ABSTRACTClostridium botulinumtypes C and D, as well as their mosaic variants C-D and D-C, are associated with avian and mammalian botulism. This study reports on the development of low-density macroarrays based on the GeneDisc cycler platform (Pall-GeneDisc Technologies) applied to the simultaneous detection of theC. botulinumsubtypes C, C-D, D, and D-C. The limit of detection of the PCR assays was 38 fg of total DNA, corresponding to 15 genome copies. Artificially contaminated samples of cecum showed a limit of detection below 50 spores/g. The tests were performed with a large variety of bacterial strains, includingC. botulinumtypes C (n= 12), C-D (n= 29), D (n= 5), and D-C (n= 10), other botulinum neurotoxin (BoNT)-producingClostridiumstrains (n= 20), non-BoNT-producing clostridia (n= 20), and other bacterial species (n= 23), and showed a high specificity. These PCR assays were compared to previously published real-time PCRs for the detection ofC. botulinumin 292 samples collected from cases of botulism events in four European regions. The majority of the samples originated from wild birds (n= 108), poultry (n= 60), and bovines (n= 56). Among the 292 samples, 144 were positive for either thebont/C-D or thebont/D-C gene by using the GeneDisc arrays. The reliability of the results tallied to 97.94%. Interestingly, only BoNT mosaics, types C-D and D-C, were found in naturally contaminated samples whatever their animal origin and their geographical location. Further investigations should now be performed in order to check that mosaic types dominate in Europe and that acquisition of mosaic types helps in survival or adaptation to particular niche.

The first two confirmed cases of type E infant botulism occurred in two 16-week-old girls in Rome, Italy. The original diagnosis for the first patient was intestinal blockage due to an ileocecal invagination, which was treated surgically. Postoperatively, the patient became unresponsive and required ventilatory assistance. A diagnosis of infant botulism was then made. The second infant presented to the same hospital 7 1/2 months later with profound weakness, hypotonicity, mydriasis, and areflexia. This case was recognized as possible botulism at admission. Both cases were confirmed by detection and identification of type E botulinal toxin in stool specimens and in enrichment cultures of those specimens. The toxigenic organisms isolated were quite different from Clostridium botulinum type E. The apparent causative organism in each case resembles Clostridium butyricum but produces a neurotoxin that is indistinguishable from type E botulinal toxin by its effects on mice and by its neutralization with type E botulinal antitoxin.

Two unconnected cases of type E botulism involving a 19-year-old woman and a 9-year-old child are described. The hospital courses of their illness were similar and included initial acute abdominal pain accompanied by progressive neurological impairment. Both patients were suspected of having appendicitis and underwent laparotomy, during which voluminous Meckel's diverticula were resected. Unusual neurotoxigenic Clostridium butyricum strains that produced botulinum-like toxin type E were isolated from the feces of the patients. These isolates were genotypically and phenotypically identical to other neurotoxigenic C. butyricum strains discovered in Italy in 1985-1986. No cytotoxic activity of the strains that might explain the associated gastrointestinal symptoms was demonstrated. The clinical picture of the illness and the persistence of neurotoxigenic clostridia in the feces of these patients suggested a colonization of the large intestine, with in vivo toxin production. The possibility that Meckel's diverticulum may predispose to intestinal toxemia botulism may warrant further investigation.

Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes.