Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type A and type B toxins in stool samples of infants with botulism

Abstract

An enzyme-linked immunosorbent assay (ELISA) for Clostridium botulinum type A and type B toxins was assessed for diagpiostic accuracy in cases of infant botulism. This test was positive in all 22 cases confirmed by the conventional tests, which included the mouse lethality assay and stool culture. Stool specimens from five cases were positive by culture, but the mouse lethality bioassay was either negative or toxicity was judged nonspecific since it could not be neutralized by specific antitoxin. The positive ELISA results in these specimens suggeste4 that this assay may be more reliable, in some cases, than the mouse bioassay. Of the 21 fecal specimens from suspected foodborne cases, 2 contained botulinal toxin demonstrable by the mouse assay and the ELISA. With regard to specificity, 35 fecal specimens from infants and 19 from suspected foodborne cases which were negative in the bioassay for botulinal toxins A and B were also negative in the ELISA. Only two fecal specimens with negative bioassay gave positive ELISA readings, providing a specificity rate of 96%. These results suggest that the ELISA may serve as a useful screening test to detect C. botu(inum toxin in clinical specimens.

Detection of type A, B, and E botulism neurotoxin genes in Clostridium botulinum and other Clostridium species by PCR: evidence of unexpressed type B toxin genes in type A toxigenic organisms

“…All except the mouse reagent were polyclonal. The assays for type A and E toxins were type specific, but the assay for type B toxin cross-reacted at a lower intensity with cultures containing strains that produced type A toxin; this cross-reactivity between ELISAs for type A and B toxins that use polyclonal reagents has been noted elsewhere (4 Administration, Washington, D.C. A set of universal primers used to amplify a conserved region of the 16S rRNA genes common to many bacterial species (30), selected by Patricia Fields, CDC (unpublished data), was also synthesized by the Biotechnology Core Facility, CDC. The primer sequences, their locations on the toxin genes, and the sizes of the PCR products amplified in their respective reactions are given in Table 1.…”

Toxigenic clostridia

“…C. botulinum isolated from a wound specimen is good evidence for wound botulism, and isolation of the organism from fecal samples is generally satisfactory for confirming foodborne or infant botulism. Although enzymelinked immunosorbent assays for detecting botulinal neurotoxins have been devised (72,224,225), the mouse bioassay is still generally used for diagnostic investigation of botulism. Detailed laboratory procedures for toxin detection and recovery of toxigenic organisms are given on the package insert distributed with the Centers for Disease Control diagnostic reagents and have been published elsewhere (120,155).…”

Update: infant botulism

“…Monovalent antitoxin preparations are obtained from CDC. Although several serological methods for toxin detection, such as enzyme-linked immunosorbent assay and PCR, have been reported (18,56,60,71), reagents for those procedures generally are not available to other interested laboratories, and no interlaboratory evaluation of these tests for diagnostic investigation has been done. Therefore, the mouse bioassay, for which common neutralization reagents are available, remains the accepted procedure for definitive diagnostic purposes.…”

“…Continuing applications of modern microbiology techniques (18,23,24) for the detection of toxin (such as enzyme-linked immunosorbent assay) and molecular techniques (such as PCR) for the detection of nucleotide sequences will facilitate future studies on the incidence, risk factors, modes of transmission, and pathogenesis of the botulinum toxin-producing pathogens causing all forms of botulism.…”