Differentiation of typical and atypical enteropathogenic Escherichia coli using colony immunoblot for detection of bundle-forming pilus expression.

AIMS: The aim of study was to develop a colony immunoblot assay to differentiate typical from atypical enteropathogenic Escherichia coli (EPEC) by detection of bundle-forming pilus (BFP) expression. METHODS AND RESULTS: Anti-BFP antiserum was raised in rabbits and its reactivity was confirmed by immunoelectron microscopy and by immunoblotting recognizing bundlin, the major pilus repeating subunit. The bacterial isolates tested in the colony immunoblot assay were grown in different media. Proteins from bacterial isolates were transferred to nitrocellulose membrane after treatment with phosphate buffer containing Triton X-100, EDTA and sodium chloride salts. When 24 typical EPEC and 96 isolates including, 72 atypical EPEC, 13 Gram-negative type IV-expressing strains and 11 enterobacteriaceae were cultivated in Dulbecco's Modified Eagle's Medium agar containing fetal bovine serum or in blood agar in the presence of CaCl(2) , they showed a positivity of 92 and 83%, and specificity of 96 and 97%, respectively. CONCLUSION: The assay enables reliable identification of BFP-expressing isolates and contributes to the differentiation of typical and atypical EPEC. SIGNIFICANCE AND IMPACT OF THE STUDY: The colony immunoblot for BFP detection developed in this study combines the simplicity of an immunoserological assay with the high efficiency of testing a large number of EPEC colonies.

Differentiation of typical and atypical enteropathogenic Escherichia coli using colony immunoblot for detection of bundle-forming pilus expression

The aim of study was to develop a colony immunoblot assay to differentiatetypical from atypical enteropathogenic Escherichia coli (EPEC) by detectionof bundle-forming pilus (BFP) expression. Anti-BFP antiserum was raised in rabbits and itsreactivity was confirmed by immunoelectron microscopy and by immunoblotting recognizing bundlin, the major pilus repeating subunit. The bacterial isolates tested in the colony immunoblot assay were grown in different media. Proteins from bacterial isolates were transferred to nitrocellulose membrane after treatment with phosphate buffer containing Triton X-100, EDTA and sodium chloride salts. When 24 typical EPEC and 96 isolates including, 72 atypical EPEC, 13 Gram-negative type IV-expressing strains and 11 enterobacteriaceae were cultivated in Dulbecco’s Modified Eagle’s Medium agar containing fetal bovine serum or in blood agar in the presence of CaCl2, they showed a positivity of 92 and 83%, and specificity of 96 and 97%, respectively. The assay enables reliable identification of BFP-expressing isolatesand contributes to the differentiation of typical and atypical EPEC.The colony immunoblot for BFP detectiondeveloped in this study combines the simplicity of an immunoserologicalassay with the high efficiency of testing a large number of EPECcolonies.

Vitamin A as adjuvant to the mouse immune response to pertussis, tetanus and diphtheria vaccines

Vitamin A was used as adjuvant, comparatively with Al(OH)3, in pertussis, tetanus and diphtheria vaccines. Both groups induced a primary immune response in mice, and one single booster dose elevated the antibodies titers in average 554 times to vitamin A groups and 104 times to Al(OH)3. These antibodies titers correlate with sera IL-4 in immunized animals, suggesting a Th2 response. Other cytokines detected in the sera and/or lymphocytes culture supernatants (IL-2 and IFN- ) indicated that vitamin A could also modulate a Th1 response in DPT and acellular pertussis vaccines.

Quantification of crotamine, a small basic myotoxin, in South American rattlesnake (Crotalus durissus terrificus) venom by enzyme-linked immunosorbent assay with parallel-lines analysis

Intraspecific variation in Crotalus durissus terrificus venom composition was studied in relation to crotamine activity. Crotamine induces paralysis in extension of hind legs of mice and myonecrosis in skeletal muscle cells. To determine whether the venom of crotamine-negative rattlesnake contains a quantity of myotoxin incapable of inducing paralysis, we have developed a very sensitivity immunological assay method, an enzyme-linked immunoabsorbent assay (ELISA), capable of detecting 0.6 ng of purified crotamine. The parallel-lines analysis of ELISA data showed to be useful because it shows the reliability of the experimental conditions. A variation in the amount of myotoxin in the crotamine-positive venom was observed, but not less than 0.1 mg of crotamine per mg of venom. It was not possible to detect it in crotamine-negative venom even at high venom concentrations.

A Bordetella pertussis acellular vaccine candidate: antigenic characterization and antibody induction

A single-step chromatography on Matrex-Gel Blue A has been employed to obtain soluble extracts containing some of the most important antigens of Bordetella pertussis, pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (69-kDa outer membrane protein), fimbriae (FIM2 and FIM3) and adenylate cyclase (AC). Two supernatants, P19 (48.8 mg PT, 6.8 mg FHA, 17.3 mg AC, 13 mg FIM2 and 4.9 mg FIM33 per liter) and P21 (0.1 mg PT, 0.07 mg FHA, 0.46 mg FIM2 and 0.94 mg FIM3 per liter), resulting from bacteria grown in Stainer-Scholte medium, were submitted to chromatography. Fractions with the antigens were obtained after stepwise elution with 60 mM sodium phosphate buffer, pH 6.0; 50 mM Tris-HC1, pH 7.4; 50 mM Tris-HC1, pH 7.4/0.75 M MgCl2; 50mM Tris-HCl, pH 7.4/4 M MgCl2 and 4 M urea. Preparations from P19 (containing 4.05 µg PT, 8.14 µg FHA, 6.3 µg AC, 3.37 µg 69-kDA, 9.54 µg FIM2 and 2.23 µg FIM3) and P21 (with 0.175 µg PT, 0.28 µg PT, 0.28 µg FHA, 0.002 µg69-kDa, 0.005 µg FIM2 and 0.122 µg FIM3) were detoxified with glutaraldehyde and tested as an acellular pertussis vaccine. These products were non-toxic for mice and induced high levels of antibodies against purified pertussis antigens, as judged by ELISA