreram em 52,5% das amostras. A. hydrophila foi a espécie mais isolada, seguida por A. allosaccharophila, A. jandaei, A. sobria e HG2. Uma toxina termolábil foi detectada em 13 dos 58 isolados analisados. Portanto, as fontes de água de consumo humano analisadas podem representar um risco para a saúde humana. É importante considerar que fontes, poços e nascentes são utilizadas como suprimento de água em áreas pobres e regiões rurais, onde predominam pessoas com problemas de desnutrição, mais suscetíveis a doenças infecciosas.
RESUMO
The heat-labile toxin (LT) is a key virulence-associated factor associated with the non-invasive secretory diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains either in humans or domestic animals. Several LT detection methods have been reported but quantification of the toxin produced by wild-type ETEC strains is usually performed by the GM1 ganglyoside enzyme-linked immunosorbent assay (GM1 ELISA). In this study we conducted the optimization of an alternative LT-quantification method, the antibody-capture ELISA (cELISA). Detailed analysis of the appropriate dilutions of capture and detecting LT-specific antibodies significantly improved the sensitivity of the method. Additionally, testing of different LT extraction techniques indicated that sonic disruption of the bacterial cells enhanced LT recovery yields, in contrast to the usual procedure based on addition of polymyxin B to the culture medium as well as extraction methods based on chloroform or Triton X-100. Moreover, the present data indicate that performance of the LT extraction method based on polymyxin B treatment can vary among wild ETEC strains.
A toxina termo-lábil (LT) é um fator de virulência associado à diarréia secretora não invasiva causada por linhagens de Escherichia coli enterotoxigênica (ETEC) em humanos ou animais domésticos. Diversos métodos de detecção de LT foram descritos na literatura, no entanto, a quantificação da toxina produzida por linhagens selvagens de ETEC é geralmente realizada por ensaio imunoenzimático com o gangliosídeo GM-1 (GM-1 ELISA). Neste estudo, conduzimos uma otimização experimental de um método alternativo de quantificação de LT, o ELISA de captura (cELISA). Análise detalhada de diluições apropriadas dos anticorpos LT específicos de captura e detecção melhorou significantemente a sensibilidade do método. Em adição, testes com diferentes técnicas de extração de LT indicaram que a ruptura das células por ultra-som, mas não o tratamento com polimixina B, clorofórmio ou Triton X-100, aumentou o rendimento da recuperação de LT. Além disto, os dados apresentados demonstram que o desempenho do método de extração de LT baseado no tratamento com polimixina B pode variar entre linhagens selvagens de ETEC.