Multiple Labeling of Compartmentalized Cortical Neurons in Microfluidic Chambers.

Neurons are complex cells with two distinct compartments: the somatodendritic and the axonal domains. Because of their polarized morphology, it is challenging to study the differential cellular and molecular mechanisms that occur in axons and impact the soma and dendrites using conventional in vitro culture systems. Compartmentalized cultures offer a solution by physically and chemically separating the axonal from the somatodendritic domain of neurons. The microfluidic chamber model presented in this work is valuable for studying these mechanisms in primary cortical cultures derived from rat and mouse. In addition, this chamber model is compatible with various microscopy methods, such as phase contrast, and fluorescence imaging of living and fixed cells. Key features • Preparation and attachment of PDMS microfluidic chambers to glass coverslips. • Primary culture of cortical neurons and plating cortical neurons in microfluidic chamber. • Confirmation of compartmentalization using the retrograde transport of the fluorescently labeled form of cholera toxin subunit B (f-Ctb). • Immunofluorescence and multilabeling of compartmentalized cortical neurons. • Retrograde transport of fluorescently labeled BDNF.

Crucial role of carbon monoxide as a regulator of diarrhea caused by cholera toxin: Evidence of direct interaction with toxin.

The present study evaluated the role of heme oxygenase 1 (HO-1)/carbon monoxide (CO) pathway in the cholera toxin-induced diarrhea and its possible action mechanism. The pharmacological modulation with CORM-2 (a CO donor) or Hemin (a HO-1 inducer) decreased the intestinal fluid secretion and Cl- efflux, altered by cholera toxin. In contrast, ZnPP (a HO-1 inhibitor) reversed the antisecretory effect of Hemin and potentiated cholera toxin-induced intestinal secretion. Moreover, CORM-2 also prevented the alteration of intestinal epithelial architecture and local vascular permeability promoted by cholera toxin. The intestinal absorption was not altered by any of the pharmacological modulators. Cholera toxin inoculation also increased HO-1 immunoreactivity and bilirubin levels, a possible protective physiological response. Finally, using fluorometric technique, ELISA assay and molecular docking simulations, we show evidence that CO directly interacts with cholera toxin, forming a complex that affects its binding to GM1 receptor, which help explain the antisecretory effect. Thus, CO is an essential molecule for protection against choleric diarrhea and suggests its use as a possible therapeutic tool.

Vibrio cholerae, classification, pathogenesis, immune response, and trends in vaccine development.

Vibrio cholerae is the causative agent of cholera, a highly contagious diarrheal disease affecting millions worldwide each year. Cholera is a major public health problem, primarily in countries with poor sanitary conditions and regions affected by natural disasters, where access to safe drinking water is limited. In this narrative review, we aim to summarize the current understanding of the evolution of virulence and pathogenesis of V. cholerae as well as provide an overview of the immune response against this pathogen. We highlight that V. cholerae has a remarkable ability to adapt and evolve, which is a global concern because it increases the risk of cholera outbreaks and the spread of the disease to new regions, making its control even more challenging. Furthermore, we show that this pathogen expresses several virulence factors enabling it to efficiently colonize the human intestine and cause cholera. A cumulative body of work also shows that V. cholerae infection triggers an inflammatory response that influences the development of immune memory against cholera. Lastly, we reviewed the status of licensed cholera vaccines, those undergoing clinical evaluation, and recent progress in developing next-generation vaccines. This review offers a comprehensive view of V. cholerae and identifies knowledge gaps that must be addressed to develop more effective cholera vaccines.

Intranasal and Intramuscular Immunization with Outer Membrane Vesicles from Serogroup C Meningococci Induced Functional Antibodies and Immunologic Memory.

BACKGROUND: Immunization is the key to prevent invasive meningococcal disease (IMD), caused by Neisseria meningitidis. Outer membrane vesicles (OMVs) can be used as meningococcal antigens. METHODS: Isogenic mice A/Sn (H2a) were immunized with low antigenic doses of OMVs of an N. meningitidis C:2a:P1.5 strain, via intranasal/intramuscular route, adjuvanted by cholera toxin subunit B (CTB) or via intramuscular route only, adjuvanted by aluminium hydroxide (AH). Mice were followed until old age and humoral and cellular responses were assessed by ELISA, Immunoblotting, Dot-blot, Serum-bactericidal assay, Immunohistochemistry and ELISpot. RESULTS: OMV+CTB and OMV+AH groups presented statistically higher antibodies titers, which persisted until middle and old ages. IgG isotypes point to a Th2 type of response. Avidity indexes were considered high, regardless of adjuvant use, but only groups immunized with OMVs and adjuvants (OMV+CTB and OMV+AH) presented bactericidal activity. The antibodies recognized antigens of molecular weights attributed to porin and cross-reactivity proteins. Although the spleen of old mice did not present differences in immunohistochemistry marking of CD68+, CD4+, CD79+ and CD25+ cells, splenocytes of immune groups secreted IL-4 and IL-17 when stimulated with OMVs and meningococcal C polysaccharide. CONCLUSION: We concluded that both adjuvants, CTB and AH, improved the immunogenicity of low doses of OMVs and contributed to a persistent immune response. Even though AH is well established in the vaccinology area, CTB seems to be a promising adjuvant candidate for meningococcal vaccines: it is suitable for mucosal delivery and supports a Th2 type of response. Therefore, OMVs are still a relevant vaccine platform.

Understanding the Anti-Diarrhoeal Properties of Incomptines A and B: Antibacterial Activity against Vibrio cholerae and Its Enterotoxin Inhibition.

Incomptines A (IA) and B (IB) are two sesquiterpene lactones with antiprotozoal, antibacterial, cytotoxic, antitumor, spermicidal, and phytotoxic properties. The antibacterial activity of IA and IB against bacteria causing diarrhoea have been reported; however, no information is available regarding their antibacterial activity on Vibrio cholerae. In this work, both compounds were evaluated for their anti-diarrhoeal potential using the bacterium V. cholerae, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis on cholera toxin, and a cholera toxin-induced diarrhoea model in male Balb/c mice. In addition, a molecular docking study was carried out to understand the interaction of IA and IB with cholera toxin. In terms of antibacterial activity, IB was three times more active than IA on V. cholerae. In the case of SDS-PAGE analysis and the in silico study, IA was most effective, revealing its potential binding mode at a molecular level. In terms of anti-diarrhoeal activity, IA was 10 times more active than IB and racecadotril, an antisecretory drug used as positive control; the anti-diarrheal activity of IB was also closer than racecadotril. The results obtained from in vitro, in vivo, and computational studies on V. cholerae and cholera toxin support the potential of IA and IB as new anti-diarrhoeal compounds.

B-Cell Epitope Mapping of the Vibrio cholera Toxins A, B, and P and an ELISA Assay.

Oral immunization with the choleric toxin (CT) elicits a high level of protection against its enterotoxin activities and can control cholera in endemic settings. However, the complete B-cell epitope map of the CT that is responsible for protection remains to be clarified. A library of one-hundred, twenty-two 15-mer peptides covering the entire sequence of the three chains of the CT protein (CTP) was prepared by SPOT synthesis. The immunoreactivity of membrane-bound peptides with sera from mice vaccinated with an oral inactivated vaccine (Schankol™) allowed the mapping of continuous B-cell epitopes, topological studies, multi-antigen peptide (MAP) synthesis, and Enzyme-Linked Immunosorbent Assay (ELISA) development. Eighteen IgG epitopes were identified; eight in the CTA, three in the CTB, and seven in the protein P. Three V. cholera specific epitopes, Vc/TxA-3, Vc/TxB-11, and Vc/TxP-16, were synthesized as MAP4 and used to coat ELISA plates in order to screen immunized mouse sera. Sensitivities and specificities of 100% were obtained with the MAP4s of Vc/TxA-3 and Vc/TxB-11. The results revealed a set of peptides whose immunoreactivity reflects the immune response to vaccination. The array of peptide data can be applied to develop improved serological tests in order to detect cholera toxin exposure, as well as next generation vaccines to induce more specific antibodies against the cholera toxin.

Epidemiological and pathogenic characteristics of Haitian variant V. cholerae circulating in India over a decade (2000-2018).

Vibrio cholerae, causative agent of the water-borne disease cholera still threatens a large proportion of world's population. The major biotypes of the pathogen are classical and El Tor. There have been recent reports of variant V. cholerae strains circulating around the world. In the present study, the epidemiological status of V. cholerae strains circulating in the country over a decade was assessed. Also, a comprehensive analysis of the difference in pathogenicity between the different biotypes of V. cholerae strains was evaluated both in-vitro and in-vivo. The amount of CT produced by different biotypes of V. cholerae strains were analyzed by GM1 ELISA and the probable reasons for the difference in toxin production was discussed. MLST analysis grouped the isolates into a single Sequence Type (ST 69) whereas PFGE analysis clustered the isolates into ten different pulsotypes revealing molecular diversity. The circulating strains were identified to produce cholera toxin and CT mRNA intermediate to the classical and prototype El Tor strains. Also, the circulating strains were identified to possess four ToxR binding sequences. In-vivo pathogenicity analysis by rabbit ileal loop fluid accumulation assay revealed the Haitian variant strains to be more hyperemic than the prototype strains.

A comprehensive review of therapeutic approaches available for the treatment of cholera.

OBJECTIVES: The oral rehydration solution is the most efficient method to treat cholera; however, it does not interfere in the action mechanism of the main virulence factor produced by Vibrio cholerae, the cholera toxin (CT), and this disease still stands out as a problem for human health worldwide. This review aimed to describe therapeutic alternatives available in the literature, especially those related to the search for molecules acting upon the physiopathology of cholera. KEY FINDINGS: New molecules have offered a protection effect against diarrhoea induced by CT or even by infection from V. cholerae. The receptor regulator cystic fibrosis channel transmembrane (CFTR), monosialoganglioside (GM1), enkephalinase, AMP-activated protein kinase (AMPK), inhibitors of expression of virulence factors and activators of ADP-ribosylarginine hydrolase are the main therapeutic targets studied. Many of these molecules or extracts still present unclear action mechanisms. CONCLUSIONS: Knowing therapeutic alternatives and their molecular mechanisms for the treatment of cholera could guide us to develop a new drug that could be used in combination with the rehydration solution.

Identification of Immunogenic Antigens of Naegleria fowleri Adjuvanted by Cholera Toxin.

The intranasal administration of Naegleria fowleri lysates plus cholera toxin (CT) increases protection against N. fowleri meningoencephalitis in mice, suggesting that humoral immune response mediated by antibodies is crucial to induce protection against the infection. In the present study, we applied a protein analysis to detect and identify immunogenic antigens from N. fowleri, which might be responsible for such protection. A Western blot assay of N. fowleri polypeptides was performed using the serum and nasal washes from mice immunized with N. fowleri lysates, either alone or with CT after one, two, three, or four weekly immunizations and challenged with trophozoites of N. fowleri. Immunized mice with N. fowleri plus CT, after four doses, had the highest survival rate (100%). Nasal or sera IgA and IgG antibody response was progressively stronger as the number of immunizations was increased, and that response was mainly directed to 250, 100, 70, 50, 37, and 19 kDa polypeptide bands, especially in the third and fourth immunization. Peptides present in these immunogenic bands were matched by nano-LC-ESI-MSMS with different proteins, which could serve as candidates for a vaccine against N. fowleri infection.

Emergence of Haitian variant genotype and altered drug susceptibility in Vibrio cholerae O1 El Tor-associated cholera outbreaks in Solapur, India.

It is evident from previous cholera epidemics/outbreaks in India, Africa and America that isolates of the seventh pandemic Vibrio cholerae El Tor (7PET) with Haitian cholera toxin (HCT) genotype were associated with increased mortality. The present study highlights the emergence of 7PET-HCT isolates causing two cholera outbreaks in Walsang and Wagdari (Solapur, India) in 2016. Molecular analyses revealed that 7PET strains from earlier outbreaks (2010 and 2012) were progenitors of the current 7PET-HCT isolates. Isolates from the 2016 outbreaks carried qnrVC and floR genes and showed reduced susceptibility to tetracycline, ciprofloxacin and azithromycin, drugs recommended by the World Health Organization (WHO) for the treatment of cholera. Remarkably, protein profiling and mass spectrometry revealed disappearance of the outer membrane protein U (OmpU) porin in 7PET-HCT isolates from the second outbreak in 2016. Downregulation of ompU gene expression was also confirmed at the transcriptional level. Strains with downregulated OmpU showed reduced minimum inhibitory concentrations (MICs) for polymyxin B, which is a pore-forming antimicrobial agent. A multipronged approach is of utmost importance to prevent further spread of circulating 7PET-HCT strains. There is a pressing need for the formulation and implementation of international policies to closely monitor the effective use of antibiotics in order to prevent the further rise and spread of antimicrobial resistance.

Vibrio cholerae: doença, manifestações clínicas e microbiologia / Vibrio cholerae: disease, clinical manifestations and microbiology / Vibrio cholerae: enfermedad, manifestaciones clínicas y microbiologia

Justificativa e Objetivos: A cólera é uma doença infecciosa intestinal aguda causada pela toxina do Vibrio cholerae. A transmissão é oral-fecal e ocorre predominantemente em ambientes aquáticos contaminados. Pode ser fatal, mas é facilmente evitada e tratada. Associa-se a sua propagação com a falta de saneamento básico pois cresce exponencialmente nesses ambientes. O diagnóstico é clínico-epidemiológico, laboratorial com meios seletivos para o microrganismo e teste rápido, este último não é sensível e nem especifico. O tratamento é com sais de reidratação oral e antibioticoterapia, indica-se a suplementação com zinco. Existe vacinação para Vibrio cholerae, e essa é a melhor medida para o controle da doença. As pesquisas em cólera já não são mais frequentes, pois ela é considerada uma doença de países que não são desenvolvidos. Conclusão: Nesse contexto, ainda são necessárias pesquisas sobre sistemas de esgoto, monitorização de efluentes, vigilância da qualidade da água de abastecimento público e vigilância da qualidade de alimentos.(AU)

Background and Objectives: Cholera is an acute intestinal infectious disease caused by the toxin of Vibrio cholerae. Transmission is oral-fecal and occurs predominantly in contaminated aquatic environments. It can be fatal, but it is easily avoided and treated. Its propagation is associated with lack of basic sanitation because it grows exponentially in environments. The diagnosis is clinical-epidemiological, laboratory with selective media for the micro-organism and rapid test, the latter is not sensitive or specific. Treatment is with oral rehydration salts and antibiotic therapy, zinc supplementation is indicated. There is vaccination for Vibrio cholerae, and this is the best measure for the control of the disease. Conclusion: Cholera research is no longer frequent, as it is considered a disease of countries that are not developed. In this context, research is still needed on sewage systems, effluent monitoring, public water supply quality surveillance and food quality monitoring.(AU)

Justificación y objetivos: La cólera es una enfermedad infecciosa intestinal aguda causada por la toxina del Vibrio cholerae. La transmisión es oral-fecal y ocurre predominantemente en ambientes acuáticos contaminados. Puede ser fatal, pero es facilmente evitada y tratada. Se asocia su propagación con la falta de saneamiento básico pues crece exponencialmente en ambientes. El diagnóstico es clínico-epidemiológico, de laboratorio con medios selectivos para el microorganismo y la prueba rápida, este último no es sensible ni específico. El tratamiento es con sales de rehidratación oral y antibioticoterapia, se indica la suplementación con cinc. Hay vacunación para Vibrio cholerae, y esa es la mejor medida para el control de la enfermedad. Conclusión: Las investigaciones en cólera ya no son más frecuentes, ya que se considera una enfermedad de países que no se desarrollan. En este contexto todavía son necesarias investigaciones sobre sistemas de alcantarillado, monitorización de efluentes, vigilancia de la calidad del agua de abastecimiento público y vigilancia de la calidad de alimentos.(AU)

Retinofugal Projections Into Visual Brain Structures in the Bat Artibeus planirostris: A CTb Study.

A well-developed visual system can provide significant sensory information to guide motor behavior, especially in fruit-eating bats, which usually use echolocation to navigate at high speed through cluttered environments during foraging. Relatively few studies have been performed to elucidate the organization of the visual system in bats. The present work provides an extensive morphological description of the retinal projections in the subcortical visual nuclei in the flat-faced fruit-eating bat (Artibeus planirostris) using anterograde transport of the eye-injected cholera toxin B subunit (CTb), followed by morphometrical and stereological analyses. Regarding the cytoarchitecture, the dorsal lateral geniculate nucleus (dLGN) was homogeneous, with no evident lamination. However, the retinal projection contained two layers that had significantly different marking intensities and a massive contralateral input. The superior colliculus (SC) was identified as a laminar structure composed of seven layers, and the retinal input was only observed on the contralateral side, targeting two most superficial layers. The medial pretectal nucleus (MPT), olivary pretectal nucleus (OPT), anterior pretectal nucleus (APT), posterior pretectal nucleus (PPT) and nucleus of the optic tract (NOT) were comprised the pretectal nuclear complex (PNT). Only the APT lacked a retinal input, which was predominantly contralateral in all other nuclei. Our results showed the morphometrical and stereological features of a bat species for the first time.

Sporothrix schenckii Immunization, but Not Infection, Induces Protective Th17 Responses Mediated by Circulating Memory CD4+ T Cells.

Sporotrichosis is a chronic subcutaneous mycosis caused by the Sporothrix schenckii species complex and it is considered an emerging opportunistic infection in countries with tropical and subtropical climates. The host's immune response has a main role in the development of this disease. However, it is unknown the features of the memory cellular immune response that could protect against the infection. Our results show that i.d. immunization in the ears of mice with inactivated S. schenckii conidia (iC) combined with the cholera toxin (CT) induces a cellular immune response mediated by circulating memory CD4+ T cells, which mainly produce interleukin 17 (IL-17). These cells mediate a strong delayed-type hypersensitivity (DTH) reaction. Systemic and local protection against S. schenckii was mediated by circulating CD4+ T cells. In contrast, the infection induces a potent immune response in the skin mediated by CD4+ T cells, which have an effector phenotype that preferentially produce interferon gamma (IFN-γ) and mediate a transitory DTH reaction. Our findings prove the potential value of the CT as a potent skin adjuvant when combined with fungal antigens, and they also have important implications for our better understanding of the differences between the memory immune response induced by the skin immunization and those induced by the infection; this knowledge enhances our understanding of how a protective immune response against a S. schenckii infection is developed.

Persistent diarrhoea in a 5-month-old baby carrying Vibrio cholerae nonO1/nonO139 producing Haitian cholera toxin.

Cholera toxin (CT) is the principal virulence factor of Vibrio cholerae for fatal cholera diarrhoea. Serogroups O1 and O139 harbour CT and are known to be epidemic strains. The remaining serogroups (nonO1/nonO139) are non-toxigenic and may be associated with mild disease. O1 serogroup emerged with a variant of CT known as Haitian cholera toxin (HCT). The HCT strains are hypervirulent and have been associated with severe cholera outbreaks in India, Western Africa and Haiti. Here, we report the presence of HCT (ctxB7) in a nonO1/nonO139 isolate causing persistent diarrhoea.

The corn smut-made cholera oral vaccine is thermostable and induces long-lasting immunity in mouse.

The use of corn smut for the production of recombinant vaccines has been recently implemented by our group. In this study, the stability and immunogenic properties of the corn smut-based cholera vaccine, based on the cholera toxin B subunit (CTB), were determined in mouse. The immunogenic potential of distinct corn smut CTB doses ranging from 1 to 30µg were assessed, with maximum humoral responses at both the systemic (IgG) and intestinal (IgA) levels at a dose of 15µg. The humoral response last for up to 70days after the third boost. Mice were fully protected against a challenge with cholera toxin after receiving three 15µg-doses. Remarkably, the corn smut-made vaccine retained its immunogenic activity after storage at room temperature for a period of 1year and no reduction on CTB was observed following exposure at 50°C for 2h. These data support the use of the corn smut-made CTB vaccine as a highly stable and effective immunogen and justify its evaluation in target animal models, such as piglet and sheep, as well as clinical evaluations in humans.

Age-related changes in neurochemical components and retinal projections of rat intergeniculate leaflet.

Aging leads to several anatomical and functional deficits in circadian timing system. In previous works, we observed morphological alterations with age in hypothalamic suprachiasmatic nuclei, one central component of this system. However, there are few data regarding aging effects on other central components of this system, such as thalamic intergeniculate leaflet (IGL). In this context, we studied possible age-related alterations in neurochemical components and retinal projections of rat IGL. For this goal, young (3 months), adult (13 months), and aged (23 months) Wistar rats were submitted to an intraocular injection of neural tracer, cholera toxin subunit b (CTb), 5 days before a tissue fixation process by paraformaldehyde perfusion. Optical density measurements and cell count were performed at digital pictures of brain tissue slices processed by immunostaining for glutamic acid decarboxylase (GAD), enkephalin (ENK), neuropeptide Y (NPY) and CTb, characteristic markers of IGL and its retinal terminals. We found a significant age-related loss in NPY immunoreactive neurons, but not in immunoreactivity to GAD and ENK. We also found a decline of retinal projections to IGL with age. We conclude aging impairs both a photic environmental clue afferent to IGL and a neurochemical expression which has an important modulatory circadian function, providing strong anatomical correlates to functional deficits of the aged biological clock.

Saccharomyces boulardii and bismuth subsalicylate as low-cost interventions to reduce the duration and severity of cholera.

We conducted a randomised single-blinded clinical trial of 100 cholera patients in Port-au-Prince, Haiti to determine if the probiotic Saccharomyces cerevisiae var. boulardii and the anti-diarrhoeal drug bismuth subsalicylate (BS) were able to reduce the duration and severity of cholera. Subjects received either: S. boulardii 250 mg, S. boulardii 250 mg capsule plus BS 524 mg tablet, BS 524 mg, or two placebo capsules every 6 hours alongside standard treatment for cholera. The length of hospitalisation plus the number and volume of emesis, stool and urine were recorded every 6 hours until the study subject was discharged (n = 83), left against medical advice (n = 11), or requested removal from the study (n = 6). There were no reported deaths or adverse study-related events. There were no statistically significant differences between the study arms and the outcomes of interest.

Vaccines for viral and bacterial pathogens causing acute gastroenteritis: Part I: Overview, vaccines for enteric viruses and Vibrio cholerae.

Efforts to develop vaccines for prevention of acute diarrhea have been going on for more than 40 y with partial success. The myriad of pathogens, more than 20, that have been identified as a cause of acute diarrhea throughout the years pose a significant challenge for selecting and further developing the most relevant vaccine candidates. Based on pathogen distribution as identified in epidemiological studies performed mostly in low-resource countries, rotavirus, Cryptosporidium, Shigella, diarrheogenic E. coli and V. cholerae are predominant, and thus the main targets for vaccine development and implementation. Vaccination against norovirus is most relevant in middle/high-income countries and possibly in resource-deprived countries, pending a more precise characterization of disease impact. Only a few licensed vaccines are currently available, of which rotavirus vaccines have been the most outstanding in demonstrating a significant impact in a short time period. This is a comprehensive review, divided into 2 articles, of nearly 50 vaccine candidates against the most relevant viral and bacterial pathogens that cause acute gastroenteritis. In order to facilitate reading, sections for each pathogen are organized as follows: i) a discussion of the main epidemiological and pathogenic features; and ii) a discussion of vaccines based on their stage of development, moving from current licensed vaccines to vaccines in advanced stage of development (in phase IIb or III trials) to vaccines in early stages of clinical development (in phase I/II) or preclinical development in animal models. In this first article we discuss rotavirus, norovirus and Vibrio cholerae. In the following article we will discuss Shigella, Salmonella (non-typhoidal), diarrheogenic E. coli (enterotoxigenic and enterohemorragic), and Campylobacter jejuni.

Vaccines for viral and bacterial pathogens causing acute gastroenteritis: Part II: Vaccines for Shigella, Salmonella, enterotoxigenic E. coli (ETEC) enterohemorragic E. coli (EHEC) and Campylobacter jejuni.

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.

A universal polysaccharide conjugated vaccine against O111 E. coli.

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.