ABSTRACT
The latex agglutination test using single-chain antibody fragments (scFvStx1 and scFvStx2) coupled to latex particles, was compared with the gold standard Vero cell assay for Shiga toxin (Stx) detection, aiming to estimate the diagnosis potential of these scFv fragments in a rapid and straightforward test. The latex complexes identified the presence of the toxins up to a 1:8 dilution in the majority of the evaluated strains. Moreover, the Stx concentration was indirectly determined in Stx-producing Escherichia coli (STEC) strains, allowing detection limit inference. A Stx dilution curve was constructed, and the data was analyzed in a non-linear model by second-order polynomial regression for prediction (p-value of 0.001 and a R2 above 0.98 were considered for correlations). The detection limit was 30ng/mL for Stx1 and 10ng/mL for Stx2. The scFvStx1 and scFvStx2 coupled to latex nanoparticles provide a toxin assay with a competitive Stx detection limit, which has a low cost and short execution time. The diagnostic method proposed here, using, for the first time, recombinant antibody fragments, raises the possibility of developing a more affordable test to be used in the routine detection and surveillance of STEC infections.
ABSTRACT
The latex agglutination test using single-chain antibody fragments (scFvStx1 and scFvStx2) coupled to latex particles, was compared with the gold standard Vero cell assay for Shiga toxin (Stx) detection, aiming to estimate the diagnosis potential of these scFv fragments in a rapid and straightforward test. The latex complexes identified the presence of the toxins up to a 1:8 dilution in the majority of the evaluated strains. Moreover, the Stx concentration was indirectly determined in Stx-producing Escherichia coli (STEC) strains, allowing detection limit inference. A Stx dilution curve was constructed, and the data was analyzed in a non-linear model by second-order polynomial regression for prediction (p-value of 0.001 and a R2 above 0.98 were considered for correlations). The detection limit was 30ng/mL for Stx1 and 10ng/mL for Stx2. The scFvStx1 and scFvStx2 coupled to latex nanoparticles provide a toxin assay with a competitive Stx detection limit, which has a low cost and short execution time. The diagnostic method proposed here, using, for the first time, recombinant antibody fragments, raises the possibility of developing a more affordable test to be used in the routine detection and surveillance of STEC infections.
ABSTRACT
Food-producing animals can harbor Escherichia coli strains with potential to cause diseases in humans. In this study, the presence of enteropathogenic E. coli (EPEC) was investigated in fecal samples from 130 healthy sheep (92 lambs and 38 adults) raised for meat in southern Brazil. EPEC was detected in 19.2% of the sheep examined, but only lambs were found to be positive. A total of 25 isolates was characterized and designated atypical EPEC (aEPEC) as tested negative for bfpA gene and BFP production. The presence of virulence markers linked to human disease as ehxA, paa, and IpfAO(113) was observed in 60%, 24%, and 88% of the isolates, respectively. Of the 11 serotypes identified, eight were described among human pathogenic strains, while three (O1:H8, O11:H21 and O125:H19) were not previously detected in aEPEC. Associations between intimin subtypes and phylogroups were observed, including eae-theta 2/A, eae-beta 1/B1, eae-alpha 2/B2 and eae-gamma 1/D. Although PFGE typing of 16 aEPEC isolates resulted in 14 unique pulsetypes suggesting a genetic diversity, specific clones were found to be distributed in some flocks. In conclusion, potentially pathogenic aEPEC strains are present in sheep raised for meat, particularly in lambs, which can better contribute to dissemination of these bacteria than adult animals
Subject(s)
Microbiology , BacteriologySubject(s)
Toxicology , Toxicology , Biodiversity , Poisons , Poisoning , Toxins, Biological , ToxicologySubject(s)
Toxicology , Toxicology , Poisons , Poisoning , Toxins, Biological , Toxicology , BiochemistrySubject(s)
Toxicology , Toxicology , Poisons , Poisoning , Toxins, Biological , Toxicology , BiochemistryABSTRACT
The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT+ (25 strains) only or LT+/ST+ (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the elt operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.
