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Applied and Environmental Microbiology, December 2002, p. 5870-5876, Vol. 68, No. 12
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.12.5870-5876.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Detection by PCR-Enzyme-Linked Immunosorbent Assay of Clostridium botulinum in Fish and Environmental Samples from a Coastal Area in Northern France

Patrick Fach,1* Sylvie Perelle,1 Françoise Dilasser,1 Joël Grout,1 Claire Dargaignaratz,2 Lucien Botella,2 Jean-Marie Gourreau,3 Frédéric Carlin,2 Michel R. Popoff,4 and Véronique Broussolle2

Laboratoire d'Etudes et de Recherches sur l'Hygiène et la Qualité des Aliments (LERHQA), Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Unité ATB, 94700 Maisons-Alfort,1 Institut National de la Recherche Agronomique (INRA), UMR A408 Sécurité et Qualité des Produits d'Origine Végétale, 84914 Avignon cedex 9,2 Laboratoire d'Etudes et de Recherches en Pathologie Animale et Zoonoses (LERPAZ), AFSSA, 94703 Maisons-Alfort,3 Institut Pasteur, Centre National de Référence des Anaérobies, 75015 Paris, France4

Received 20 February 2002/ Accepted 24 August 2002


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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.


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INTRODUCTION
 
Botulinum neurotoxins (BoNT) are produced by phenotypically and genetically different Clostridium species including Clostridium botulinum and some strains of C. baratii and C. butyricum. BoNTs are the most potent biological and chemical substances known and are responsible for botulism, which is characterized by severe flaccid paralysis. BoNTs are divided into seven toxin types (A to G) according to their antigenic properties. Toxin types A, B, E, and, more rarely, F cause human botulism, whereas toxin types C and D are mainly responsible for animal botulism (24, 37, 40).

The possible presence of C. botulinum spores in marine sediments and subsequent contamination of fish and other seafood are potential sources of human botulism. Nonproteolytic strains of C. botulinum, mainly type E, can grow and produce toxin in fish products at temperature as low as 5°C, and temperature abuse in food preservation can permit the growth and toxinogenesis of proteolytic C. botulinum strains (23, 35). Fish and fish products preserved at 5°C for a long period or fermented and stored at room temperature are important and severe food poisoning hazards (35). Surveys of C. botulinum in fish have been performed in Nordic countries (25-27, 29, 30), but few data are available on its prevalence in environmental and food samples from other European countries. Information on the prevalence of C. botulinum in the environment and food is critical for an assessment of botulism hazards. In a coastal area of northern France, near the Canche river estuary, a severe outbreak of wild avian botulism related to type E toxin occurred in 1996, suggesting a potentially high local prevalence of C. botulinum and fish products as a possible source of contamination (20). The objective of this study was to investigate the prevalence of C. botulinum in this area.

Standard bacteriological methods for C. botulinum isolation and identification are not practicable in routine analysis, since equipment for anaerobic bacteriology is required and since no efficient selective media for isolation and counting of C. botulinum are currently available. Mouse bioassay, which is a reliable and more sensitive test than the immunological techniques, is still the reference method for BoNT detection, but animal testing is increasingly restricted. DNA-based methods have been described for detection of C. botulinum in samples (1-4, 10, 13, 14, 16, 17, 19, 26, 31, 34, 42-44, 46) but are not commercially available; they use conventional procedures of PCR product detection such as agarose gel electrophoresis or Southern hybridization, which are not convenient for processing large numbers of samples. A microtiter plate technique for the detection of PCR products targeting C. botulinum type B has been reported by Szabo et al. (45), and recently a new quantitative PCR method has been investigated for detection of C. botulinum type E (33). Alternatively, several techniques involving microtiter plates and capture of PCR products with specific probes have been successfully developed for the detection of other pathogens (5, 7, 15, 18, 21, 32, 38, 39, 47). This method can be automated and can be readily implemented on a large scale.

Investigations of C. botulinum prevalence in fish and environmental samples in northern France were carried out with a new PCR-enzyme-linked immunosorbent assay (ELISA) detection system, and the results were compared with results obtained by the standard method. This PCR technique, based on identification of the most highly conserved region of bont genes, permits the simultaneous detection of C. botulinum types A, B, E, and F.


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MATERIALS AND METHODS
 
Bacterial strains.
The bacterial strains used in this study are listed in Table 1. Clostridium strains were maintained on cooked meat medium (CMM) (Oxoid, Basingstoke, United Kingdom) and stored at 5°C. Spores of Bacillus strains were produced as previously described (8, 9) and kept in a 30% (vol/vol) glycerol solution at -20°C.


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  		TABLE 1. Results of PCR-ELISA and PCR toxinotyping on pure cultures of C. botulinum and other species

Preparation of C. botulinum spores.
Five strains of C. botulinum (two type A [ATCC 7948 and CIP 104310T], two proteolytic type B [NCTC 7373 and ATCC 7949], and one type E [NCTC 8266]) were used. Spores were produced in two-phase CMM by the method of Peck et al. (36). After incubation for 7 to 10 days at 30°C, spores were harvested by centrifugation (5,000 x g at 4°C for 10 min), washed four times in sterile distilled water, suspended again in 4 ml of sterile distilled water, and stored at 4°C until use. The concentrations of the spore suspensions were determined by plating 100 µl of 10-fold dilutions in water on TYG agar plates (tryptone [Biokar, Beauvais, France], 30 g; yeast extract [Biokar], 20 g; glucose, 5 g; cysteine-HCl, 0.5 g; resazurin, 4 mg; agar [Biokar], 15 g; water, 1 liter [pH 7.3]). Colonies were counted after 48 to 72 h of incubation at 30°C under anaerobic conditions.

Cultures.
Strains of Clostridium spp. were anaerobically grown overnight at 30°C by transfer of 100-µl CMM cultures to 10 ml of TYG broth. The strains were then subcultured for 9 h at 30°C by transferring 100 µl of the cultures to 10 ml of TYG broth. Cultures of Bacillus strains were grown in J agar (J broth containing 5 g of tryptone, 15 g of yeast extract, 3 g of K2HPO4, 2 g of glucose, and 1 liter of water, [pH 7.4], with 15 g of agar) (8). They were grown by transfer of a single 24- to 48-h colony into 10 ml of J broth.

Samples.
A total of 214 samples (25 sediments, 175 seawater fish, 4 freshwater fish, 2 shellfish, and 8 waste dump samples) were collected near the Canche river estuary between Boulogne-sur-Mer and Berck in the north of France (Fig. 1). Fish samples included plaice (Pleuronectes platessa) (n = 33), common dab (Limanda limanda) (n = 32), whiting (Merlangius merlangus) (n = 30), bib (Trisopterus luscus) (n = 21), mackerel (Scomber scrombus) (n = 16), red mullet (Mullus barbatus) (n = 12), yellow gurnard (Trigla lucema) (n = 8), red gurnard (Aspitrigla cuculus) (n = 8), common sole (Solea solea) (n = 6), herring (Clupea harengus) (n = 5), horse mackerel (Trachurus trachurus), flounder (Platichthys flesus), sea bream (Chrysophrys aurata), roach (Rutilus rutilus), perch (Percea fleuviatilis), and eel (Anguilla anguilla) (n = 8). Waste dump samples were liquids released and mud taken from two local waste dumps (Fig. 1). The samples were kept refrigerated and sent by rapid transport to Institut National de la Recherche Agronomique in Avignon, France. Fish and shellfish were treated on arrival in the laboratory as described below. Other samples (sediments and waste dump samples) were kept refrigerated at +4°C until analyzed. Fresh fish used for artificial contamination was purchased from a local supermarket.



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  		FIG. 1. Map of the survey area. Seawater fish (SW) are found in the gray area. Freshwater fish (FW) and sediments were taken in the Canche River estuary, marked with an arrow. Waste dump samples were taken at Dannes (WDD) and La Caloterie (WDC).

Preparation of fish and shellfish.
Skin and muscles were aseptically removed from fresh fish samples in a laminar-flow hood by using sterile instruments, cut, placed in individual plastic pouches, homogenized in a blender (Stomacher; Seward Medical, London, United Kingdom) for 2 min, and stored at -20°C until use. Flesh of shellfish was aseptically taken and treated in the same way as for fresh fish. Approximately 100 g was prepared for each sample.

Enrichment procedure.
Samples of 25 or 50 g of raw material were diluted 10-fold (wt/vol) in prereduced TYG broth containing 200 mg of D-cycloserine per liter as described by Sebald and Petit (41) and under a gas flow of N2-H2 (95:5, vol/vol). After a 72-h incubation at 30°C, a 40-ml aliquot of the enrichment broth was collected and kept frozen at -20°C for further mouse bioassay, and a 1-ml aliquot of the same enrichment broth was anaerobically transferred to 10 ml of TYG broth and subcultured at 30°C for 16 h (2). A 1-ml sample this subculture was collected and centrifuged at 9,000 x g for 5 min, and the supernatant was discarded. The cell pellets were stored at -80°C until used for PCR-ELISA testing.

Artificially inoculated samples.
Homogenized samples of 25 g of fish were mixed with 225 ml of TYG broth and inoculated with suspensions of spores obtained by 10-fold dilutions in ice-cold distilled water from stock solutions at 107 to 109 spores ml-1. Artificially inoculated samples were prepared to obtain target concentrations of 5,000, 500, 50, and 5 spores per 25 g of inoculated fish sample. Actual concentrations of the inoculated spore suspensions were checked as described in "Preparation of C. botulinum spores" (see above). Each type of C. botulinum was separately inoculated. Uninoculated samples were used as negative controls. The enrichment procedure was performed as previously described.

DNA extraction.
The cell pellets were washed in 1 ml of phosphate-buffered saline and mixed with 200 µl of InstaGene Matrix (Bio-Rad Laboratories, Marnes-La-Coquette, France). Bacterial DNA was released by heating the sample for 30 min at 56°C and 10 min at 100°C. After vortexing and centrifugation (12,000 x g at 4°C for 2 min), 10 µl of the supernatant was used in the PCR amplifications. In cases of inhibition of PCR amplifications, extracted DNA was further diluted 2-, 10-, and 50-fold in a sterile 3% (wt/vol) bovine serum albumin solution in water and tested again.

Primers and probes.
Primers and probes were designed by alignment of the DNA sequences of the C. botulinum neurotoxin genes (bont/A, bont/B, bont/E, and bont/F) by using the Multalin program (http://prodes.toulouse.inra.fr/multalin/multalin.html). The most highly homologous regions of the four sequences allowed the CB1 and P261 primers and the CB and P260 probes to be designed for simultaneous detection of bont genes in PCR-ELISA. The CBA, CBB, CBE, and CBF primers, specific to each bont gene, were designed for PCR toxinotyping. The CatCap and CatRev probes have been described previously (15). The sequences and corresponding sequence locations of these oligonucleotides and the expected sizes of the amplicons are shown in Table 2.


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  		TABLE 2. Location of primers and probes within the specified genes

Cloned sequences.
Each CB1-P261 amplified fragment from the bont/A, bont/B, bont/E, and bont/F genes was cloned into the pMOS Blue vector by using the pMOS Blue blunt-ended cloning kit (Amersham Biosciences, Saclay, France). Recombinant plasmid DNAs were purified using plasmid kits (Qiagen, Courtaboeuf, France) as specified by the manufacturer instructions. Plasmid DNA concentrations were determined by fluorimetry, and the copy number of each plasmid was calculated on the basis of its size. Cloned sequences were used as positive controls and to determine the detection limit (cutoff) of the PCR-ELISA. The threshold selection criteria determining positive versus negative samples were established by testing triplicates of serial 10-fold dilutions (106 to 1 copy ml-1) of each sample in PCR-ELISA. The mean values were calculated for each dilution and for the negative controls, and standard deviations were also determined.

PCR conditions.
Amplification reactions with the CB1 and P261 primers were performed in a total volume of 100 µl in 15 mM Tris-HCl (pH 8.0)-50 mM KCl buffer containing 2% glycerol, 1.5 mM MgCl2, 0.8 µM each degenerated primer CB1 and P261, 100 µM each deoxynucleoside triphosphate, 2.5 U of AmpliTaq Gold (Applied Biosystems, Courtaboeuf, France), and five copies of a synthetic internal control (IC). IC is a recombinant pMOS Blue plasmid DNA with CB1 and P261 primer binding regions, flanking a DNA sequence of the chloramphenicol resistance gene (Cmr) from Tn 9. This feature allowed the CB1 and P261 primers (Table 2) to coamplify the bont/A, bont/B, bont/E, and bont/F genes and IC in the same reaction. IC was incorporated in each test sample to monitor PCR inhibitors and ensure successful amplification. Amplification was carried out in the GeneAmp PCR system 9600 (Applied Biosystems) with the following temperature profile: 1 cycle of 94°C for 10 min; 5 cycles of 95°C for 15 s, 45°C for 15 s, and 72°C for 30 s; 35 cycles of 95°C for 15 s, 48°C for 15 s, and 72°C for 30 s; and 1 cycle of 72°C for 10 min. Positive controls using appropriate purified plasmid DNA and two negative controls containing all reagents except DNA template were included with each amplification set. To avoid contamination, sample preparation, PCR amplification, and PCR detection were performed in three different rooms.

ELISA conditions.
After amplification, the PCR products were alkali denatured by adding 100 µl of the denaturing solution containing 0.2 M NaOH and 0.05 M EDTA directly to the PCR tube. Denaturation was carried out for 10 min at room temperature, and denatured PCR products were detected in a sandwich hybridization assay using microwells coated with streptavidin (Roche Diagnostics, Meylan, France). This step was performed in parallel on two microtiter plates: one for the specific detection of bont genes and one for the IC detection. The set of internal capture and detection probes for detection of bont genes was CB oligonucleotide 5'-end labeled with biotin and P260 oligonucleotide 3'-end labeled with digoxigenin (Table 2). The set of internal capture and detection probes for detection of the Cmr gene present in IC was CatCap oligonucleotide 5'-end labeled with biotin and CatRev oligonucleotide 3'-end labeled with digoxigenin (15) (Table 2). Thus, using two different sets of internal capture and detection probes in two distinct microtiter plates, it was possible to differentiate amplicons derived from C. botulinum from those derived from the IC. For each hybridization, 50-µl aliquots of denatured amplified products were deposited in each microtiter plate well and incubated at 37°C for 1 h with 200 µl of the hybridization solution containing 5x SSC, (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% N-lauroylsarcosine, 0.02% sodium dodecyl sulfate, 1% blocking reagent (Roche Diagnostics), and 12 pmol of each capture and detection probe ml-1. The microwells were then washed six times at room temperature with a solution of 0.1 M Tris-HCl (pH 7.5)-0.15 M NaCl containing 0.5 g of Tween 20 liter-1, 0.5% blocking reagent (Roche Diagnostics), and 100 mg of fish sperm DNA liter-1. Then 200 µl of 0.1 M maleic acid (pH 7.5), 0.15 M NaCl buffer containing 1% blocking reagent (Roche Diagnostics), and 0.1 U ml-1 peroxidase-labeled anti-digoxigenin antibody (Roche Diagnostics) were added per well, and the strips were incubated at 37°C for 30 min. After six washings at room temperature as previously described, 200 µl of 3,3',5,5'-tetramethylbenzidine solution (Roche Diagnostics) was added and the enzyme reaction was carried out by incubation at 37°C for 30 min. Finally, the reaction was stopped with 100 µl of 1.5 M H2SO4 and absorbance was measured at 450 nm (against the reference wavelength 620 nm) using a microtiter plate reader (SLT Spectra; BMG Labtechnologies, Champigny, France).

PCR toxinotyping of bont genes.
PCR toxinotyping of bont genes was conducted on PCR-ELISA-positive samples. The BoNT type was identified using a heminested PCR. Thus, amplicons derived from amplifications with CB1 and P261 primers were diluted 10-fold in sterile water. The diluted amplicons (5 µl) were used for four subsequent PCR reamplifications with the CB1-CBA, CB1-CBB, CB1-CBE, and CB1-CBF sets of primers (Table 2), specifically amplifying the bont/A, bont/B, bont/E, and bont/F genes, respectively. The positive and negative controls used in the amplifications with CB1 and P261 primers were treated in heminested PCR like the other samples. Additional positive controls using appropriate purified plasmid DNA and two negative controls containing all reagents except the DNA template were also included with each reamplification set. Amplification reactions were performed as described above and according to the following temperature profile: 1 cycle of 94°C for 10 min; 5 cycles of 95°C for 15 s, 58°C for 15 s, and 72°C for 30 s; 30 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 30 s; and 1 cycle of 72°C for 10 min. The PCR products were visualized by agarose gel electrophoresis (2% agarose gel stained with ethidium bromide) as previously described (14). To avoid contamination, sample preparation, PCR amplification, and PCR detection were performed in three different rooms.

Mouse bioassay.
The PCR-ELISA-positive samples were further characterized at Institut Pasteur (Paris, France) by the standard mouse bioassay. A 2-ml sample of each first enrichment culture (72 h) was centrifuged, and 1 ml of the culture supernatant was incubated with 200 µg of trypsin ml-1 for 20 min at room temperature. A volume of 0.5 ml was then injected intraperitoneally into mice (two mice per sample), and the mice were watched for the characteristic symptoms of botulism (labored breathing, pinching of the waist, and paralysis) and for death for 4 days. Botulinum toxins were confirmed and types were identified by a seroneutralization test on mice by using specific botulinum antitoxins (Institut Pasteur) (13).

MPN counts.
The survey data were converted to most-probable-number (MPN) counts by the method of Halvorson and Ziegler (22), and Dodds (11, 12), who used the same conversion to facilitate a comparison of survey results. The MPN values in tested samples were calculated by the formula MPN = ln(n/q), where n is the number of samples tested and q is the number of negative samples.


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RESULTS
 
PCR-ELISA with cloned sequence of the bont genes and pure culture of bacteria.
PCR-ELISA results were obtained spectroscopically by using a microplate reader that provided an absorbance value corresponding to the amount of PCR products. The threshold sensitivity was determined by testing 10-µl serial dilutions of the cloned sequence of bont genes (bont/A, bont/B, bont/E, and bont/F) in PCR-ELISA. DNA plasmid concentrations of 106 and 105 copies ml-1 (104 and 103 copies in the PCR tube) gave an absorbance value of 4.0, and DNA plasmid concentrations ranging from 104 to 103 copies ml-1 (102 to 10 copies in the PCR tube) gave absorbance values between 0.215 and 3.350. Concentrations of 102 copies ml-1 (approximately one copy in the PCR tube) usually yielded absorbance values lower than 0.03, except for four samples, which were 0.110 (bont/E), 0.139 (bont/A), 0.153 (bont/F), and 0.168 (bont/B). For concentrations lower than 10 copies ml-1 (less than 1 copy in the PCR tube), absorbance values were lower than 0.03, except for one that was 0.102 (bont/A). Mean values and standard deviations of PCR-negative controls (sterile water) were also measured, and the cutoff of the test was established on the basis of mean values plus three standard deviations. Accordingly, absorbance values greater than or equal to 0.100 were considered positive and values lower than 0.100 were considered negative. Taken together, our results indicate that the sensitivity level of the PCR-ELISA allows the detection of a small number of bont gene copies (less than 10 gene copies) in the PCR tube.

Specificity was then estimated with 38 C. botulinum strains and 23 other Clostridium and Bacillus strains. As shown in Table 1, the C. botulinum strains included types A, B, E, and F, responsible for human botulism, and types C and D, mainly responsible for animal botulism. The absorbance readings obtained with the 23 non-C. botulinum strains, including a C. tetani strain, were lower than 0.033, mostly between 0.009 and 0.020 (data not shown). The 4 nontoxigenic C. botulinum strains in the mouse bioassay and the 10 isolates of C. botulinum types C and D tested negative in PCR-ELISA, whereas the absorbance readings obtained with the 24 toxigenic C. botulinum strains of types A, B, E, and F were greater than the cutoff, indicating that the PCR test was fully reliable. C. botulinum types A, E, and F and proteolytic C. botulinum type B strains gave high absorbance values (3.0 to 4.0), but the nonproteolytic C. botulinum type B strains yielded lower values, ranging from 0.60 to 0.88 (data not shown). Furthermore, PCR products from amplifications with CB1-P261 and toxinotyping primers migrated at the expected sizes on gel electrophoresis (Fig. 2).



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  		FIG. 2. Agarose gel electrophoresis of PCR products obtained by PCR with primers used to detect the bont/A, bont/B, bont/E and bont/F genes. (A) PCR with the CB1 and P261 primers. (B) PCR toxinotyping with the CB1 and CBA primers. (C) PCR toxinotyping with the CB1 and CBB primers. (D) PCR toxinotyping with the CB1 and CBE primers. (E) PCR toxinotyping with the CB1 and CBF primers. Lanes: M, DNA molecular weight marker VI (Roche Diagnostics); 1, C. botulinum type A (strain CIP 104310T); 2, C. botulinum type A (strain IP PPA); 3, C. botulinum type B (strain IFR 83-1); 4, C. botulinum type B (strain ATCC 7949); 5, C. botulinum type C (strain CIP 1663); 6, C. botulinum type C (strain CIP 4165); 7, C. botulinum type D (strain CIP 1873); 8, C. botulinum type D (strain CIP 7296); 9, C. botulinum type E (strain IFR 81-26); 10, C. botulinum type E (strain IFR 93-07); 11, C. botulinum type F (strain IFR 86-32); 12, C. botulinum type F (strain IFR 86-33); 13, nontoxigenic C. botulinum (strain IFR 87-4); 14, nontoxigenic C. botulinum (strain IP 331/81-23); 15, C. sporogenes (strain IFR 84-17); 16, C. sporogenes (strain CIP 793); 17, C. tetani (strain CIP E19406); 18, C. butyricum (strain CIP 6051); 19, B. cereus (strain CIP 5127); 20, B. cereus (strain INRAAV P14-1); 21, negative control (sterile water).

PCR-ELISA in artificially contaminated fish products.
The study was carried out with 62 fish products including 46 artificially contaminated and 16 noncontaminated samples (Table 3). All 16 noninoculated samples gave a negative result by PCR-ELISA and mouse bioassay. Among 11 samples artificially contaminated with three to eight spores per 25 g, 9 were positive by both PCR-ELISA and mouse bioassay and 2 were negative by both methods. At higher contamination levels (30 to 82 and 235 to 820 spores per 25 g), the PCR-ELISA detected all inoculated samples whereas the mouse bioassay failed to identify 3 of 14 and 2 of 15 respectively. When fish products were artificially contaminated with more than 1,000 spores per 25 g, positive results were obtained by both PCR-ELISA and mouse bioassay. The results with artificially contaminated fish samples indicated that PCR-ELISA is a sensitive method that is able to detect fewer than 10 C. botulinum spores in 25 g of products in 80% of the samples. Moreover, all samples contaminated with 30 to 820 spores per 25 g were detected by PCR-ELISA, whereas the mouse bioassay was positive in only 24 of 29 samples.


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  		TABLE 3. Detection of C. botulinum in artificially contaminated fish samples

Evaluation of the prevalence of C. botulinum in fish and environmental samples from a coastal area of northern France.
A total of 214 samples were collected from a coastal area of northern France and surveyed for C. botulinum by PCR-ELISA and mouse bioassay. PCR-ELISA was performed as described in Materials and Methods with an IC to monitor false-negative results due to sample PCR inhibitors. PCR inhibition occurred in 95 (54.3%) of 175 seawater fish, 4 (16%) of 25 sediments, 6 of 8 waste dump samples, and 2 of 4 freshwater fish samples, whereas the 2 shellfish samples showed no PCR inhibition. Reanalysis of a twofold dilution of the inhibited DNA samples still gave inhibition in 29 (30.5%) of 95 seawater fish, 1 of 4 sediments, 5 of 6 waste dump samples, and both freshwater fish samples. After a 10-fold dilution of the crude extracted DNA, PCR inhibition was no longer detected, except in one seawater fish and one sediment sample that required a 50-fold dilution to avoid an inhibitory effect. Among the 31 C. botulinum-positive samples, 19 contained PCR inhibitors. Twofold and 10-fold DNA dilutions from 14 and 5 inhibited samples, respectively, were necessary to bypass PCR inhibitors and subsequently detect the presence of bont genes.

The prevalence of C. botulinum was 4% (1 of 25) in sediment, 16.6% (29 of 175) in seawater fish, and 25% (1 of 4) in freshwater fish, whereas the 2 shellfish samples and 8 waste dump samples were negative. The MPN counts of C. botulinum, estimated by the method of Halvorson and Ziegler (22), was 3 to 6 spores kg-1 (Table 4).


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  		TABLE 4. Detection of C. botulinum in fish and sediments samples from a coastal area in northern France

PCR toxinotyping identified bont/A in 7 seawater fish samples, bont/B in 21 samples (20 seawater fish and 1 freshwater fish), and bont/E in 2 samples (1 sediment and 1 seawater fish). One sample (seawater fish) was positive for both bont/B and bont/E. The bont/F gene was not found. Among the 31 PCR-ELISA-positive samples, 27 were tested for the presence of BoNT by the mouse bioassay and 24 were positive. Thus, PCR-ELISA yielded a greater number of positive samples (three more seawater fish samples) than did the mouse bioassay. The percent agreement was 88.9%. Considering the toxin type, the two methods gave identical results in 23 of 24 samples. The concordance between PCR toxinotyping and the seroneutralization assay was 95.8%.


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DISCUSSION
 
In this study, we developed a PCR-ELISA method to detect C. botulinum in fish and sediment samples. An IC was included to monitor false-negative samples. The PCR-ELISA proved specific and highly sensitive; a positive signal was detected in most samples containing fewer than 10 spores of C. botulinum per 25 g of fresh fish. Negative artificially inoculated samples may be explained by poor multiplication in enrichment broth unable to reach PCR-ELISA-detectable levels. The PCR-ELISA was more sensitive than the mouse bioassay and was able to detect additional positive samples (44 and 39), respectively.

In naturally contaminated samples, the PCR-ELISA detected C. botulinum in 31 samples among the 214 tested. The prevalence of C. botulinum was 16.6% in seawater fish and 4% in sediment samples. Freshwater fish, shellfish, and waste dump samples were negative, but their numbers were insufficient to enable us to evaluate C. botulinum prevalence in these sample types. PCR-ELISA also produced more positive results than the mouse bioassay did, but the percent agreement between the two methods was high (88.9%). The discordance between PCR-ELISA and mouse bioassay may be due to possible PCR detection of injured bacteria or neurotoxin inactivation by proteases from fish products. Another possibility is that some strains contain silent bont genes that can be detected by PCR-ELISA but not by the mouse bioassay. Such strains as C. botulinum A containing the bont/B gene have been described previously (28). However, nontoxigenic strains harboring a defective bont gene have been reported. The MPN counts of C. botulinum detected in naturally contaminated samples indicated a contamination of 3 to 6 C. botulinum MPN per kg, which is among the lower range of contamination in fish or fish products (12).

PCR toxinotyping showed that type B was the major type (more than 70% of the positive naturally contaminated samples [22 of 31]). Type A represented about 22.5% of the positive samples (7 of 31), and type E was less frequent (9.6% [3 of 31]). Type F was not detected. Of the 24 positive samples which were analysed by both PCR toxinotyping and seroneutralization on mouse bioassay, 23 gave the same toxin type with both methods. Thus, PCR toxinotyping of bont genes and mouse bioassay followed by seroneutralization showed close concordance.

This survey of C. botulinum in fish and environment samples from northern France showed a significant occurrence of C. botulinum (about 14.5% [31 of 214 samples]), with a high prevalence of type B. This high prevalence of C. botulinum type B in fish samples is relatively unusual; most worldwide surveys, mainly in northern Europe, report a high prevalence of C. botulinum type E (12, 26, 29). The prevalence of C. botulinum in European sediments is variable; in Finland and in other Scandinavian countries, C. botulinum type E is the most frequent type (25, 26), while type B is predominant in British sediments (11). The contamination of fish products in the surveyed area is not itself a serious hazard. It is very similar to what is reported elsewhere, and fish processing and fish preparation in France have not been identified as a significant botulism hazard. The most common source of type B human botulism in France is processed pork products; fish products have never yet been reported to be involved in type B human botulism in France (6). However, this survey shows that C. botulinum constantly enters the food chain and that this hazard must always be considered in the fish market and fish-processing industry. The development of rapid methods such as PCR-ELISA to evaluate the prevalence of C. botulinum in food materials is an improvement which should eventually aid in the risk assessment of botulism in foods.


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ACKNOWLEDGMENTS
 
This work was supported by the French Ministry of Research and Technology grant EN98-03 under the program "Health and Environment."


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire d'Etudes et de Recherches sur l'Hygiène et la Qualité des Aliments (LERHQA), Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Unité ATB, 1-5 rue de Belfort, 94700 Maisons-Alfort, France. Phone: 33 (0) 1 43 76 30 99. Fax: 33 (0) 1 43 76 26 30. E-mail: p.fach{at}afssa.fr. Back


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Applied and Environmental Microbiology, December 2002, p. 5870-5876, Vol. 68, No. 12
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.12.5870-5876.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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