Evaluation of lateral-flow Clostridium botulinum neurotoxin detection kits for food analysis

The suitability and sensitivity of two in vitro lateral-flow assays for detecting Clostridium botulinum neurotoxins (BoNTs) in an assortment of foods were evaluated. Toxin extraction and preparation methods for various liquid, solid, and high-fat-content foods were developed. The lateral-flow assays, one developed by the Naval Medical Research Center (Silver Spring, MD) and the other by Alexeter Technologies (Gaithersburg, MD), are based on the immunodetection of BoNT types A, B, and E. The assays were found to be rapid and easy to perform with minimum requirements for laboratory equipment or skills. They can readily detect 10 ng/ml of BoNT types A and B and 20 ng/ml of BoNT type E. Compared to other in vitro detection methods, these assays are less sensitive, and the assessment of a result is strictly qualitative. However, the assay was found to be simple to use and to require minimal training. The assays successfully detected BoNT types A, B, and E in a wide variety of foods, suggesting their potential usefulness as a preliminary screening system for triaging food samples with elevated BoNT levels in the event of a C. botulinum contamination event.     

Detection of Type A, B, E, and F Clostridium botulinum Neurotoxins in Foods by Using an Amplified Enzyme-Linked Immunosorbent Assay with Digoxigenin-Labeled Antibodies

An amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium botulinum complex neurotoxins was evaluated for its ability to detect these toxins in food. The assay was found to be suitable for detecting type A, B, E, and F botulinum neurotoxins in a variety of food matrices representing liquids, solid, and semisolid food. Specific foods included broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meat products, and dairy foods. The detection sensitivity of the test for these botulinum complex serotypes was found to be 60 pg/ml (1.9 50% lethal dose [LD₅₀]) for botulinum neurotoxin type A (BoNT/A), 176 pg/ml (1.58 LD₅₀) for BoNT/B, 163 pg/ml for BoNT/E (4.5 LD₅₀), and 117 pg/ml for BoNT/F (less than 1 LD₅₀) in casein buffer. The test could also readily detect 2 ng/ml of neurotoxins type A, B, E, and F in a variety of food samples. For specificity studies, the assay was also used to test a large panel of type A C. botulinum, a smaller panel of proteolytic and nonproteolytic type B, E, and F neurotoxin-producing Clostridia, and nontoxigenic organisms using an overnight incubation of toxin production medium. The assay appears to be an effective tool for large-scale screening of the food supply in the event of a botulinum neurotoxin contamination event.     

Monoclonal antibodies to type A, B, E and F botulinum neurotoxins

Mouse monoclonal antibodies against the most acutely toxic substances, botulinum neurotoxins (BoNTs) of types A, B, E, and F, was generated and characterized, that recognize their respective toxins in natural toxin complex. Based on these antibodies, we developed sandwich-ELISA for quantitative detection of these toxins. For each respective toxin the detection limit of the assay was: BoNT/A - 0.4 ng/ml, BoNT/B - 0.5 ng/ml; BoNT/E - 0.1 ng/ml; and for BoNT/F - 2.4 ng/ml. The developed assays permitted quantitative identification of the BoNTs in canned meat and vegetables. The BNTA-4.1 and BNTA-9.1 antibodies possessed neutralizing activity against natural complex of the botulinium toxin type A in vivo, both individually and in mixture, the mixture of the antibodies neutralized the higher dose of the toxin. The BNTA-4.1 antibody binds specifically the light chain (the chain with protease activity) of the toxin, whereas BNTA-9.1 interacts with the heavy chain. We believe that the BNTA-4.1 and BNTA-9.1 monoclonal antibodies are prospective candidates for development of humanized therapeutic antibodies for treatment of BoNT/A-caused botulism.     

Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples

Botulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 10³ and 10⁵ CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F.Botulinum neurotoxins (BoNTs), the causative agents of botulism, are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven serotypes, A to G. While the botulinum neurotoxins BoNT/A, BoNT/B, BoNT/E, and BoNT/F are known to cause botulism in humans, BoNT/C and BoNT/D are frequently associated with botulism in cattle and birds. Despite its toxicity, BoNT/G has not yet been linked to naturally occurring botulism (26).Botulism is a life-threatening illness caused by food contaminated with BoNT (food-borne botulism), by the uptake and growth of C. botulinum in wounds (wound botulism), or by colonization of the intestinal tract (infant botulism) (14). In addition, C. botulinum and the botulinum neurotoxins are regarded as potential biological warfare agents (8).The gold standard for the detection of BoNTs from food or clinical samples is still the mouse lethality assay, which is highly sensitive but rather time-consuming. In addition to various immunological assays for BoNT detection, several conventional and real-time PCR-based assays for the individual detection of bont genes have been reported (2, 9-12, 15, 20, 23, 27-30). A major improvement is the simultaneous detection of more than one serotype, which results in a reduction of effort and in the materials used. In recent years, both conventional and real-time PCR-based multiplex assays have been developed for the simultaneous detection of C. botulinum serotypes (1, 6, 22, 24). To date, however, no internally controlled multiplex real-time PCR assay for the simultaneous detection and differentiation of all four serotypes relevant for humans has been reported.We describe here a highly specific and sensitive multiplex real-time PCR assay based on the 5′-nuclease TaqMan chemistry (17) for the simultaneous detection of the C. botulinum types A, B, E, and F, including an internal amplification control (IAC). Furthermore, we developed six different singleplex assays based on the TaqMan chemistry for the detection of C. botulinum serotypes A to F. Assays were validated on 42 C. botulinum strains, 57 non-C. botulinum strains, on spiked food samples, and on real samples from cases of botulism in Germany.     

Rapid, Quantitative PCR Monitoring of Growth of Clostridium botulinum Type E in Modified-Atmosphere-Packaged Fish

A rapid, quantitative PCR assay (TaqMan assay) which quantifies Clostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C. botulinum type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of C. botulinum type E and 18 genera of 42 non-C. botulinum type E strains, including strains of C. botulinum types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of C. botulinum DNA could be monitored (the quantifiable range was 10² to 10⁸ CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of C. botulinum cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.     

Gene Organization and Sequence Determination of the Two Botulinum Neurotoxin Gene Clusters in Clostridium botulinum Type A(B) Strain NCTC 2916

The gene organization and nucleotide sequence of the type A and B BoNT-gene clusters in Clostridium botulinum strain NCTC 2916 were studied. The aim was to clarify the organization of genes within C. botulinum type A strains possessing an unexpressed BoNT/B gene. The BoNT/A-gene cluster includes genes encoding BoNT, NTNH and a part of P-47 (the gene for this protein was reported in strains of C. botulinum types E and F). Clustered with the silent BoNT/B gene were genes encoding NTNH, P-21 and HA-33. Sequencing analysis of the NTNHs revealed the presence of 471 amino acids identical in the type B and A gene clusters. This gene organization contrasts markedly with the purported organization in strain NCTC 2916 described by Henderson et al. (FEMS Microbiol. Lett. 140, 151–158). In type A(B) strain NCTC 2916, the neurotoxin gene is of type BoNT/A1 within a gene cluster that has identical organization to that found in BoNT/A2 type strains; these observations may be significant in establishing the origin of the BoNT-gene cluster. Received: 28 July 1997 / Accepted: 15 October 1997     

Differentiation of Clostridium botulinum Serotype A Strains by Multiple-Locus Variable-Number Tandem-Repeat Analysis

Ten variable-number tandem-repeat (VNTR) regions identified within the complete genomic sequence of Clostridium botulinum strain ATCC 3502 were used to characterize 59 C. botulinum strains of the botulism neurotoxin A1 (BoNT/A1) to BoNT/A4 (BoNT/A1-A4) subtypes to determine their ability to discriminate among the serotype A strains. Two strains representing each of the C. botulinum serotypes B to G, including five bivalent strains, and two strains of the closely related species Clostridium sporogenes were also tested. Amplified fragment length polymorphism analyses revealed the genetic diversity among the serotypes and the high degree of similarity among many of the BoNT/A1 strains. The 10 VNTR markers amplified fragments within all of the serotype A strains but were less successful with strains of other serotypes. The composite multiple-locus VNTR analysis of the 59 BoNT/A1-A4 strains and 3 bivalent B strains identified 38 different genotypes. Thirty genotypes were identified among the 53 BoNT/A1 and BoNT/A1(B) strains, demonstrating discrimination below the subtype level. Contaminating DNA within crude toxin preparations of three BoNT/A subtypes (BoNT/A1 to BoNT/A3) also supported amplification of all of the VNTR regions. These markers provide clinical and forensics laboratories with a rapid, highly discriminatory tool to distinguish among C. botulinum BoNT/A1 strains for investigations of botulism outbreaks.     

Domain Organization in Clostridium botulinum Neurotoxin Type E Is Unique: Its Implication in Faster Translocation

Clostridium botulinum produces seven antigenically distinct neurotoxins [C. botulinum neurotoxins (BoNTs) A-G] sharing a significant sequence homology. Based on sequence and functional similarity, it was believed that their three-dimensional structures will also be similar. Indeed, the crystal structures of BoNTs A and B exhibit similar fold and domain association where the translocation domain is flanked on either side by binding and catalytic domains. Here, we report the crystal structure of BoNT E holotoxin and show that the domain association is different and unique, although the individual domains are similar to those of BoNTs A and B. In BoNT E, both the binding domain and the catalytic domain are on the same side of the translocation domain, and all three have mutual interfaces. This unique association may have an effect on the rate of translocation, with the molecule strategically positioned in the vesicle for quick entry into cytosol. Botulism, the disease caused by BoNT E, sets in faster than any other serotype because of its speedy internalization and translocation, and the present structure offers a credible explanation. We propose that the translocation domain in other BoNTs follows a two-step process to attain translocation-competent conformation as in BoNT E. We also suggest that this translocation-competent conformation in BoNT E is a probable reason for its faster toxic rate compared to BoNT A. However, this needs further experimental elucidation.     

Clostridium botulinum Group II Isolate Phylogenomic Profiling Using Whole-Genome Sequence Data

Clostridium botulinum group II isolates (n = 163) from different geographic regions, outbreaks, and neurotoxin types and subtypes were characterized in silico using whole-genome sequence data. Two clusters representing a variety of botulinum neurotoxin (BoNT) types and subtypes were identified by multilocus sequence typing (MLST) and core single nucleotide polymorphism (SNP) analysis. While one cluster included BoNT/B4/F6/E9 and nontoxigenic members, the other comprised a wide variety of different BoNT/E subtype isolates and a nontoxigenic strain. In silico MLST and core SNP methods were consistent in terms of clade-level isolate classification; however, core SNP analysis showed higher resolution capability. Furthermore, core SNP analysis correctly distinguished isolates by outbreak and location. This study illustrated the utility of next-generation sequence-based typing approaches for isolate characterization and source attribution and identified discrete SNP loci and MLST alleles for isolate comparison.     

Optimization of peptide substrates for botulinum neurotoxin E improves detection sensitivity in the Endopep–MS assay

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous substances known to humankind. It is essential to have a simple, quick, and sensitive method for the detection and quantification of botulinum toxin in various media, including complex biological matrices. Our laboratory has developed a mass spectrometry-based Endopep–MS assay that is able to rapidly detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Botulinum neurotoxin type E (BoNT/E) is a member of a family of seven distinctive BoNT serotypes (A–G) and is the causative agent of botulism in both humans and animals. To improve the sensitivity of the Endopep–MS assay, we report here the development of novel peptide substrates for the detection of BoNT/E activity through systematic and comprehensive approaches. Our data demonstrate that several optimal peptides could accomplish 500-fold improvement in sensitivity compared with the current substrate for the detection of both not-trypsin-activated and trypsin-activated BoNT/E toxin complexes. A limit of detection of 0.1 mouse LD₅₀/ml was achieved using the novel peptide substrate in the assay to detect not-trypsin-activated BoNT/E complex spiked in serum, stool, and food samples.     

Real-time PCR detection of the nontoxic nonhemagglutinin gene as a rapid screening method for bacterial isolates harboring the botulinum neurotoxin (A–G) gene complex

Botulinum neurotoxin (BoNT) producing clostridia contain genes encoding a specific neurotoxin serotype (A–G) and nontoxic associated proteins that form the toxin complex. The nontoxic nonhemagglutinin (NTNH) is a conserved component of the toxin complex in all seven toxin types. A real-time PCR assay that utilizes a locked nucleic acid hydrolysis probe to target the NTNH gene was developed to detect bacterial strains harboring the botulinum neurotoxin gene cluster. The specificity of the assay for Clostridium botulinum types A–G, Clostridium butyricum type E and Clostridium baratii type F was demonstrated using a panel of 73 BoNT producing clostridia representing all seven toxin serotypes. In addition, exclusivity of the assay was demonstrated using non-botulinum toxin producing clostridia (7 strains) and various enteric bacterial strains (n = 27). Using purified DNA, the assay had a sensitivity of 4–95 genome equivalents. C. botulinum type A was detected directly in spiked stool samples at 10²–10³ CFU/ml. Stool spiked with 1 CFU/ml was detected when the sample was inoculated into enrichment broth and incubated for 24 h. These results indicate that the NTNH real-time PCR assay can be used to screen enrichment cultures of primary specimens at earlier time points (24 h) than by toxin detection of unknown culture supernatants (up to 5 days).     

Elimination of Botulinum Neurotoxin (BoNT) Type B from Drinking Water by Small-Scale (Personal-Use) Water Purification Devices and Detection of BoNT in Water Samples

Seven small-scale drinking water purification devices were evaluated for their capacity to eliminate botulinum neurotoxin (BoNT) type B from drinking water. Influent water inoculated with toxic Clostridium botulinum cultures and effluent purified water samples were tested for the presence of BoNT by using a standard mouse bioassay and two commercial rapid enzyme immunoassays (EIAs). The water purification devices based on filtration through ceramic or membrane filters with a pore size of 0.2 to 0.4 μm or irradiation from a low-pressure UV-lamp (254 nm) failed to remove BoNT from raw water (reduction of <0.1 log₁₀ units). A single device based on reverse osmosis was capable of removing the BoNT to a level below the detection limit of the mouse bioassay (reduction of >2.3 log₁₀ units). The rapid EIAs intended for the detection of BoNT from various types of samples failed to detect BoNT from aqueous samples containing an estimated concentration of BoNT of 396,000 ng/liter.     

Detection of type A, B, E, and F Clostridium botulinum neurotoxins in foods by using an amplified enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies

An amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium botulinum complex neurotoxins was evaluated for its ability to detect these toxins in food. The assay was found to be suitable for detecting type A, B, E, and F botulinum neurotoxins in a variety of food matrices representing liquids, solid, and semisolid food. Specific foods included broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meat products, and dairy foods. The detection sensitivity of the test for these botulinum complex serotypes was found to be 60 pg/ml (1.9 50% lethal dose [LD50]) for botulinum neurotoxin type A (BoNT/A), 176 pg/ml (1.58 LD50) for BoNT/B, 163 pg/ml for BoNT/E (4.5 LD50), and 117 pg/ml for BoNT/F (less than 1 LD50) in casein buffer. The test could also readily detect 2 ng/ml of neurotoxins type A, B, E, and F in a variety of food samples. For specificity studies, the assay was also used to test a large panel of type A C. botulinum, a smaller panel of proteolytic and nonproteolytic type B, E, and F neurotoxin-producing Clostridia, and nontoxigenic organisms using an overnight incubation of toxin production medium. The assay appears to be an effective tool for large-scale screening of the food supply in the event of a botulinum neurotoxin contamination event.     

High-throughput assays for botulinum neurotoxin proteolytic activity: serotypes A, B, D, and F

Botulinum neurotoxins (BoNT) are zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. Because the paralytic effect of BoNT is a consequence of its enzymatic activity, selective inhibitors may be useful as drugs or as tools for further research. To expedite inhibitor discovery, we developed high-throughput, solid-phase protease activity assays for four of the seven BoNT serotypes: A, B, D, and F. Each assay consisted of a cleavable oligopeptide, based on the natural substrate sequence, labeled with fluorescein and covalently attached to maleimide-activated multiwell plates. Solutions of holotoxin or nontoxic catalytic domain of BoNT were incubated in substrate-coated wells, with or without test compounds, followed by transfer and assay of solubilized product in a multiwell fluorometer. Routine toxin concentrations ranged from 10 to 100 ng/ml, but concentrations as low as 2 ng/ml gave reproducible signals. The fluorescence assays were selective, gave very low background readings, and were stable upon prolonged storage. Using the nontoxic catalytic domain of BoNT A, we determined the relative inhibitory potencies of a family of structurally related pseudotripeptide compounds. Unlike previous methods, our assays did not employ antibodies or reverse-phase extraction steps, only well-to-well transfers, and were easily adapted to a high-throughput automated environment.     

Multiplex PCR Assay for Detection and Identification of Clostridium botulinum Types A, B, E, and F in Food and Fecal Material

Botulism is diagnosed by detecting botulinum neurotoxin and Clostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 10² cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10⁻² spore/g for types A, B, and F to 10⁻¹ spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.     

Gene arrangement in the upstream region of Clostridium botulinum type E and Clostridium butyricum BL6340 progenitor toxin genes is different from that of other types

The cluster of genes encoding the botulinum progenitor toxin and the upstream region including p21 and p47 were divided into three different gene arrangements (class I–III). To determine the gene similarity of the type E neurotoxin (BoNT/E) complex to other types, the gene organization in the upstream region of the nontoxic-nonhemagglutinin gene (ntnh) was investigated in chromosomal DNA from Clostridium botulinum type E strain Iwanai and C. butyricum strain BL6340. The gene cluster of type E progenitor toxin (Iwanai and BL6340) was similar to those of type F and type A (from infant botulism in Japan), but not to those of types A, B, and C. Though genes for the hemagglutinin component and P21 were not discovered, genes encoding P47, NTNH, and BoNT were found in type E strain Iwanai and C. butyricum strain BL6340. However, the genes of ORF-X₁ (435 bp) and ORF-X₂ (partially sequenced) were present just upstream of that of P47. The orientation of these genes was in inverted direction to that of p47. The gene cluster of type E progenitor toxin (Iwanai and BL6340) is, therefore, a specific arrangement (class IV) among the genes encoding components of the BoNT complex.     

Colloidal gold-based immunochromatographic assay for detection of botulinum neurotoxin type B

A rapid immunochromatographic assay was developed to detect botulinum neurotoxin type B (BoNT/B). The assay was based on the sandwich format using polyclonal antibody (Pab). The thiophilic gel purified anti-BoNT/B Pab was immobilized to a defined detection zone on a porous nitrocellulose membrane and conjugated to colloidal gold particles that served as a detection reagent. The BoNT/B-containing sample was added to the membrane and allowed to react with Pab-coated particles. The mixture was then passed along the porous membrane by capillary action past the Pab in the detection zone, which will bind the particles that had BoNT/B bound to their surface, giving a red colour within this detection zone with an intensity proportional to BoNT/B concentration. In the absence of BoNT/B, no immunogold was bound to the solid-phase antibody. With this method, 50 ng/ml of BoNT/B was detected in less than 10 min. The assay sensitivity can be increased by silver enhancement to 50 pg/ml. The developed BoNT/B assay also showed no cross reaction to type A neurotoxin (BoNT/A) and type E neurotoxin (BoNT/E).     

Development of a Combined Selection and Enrichment PCR Procedure for Clostridium botulinum Types B, E, and F and Its Use To Determine Prevalence in Fecal Samples from Slaughtered Pigs

A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymerase rTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70°C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30°C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 × 10³ spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.     

Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System

Clostridium botulinum subtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producing C. botulinum strain 657Ba at 100× lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenic C. botulinum expression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided in trans by the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed in Escherichia coli has indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of the in vitro, cellular, and in vivo activities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin.     

Characterization of the Genes Encoding the Botulinum Neurotoxin Complex in a Strain of Clostridium botulinum Producing Type B and F Neurotoxins

The organization of the clusters of genes encoding proteins of the botulinum neurotoxin (BoNT) progenitor complex was elucidated in a strain of Clostridium botulinum producing type B and F neurotoxins. With PCR and sequencing strategies, the type B BoNT-gene cluster was found to be composed of genes encoding BoNT/B, nontoxic nonhemagglutinin component (NTNH), P-21, and the hemagglutinins HA-33, HA-17, and HA-70, whereas the type F BoNT-gene cluster has genes encoding BoNT/F, NTNH, P-47, and P-21. Comparative sequence analysis showed that BoNT/F in type BF strain 3281 shares highest homology with BoNT/F of non-proteolytic (group II) C. botulinum whereas NTNH and P-21 in the type F cluster of strain 3281 are more similar to the corresponding proteins in proteolytic (group I) type F C. botulinum. These findings indicate diverse evolutionary origins for genes encoding BoNT/F and its associated non-toxic proteins, although the genes are contiguous. By contrast, sequence comparisons indicate that genes encoding BoNT/B and associated non-toxic proteins in strain 3281 possess a similar evolutionary origin. It was demonstrated that the genes present in the BoNT/B gene cluster of this type BF strain show exceptionally high homology with the equivalent genes in the silent BoNT/B gene cluster of C. botulinum type A(B), possibly indicating their common ancestry. Received: 30 March 1998 / Accepted: 21 May 1998