Purification and characterization of the fimbria F18ac (2134P) isolated from enterotoxigenic Escherichia coli (ETEC)

Amorim, Claudio Roberto Nobrega; Matsuura, Maria Sumiko Arita; Rosa, JP; Greene, Lewis J.; Leite, Domingos S.; Yano, Tomomasa

doi:10.1016/s0378-1135(00)00216-9

Cited by 4 publications

(3 citation statements)

References 23 publications

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“…The higher the values, the greater the genetic variability, the greater the adaptability to the environment, the greater the selection potential and the better the effect of genetic breeding. Conversely, less heterozygosity and high genetic purity in the population can be regarded as a pure line to be utilized [23]. The PICs of the TAP1 gene in this study were in the range of 0.25-0.5, and all of them were moderately polymorphic.…”

Section: Polymorphism Of Antidiarrheal Genesmentioning

confidence: 62%

Detection and Analysis of Antidiarrheal Genes and Immune Factors in Various Shanghai Pig Breeds

Zhou,

Liu,

et al. 2024

Biomolecules

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The aim of this study was to identify effective genetic markers for the Antigen Processing Associated Transporter 1 (TAP1), α (1,2) Fucosyltransferase 1 (FUT1), Natural Resistance Associated Macrophage Protein 1 (NRAMP1), Mucin 4 (MUC4) and Mucin 13 (MUC13) diarrhea-resistance genes in the local pig breeds, namely Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs, to provide a reference for the characterization of local pig breed resources in Shanghai. Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLR) and sequence sequencing were applied to analyze the polymorphisms of the above genes and to explore the effects on the immunity of Shanghai local pig breeds in conjunction with some immunity factors. The results showed that both TAP1 and MUC4 genes had antidiarrheal genotype GG in the five pig breeds, AG and GG genotypes of the FUT1 gene were detected in Pudong white pigs, AA antidiarrheal genes of the NRAMP1 gene were detected in Meishan pigs, the AB type of the NRAMP1 gene was detected in Pudong white pigs, and antidiarrheal genotype GG of the MUC13 gene was only detected in Shanghai white pigs. The MUC13 antidiarrhea genotype GG was only detected in Shanghai white pigs. The TAP1 gene was moderately polymorphic in Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs, among which TAP1 in Shanghai white pigs and Shawutou pigs did not satisfy the Hardy–Weinberg equilibrium. The FUT1 gene of Pudong white pigs was in a state of low polymorphism. NRAMP1 of Meishan pigs and Pudong white pigs was in a state of moderate polymorphism, which did not satisfy the Hardy–Weinberg equilibrium. The MUC4 genes of Shanghai white pigs and Pudong white pigs were in a state of low polymorphism, and the MUC4 genes of Fengjing pigs and Shawutou pigs were in a state of moderate polymorphism, and the MUC4 genes of Fengjing pigs and Pudong white pigs did not satisfy the Hardy–Weinberg equilibrium. The MUC13 gene of Shanghai white pigs and Pudong white pigs was in a state of moderate polymorphism. Meishan pigs had higher levels of IL-2, IL-10, IgG and TNF-α, and Pudong white pigs had higher levels of IL-12 than the other pigs. The level of interleukin 12 (IL-12) was significantly higher in the AA genotype of the MUC13 gene of Shanghai white pigs than in the AG genotype. The indicator of tumor necrosis factor alpha (TNF-α) in the AA genotype of the TAP1 gene of Fengjing pigs was significantly higher than that of the GG and AG genotypes. The indicator of IL-12 in the AG genotype of the Shawutou pig TAP1 gene was significantly higher than that of the GG genotype. The level of TNF-α in the AA genotype of the NRAMP1 gene of Meishan pigs was markedly higher than that of the AB genotype. The IL-2 level of the AG type of the FUT1 gene was obviously higher than that of the GG type of Pudong white pigs, the IL-2 level of the AA type of the MUC4 gene was dramatically higher than that of the AG type, and the IgG level of the GG type of the MUC13 gene was apparently higher than that of the AG type. The results of this study are of great significance in guiding the antidiarrhea breeding and molecular selection of Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs and laying the foundation for future antidiarrhea breeding of various local pig breeds in Shanghai.

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Section: Polymorphism Of Antidiarrheal Genesmentioning

confidence: 62%

Detection and Analysis of Antidiarrheal Genes and Immune Factors in Various Shanghai Pig Breeds

Zhou,

Liu,

et al. 2024

Biomolecules

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“…Precipitation with 15% ammonium sulfate similarly precipitated the major fimbrial protein of CS4, whereas many of the other protein contaminants remained in solution (Figure 1A, lanes 6,7). The prior referenced methods that employed ammonium sulfate to precipitate fimbriae used conditions of 40 – 60% saturation [20–22]. Selective precipitation from contaminants at the minimal level of saturation is preferable however, as protein contaminants that remain soluble are readily removed in the supernatant.…”

Section: Resultsmentioning

confidence: 99%

“…One method made use of sequential fractionation by 2-step differential ultra-centrifugation, size-exclusion chromatography and density ultracentrifugation [18, 19]. Alternate methods have employed ammonium sulfate precipitation, followed by fractionation of the precipitated material by size exclusion chromatography or negative chromatography through a weak anion exchange resin [20–22]. Neither of these prior reported methods is ideal.…”

Section: Introductionmentioning

confidence: 99%

Simple method for purification of enterotoxigenic Escherichia coli fimbriae

Curtis

Grassel

Laufer

et al. 2016

Protein Expression and Purification

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Enterotoxigenic E. coli (ETEC) are endemic pathogens in the developing world. They frequently cause illness in travelers, and are among the most prevalent causes of diarrheal disease in children. Pathogenic ETEC strains employ fimbriae as adhesion factors to bind the luminal surface of the intestinal epithelium and establish infection. Accordingly, there is marked interest in immunoprophylactic strategies targeting fimbriae to protect against ETEC infections. Multiple strategies have been reported for purification of ETEC fimbriae, however none is ideal. Purification has typically involved the use of highly virulent wild-type strains. We report here a simple and improved method to purify ETEC fimbriae, which was applied to obtain two different Class 5 fimbriae types of clinical relevance (CFA/I and CS4) expressed recombinantly in E. coli production strains. Following removal from cells by shearing, fimbriae proteins were purified by orthogonal purification steps employing ultracentrifugation, precipitation, and ion-exchange membrane chromatography. Purified fimbriae demonstrated the anticipated size and morphology by electron microscopy analysis, contained negligible levels of residual host cell proteins, nucleic acid, and endotoxin, and were recognized by convalescent human anti-sera.

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Efficacy of a single oral dose of a live bivalent E. coli vaccine against post-weaning diarrhea due to F4 and F18-positive enterotoxigenic E. coli

Nadeau

Fairbrother

Zentek

et al. 2017

The Veterinary Journal

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F4- and F18-positive enterotoxigenic E. coli strains (F4-ETEC and F18-ETEC) are important causes of post-weaning diarrhea (PWD) in pigs. F4 (antigenic variant ac) and F18 (ab and ac) fimbriae are major antigens that play an important role in the early stages of infection. Herein, the efficacy of a live oral vaccine consisting of two non-pathogenic E. coli strains, one F4ac- and one F18ac-positive, was evaluated using F4ac-ETEC and F18ab-ETEC challenge models. A randomized, masked, placebo-controlled, block design, parallel-group confirmatory study with two different vaccination-challenge intervals (7 and 21 days) was conducted for each challenge model. The vaccine was administered in one dose, to ≥18-day-old piglets via drinking water. Efficacy was assessed by evaluating diarrhea, clinical observations, weight gain and fecal shedding of F4-ETEC or F18-ETEC. Anti-F4 and anti-F18 immunoglobulins in blood were measured. The vaccination resulted in significant reductions in clinical PWD and fecal shedding of F4-ETEC and F18-ETEC after the 7- and 21-day-post-vaccination heterologous challenges, except for after the 21-day-post-vaccination F4-ETEC challenge, when the clinical PWD was too mild to demonstrate efficacy. A significant reduction of mortality and weight loss by vaccination were observed following the F18-ETEC challenge. The 7-day protection was associated with induction of anti-F4 and anti-F18 IgM, whereas the 21-day protection was mainly associated with anti-F4 and anti-F18 IgA. The 7-day onset and 21-day duration of protection induced by this vaccine administered once in drinking water to pigs of at least 18days of age were confirmed by protection against F4-ETEC and F18-ETEC, and induction of F4 and F18-specific immunity. Cross protection of the vaccine against F18ab-E. coli was demonstrated for both the 7- and 21-day F18-ETEC challenges.

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Purification and characterization of the fimbria F18ac (2134P) isolated from enterotoxigenic Escherichia coli (ETEC)

Cited by 4 publications

References 23 publications

Detection and Analysis of Antidiarrheal Genes and Immune Factors in Various Shanghai Pig Breeds

Detection and Analysis of Antidiarrheal Genes and Immune Factors in Various Shanghai Pig Breeds

Simple method for purification of enterotoxigenic Escherichia coli fimbriae

Efficacy of a single oral dose of a live bivalent E. coli vaccine against post-weaning diarrhea due to F4 and F18-positive enterotoxigenic E. coli

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