Production and characterization of rabbit polyclonal sera against Shiga toxins Stx1 and Stx2 for detection of Shiga toxin‐producing Escherichia coli

Abstract

STEC has emerged as an important group of enteric pathogens worldwide. In this study, rabbit polyclonal Stx1 and Stx2 antisera were raised and employed in the standardization of immunoassays for STEC detection. Using their respective antisera, the limit of detection of the toxin was 35.0 pg for Stx1 and 5.4 pg for Stx2. By immunoblotting, these antisera recognized both toxin subunits. Cross-reactivity was observed in the A subunit, but only Stx2 antiserum was able to neutralize the cytotoxicity of both toxins in the Vero cell assay. Six stx-harboring E. coli isolates were analyzed for their virulence traits. They belonged to different serotypes, including the O48:H7, described for the first time in Brazil. Only three strains harbored eae, and the e-hly gene and hemolytic activity was detected in five strains. Three isolates showed new stx2 variants (stx 2v−ha and stx 2vb−hb ). The ELISA assay detected all six isolates, including one VCA-negative isolate, while the immunodot assay failed to detect one isolate, which was VCA-positive. In contrast, the colony-immunoblot assay detected only one VCA-positive isolate. Our results demonstrate that among the immunoassays developed in this study, the immunodot, and particularly the ELISA, appear as perspective for STEC detection in developing countries.Key words ELISA, immunodot, polyclonal rabbit antisera, Shiga toxin-producing E. coli.STEC constitutes an important group of emerging enteric pathogens. STEC was first discovered in 1977 (1) and first associated with HUS in 1983 (2). Since then, STEC has been identified as a major food-borne pathogen in different countries all around the world. Epidemiological studies have shown that STEC are present in numerous serotypes in cattle and other domestic animals worldwide, independent of geographic region, species, husbandry methods and climate (3-5). Prevalence rates and types of STEC in humans also vary among different regions, even in the same country (4). List of Abbreviations: BSA, bovine serum albumin; DAB, diaminobenzidine; DEC, diarrheagenic E. coli; eae, intimin; E. coli, Escherichia coli; EHEC, enterohemorrhagic E. coli; e-hly, enterohemolysin; ELISA, enzyme-linked immunosorbent assay; H 2 O 2 , hydrogen peroxide; HUS, hemolytic-uremic syndrome; NBT/BCIP, nitro blue tetrazolium chloride/ 5-bromo-4-chloro-3-indolyl phosphate toluidine salt; OPD, σ-phenylenediamine; PBS, phosphatebuffered saline; SDS, sodium dodecylsulfate; STEC, Shiga toxin-producing Escherichia coli; Stx1, Shiga toxin 1; Stx2, Shiga toxin 2; TSA, tryptic soy agar; TSB, tryptic soy broth; VCA, Vero cell assay Among the DEC pathotypes, STEC has been considered emergent in Brazil. In different regions of our country, the isolation rate of STEC strains ranges from 12 to 71% in animals from dairy farms, beef farms and slaughterhouses for cattle (6-11) and also for sheep (12). In spite of the low prevalence (13-16), the occurrence of clinically important STEC strains associated with disease in humans, including bloody diarrhea, hemolytic anemia and...

First description of a Shiga toxin-producing Escherichia coli O103:H2 strain isolated from sheep in Brazil

“…The stx1 eae-ε ehxA virulence gene profile is commonly observed among STEC O103:H2 strains [4,5,7,9]. Stx1 expression was confirmed by cytotoxicity and neutralization assays on Vero cells [17], and enterohemolysin production was evidenced by the appearance of lysis zone on washed sheep blood agar plates [17]. The genotypic and phenotypic characteristics observed were similar to those of O103:H2 strains previously isolated from patients with diarrhea and hemolytic anemia in Brazil (Table 1).…”

Diarrheagenic Escherichia coli

“…Moreover, these commercially available tests are not affordable for developing countries. Thus, to outline this, previous works have established different formats of immunoassays, employing either a mixture of rabbit anti-Stx1 and anti-Stx2 sera by indirect ELISA or polyclonal and monoclonal antibodies in a capture ELISA assay for the detection of STEC 207, 208, 209. The standardized methods are reproducible, fast, easy to perform, showing high sensitivity in detecting Stx by capture ELISA, even in low-producing isolates.…”

Presence of Shiga toxin 2e‐producing Escherichia coli and atypical enteropathogenic E. coli in an asymptomatic child

“…The aEPEC isolates produced intimin (Menezes et al , 2009), but not BFP, as evidenced by immunoblotting (Nara et al , 2010). Stx2e production was confirmed by cytotoxicity and neutralization assays on Vero cells (Mendes–Ledesma et al , 2008). Table 1 summarizes the genotypic and phenotypic characteristics of aEPEC and STEC.…”