Identification and Characterization of <i>Clostridium perfringens</i> Atypical CPB2 Toxin in Cell Cultures and Field Samples Using Monoclonal Antibodies
Anna Serroni,
Claudia Colabella,
Deborah Cruciani,
Marcella Ciullo,
Silvia Crotti,
Paola Papa,
Antonella Di Paolo,
Marco Gobbi,
Katia Forti,
Martina Pellegrini,
Romolo Salini,
Nicoletta D’Avino,
Monica Cagiola,
Giovanni Pezzotti,
Antonio De Giuseppe
Affiliations
Anna Serroni
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale” (IZSAM), Campo Boario, 64100 Teramo, Italy
Claudia Colabella
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati” (IZSUM), Via G. Salvemini 1, 06126 Perugia, Italy
Deborah Cruciani
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati” (IZSUM), Via G. Salvemini 1, 06126 Perugia, Italy
Marcella Ciullo
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati” (IZSUM), Via G. Salvemini 1, 06126 Perugia, Italy
Silvia Crotti
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati” (IZSUM), Via G. Salvemini 1, 06126 Perugia, Italy
Paola Papa
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati” (IZSUM), Via G. Salvemini 1, 06126 Perugia, Italy
Antonella Di Paolo
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati” (IZSUM), Via G. Salvemini 1, 06126 Perugia, Italy
Marco Gobbi
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati” (IZSUM), Via G. Salvemini 1, 06126 Perugia, Italy
Katia Forti
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati” (IZSUM), Via G. Salvemini 1, 06126 Perugia, Italy
Martina Pellegrini
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati” (IZSUM), Via G. Salvemini 1, 06126 Perugia, Italy
Romolo Salini
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale” (IZSAM), Campo Boario, 64100 Teramo, Italy
Nicoletta D’Avino
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati” (IZSUM), Via G. Salvemini 1, 06126 Perugia, Italy
Monica Cagiola
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati” (IZSUM), Via G. Salvemini 1, 06126 Perugia, Italy
Giovanni Pezzotti
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati” (IZSUM), Via G. Salvemini 1, 06126 Perugia, Italy
Antonio De Giuseppe
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati” (IZSUM), Via G. Salvemini 1, 06126 Perugia, Italy
A direct sandwich enzyme-linked immunosorbent assay (sELISA) was developed for the detection of the atypical β2-toxin (CPB2) of Clostridium perfringens. Polyclonal (PAbs) and monoclonal (MAbs) antibodies were previously obtained employing recombinant CPB2 produced in the baculovirus system as antigen. In the current study, PAbs were used as capture molecules, while purified MAbs conjugated to horseradish peroxidase (MAbs-HRP) were used for the detection of atypical CPB2 toxin. MAbs 5C11E6 and 2G3G6 showed high reactivity, sensitivity and specificity when tested on 232 C. perfringens cell culture isolates. In addition, a reactivity variation among different strains producing atypical CPB2 toxin was observed using the conformation-dependent MAb 23E6E6, suggesting the hypothesis of high instability and/or the existence of different three-dimensional structures of this toxin. Results obtained by sELISA and Western blotting performed on experimentally CPB2-contaminated feces revealed a time-dependent proteolytic degradation as previously observed with the consensus allelic form of CPB2. Finally, the sELISA and an end-point PCR, specific for the atypical cpb2 gene, were used to test field samples (feces, rectal swabs and intestinal contents) from different dead animal species with suspected or confirmed clostridiosis. The comparison of sELISA data with those obtained with end-point PCR suggests this method as a promising tool for the detection of atypical CPB2 toxin.
