Detection by PCR-Enzyme-Linked Immunosorbent Assay ofClostridium botulinumin Fish and Environmental Samples from a Coastal Area in Northern France

Abstract

The prevalence of Clostridium botulinum types A, B, E, and F was determined in 214 fresh fish and environmental samples collected in Northern France. A newly developed PCR-enzyme-linked immunosorbent assay (ELISA) used in this survey detected more than 80% of samples inoculated with fewer than 10 C. botulinum spores per 25 g and 100% of samples inoculated with more than 30 C. botulinum spores per 25 g. The percent agreement between PCR-ELISA and mouse bioassay was 88.9%, and PCR-ELISA detected more positive samples than the mouse bioassay did. The prevalence of C. botulinum in seawater fish and sediment was 16.6 and 4%, respectively, corresponding to 3.5 to 7 and 1 to 2 C. botulinum most-probable-number counts, respectively, and is in the low range of C. botulinum contamination reported elsewhere. The toxin type identification of the 31 naturally contaminated samples was 71% type B, 22.5% type A, and 9.6% type E. Type F was not detected. The high prevalence of C. botulinum type B in fish samples is relatively unusual compared with the high prevalence of C. botulinum type E reported in many worldwide and northern European surveys. However, fish processing and fish preparation in France have not been identified as a significant hazard for human type B botulism.

Detection of Clostridium botulinum Type C Cells in the Gastrointestinal Tracts of Mozambique Tilapia (Oreochromis mossambicus) by Polymerase Chain Reaction

“…Fish are suspected of harboring the bacteria in their gastrointestinal tracts, where toxin is subsequently formed, perhaps under certain stressful conditions. In the past, detection of Clostridium botulinum within an animal has involved inoculation of growth media with tissue, intestinal contents, or fecal material, followed by DNA extraction of the media and finally polymerase chain reaction (Szabo et al, 1994;Hielm et al, 1996Hielm et al, , 1998Kimura et al, 2001;Fach et al, 2002). This method of sample preparation does not distinguish between spores, the resting phase, and cells, the active phase of the organism.…”

Prevalence of Neurotoxic Clostridium Botulinum Type C in the Gastrointestinal Tracts of Tilapia (Oreochromis Mossambicus) in the Salton Sea

“…This finding links tilapia to the deaths of pelicans and other piscivorous birds by type C avian botulism at Salton Sea. To our knowledge, this association has not been demonstrated in any other bird or fish population, although similar work has looked for other types of C. botulinum (Fach et al, 2002). The intestines of healthy fish generally would be considered inhospitable to C. botulinum, but Riedel and Costa-Pierce (2001) showed that Salton Sea tilapia consume significant amounts of sediment during the year.…”

In-house Validation of PCR Based Procedure for Specific Detection of Clostridium Botulinum Types C and D

“…Generally, the data connected with detection of C and D C. botulinum types with PCR based method is rarely presented in the literature. An extensive part of the available literature concerns the occurrence of this microorganism in food, faecal, and environmental samples (6,8,15,18). Furthermore, the scale of C. botulinum and especially the occurrence of C and D types in feed is underestimated.…”

“…The inhibitory effect could be caused by natural microflora, such as: other Clostridium strains, which reveal faster growth (C. perfringens, C. tetani), Staphylococcus, and Bacillus. Moreover, the PCR amplification could be affected by heavy metals occurring in inoculated silage samples (6,8,15,20). The inhibitory effect of matrix ingredients was also described by other authors who detected C. botulinum in different types of samples.…”